Food Safety and Preservation: Modern Biological Approaches to Improving Consumer Health [First edition] 0128149566, 9780128149560

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Food Safety and Preservation

Food Safety and Preservation Modern Biological Approaches to Improving Consumer Health Edited by Alexandru Mihai Grumezescu Alina Maria Holban

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1800, San Diego, CA 92101-4495, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom © 2018 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN 978-0-12-814956-0 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Wolff, Andre Gerhard Acquisition Editor: Osborn, Patricia Editorial Project Manager: Truesdell, Jaclyn A. Production Project Manager: Krishna Kumar, Divya Cover Designer: Pearson, Victoria Typeset by SPi Global, India

Contributors Hikmate Abriouel  University Jaen, Jaén, Spain Nurul Absar  University of Science and Technology Chittagong (USTC), Chittagong, Bangladesh Cristóbal N. Aguilar  Autonomous University of Coahuila, Saltillo, Mexico Naveed Ahmad  University of Agriculture, Faisalabad, Pakistan Thonas Alexiou  Novel Global Community Educational Foundation, Hebersham, NSW, Australia Muhammad Ali  Quaid-i-Azam University, Islamabad, Pakistan Gjumrakch Aliev  “GALLY” International Biomedical Research Consulting LLC, San Antonio, TX; University of Atlanta, Johns Creek, GA, United States; Institute of Physiologically Active Compounds Russian Academy of Sciences, Chernogolovka, Russia Mohammad AL-Mamun  University of Science and Technology Chittagong (USTC), Chittagong, Bangladesh Ali Asghar  University of Agriculture, Faisalabad, Pakistan Ghulam Ashraf  King Abdulaziz University, Jeddah, Saudi Arabia Mukerrem B.Y. Aycan  University of Erciyes, Kayseri, Turkey Nurgul K. Bakirhan  Ankara University, Ankara, Turkey Sonia Barberis  Universidad Nacional de San Luis, San Luis, Argentina; Instituto de Física Aplicada (INFAP), San Luis, Argentina Cristina Barcia  Universidad Nacional de San Luis, San Luis, Argentina Md. Latiful Bari  University of Dhaka, Dhaka, Bangladesh Samina Bashir  Quaid-i-Azam University, Islamabad, Pakistan Karsten Becker  University Hospital Münster, Institute of Medical Microbiology, Münster, Germany Nabil Benomar  University Jaen, Jaén, Spain Anupam Bishayee  Larkin Health Sciences Institute, Miami, FL, United States Baishakhi Biswas  University of Science and Technology Chittagong (USTC), Chittagong, Bangladesh Giuseppe Blaiotta  University of Naples Federico II, Portici, Italy Romina Brasca  Universidad Nacional del Litoral-CONICET, Ciudad Universitaria, Santa Fe, Argentina Shalini Chandel  Directorate of Mushroom Research, Solan, India Daniele Chieffi  National Research Council of Italy, Institute of Sciences of Food Production (CNR-ISPA), Bari, Italy

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Contributors Gyu-Sung Cho  Max Rubner-Institute, Kiel, Germany Tuhina Chowdhury  University of Science and Technology Chittagong (USTC), Chittagong, Bangladesh María J. Culzoni  Universidad Nacional del Litoral-CONICET, Ciudad Universitaria, Santa Fe, Argentina Reynaldo De la Cruz-Quiroz  Monterrey Institute of Technology, Monterrey, Mexico Nora Debattista  Universidad Nacional de San Luis, San Luis, Argentina K.D. Devi Nelluri  KVSR Siddhartha College of Pharmaceutical Sciences, Vijayawada, Andhra Pradesh, India Tuba Dilmaçünal  Süleyman Demirel University, Isparta, Turkey Adriana C. Flores-Gallegos  Autonomous University of Coahuila, Saltillo, Mexico Charles M.A.P. Franz  Max Rubner-Institute, Kiel, Germany Vincenzina Fusco  Institute of Sciences of Food Production, National Research Council of Italy (CNR-ISPA), Bari, Italy Antonietta M. Gatti  Health, Law and Science, Geneve, Switzerland Gunjan Goel  Jaypee University of Information Technology, Solan, India Héctor C. Goicoechea  Universidad Nacional del Litoral-CONICET, Ciudad Universitaria, Santa Fe, Argentina Amanda G. Gonçalves  Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil Avinash S. Hanumakonda  National Institute of Food Technology Entrepreneurship and Management (NIFTEM), Sonipat, Haryana, India Gassan Hodaifa  University of Pablo de Olavide, Seville, Spain Humberto M. Hungaro  Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil Irum Iqrar  Quaid-i-Azam University, Islamabad, Pakistan Jan Kabisch  Max Rubner-Institute, Kiel, Germany Mohammad A. Kamal  King Abdulaziz University, Jeddah, Saudi Arabia; Novel Global Community Educational Foundation, Hebersham, NSW, Australia Oya B. Karaca  Cukurova University, Adana, Turkey Ali T. Khalil  Qarshi University, Lahore, Pakistan Ali H. Khalil  University of Engineering and Technology, Peshawar, Pakistan Siva K. Korada  National Institute of Food Technology Entrepreneurship and Management (NIFTEM), Sonipat, Haryana, India Hakan Kuleaşan  Süleyman Demirel University, Isparta, Turkey Sevinc Kurbanoglu  Ankara University, Ankara, Turkey Dhananjaya B. Lakkappa  Jain University, Ramanagara, India Alain Largeteau  CNRS, Université de Bordeaux, ICMCB, Pessac, France Da-Yong Lu  Shanghai University, Shanghai, China Rishi Mahajan  Jaypee University of Information Technology, Solan, India Eduardo Martínez-Terrazas  Autonomous University of San Luis Potosí, Ciudad Valles, Mexico Stefano Montanari  Nanodiagnostics, Modena, Italy

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Contributors Muhammad Nadeem  University of Agriculture, Faisalabad, Pakistan Karen Nathiely Ramírez-Guzmán  Autonomous University of Coahuila, Saltillo, Mexico Leopoldo M. Nieto  University of Granada, Granada, Spain Celile A. Oluk  Eastern Mediterranean Agricultural Research Institute, Adana, Turkey Sibel A. Ozkan  Ankara University, Ankara, Turkey Maura Palmery  “La Sapienza” University of Rome, Rome, Italy Ilaria Peluso  Research Centre for Food and Nutrition (CREA-AN), Rome, Italy Mythili Prakasam  CNRS, Université de Bordeaux, ICMCB, Pessac, France Swathi Putta  Andhra University, Vishakhapatnam, India Héctor G. Quiroga  Universidad Nacional de San Luis, San Luis, Argentina Muhammad Atif Randhawa  University of Agriculture, Faisalabad, Pakistan Raffaella Reggi  “La Sapienza” University of Rome, Rome, Italy Raúl Rodríguez-Herrera  Autonomous University of Coahuila, Saltillo, Mexico Luciana Scotti  Federal University of Paraiba, João Pessoa, Brazil Marcus T. Scotti  Federal University of Paraiba, João Pessoa, Brazil Mahdi Seyedsalehi  School of Environment, Tsinghua University, Beijing, China Zabta K. Shinwari  Qarshi University, Lahore, Pakistan Sidrah  University of Agriculture, Faisalabad, Pakistan Willian C. Silva  University of Campinas, Campinas, São Paulo, Brazil Marcio R. Silva  Brazilian Agricultural Research Corporation (Embrapa Dairy Cattle), Juiz de Fora, Minas Gerais, Brazil Juan M. Talia  Universidad Nacional de San Luis, San Luis, Argentina Brenda N. Targino  Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil Navya Sree Thota  KVSR Siddhartha College of Pharmaceutical Sciences, Vijayawada, Andhra Pradesh, India Cristian Torres-León  Autonomous University of Coahuila, Saltillo, Mexico Bengi Uslu  Ankara University, Ankara, Turkey Sabina Yeasmin  University of Dhaka, Dhaka, Bangladesh Nagendra S. Yarla  City University of New York Medical School, New York, NY, United States

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Preface With the progress in food design and processing technologies, the research in preservation and safety has also advanced. One of the main focuses of the food industry is to obtain high amounts of foods with a particular quality. To achieve this goal, various techniques have been recently developed to ensure efficient and durable food preservation. Food safety includes numerous approaches, starting with regulatory constraints, food production and processing, to storage, contamination control, quality assurance, and preservation. Modern safety and preservation tools are investigating the biological means to ensure quality standards, avoid or detect food contamination and spoilage, and also to ensure prolonged and safe preservation. The emergence of innovative tools has allowed to control foods efficiently and to identify any biological or nonbiological factors, which may interfere with the quality and safety of food products. Such tools refer to better food management approaches, faster strategies used for food biosecurity, hazard detection, contamination control, investigation and prevention of food-borne diseases, and reduction of food degradation. The aim of this book was to bring together the most recent progress in the field of food safety by discussing the traditional and novel biological approaches to improving consumer health. Biopreservation and biocontrol tools are highlighted in this volume, which is mainly aimed at food scientists, microbiologists, biochemists, biotechnologists, industrial companies, and also any readers interested in learning about recent advances in the field of food safety and preservation tools. The volume contains 20 chapters prepared by outstanding authors from India, USA, Brazil, Russia, Australia, China, Pakistan, Turkey, Argentina, Mexico, Bangladesh, Italy, Spain, Germany, and France. Chapter 1, A Critical Appraisal of Different Food Safety and Quality Management Tools to Accomplish Food Safety, by Siva Kumar Korada and coworkers, gives a critical appraisal on the present situation and different food safety certification schemes and quality management standards in order to bring out quality products from a food industry. Expansion of the food supply chain takes place from the regional level to the international level; therefore, there must be a mutual understanding and an efficient communication between the government bodies, manufacturers, and customers. Chapter 2, Food Safety: Benefits of Contamination Control on Consumers’ Health, by Muhammad Atif Randhawa et al., tried to cover the most common possible causes of xxi

Preface food contamination, health effects, types of contaminants, and their entry points in foods. Increasing awareness will be effective to cope with food-related hazards. Chapter 3, Preemptive and Proactive Strategies for Food Control and Biosecurity, by Ali Talha Khalil and collaborators, discusses proactive and dynamic strategies with stringent biosecurity measures which are required to meet the challenges of Sustainable Development Goal 2 “zero hunger” adopted in recent years. Chapter 4, Validation of Analytical Methods for the Assessment of Hazards in Food, by Sevinc Kurbanoglu et al., discusses the validation methods and their acceptance criteria for the quality and safety of foods. The reliability of a method is determined by the validation results, where specificity, accuracy, precision, limit of detection and determination, and sensitivity and applicability are reported. Validation of a method can be achieved in three steps: by identification of appropriate and necessary validation parameters, design of experiments for parameter evaluation, and by determination of the acceptance criteria. Chapter 5, The Detection of Pesticide in Foods Using Electrochemical Sensors, by Nurgul K. Bakirhan and coworkers, presents sensitive and selective electrochemical sensors developed to control the level of pesticides in food samples. Electrochemical method is one of the analytical methods which can provide a sensitive, reliable, short-time analysis, with no pretreatment steps, and can be easily miniaturized and integrated compared to other analytical methods. Chapter 6, Multiway Calibration Approaches for Quality Control of Food Samples, by Romina Brasca et al., discusses the state-of-the-art multiway calibration methods used to perform quality control of food samples. Several examples of multidimensional chemometric methods developed to quantitate directly the analytes of interest in complex systems even in the presence of interferences, which is known as the “second-order advantage,” are commented. Chapter 7, Biocontrol as an Efficient Tool for Food Control and Biosecurity, by K. Nathiely Ramírez-Guzmán and coworkers, addresses the importance of standards and rules of food biosafety, and also the different strategies and specific characteristics of control in traditional food production, including relevant information on the emerging trends and alternatives for their conservation. Chapter 8, Foodborne Diseases and Responsible Agents, by Latiful Bari and Sabina Yeasmin, briefly describes the possible causative agents responsible for food-borne diseases. This chapter reviews the microbial agents, for example, viruses, bacteria, parasites, but also toxins, helminths, and unconventional infectious agents, such as prions; chemical contaminants including agrochemicals, pesticides, and veterinary drugs residues; and environmental contaminants (water, air, or soil pollution), which include dioxins, chlorinated biphenyls, furans, and heavy metals which may contaminate the environment as a result of industrial activities and thus entering into the food chain. Furthermore, food-processing-induced contaminants including acrylamide, 3-MCPD (3-monochloropropane-1,2-diol), etc.; migration from food packaging materials xxii

Preface (i.e., bisphenol A or phthalates from plastic materials, 4-methylbenzophenone from inks); presence or/and use of unapproved food additives and adulterants and intentional contaminants and cross-contamination during food preparation are responsible for food allergies and food intolerances and cause severe illness. Chapter 9, Challenges in Emerging Food-Borne Diseases, by Nelluri Kanaka Durga Devi and Navya Sree Thota, reveals the importance of the generation and maintenance of constructive dialogue and collaboration between the public health, veterinary and food safety experts and multi-pathogen expertise to monitor the changing trends of well-recognized diseases and also to detect emerging pathogens in a timely manner. Chapter 10, Opportunistic Food-Borne Pathogens, by Vincenzina Fusco and coworkers, provides an overview of the main opportunistic pathogens occurring in foods and the most promising strategies to control their occurrence in food products. Chapter 11, Food Poisoning and Intoxication: A Global Leading Concern for Human Health, by Mohammad AL, Mamun et al., discusses the main food poisoning and intoxication types, causing agents, and common occurring conditions. The main preventive options are also presented. Chapter 12, Staphylococcal Food Poisoning, by Vincenzina Fusco et al., provides an overview on the staphylococcal food poisoning and its causative factors. Culture-dependent and culture-independent methods to detect coagulase-positive cocci and the most attractive perspectives for future research to ensure the identification, quantitative detection, and typing of these important pathogenic bacteria are described. Moreover, classical and advanced methods to detect and quantify staphylococcal enterotoxins are provided in this chapter. Chapter 13, Campylobacter: An Important Food Safety Issue, by Willian Cruzeiro Silva and collaborators, organized a general overview on Campylobacter as a food-borne pathogen, including the characteristics of this bacterial group, epidemiological aspects of the disease, contamination sources, analytical methods, and perspectives for control and prevention. Chapter 14, Food Contamination: From Food Degradation to Food-Borne Diseases, by Antonietta M. Gatti and Stefano Montanari, discusses diverse diseases of the digestive system that can be induced by food and their current investigation methods. Through direct investigations on food samples by means of an environmental scanning electron microscope coupled with the X-ray microprobe of an energy-dispersive system, “foreign bodies,” without any nutritive effect but potentially toxic, are looked for and characterized by their size, shape, and chemistry. Also, in this chapter the correlation and the ensuing mechanisms between the ingestion of those contaminants and the development of a disease are shown. Chapter 15, A Review on the Implications of Interaction Between Human Pathogenic Bacteria and the Host on Food Quality and Disease, by Rishi Mahajan et al., emphasizes on understanding how fresh products can be an important source of food-borne diseases. Since plants can represent an important food biocontamination source and vector of infectious agents, interactions between the pathogen and the plant host, the evaluation of human health risk by the presence of cross-domain pathogens, and influence of ecological xxiii

Preface factors on survival and growth of contaminant pathogens on fresh food products can lead to better management of food-borne infectious diseases. Chapter 16, Novel Strategies for the Reduction of Microbial Degradation of Foods, by Tuba Dilmaçünal and Hakan Kuleaşan, discusses the various methods to prevent microbial contamination and food degradation such as the application of proper storage conditions, using advanced processes and packaging materials, rapid detection of microbial contaminants with new technologies which may extend the shelf life and quality of fresh and processed foods, fruits, and vegetables and also reduce postharvest losses. Chapter 17, Relevance and Legal Frame in Novel Food Preservation Approaches for Improving Food Safety and Risks Assessment, by Gassan Hodaifa and Leopoldo Martínez Nieto, presents the main types of pesticides used as preservation agents, processing factors, and the main degradation agents associated with preservatives, by exemplifying the olive oil production situation. The current legal frame of the preservation agents used and the use of preservation agents to pest control and its transfer to food are also discussed here. Chapter 18, The Current Approaches and Challenges of Biopreservation, by Celile Aylin Oluk and Oya Berkay Karaca, deals with the current biopreservation technologies used in the food industry, empathizing on the most recent challenges. Biopreservation is one of the alternative food preservation technologies that improves product shelf life, hygienic quality, and minimizes the impact on the sensory and nutritional properties of perishable food products. Technologies involving the use of lactic acid bacteria, bacteriophages, bacteriocins, and lysozyme-based products with antibiofilm properties are discussed. Chapter 19, Modern Preservation Tools Through Packaging for High Hydrostatic Pressure Processing, by Mythili Prakasam and Alain Largeteau, describes the various characteristics of the packaging materials required for high hydrostatic pressure and their great impact on the food industry, empathizing on the new developments and applications in food preservation and safety. Chapter 20, Natural Food Preservatives Against Microorganisms, by Sonia Barberis et al., reviews the natural components found in different sources to be used as potential natural food preservatives. The merging biotechnological means used for natural food preservation, such as nanoparticles, bacteriophages and endolysins, bacterial Quorum sensing inhibitors, and novel phytoproteases are also dissected in this chapter. Finally, the regulatory status, activity validation methods, toxicological and allergenic effects, antimicrobial–food component interactions, and production costs are also revealed, focusing on the implementation of the natural preservatives in food industry.

Alina Maria Holban University of Bucharest, Bucharest, Romania

Alexandru Mihai Grumezescu University Politehnica of Bucharest, Bucharest, Romania

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CHAPTE R 1

A Critical Appraisal of Different Food Safety and Quality Management Tools to Accomplish Food Safety Siva K. Korada⁎, Nagendra S. Yarla†, Swathi Putta‡, Avinash S. Hanumakonda⁎, Dhananjaya B. Lakkappa§, Anupam Bishayee¶, Luciana Scottiǁ, Marcus T. Scottiǁ, Gjumrakch Aliev#,⁎⁎,††, Mohammad A. Kamal‡‡,§§, Da-Yong Lu¶¶, Mukerrem B.Y. Aycanǁǁ, Raffaella Reggi##, Maura Palmery##, Ghulam Ashraf‡‡, Thonas Alexiou§§, Ilaria Peluso⁎⁎⁎ *National Institute of Food Technology Entrepreneurship and Management (NIFTEM), Sonipat, Haryana, India †City University of New York Medical School, New York, NY, United States ‡Andhra University, Vishakhapatnam, India §Jain University, Ramanagara, India ¶Larkin Health Sciences Institute, Miami, FL, United States ǁFederal University of Paraiba, João Pessoa, Brazil #“GALLY” International Biomedical Research Consulting LLC, San Antonio, TX, United States **University of Atlanta, Johns Creek, GA, United States ††Institute of Physiologically Active Compounds Russian Academy of Sciences, Chernogolovka, Russia ‡‡King Abdulaziz University, Jeddah, Saudi Arabia §§Novel Global Community Educational Foundation, Hebersham, NSW, Australia ¶¶Shanghai University, Shanghai, China ǁǁUniversity of Erciyes, Kayseri, Turkey ##“La Sapienza” University of Rome, Rome, Italy ***Research Centre for Food and Nutrition (CREA-AN), Rome, Italy

1.1 Introduction Food safety always talks about the safe food that should be free from unintentionally added components like contaminants (physical, chemical, and biological contaminants) or intentionally added components like adulterants. Both adulterants and contaminants can make the food unsafe for human consumption and can cause health hazards (Aggarwal, n.d.). Foodborne threats like microbial and chemical contaminants can continuously associate with public health risks and can lead to a decrease in food trade with significant financial losses and social costs.1 Controlling of entry of contaminants into the food chain is a difficult task. It may enter at any point from the harvesting stage to the consumption stage. Several issues are responsible for contaminant/hazard entry into a food production process flow, which include poor sanitary 1

http://www.fao.org/fileadmin/templates/agns/pdf/factsheets/improving_food_safety_along_food_chain.pdf.

Food Safety and Preservation. https://doi.org/10.1016/B978-0-12-814956-0.00001-9 © 2018 Elsevier Inc. All rights reserved.

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2  Chapter 1 practices, poor handling practices, poor manufacturing practices, poor transportation and storage practices, and usage of contaminated primary commodities like raw materials, the absence of proper control and preventive measures at each and every stage of the food chain, etc.2 Supplying/exporting of poor quality and contaminated food to foreign countries may lead to the cancellation of consignments and it creates a big barrier to trade, reflecting decreases in foreign exchange. In order to facilitate the global trade of Indian products and making their availability in major leading retailer chains, food safety industry standards play a key role in producing the product safely in a consistent manner. In 2006, Food Safety and Standard Authority of India (FSSAI)—a regulatory body under the Ministry of Human Health and Welfare—came into force to implement the science-based standards to provide safe food throughout the food supply chain for customer consumption. Initially India is supported by different sector-based acts. After implementation of FSSAI everything got canceled and was laid on a single platform.3 It is observed that most of the food business operators are not aware of the different standards of demand by the exporting countries and even about our Indian standards. In a survey conducted by FICCI recently it came to be known that nearly 30% of food business operators and industry holders are unaware of compulsory implementation of FSSAI standards to run a business (The Financial Express, 2010). This information is an attempt to create awareness among food business operators, primary producers, manufacturers, and retailers related to the different food safety certification schemes and standards to bring out safe and quality products from a food industry.

1.2  The Emerging Scenario of Contaminants and Residues Related to Food Safety These days food safety has already become a global concern. Chemical residues and biological contaminants are constantly pushed to cause food safety risks and are also continuously disrupting economic growth and international trade. Food safety regulators all over the globe have shown an increased concern toward antibiotic residues, pesticides, and biological contaminants because of rampant of incidents happened during food exporting. For instance, a recent issue happened to be the ban on import of Indian Alphonso mango in the European Union (EU) temporarily because of pest infestation which resulted in the development of trade barriers for exporters (EU bans Indian Alphonso mangoes, 4 vegetables from May 1, 2014). Another case of ban happened is with the exporting of Indian honey to the EU because of high doses of antibiotic application for the purpose of beekeeping which resulted in high levels of antibiotic residues in honey compared to the set standards. Upon shipment to EU and followed by inspection with those food safety investigators it has been found that the presence of cross-contaminated antibiotics in honey is at higher 2

http://www.wpro.who.int/foodsafety/documents/docs/English_Guidelines_Food_control.pdf. http://www.fssai.gov.in/AboutFSSAI/introduction.aspx.

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A Critical Appraisal of Different Food Safety  3 levels compared to those EU norms. In addition, they also found lead contamination in that respective shipment. Hence, it leads to the cancellation of consignment and now EU is showing no more interest in accepting Indian honeys (Schneider, 2011). Similarly, after the ban of Indian Alphonso mango temporarily by the EU another product under examination for banning by the importers is Indian green chilies by Saudi Arabia. They recently expressed their concern about the quality products they are importing from India to Agricultural and Processed Food Products Export Development Authority (APEDA) stating that as Indian green chilies are contaminated with unacceptable levels of pesticide residues, which is not complying with their norms (OUR BUREAU, 2014). The global presence of pesticide and antibiotic residues are always under stringent regulation. In India, FSSAI is the technical standard setting body and it establishes values of maximum residue levels (MRLs) with the help of the Medical Toxicology Unit of Central Insecticides Board & the Registration Committee (CIB&RC). Once the values of MRLs are established, they will be incorporated into the Food Safety and Standard Regulation (Gain Report. Global Agricultural Information Network. USDA Foreign Agricultural Service. IN1104, 2011). Before exporting the products to countries like the EU, Australia, United States, and other developed countries several quality evaluations have to be undertaken at the direction of their specified quality limits (Smriti et al., 2012). Understanding the exporting countries quality demands and requirements will help us in a smooth trade facility. A notable example happened with Indian snack products, the US Food and Drug Administration (FDA) had rejected the number of snack product imports from India because of high level contaminant presence (News Desk, 2015). Biological contamination is also a major factor in disrupting international trade, decreasing foreign exchange rate, and establishing a barrier between the trading countries. The World Health Organization’s (WHO) recently released data show that contaminated food is associated with deaths of nearly 2 million people annually (WHO campaigns, 2015). Salmonella is a pathogen which causes vomiting, nausea, and diarrhea in humans and its contamination is frequently associated with fresh produces (Lawley, 2013). A case of under scan of the ban is linked with Indian betel leaves because of consistent reports of salmonella contamination since 2011 by the EU Rapid Alert System for Food and Feed (RASFF). China occupies the first place in receiving RASFF notification in this regard, and now India is in the second position.4 Before shipping the produce to exporting countries it should be ensured that strong microbiological compliance is required in accordance with the demands of the exporting country. Hence, it is recommended that before forwarding a shipment to foreign countries it is important to understand the quality norms set by the exporters, their country regulatory requirements, product hygiene, quality and choosing the right scientific testing service to know the analytical status of the commodity and microbial compliance, then only it allows the food industry to supply a food product with high-quality standards that constantly meet their demands. 4

http://timesofindia.indiatimes.com/world/uk/Now-Indian-paan-leaves-under-EU-scanner/articleshow/36520487.cms.

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1.3  Different Food Safety Certification Schemes, Quality Systems, and Other Popular Standards Globalization of a food supply chain requires an organization to be certified by different international standards.5 Crafting the following basic food safety requirements and standards will help us in building the technical proficiency in food safety risk management and improves the quality. The following are the generic controls and standards considered moderately to build quality into the product in order to improve food safety and increase consumer confidence toward the brand: 1. 2. 3. 4. 5. 6. 7.

Prerequisite programs (PRPs). Hazard analysis and critical control point (HACCP). Quality management systems (QMS): ISO 9001. Food safety management systems (FSMS): ISO 22000. Laboratory management system: ISO 17025. Retailer standards. Technical barrier for exporting.

1.4  Prerequisite Programs These are the basic requirements for a food-related sector. Their scope and concept is wide and they cover and integrate a set of activities such as good manufacturing practices (GMP), good hygienic practices (GHP), standard operating procedures (SOP), (sanitation standard operating procedures (SSOPs), and good handling practices (GHP) to ensure the safety of food. All these sets of practices and activities are a part of the quality assurance system. Before implementing a HACCP system in a food industry, they have to strengthen their PRPs effectively at a place.6 Following these practices and activities will ensure them in producing the product consistently in a controlled manner.

1.4.1  Hazard Analysis Critical Control Point (HACCP) The concept of HACCP had a history of nearly half-a-century. Since its launch by the three different organizations which include NASA (U.S. National Aeronautics Space Administration—An International Space Station), Natick Army Lab, and Pillsbury group company it has combinely undertaken a project aiming to produce safe food for space travelers. Initially it consists of three principles and later it has undergone constant improvements in the 5

http://www.sgs.com/~/media/Global/Documents/Brochures/SGS%20CTS%20Food%20Brochure%20Hyb%20 EN%202013.pdf. 6 http://www.foodsafetyindia.com/2009/12/prerequisite-programmes-for-haccp.html.

A Critical Appraisal of Different Food Safety  5 system and finally it came up with seven principles. Again in 1993, the Codex Alimentarius Commission has given its first HACCP standard and also launched the definition of HACCP worldwide. Now it has been applied globally by many food industries because of its importance in managing food safety risks (John, n.d.). The application of HACCP is multifactorial. It can be applied to any sector from the farm to folk. It involves identifying and controlling of possible hazards in a food chain and analyzes the risks associated with food safety by performing risk assessments. Risk assessment will define the severity of the hazard by calculating the likelihood of the hazard occurring and its consequences in terms of its severity. Table 1.1 demonstrates about the seven principles of HACCP, which could lead the successful production of food by minimizing health causing hazards. The ultimate goal of implementing HACCP in food industries is to reduce/control the food-borne illness risk factors such as contaminants, hazards in a food chain and to ensure the quality requirements of the product (A Training Manual on Food Hygiene and the Hazard Analysis and Critical Control Point (HACCP) System, n.d.). Table 1.1: Seven principles of HACCP S. no. 1. 2.

3.

4.

5. 6.

7.

Principle

Description

• Identify and collect all major hazards (biological, chemical, and physical) in a food chain which are significant for food safety Determine critical control • In order to control the hazard CCP identification is needed. It is a point (CCP) step to control/eliminate/bringing down the identified hazard to an acceptable range. • Not each and every step associated with hazard is considered as CCP. For to decide whether it is a CCP or not, it has to undergo the logical decision process (contain sets of logical questions we have to answer it in a sequential mode). Establish critical limit for • A critical limit must be a maximum/minimum measurable value at critical control point which a hazard must be controlled at a CCP to prevent/eliminate/ bringing down to an acceptable level the occurrence of a food-safety hazard. Established control point shall be scientifically sound and validated. Establishing ways to • Once limits are established for CCP, there must be continuous monitor CCP monitoring of a person is needed to know whether those limits working properly or not in a food chain process. Establishing corrective • React upon CCP limit deviation and take necessary action before actions causing food safety risk. Establish verification • Cross verifies the CCP limits in its functionality by applying procedures procedures, steps, tests, and other experiments to determine a HACCP plan working efficiently. Establish record and • Maintain proper record keeping and documentation wherever it is documentation required to make the system more effective. For instance conducted tests, experimental results, and validation report keeping, CCP log sheets, checklists, designed HACCP plans, etc. Conduct hazard analysis

Source: A Training Manual on Food Hygiene and the Hazard Analysis and Critical Control Point (HACCP) System. A Report of a FAO Information Division. ISBN 92-5-104115-6.

6  Chapter 1

1.4.2  Quality Management Systems (QMS): ISO 9001 This system was designed to meet the customer need, satisfaction, and requirements in terms of quality. In order to improve the performance of an organization, continual improvement is considered as a key quality parameter which will bring you out when the organization is implemented with QMS. If an organization is supported with ISO 9001 it means that it is committed to deliver quality service to the customer.7 Generally, ISO 9001:2008 sets out quality requirements according to the customer expectation and requirements. Recently, ISO 9001:2008 underwent revision and launched ISO 9001:2015 with the latest upgradations in order to maintain consistency and make the organization more competent.8

1.4.3  Food Safety Management Systems (FSMS): ISO 22000 ISO standards help organizations by promoting the quality and safety of food and also by increasing the efficiency of the food supply chain from farm to table by managing or eliminating food safety risks. Globalization of food chains leads to increased demands in the safety and quality of the supplied food from farm to consumer. Consequences of exporting unsafe foods are more severe and are concerned with brand reputation and financial loss due to food recalls. World-wide recognized requirements and international standards are required these days in order to guarantee product safety along the food chain. Food safety management standards help in identifying and controlling the potential hazards in the food chain. In order to ensure food safety, ISO 22000 merged with HACCP and with other preventive procedures.9,10 ISO 22000 standards undergo reconsideration and revision at least once in every 5 years. Table 1.2 demonstrates about various document associated with ISO family, which briefs about their individual document. Meanwhile, the ISO working group collects the information about the existing gaps by considering experts, modifications, latest updates, and requirements which will be incorporated in the next revision.11

1.4.4  Laboratory Management System: ISO/IEC 17025:2005 Food safety parameters and analytical challenges involved in the ascertaining food safety risks. Performing and documenting analytical studies is one of the major requirements to ensure food safety by continuously meeting the country’s own regulatory requirements and the exporting country’s regulatory demands, crossing obstacle trade barriers like SPS (Sanitary and Phytosanitary Measures Agreement), and by giving food safety assurance to the consumer (Aggarwal, n.d.; Agreement on the Application of Sanitary and Phytosanitary  7

http://www.sgs.com/~/media/Global/Documents/Brochures/SGS_SSC_NG_ISO_9001_web_LR.pdf. http://www.iso.org/iso/iso_9001_-_moving_from_2008_to_2015.pdf.  9 http://www.qualitydigest.com/magazine/2009/apr/article/gfsi-food-safety-standards.html. 10 http://www.iso.org/iso/home/standards/management-standards/iso22000.htm. 11 http://www.insidestandards.com/iso-22000-revision-underway/.  8

A Critical Appraisal of Different Food Safety  7 Table 1.2: Overview of ISO 22000-associated documents S. No ISO 22000 Family 1

ISO 22000:2005

2

ISO 22004:2014

3

ISO 22005:2007

4

ISO/TS 22002–1:2009

5

ISO/TS 22002–2:2013

6

ISO/TS 22002–3:2011

7

ISO/TS 22002–4:2013

8

ISO/TS 22003:2013

Importance • This document contains specific requirements for an organization to implement food safety management systems • It also addresses the organizations ability about how they are committed toward controlling food safety hazards in order to produce safe food for human consumption • This document contains guidance and assistance for application and fulfillment of ISO 22000 requirement • This document is supported with some basic requirements and steps that helps in establishing effective traceability system along the feed and food chain • This document contains detailed technical information about how to implement, design, and maintain prerequisite programmes (PRPs) for food manufacturers • Helps in managing food safety risks along food supply chain • Addresses the requirements in compliance to ISO 22000:2005 • This document contains detailed technical information about how to implement, design, and maintain prerequisite programmes (PRPs) in catering field • Helps in managing food safety risks • Also fulfill the requirements in compliance to ISO 22000:2005 • These technical specifications assist the farmers in implementing and designing pre-requisite programs (PRPs) to control their farming by food safety hazards • It assists in building and designing prerequisite programs (PRPs) for food packaging industries by managing food safety hazards • Contain in detailed technical information and procedures about how to conduct audit and gain certification from bodies by fulfilling ISO 22000 requirements

Source: http://www.iso.org/iso/home/standards/management-standards/iso22000.htm.

Measures (SPS) and Agreement on Technical Barriers to Trade (TBT), 2000). Meeting these demands need state-of-the-art laboratory infrastructure. In the quality control division, testing of a product and calibration and validation of the equipment and parameters are the two key important aspects for successful quality assurance. To increase the confidence toward their product testing, most of the suppliers, retailers, and even food manufacturers supported by the quality lab always choose testing and calibration results from laboratories which have ISO/ IEC 17025:2005 accreditations. ISO/IEC 17025 is a general requirement for a testing and calibration laboratory to show their technical capability and competency toward their analyses (ISO/IEC 17025, 2005). Data or results generated by ISO 17025-accredited laboratories are more readily accepted because of the accuracy in their results (Honsa and McIntyre, 2003). For a laboratory to undergo formal recognition with ISO17025:2005, it has to maintain the documented quality management

8  Chapter 1 system, technical management in order to show their competency and administrative operations (ISO/IEC 17025, 2005). In India, the National Accreditation of Board of Laboratories (NABL) is the third-party accreditation body. If any laboratory wants to be accredited by NABL, it has to prove compliance with the entire clause of ISO 17025:2005.12 Moreover, international trade always demands laboratory-based data for their exporting product and also at the same time the laboratory has to be recognized by the importing country to meet their technical compliances. This concern leads to the implementation of the Mutual Recognition Arrangements (MRAs) between the accreditation bodies of other countries. Hence, MRAs will act as a channel for international trades and also increase the cross-border stakeholder confidence and the acceptance of recognized compliance assessment bodies (Complying with ISO 17025, 2009).

1.5  Retail Standards In a food supply chain generally the food product moves from manufacturers to distributors, distributors to wholesalers and from wholesalers to retailers and finally from a retailer the product will directly reach to the consumer.13 During the journey, the product may lack its safety, quality, and hygiene due to extreme storage conditions and improper handling results in the entry of various kinds of contaminants which in turn influences the retailer’s brand value. By keeping this point in view, the retailers started demanding their food product manufacturers/suppliers to follow certain food safety and quality standards in order to bring out safe and quality food and they also started auditing toward their respective manufacturers/suppliers, so that they believe in their abilities to meet their needs and requirements. These days compared with the suppliers, the retailers are becoming more powerful and thus these kinds of reasons lead to the retailers to create their own brand standard to guarantee food safety across the supply chain and to preserve their own brand forever in the market. To meet their requirements, the Global Food Safety Initiative (GFSI), a nonprofit organization, was established in 2000 by a group of food safety experts from different disciplines to increase the safety of food and enhance customer trust.14 The GFSIrecognized standards are the British Retail consortium (BRC), the Safe Quality Food (SQF) code, the International Feautured Standard (IFS), FSSC 22000, Global GAP, and many more.15

1.5.1  British Retail Consortium The BRC is an important global food safety standard intended to design for suppliers to the retail sector to ensure safe product delivery to the final consumer. It released its first technical standard version in 1998 for their food suppliers.16 This standard works in close combination with Hazard Analysis Critical Control Point (HACCP) principles in order to manage safety, 12

http://www.nablindia.org/index.php?option=com_content&view=article&id=137&Itemid=73. http://smallbusiness.chron.com/differences-between-wholesalers-distributors-retailers-30836.html. 14 http://www.mygfsi.com/. 15 https://en.wikipedia.org/wiki/Global_Food_Safety_Initiative. 16 https://en.wikipedia.org/wiki/British_Retail_Consortium. 13

A Critical Appraisal of Different Food Safety  9 hygiene, and quality of products.17 Initially, these standards were followed by the United Kingdom (UK) only, but now these standards are applied all over the world. BRC covers its standard in four different aspects. (1) BRC Global Standard for Packaging and Packaging Materials (2) BRC Global Standard for Storage and Distribution (3) BRC Global Standard for Food Safety, and (4) BRC global standard for consumer products.16

1.5.2  The Safe Quality Food (SQF) Code This food safety management tool is initiated to minimize food safety-related risks along the food supply chain and to provide a one-stop solution for both suppliers and buyers. It is also a GFSI-recognized standard and it utilizes HACCP-based approaches to attain the quality and safety of food.18 The concept of SQF is not new, it has nearly 20 years of history. It was initially introduced by the Australian Department of Agriculture and was later taken over by the US Food Marketing Institute (FMI) and now it became an internationally recognized one. This scheme existed in two codes which include SQF 1000 and SQF 2000. SQF 1000 code targets only primary producers, especially farmers, whereas SQF 2000 code targets all sectors in a food supply chain to attain product quality.18,19

1.5.3  International Featured Standard (IFS) If you go back to the history of the origin of this standard in the beginning most of the retailers and wholesalers developed their own quality assurance systems to improve their product quality standard. Later increased consumer concern toward safety and quality of products and increasing pressure of legal requirements led to the development of a unique standard. Hence, this standard was designed to support and to satisfy the consumer need and also meet the key objectives of retailers. Initially IFS scheduled their entry as IFS publication, later they came up with a standard and now it is covering all major activities like IFS Food, IFS Broker, IFS Logisics, IFS Cash and Carry Wholesales and IFS Packaging Guidelines, etc. Attainment of this standard will reduce the burden of audits, time and cost saving for both suppliers, retailers, and wholesalers.20

1.5.4  Food Safety System Certification (FSSC 22000) FSSC 22000 is a recently evolved standard after incorporating additional requirements to the existing ISO 22000 standards. As ISO 22000 is not a GFSI recognized one because it lacks some requirements related to the prerequisite programs to become a benchmark standard. In 2010, the upgraded version of ISO 22000 food safety management system was launched as 17

http://www.bureauveritas.co.uk/35a8b300495b9e939af6dbce4d2e6585/SERVICE+SHEET_British+retail+ consortium+global+standards.pdf?MOD=AJPERES&CACHEID=35a8b300495b9e939af6dbce4d2e6585. 18 http://www.qualitydigest.com/magazine/2009/apr/article/gfsi-food-safety-standards.html. 19 http://www.australianoilseeds.com/__data/assets/pdf_file/0019/946/Fast_Facts_4_- Quality_Assurance.pdf. 20 http://www.ifs-certification.com/index.php/en/retailers-en/introduction-to-ifs/ifs-history.

10  Chapter 1 FSSC 22000, which includes additional document concerned with the prerequisite programs (PAS 220—A Publicly Available Specification) and completed the requirements and now it is recognized by the GFSI-certified standard upon full-fulfillment of the requirement.21 The advantage of adopting this certification scheme is that it utilizes the typical management system to control food safety hazards thereby attaining food safety. Food manufacturers and some large retailers encourage only GFSI-recognized schemes to get globalwide recognition and acceptance toward their product.22 FSSC 22000 Integrates · Integrates PRPs, seven principles of HACCP and ISO 9001(Quality Management Standard) ISO 22000

PAS 220

· Lacks some pre-requisite programmes for fulfilment of GFSI recognized standard

· Contain additional pre-requisite requirements to full fill the ISO 22000 lacking and to be recognised as GFSI · Fills the quality gap · Now it is replaced by ISO/TS 22002-1 · Helpful for food manufacturers and retailers

1.5.5  Global GAP Day-to-day consumer concern toward product quality and safety has been increasing rapidly. Legislative bodies also started forcing farmers to produce agricultural products with less agrochemical usage in an environmentally sustainable manner. Countries like the EU, Japan, United States, New Zealand, and many other developed ones have their own residue limits for chemical residues, pesticides, and environmental contaminants. Constantly meeting each country’s demand is a difficult task. It is well known that every food-related raw material started from the farm level only. Implementing and attaining good agriculture practices will ensure to manage those food safety risks at the farm level. Implementing standards like GLOBAL GAP will assure your farm that the produce is at a tolerable level and also increases the market access as well as trade access in an assured manner. Global GAP stands for Global Good Agricultural Practices. It targets only primary producers to produce better quality of raw materials in an eco-friendly manner. It is an initiative taken by the major European retailers, supermarket holders, and suppliers, who developed the scheme aiming 21 22

http://trade.ec.europa.eu/doclib/docs/2011/april/tradoc_147836.pdf. http://www.22000-tools.com/what-is-fssc-22000.html.

A Critical Appraisal of Different Food Safety  11 to produce safe and quality output from the fields. Initially, it is named as EUREPGAP, later in 2007 they changed the name to GLOBAL GAP because of its global wide acceptance (GLOBAL G.A.P., 2015).23,24

1.6  Technical Barriers for Exporting Technical obstacles are created in order to restrict the shipment of unsafe food to another country. As per the World Trade Organization (WTO) agreements in order to preserve the health and safety of humans, animals and plants and to safeguard the environment, they have undertaken some steps like the Technical Barrier Trade (TBT) and Sanitary and Phytosanitary (SPS) measures while the product crosses national borders. The aim of these barriers is to facilitate smooth trading without any obstacles.25 TBT agreement is concerned with strong technical compliance toward the mandatory standards they have adopted and it must be supported with scientific information for their compliance toward confirmatory assessments, whereas SPS agreements deal with the restriction of unsafe imported agricultural products in order to protect the health of plants, animals, and humans. If any country wishes to import agricultural products it has to undergo strong compliance toward their national SPS measures (Andrew, edewa. WTO TBT, and SPS AGREEMENTS. United Nations Industrial Development Organization, n.d.).

1.7 Conclusions Implementing standards, strict adherence to government food safety regulations and adapting quality assurance divisions in food industries allows us to run the food business successfully by managing the food safety risks and thereby increases the food manufacturer’s ability and confidence to produce a quality product consistently with increased quality and safety. Meeting the exporting country regulatory requirements, our Indian products can enhance the competitiveness in the worldwide market. Attaining standards through certifications will allow the organizations to meet food safety and quality requirements, thereby increase the customer trust and brand values. Seeking the help of inspection bodies will help us in identifying and controlling hazards through their highly experienced audits.

References A Training Manual on Food Hygiene and the Hazard Analysis and Critical Control Point (HACCP) System. A Report of a FAO Information Division. ISBN 92-5-104115-6. Aggarwal, M., Food safety parameters and analytical challenges involved in ascertaining food safety risks. Presentation Available at: http://www.groenecirkels.nl/web/ file?uuid=6f084364-4ccb-422f-86de-f644b38363d3&owner=07cf9470-9ddf-4600-a042-25a2c8bcf791. 23

http://www.globalgap.org/uk_en/who-we-are/about-us/history/. http://www.bureauveritas.com/468c298047e94b9cb47ebcafdca0d0a3/GLOBALGAP. pdf?MOD=AJPERES&CACHEID=468c298047e94b9cb47ebcafdca0d0a3. 25 https://www.wto.org/english/tratop_e/tbt_e/tbt_e.htm. 24

12  Chapter 1 Agreement on the Application of Sanitary and Phytosanitary Measures (SPS) and Agreement on Technical Barriers to Trade (TBT), 2000. A Resource manual on multilateral trade negitiations on agriculture. Food and Agriculture Organization of the United Nations, Rome. Available at: http://www.fao.org/docrep/003/x7354e/ x7354e02.htm. Andrew, edewa. WTO TBT & SPS AGREEMENTS. United Nations Industrial Development Organization, www. unido.org. http://www.tradecom-acpeu.org/fileadmin/user_upload/05_E-Library/SPS_and_TBT/Work-shop_ for_Kenyan_Diplomats_Module_SPS_and_TBT.pdf. Complying with ISO 17025, 2009. A practical guidebook for meeting the requirements of laboratory accreditation schemes based on ISO 17025:2005 or equivalent national standards. United Nations Industrial Development Organization, Vienna. Available at: https://www.unido.org/fileadmin/user_media/Publications/Pub_free/ Complying_with_ISO_17025_A_practical_guidebook.pdf. EU bans Indian Alphonso mangoes, 4 vegetables from May 1, 2014. The Hindu, 16:37 IST | updated: April 28, 2014 16:37 IST London. April 28, 2014. Available at: http://www.thehindu.com/news/international/world/eubans-indian-alphonso-mangoes-4-vegetables -from-may-1/article5956482.ece. Gain Report. Global Agricultural Information Network, 2011. USDA Foreign Agricultural Service. IN1104, January. Available at: http://agriexchange.apeda.gov.in/MarketReport/Reports/India_regulation_on_MRL.pdf. GLOBAL G.A.P (2015, October 15). In Wikipedia, The Free Encyclopedia. Retrieved 11:26, November 11, 2015, From https://en.wikipedia.org/w/index.php?title=GLOBALG.A.P&oldid=685859102. Honsa, Julie D, Deborah A. McIntyre (2003). “ISO 17025: Practical Benefit of Implementing a Quality system. J. AOAC Int. 86(5): 1038–1044. Retrieved 28 Febuary 2012. ISO/IEC 17025, 2005. General requirements for the competence of testing and calibration laboratories. John G. Surak . The Evolution of HACCP. Manufacturing & Distribution. February 1, 2009.Available at: http:// www.foodqualityandsafety.com/article/the-evolution-of-haccp/. Lawley, R., 2013. Salmonella. Food Safety Watch 2. Available at: http://www.foodsafetywatch.org/factsheets/ salmonella/. News Desk, 2015. FDA rejects several snack products from India for contaminants. Food Safety News. June 16. http://www.foodsafetynews.com/2015/06/fda-rejects-several-snack-products-from-india-for-contaminants/#. VlP_NBGqqko. OUR BUREAU, 2014. Saudi Arabia bans Indian green chilli. THE HINDU. Business Line. June 2. http://www. thehindubusinessline.com/markets/commodities/saudi-arabia-bans-indian-green-chilli/article6075698.ece. Schneider, A., 2011. Asian Honey, Banned in Europe, Is Flooding U.S. Grocery Shelves. In: Food Safety News. August 15. Available at: http://www.foodsafetynews.com/2011/08/honey-laundering/#.VlDQ2RGqqko. Smriti S, Rajavally Prem, V.S. Rawat, Manjeet Aggarawal, R.K. Khandal. 2012. Food Safety Issues Related to Residues and Contaminants:Emerging Scenario in India. Food and Beverage News. Friday, 06, 2012, Available at: http://www.fnbnews.com/article/detnew.asp?articleid=31150§ionid=23. The Financial Express, 2010. Almost 30% of food industry unaware of safety standards: Ficci. 14th June. WHO Campaigns, 2015. World Health Day 2015. Available at: http://www.who.int/campaigns/world-healthday/2015/event/en/#. Food safety 7 April 2015.

CHAPTE R 2

Food Safety: Benefits of Contamination Control on Consumers’ Health Muhammad Atif Randhawa, Ali Asghar, Muhammad Nadeem, Naveed Ahmad, Sidrah University of Agriculture, Faisalabad, Pakistan

2.1 Introduction Both developed and developing countries are facing the challenges of foodborne diseases (De Cunha et al., 2012), and these diseases have become a major cause of death in the developing countries. Despite the cooperated efforts for a number of decades, foodborne diseases are still a major global public health problem with considerable illnesses and mortality linked with the intake of the contaminated produce (Havelaar et al., 2010). Food impurities are taken as the materials which may come in interaction with foodstuff as a result of environmental pollution or due to some wrong practices in farming methodologies. If these impurities are present in an amount more than certain specific levels, then these materials can damage human health. Impurities may form naturally or they may be carried by the foodstuff from water, air, or soil. However, some impurities may be produced during the production of food or during its processing; for instance, acrylamides, a chemical found in potato crisps. It may be produced during the cooking processes, while the mycotoxins are mostly produced by the fungi which can be present in nuts (Danyi et al., 2009). Contamination occurs when something, not normally present in food, gets added to it. Contamination indicates that the addition is not planned. The materials added may possibly cause problems. The three main causes of contamination are from chemical, physical, and microbial sources. Physical toxins may become the part of food mixtures and they may not alter or harm the food itself, although their incidence may produce health risks for its consumer. For example, metal pieces and broken bits of glass may accidently pass into foods. These substances would not ruin the food, but could be dangerous if swallowed as it can harm the internal organs. Other physical contaminants are insects, packaging materials, and rodent stools. Physical adulteration can be occurred at any level of the food chain, so all reasonable defenses must be applied to avoid this type of contamination. Physical impurities are considered as extra material or unfamiliar things that are usually not present in the food and can cause damage, illness, or psychological shock to the end user (West and Meek, 2006). Food Safety and Preservation. https://doi.org/10.1016/B978-0-12-814956-0.00002-0 © 2018 Elsevier Inc. All rights reserved.

13

14  Chapter 2 Chemical adulteration is another dilemma. The chemicals are used to protect crops by reducing losses inflicted by insects. Herbicides are also used to control weeds. Both substances are considered as pesticides. If pesticides remain on food, they enter the food supply chain. Another main way through which chemical pollutants can enter the food supply is water. Water is used to process nearly every food. Water is a very good solvent. Thus, many toxic substances will get dissolved in it and pollute the water sources. Adverse responses to chemicals found in food can be categorized as food intoxication and food sensitivity. Usually, every one of us is susceptible to food intoxication which can occur after consuming spoiled food. Food allergies are examples of sensitivity of food components to our immune system. Our immune system when considers any food constituent as a foreign invader produces response in the form of skin rashes, vomiting, nausea, and allied symptoms. Various categories of food substances such as pesticide residues, additives, colors, and flavors can evoke adverse responses in cells. Hence, these are put under contaminant category (Snyder et al., 2014). Food itself is not changed by the physical and chemical impurities. They are the potential threats when consumed with food. The food is considered as ruined when some undesired alterations occur in food itself. Mostly, a food is considered spoiled when its look, feel, taste, or aroma has changed badly. Fungi, bacteria, and mold can be the reason of undesirable alterations in food. They can also produce some undesirable changes. Mostly the enzymes work together with microorganisms to spoil the food. Viral infections caused by food and water are gradually known as reasons of diseases in humans. This is somewhat elucidated by variations in the processing of food and patterns of consumption, for example, by the enhanced intake of organically produced food. They become the reason of human and animal infections through the gastrointestinal (GI) tract and are spread basically through the fecal-oral path or through unclean water. The safety measurement of foods depends on the assessment of the microbiological quality and safety standards of foods (Jacxsens et al., 2010). Foodborne illnesses include a broad range of illnesses and are the reason of considerable morbidity and mortality worldwide. It is a rising public health issue in developed as well as in developing countries. It is hard to estimate the exact deaths related to foodborne illnesses. Although, during 2005, 2 million estimated deaths occurred because of GI illness worldwide. More than 250 various foodborne sicknesses and diseases are caused by different toxins or by pathogens. Foodborne diseases are a chief health problem leading to high mortality and morbidity. The issue of infectious diarrhea includes 3–5 billion cases and 1.8 million deaths yearly, mostly in young children, caused by adulterated food and water (Linscott, 2011). The pathogens present in food such as viruses, bacteria, parasites, biotoxins, and chemicals cause foodborne illnesses. Although common foodborne illness issues are minor and selflimiting, severe issues can happen in high-peril groups, causing high morbidity and mortality. The high-peril groups for foodborne illnesses are newborns, toddlers, the elderly, and immunocompromised people.

Benefits of Contamination Control on Consumers’ Health  15 The social and economic concerns of food alteration is of great importance, and for the countries with restricted assets they may be terrible. Foodborne diseases cause social and economic issues, for example, income loss, loss of manpower, and medical-care losses. The influence of food losses is also substantial. The estimated loss of legumes and grains is at least 10% worldwide. With no grain staples, vegetable, and fruit the loss is supposed to be 50%. A substantial amount of these losses is because of contamination. It is assessed that about 1000 million tons of agricultural produce is at impurity risk due to mycotoxins every year. Food contamination disturbs the trade by two means. First, the contaminated food products may be rejected if the level of impurities is more than the bounds declared by the country which imports. Second, the reputation of a country for its poor food safety can cause reduction in its trade and tourism. Failure to fulfill the minimum safety standards may cause food losses and, in severe cases, has influence on a country’s food security status. Numerous factors can lead to a food being perilous, for example, toxins naturally present in food, polluted water, insecure use of insecticides, and animal drug abrupt use. Poor and unhealthy conduct and storage situations, and lack of suitable temperature control, also cause food deterioration. Prevention improves the capacity and knowledge of food chain workers to put on safe handling practices. Food chain operators must be experts and well informed about production of safe food. Foods are expected to be prepared, stored, and handled according to the standards of food safety. This expectation requires good hygienic and production practices for its fulfillment, to ensure that the final product is safe for the consumers. (Godfray et al., 2010).

2.2  Sources of Contamination The occurrence of viruses, pathogenic bacteria, and parasites on fresh vegetables and fruits has been extensively reported. The contamination of a product may happen in the orchard or field, during the harvest, after the harvest, during processing, during delivery, or in home.

2.2.1  Harvesting and Handling Postharvest contaminant sources include soil; feces of animals which may contaminate soil, water used; chemicals such as fungicides, insecticides; and animals, workers, and equipment used for harvesting (Matthews et al., 2014). Pathogenic strains of microbes such as Listeria, Salmonella, and Escherichia coli cause outbreak of various food-related diseases. These dangerous organisms survive in water and soil for an extended period of time. The behavior of enteric bacteria in the production environment can be better understood now because of the studies which are conducted under open field conditions rather than under greenhouse conditions (Matthews, 2013). According to researches viruses, bacteria, and parasites present in animal feces used for soil fertility are considered as a health concern. Wildlife feces are also considered as a health concern. Manure is applied for soil adjustments as a solid, semisolid, or liquid all over the

16  Chapter 2 field, while the feces of wildlife are scattered in the field at different locations which can pose a problem. In the United States during an intensive outbreak of E. coli, environmental investigations exposed through molecular essays found that the feces of wild swine harmonized the outbreak strain (Jay et al., 2007). Approaches to avoid wildlife interference of a farm can be expensive and difficult to cope with as it is costly to put a fence around the field and often it is not practical and if it is not done properly the wild animals can burrow through the fence. Animal traps and crop fencing help to monitor field interruptions. Cost-effective methods are needed to diminish this type of contamination. Manure pits are extensively used for soil adjustments. Manure pits help to reduce the smell of manure and also makes the storage and transportation easy (Chen and Jiang, 2014). The chicken litter is known to harbor a wide range of pathogens and if not correctly treated before its application in field, it can cause contamination of crops (Chinivasagam et al., 2010). A thermal process is used to prepare the manure pits; however, some studies have been done to authenticate their ability to inactivate microbes which are a concern to human health. Such studies would help to develop the standard practices to enhance safe manure pits. Fish emulsion is becoming popular to increase the nitrogen level of soil. Thermal processes are also used to prepare fish emulsions. Like the pelletized manure, research is essential to authenticate the inactivation of e human pathogens. The method of irrigation is also responsible for the microbiological impurity of the edible portion of a crop as it is applied on the surface groove, subsurface drip, or by overhead sprayers. According to researches, the crop contamination risk is enhanced by the sprinkle irrigation method as compared with subsurface and surface furrow methods. Studies conducted in the laboratory and in greenhouses reported that spray-irrigated lettuce leaves contained E. coli O157:H7 for 20 days, and repeated use of the contaminated water enhanced the pathogen level on the edible part of the lettuce up to a dangerous level (Fonseca et al., 2011). Climate change and food safety are associated with each other and this association has received much attention (Lake et al., 2012). Vegetables are produced and harvested under variable environmental conditions. Genetic, physiological, and phenotypic properties of microbes are influenced by the climatic change; thus, when it comes to vegetables’ microbial safety the climatic conditions must be taken into account (Liu et al., 2013). Temperature and rainfall patterns change annually. The average temperature of the world has been increased gradually (Camuffo and Bertolin, 2012). Environmental changes are becoming drastic effecting affecting the rainfall. Some territories are becoming rain deficient which affect the food production and diversity of plants negatively. The fate of viruses in manure, water, soil, and preharvest contamination of crops is unknown. Research done on this issue is lacking in a broad review on the sources of the viruses and viral impurity (Wei and Kniel, 2010). Viruses from the animal and human feces can contaminate water, soils, and manure. Viruses leaked from septic tanks, animal lagoons, or viruses, which survive the manure treatment can pollute vegetable fields and the resultant

Benefits of Contamination Control on Consumers’ Health  17 crops. Liquid, semiliquid, and semisolid type of manure is applied to the agricultural land and it is provided by the manure management systems. Unluckily, the virus is not eliminated completely by these systems (Wei and Kniel, 2010). A minute amount of viruses, that is, 10–100 particles, can be infectious, so preharvest vegetables are significantly concerned with the contamination. It has become a major goal to reduce the postharvest losses of food. Highly perishable foods such as mango, banana, tomatoes, and peaches are 30% lost after harvest before reaching the consumer. It is better to invest in food safety after the harvesting of crop rather than making efforts to enhance the production. On the basis of the results, the investments done to save food are less costly as compared with the investments done to increase production. A minute decrease in postharvest losses reduces the overall production cost in a significant way and it also reduces the problems of few land resources. A product from the field makes its way toward the society through harvesting. After harvesting, procuring is done. Usually scythe, collector, or sickles are used for this purpose. Harvesting is declared as the most work-focused action of the reaping season. On widespread automatic ranches, reaping uses the most expensive and up-to-date tools, for instance, the consolidate collector. The word “reaping” usually involves care of the crops after the harvest, which includes cleaning, arranging, pressing, and chilling (Kitinoja et al., 2011). When the vegetables come in contact with the harvesting equipment cross contamination occurs. These equipment may include knives, gloves and hands of the handler and containers, etc. (Matthews, 2013). Lettuce is now being trimmed in the fields during its harvest to reduce the shipping and waste disposal cost. Lettuce core cutting can cause tissue damage which invites growth of microbes. Contaminated knives can disseminate E. coli O157:H7 when used for cutting such produce (Taormina et al., 2009). These knives when come in contact with gloves of workers also get contaminated with enteric pathogens. Machine harvesting causes more surface contact exposure (Fallon et al., 2009). Spinach is harvested by using the lawn mower-type equipment. This type of machine can also introduce microbes and these microbes when introduced into foods, contaminate foods and cause health hazards (Buchholz et al., 2012). Therefore, harvesting equipment and machines must be kept clean by washing and disinfecting using chemicals so that the risk of contamination may be prevented. Vegetable and fruits get pathogens on their surface transferred by hands. In a study two-way transfer of Salmonella typhimurium between the hands and green bell pepper was revealed. The study exhibited that hand washing gel or any other alcohol-based gel used for cleaning hands can reduce S. typhimurium by many logs. Work sites should be provided with better washroom facilities in such a way that they may not harm the crops. Vegetables after harvesting can get contaminated due to poor handling, storage, and shipping. Bins are used to grow baby plants. And these bins get contaminated when they are stacked upon one another or placed onto the soil before shipping

18  Chapter 2 and processing. (Matthews et al., 2014). It should be emphasized in new practices that bins should not be stacked nor placed on soil after harvesting. The quality of vegetables can be maintained after harvesting by rapid cooling of them. The refrigeration temperature if used to cool the vegetables will help to reduce pathogens as well as to decrease the ripening and respiration rates (Buchholz et al., 2010). Various precooling methods are used according to the commodity. Cooling methods are of different types, for example, normal room cooling, air cooling, cooling through water called hydrocooling and vacuum cooling. These methods are used for different commodities according to their specific needs. Leafy green vegetables are cooled by using the methods of vacuum and hydrocooling. The product is showered with cold water and water is allowed to evaporate from the surface (Buchholz et al., 2010). These processes allow the pathogens to enter the commodity or they may become the cause of cross contamination. Li et al. (2008) stated that E. coli O157:H7 may be infiltrated into lettuce through vacuum cooling (Khalil and Frank, 2010). 2.2.1.1  Food handling If food is handled safely it can also minimize the dangers related to foodborne illnesses. Foodborne illnesses can occur in the produce, cured meat, eggs, and canned foods. 2.2.1.2  Food contamination during food handling It has been estimated that 9% of all foodborne illnesses happen at homes. Researches and surveys say that 15% of these illnesses is because of poor handling of food at homes. For bacterial infections, the pollution variables may include cross contamination from raw foods, hand contact with a food, and poor cleaning of the food surfaces and utensils. A few microbiologists have analyzed the kitchen surface as a contamination source. Mostly bacteria are transferred from the wipes and kitchen sinks, these include coliforms that are also present on dish wipes, sink, ledges, and on cutting sheets. Another study has shown that flushing surfaces in combination with the use of cleanser-based cleaners is essential for cleaning, and that Salmonella grows on materials even after they were flushed. A few studies have focused on good handling practices and microbial destruction. Mixed grains when examined showed some minute amounts of microscopic organisms, and they were deeply tainted. The researchers recognized that the consumer cleaning practices in the kitchen were not satisfactory to offset the cross contamination. They presumed it on the evidence of watching that the same cutting board was used for the cutting of meat and RTE (ready-toeat) items. Cutting boards and blades were washed with cold water instead of a cleanser and kitchen surfaces and shelves were touched with contaminated hands. So all these evidence and presumptions suggest that consumers should also be aware of their role in nutrition care practices. The USDA Food Safety and Inspection Service (FSIS) constructed the Be Food Safe crusade “so that dealers may be provided with the instruments to teach the buyers about foodborne illnesses and to increase the level of awareness related to the dangers associated

Benefits of Contamination Control on Consumers’ Health  19 with undercooking of foods.” There is focus on four key points: perfect, separated, cook, and chill. An optional area was to sort out whether outside sustenance security would affect the practices inside the kitchen (Sneed et al., 2015). 2.2.1.3  Food contamination during processing Contaminants may be produced during processing operations applied on food such as fermentation or heating processes, and these contaminants are known as processing contaminants. These types of contaminants are usually absent in raw foods. These may be produced as a result of the chemical reactions happening during the processing of the food when the raw material combines with the other constituents added to the food. The entry of these contaminants in the food cannot be avoided wholly. However, for the reduction of the levels of the synthesis of these contaminants some of the processes related to technology can be used and optimized. The processing operations involve sanitizing and washing, cutting, packaging as well as storing the food due to which chances of cross contamination are decreased. Various secretions having nutrients are released during the cutting of the vegetables which help in the growth and nourishment of several enteric pathogens. So they must be removed carefully (Matthews, 2013). Cutting and shredding of lettuce leaves causes tissue damage which may result in the secretion of latex from the cut surface of the leaf as well as E. coli population increase (Brandl, 2008). It was investigated that viruses may persist or transmit during the food processing operations. After cutting and chopping, the contaminated produce including tomato, carrot, and cucumber can transmit norovirus and hepatitis-A virus to the utensils. During processing, the washing of vegetables is beneficial as it minimizes the microbial load on the produce, whereas water intended for the washing having significant microbial load or not properly sanitized can itself become a source of contamination for the food (Holvoet et al., 2012). During processing, the primary vehicle for the spread the Salmonella enteritidis to freshly cut vegetables in a homogeneous way is washing water (Pérez-Rodríguez et al., 2014). In the industry of fresh produce, chlorine-containing sanitizers are being used widely because they are of low cost. On the other hand, these chlorine-based sanitizers may cause negative impact on human health and also release some harmful side products to the environment. The research is now shifting toward finding other alternative sanitizers which are cost effective and safe for human consumption. In order to reduce the microbial load and to provide the maximum safety of the fresh produce, the design of the equipment used and practices involved in processing must be optimized for washing and sanitizing (Matthews, 2013). Substances present in any food item are usually chemical in nature. Theoretically, it is considered that any process applied on the food can cause chemical changes in the food. As a result of these chemical changes some substances are produced and pose harmful effects for human consumption. These chemical substances being produced during the manufacturing,

20  Chapter 2 cooking (including home cooking), food packaging, and other operations involved in processing are process contaminants. The process that may contribute to the changes in food are cooking, pickling, fermentation, and acid hydrolysis. 2.2.1.4 Pickling An online metaexamination for the study of pickles was reported by the British Journal of Cancer as pickles are associated with the risk of esophageal tumor. On the basis of factual metainvestigation, the report proved that the utilization of Asian salted vegetables can increase the risk of esophageal disorder to almost twofolds. This examination proved results with “high heterogeneity” and the study was supported by several other studies. Basically, some parasites boost the development of N-nitroso mixes, which are responsible for esophageal cancer. In pickles, a nonalkylating nitroso substance named Roussin red methyl ester is present which is linked with the tumor-advancing effect. Some other compounds like Fumonisin mycotoxins are also being identified in pickles causing kidney and liver tumor in rodents (Islami et al., 2009). 2.2.1.5  Dry-heat cooking Air or fat is used as a medium in dry-heat cooking. As compared with moist cooking, in which water or steam is used, a high temperature can be maintained in the process of dryheat cooking. In dry-heat cooking, contaminants such as polycyclic aromatic hydrocarbons (PAHs), heterocyclic amines (HCAs), and acrylamide are formed which can affect human health negatively, so the cooking methods must be optimized and standardized. 2.2.1.6 Pasteurization In the process of pasteurization, all microorganisms are not intended to be killed as in sterilization. But it is aimed to minimize the number or the level of the microbes so that their presence may not cause any harm to the health. Commercially, pasteurization is not used for the sterilization of foods because it can affect the quality as well as the taste of the food products. Several foods, including dairy products, are heated at high temperatures to kill the pathogenic microbes and to ensure safe consumption of food. If the food is not heated properly then microbes can grow (Montville et al., 2005). 2.2.1.7  Fermentation and acidic hydrolysis Fermentation causes the decomposition of the food due to which certain by-products are produced due to the action of some bacteria. Alcohol is one of these by-products. Fermented foods including soy sauce contain a significant volume of alcohol. In fermented foods alcohol is present in very small amounts which can also affect the human body cells. During fermentation fungal toxins are also produced which cause hazardous effects on the health. For the breakdown of food, the two basic processes which are used in food industry are fermentation and acid hydrolysis. As a result of these two processes, many products are

Benefits of Contamination Control on Consumers’ Health  21 formed. Some products like ethyl carbamates are undesirable products and these are classified as process contaminants. It is unavoidable to avoid the formation of process contaminants in the food completely during processing, but the level of these contaminants can be minimized by reducing the cooking temperature and time. Several international and national food safety authorities and other food industries are working in order to evaluate the exact mechanism of the formation of process contaminants and for the development of such ways which can be used to reduce the formation of process contaminants in both home and industrial settings. These authorities also work to conduct various product surveys and updated the risk assessment methodologies. From a consumer point of view, a well-balanced diet without any interruption of specific foods such as deep-fried items, alcoholic beverages, fermented, or barbecued can help to reduce the exposure to the process contaminants. The Centre for Food Safety is working on a worldwide basis to develop and investigate the risk assessment studies on process contaminants (WHO, 2014).

2.2.2  Contamination During Packaging Presently, food is being sold in proper packages. The packaging of the food provides several benefits including physical protection and barrier protection. Food packaging also enables better preservation of food in order to increase a producťs shelf life. It also allows the safe transportation of food, enhances the shelf life of the food items in order to maintain consumer convenience as well as prevents the food from microbial and other sources of contamination. Commonly, during the formation of food packaging, different types of additives including product stabilizers, filling agents, plasticizers, or antioxidants are being used in the polymer for the improvement of the characteristics of the material. The food may come in contact with the packaging materials, whether directly or indirectly, and results in the transmission of this type of substances from the packaging materials to the food; this process is known as migration. Several chemicals are used in the synthesis of packaging materials and many of these substances can drift in the food. During the period of packaging and storing, unfavorable and poor conditions may enhance the development and survival of the microbes, which cause spoilage on the produce. Many other chemicals which are responsible for food contamination are being used in food packaging materials. There should be a legal limit for the use of these chemicals. So, where there is no specified limit for the use of these chemicals, the manufacturer of the food packaging material must make sure that this packaging material may not enter the food item and limit the suitability and safety of food. Particularly, it is very necessary to reduce the migration of these toxic substances in the food from the packaging materials. And in case if migration takes place, there should be surety that these would not cause any hazard to the human life. The possibility of the chemical transfer to the food item must be considered by the manufacturer of the food packaging material and it should be mandatory for all types of food items that come in contact with this packaging

22  Chapter 2 material. The conditions to which the packaging material and food is going to be subjected must also be kept in mind during the manufacture of the food packaging material. The substances that migrate from the packaging material to the food items may cause harm to the consumer’s health. To avoid such type of contamination that takes place from the interaction of packaging material with food and its harmful effect on consumer’s health, strict legislations have been formed in Australia, Euroasia, and in some other countries. These legislations are applicable to any consumer or industrialist (Caleb et al., 2013). The pathogens including Salmonella, Listeria monocytogenes, and E. coli may grow faster on shredded lettuce which is stored at a temperature of 25°C under some modified atmosphere packaging condition (Oliveira et al., 2010). It was observed that Salmonella and L. monocytogenes populations increase at a faster rate in vegetables like escarole, spinach, and arugula that were kept at a temperature of about 15°C (Sant'ana et al., 2012). Any change in storage temperature or packaging conditions can cause an enhanced growth of pathogens in the food items (Sharma et al., 2011). The chemical substances that would migrate from the packaging material to the food and their effect on the quality and safety of the food clearly depends on the kind and nature of the food packaging material. There is an increased level of risk to the human health due to the constant introduction of more unique type packaging material. Typically, the availability of synthetic polymers is almost negligible due to their high molecular weights, that is, many thousand Daltons. However, polymers having low molecular weights are used and these low molecular weight polymers have a finite potential to harm human health. Chemical substances that drift from the plastic material used for the packaging of the food include catalysts, solvents, monomers, and additives. And these additives contain antioxidants, antifogging agents, antistatic agents, heat stabilizers, filling additives, pigments, and dyes. Board and paper is also used in food packaging which consists of the pulp obtained from the different vegetable sources and this type of packaging mostly affect the dry food items. In the preparation of this type of material, usually wet strength sizing agents, retention aids, fillers, biocides, grease-proofing agents, fluorescent-whitening agents, and starch are used. Waxes or polymers of polythene are also used as coating on paper and board for proper packaging of food. One of the major sources of the migrant is recycled fiber. Inks are also considered as the major contributor of migrants and specially include photoinitiators like benzophenone (BP) or 2-isopropylthioxanthone (ITX), which comes from UV curable inks. Owing to migratory nature of ink components from packages, recently remedial measures are being considered. The use of metallic cans for food packaging causes the occurrence of corrosion process on the surface of the metal and as a result migration of metallic ions like iron and tin from the packaging materials to the food takes place. In order to avoid the corrosion in metallic packaging material, the inner surface of the cans is coated with varnishes like epoxy resins. Some other minor side products such as bisphenol-A, diglycidyl ether (BADGE),

Benefits of Contamination Control on Consumers’ Health  23 or cyclodiBADGE can also drift from the packaging material to the food. Mostly the metal cans are made by using tinplate (in which steel is coated with tin), aluminum, or tin-free steel (in which steel is usually coated with chromium and chromium oxides). Food cans are mostly composed of tinplate while the beverage cans are made of aluminum. A polymeric layer covers the internal lining of the cans, in this way food directly comes in contact with the lacquer and not with the metal. So in the can system metal ions as well as the migrating components from the coatings derivatives are considered. The migrating components from the coating includes small amounts of additives, monomers, and oligomers and a large number of various other undescribed or unknown components (Buculei et al., 2012). A common material which is being used in the packaging of jams, marmalades, beans, vegetables, or sauces is glass. The metallic lids that are used to close the jars cause migration. For the assurance of better seal, a PVC jacket is used in these lids. Several authors have been reported that an additive, epoxidized soybean oil (ESBO), is being used as a plasticizer in the formation of PVC and may migrate into the food items. The major components of glass packaging are sodium, calcium oxide, and silica. Some these constituents may not cause any significant harm to human health. Food-contact ceramics contain silica as a major component. While another important raw material for the formation of ceramics is clay and clay is composed of silica, alumina, and water. However, substances that may concern with the health aspect originate from the printing ink and glazes. So lead and cadmium are two major components which are present as a source of contamination should be controlled frequently. A comprehensive overview of the ability of the migration of elements for the different types of the glass is promoted by the Food Standards Agency (United Kingdom) to make people aware about the packaging problems (Nerín et al., 2016). In New Zealand and Australia legislative requirements include Territory and State Food Acts, which are formed to ensure the safety and suitability of food. The New Zealand Food Act and State and Territory Food Acts consist of some usual requirements for packaging to minimize the sale of food handling or packaging material that is not safe for human consumption. In the same way, it is mandatory for the food business operator to comply with the requirements given in the Australian and New Zealand Food Standards Code. Several requirements related to the specific levels of the contaminants related to the food packaging are included in this code. This code for other chemicals related to packaging ensures that the safe packaging responsibility relies on the food manufacturer and retailers (Tager, 2014).

2.2.3  Contamination During Storage and Transport The bacteria that cause food poisoning grow and multiply only on the temperature danger zone of between 5°C and 60°C, while these bacteria causing food poisoning may not multiply continuously with the same rate at this temperature range. The temperature range at which

24  Chapter 2 they multiply most quickly is between 36°C and 38°C (around the temperature of human body). And when the temperature exceeds 60°C almost all germs causing food poisoning are killed. While the germs stay alive below 5°C they cannot multiply and grow. In order to minimize the growth and multiplication of these bacteria, food must be stored out of this temperature danger zone. Bacteria cannot grow in some foods like flours, cereals, dried products, spices, sauces, sugar, and unopened canned foods. But if they are kept in storage for longer periods of time, their quality can be affected and pests are the main disturbing factor in such food. When the canned high-moisture foods are kept in storage for longer periods or when their cans are damaged or broken during storage, during production or transport bacterial contamination may take place (Godfray et al., 2010). 2.2.3.1  Cross contamination The transmission of microbes from the raw food, unclean surfaces, or unclean utensils to the cooked food, clean surfaces, or clean utensils is commonly known as cross contamination. Raw food items, when not properly stored at a separate place, may contaminate the cooked or RTE items. In case it is necessary to store raw and cooked food items at the same place, then the raw food items should be placed in the shelves below in the refrigerator and the cooked food should be stored in the shelves above. In this way, the liquid dripping from the raw food can be avoided. During storage, the food should be properly covered with some plastic film or foil or the lid of the container should be tightly fitted (Council, 2008). Perishable food items are usually kept in the refrigerator for storage. Some fresh vegetables and fruits, however, can be stored out of the refrigerator at a particular cool temperature. Refrigeration of the food can lower down the rate of food spoilage. At low temperatures, the rate of the chemical changes that take place in the food items and the growth of microbes slow down (Zealand, 2004). 2.2.3.2 Thawing The process in which frozen food is retuned back to its form by increasing the temperature of the food is called thawing. In thawing, the food is defrosted in a microwave oven or put in the normal compartment of the refrigerator. Defrosting the food in the microwave oven using the defrost mode is the safest way of thawing. Once the product is thawed, again this should not be kept at freezing temperature because the bacteria will grow fast in this freezing and defrosting time period and in this way food will become nonedible (Leygonie et al., 2012). 2.2.3.3  Contamination during transportation Globally, about 200 metric tons of food is being transported every year. Of these, 35% of the food is being transported by land, 60% of the food is transported by sea, while 5% by air. The quantity and variety of the food which is being transported, the mass of the container as well as the specific temperature and handling requirements necessary for food

Benefits of Contamination Control on Consumers’ Health  25 storage are some of the factors that may contribute to the susceptibility of the food industry to contamination during storage and transport. It is reported that cross contamination, tampering, interruption, and temperature abuse are the points which are considered greatly during the transportation of food, while rare cases are reported from industry experience where there is failure in food safety directly because of the storage and transportation practices (Bendickson, 2007). During transportation of food, contamination can take place. Contamination can occur due to the smoke from the exhaust of petrol or diesel vehicles, or due to the process of cross contamination which can take place in the vehicle being used for the transportation of food. A serious risk regarding the safety of food can be held due to cross contamination. In the European Economic Community, a major illness outbreak was reported in 1999 due to the use of fungicide-contaminated pallets during the storage and transportation of food. Chemicals used for disinfection can cause cross contamination during long-distance transportation. During long-distance transportation, for example, transportation by ship, food is not checked for the barrier properties besides the organic compounds as it is usually checked for permeation of gasses such as CO2, O2, and water vapors. But these types of barrier properties are not used for organic compounds. Nerín et al. (2016) conducted a study which is a good example of cross contamination of food due to the transfer of methyl bromide, ortho and paraxylenes, naphthalene and toluene via the high-barrier theoretical materials. When food is transported, it is normally packaged to provide protection to the food and to avoid any type of contamination. The packaging provides protection to the food; however, care should be taken to avoid any damage to the packaging which itself can cause contamination and can affect the suitability and safety of food. Poor handling practices, exposure to rain, and some other environmental factors may contribute to the damage of the packaging material. Packaging can also get contaminated due to the contact with some poisonous substances which in turn can threat the producťs safety and suitability. Food items should not be transported with poisonous substances at the same time, but sometimes it becomes necessary, for example, in case of groceries. If these poisonous substances are packaged separately in a proper manner, then it is acceptable. There are wide chances of contamination during transportation if the food is not properly packaged. If the food section of the transport vehicle is well cleaned and properly enclosed, it can by itself provide protection to the food. For instance, meat carcasses are usually transported within the vehicle used for transportation in an unpackaged form. Care must be taken in case when different types of foods are being transported in an unpackaged form within the same vehicle. After harvesting, raw fruits and vegetables can be transported in an open vehicle because it cannot affect their safety and suitability. During further processing raw fruits and vegetables are processed to remove any contamination. The transportation of unpackaged food using an open vehicle is not supported, unless and until the safety and suitability of the food is ensured (Ackerley et al., 2010).

26  Chapter 2

2.3  Type of Contaminants 2.3.1 Microorganisms Microorganisms play a vital role in our lives, but at the same time some types of microorganisms cause threats to human health, for example, Bacillus cereus, E. coli, Clostridium botulinum, and Salmonella. In fact, we depend on food and cannot live without it. Three major types of food contaminants include parasites, viruses, and bacteria. Another type of microorganisms are fungi, these cause spoilage of the food but cannot cause food poisoning. These microorganisms are called decomposers of dead plant and animal matter into simplest compounds that are reused in the food chain. Most microorganisms are beneficial, for example, they synthesize vitamins in the large intestine and allow nutrients to be absorbed into the blood stream. The ancient people were aware of the microbial activity involved in the processing methods as they were routinely involved in the preparation of bread, alcoholic products, and fermented products at rituals (Mishra, 2013). The major sources of microbial contamination are raw food commodities, water, animal waste matter, and soil. Water is a dominant source of contamination as it is a major human body constituent (Park et al., 2012). During handling and processing practices, food becomes contaminated through air, utensil/equipment surfaces, and dust. The entries of microbial contamination into the food chain lead the microbial hazards through production, processing, preparation environment (Havelaar et al., 2010) storage, and transportation (Gil et al., 2015) (Table 2.1). Table 2.1: Food poisoning bacteria and their health effect Causative Organism

Incubation Duration

Bacillus cereus

Etymology

Prevalence in Foods

1–6 h

Sudden onset of nausea and vomiting, with or without diarrhea

Campylobacter jejuni

2–5 days

Clostridium botulinum

12–72 h

Clostridium perfringens Enterohemorrhagic E. coli—O157:H7

8–16 h

Fever, abdominal cramping, diarrhea with or without blood; Guillan-Barre syndrome can be seen in some individuals Abdominal cramping, nausea, vomiting, diarrhea, double vision; death or long term nerve damage may be seen Diarrhea, abdominal cramping, and nausea Bloody diarrhea, abdominal pain and vomiting; fever may be absent; hemolytic uremic syndrome

Cooked foods, like meat or fried rice that have not been properly refrigerated Raw and undercooked poultry, unpasteurized milk, contaminated water Improperly canned foods, herb-infused oils, baked potatoes in aluminum foil Meat, poultry, gravy, inadequately reheated food Undercooked beef, unpasteurized milk and fruit juices, raw fruits, and vegetables

1–8 days

Reproduced from Linscott, A.J., 2011. Food-borne illnesses. Clin. Microbiol. Newsl. 33, 41–45.

Benefits of Contamination Control on Consumers’ Health  27 During food handling and processing practices, food is exposed to a range of contamination (Carlin, 2011). Soil provides an environment for the spore-forming bacteria. Sterilized, sealed and canned food products remain stable at room temperature for years, the heat treatment inactivated the mesophilic microbes. Primarily, spoilage occurs in low-acid canned products at high incubation temperatures and multiplication of spore-forming microflora occurs. These have a less pathogenic effect on humans, but is considered an industrial risk, so control can be applied by quality testing. Moreover, Bacillus licheniformis, Bacillus coagulan, Clostridium, Paenibacillus spp., and Thermonanaerobacter spp. are the major microflora in canning industries owing to their ability to resist high temperatures (André et al., 2013). The identified pathogens that cause hazards on fresh vegetables include Shigella spp., L. monocytogenes, Staphylococcus aureus, Aeromonas hydrophila and the spore formers B. cereus, C. botulinum, and C. perfringens. However, the ones implicated in most outbreaks involving fresh fruits and vegetables are Salmonella and E. coli O157:H. Among the pathogens causing foodborne disease outbreaks, the most harmful is the norovirus that is related with fresh produce (Todd and Greig, 2015). So, we should enhance the surveillance programs and workshop to eliminate the risk of foodborne outbreaks. The harmful microorganisms are affecting the human life in many ways, some microflora cause diseases and some cause spoilage of the food products, clothes and leather, etc. The microorganisms grow on the food commodity surface and produce toxic substance that make the food poisonous and unfit for human consumption. The intake of such food causes illness and this is known as “food poisoning.” B. cereus have great ability to spoil and poison the food. Vegetable, meat, milk, rice, snacks, and other types of food are associated with B. cereus foodborne illnesses. Vomiting and diarrhea are two different types of foodborne diseases caused by B. cereus (Stenfors Arnesen et al., 2008). Up to 250 types of foodborne illnesses had been described. Majority of these occur due to the consumption of such food contaminated with virus, bacteria, and parasites. Another illness caused by the consumption of harmful chemical- and toxin-containing foods are poisonous mushrooms. The foodborne toxic substance and harmful chemicals enter the human body through the GI tract. So, these diseases cause different symptoms such as nausea, vomiting, abdominal cramps, and diarrhea. According to FDA, 40% foodborne outbreak is by green leafy vegetables based on the data delivered by the “Center for Disease Control and Prevention,” (CSPI, 2009). The total calculated costs spent on foodborne illness account to $159 billion per year in the United States (Scharff, 2010). Foodborne outbreaks confirmed in the EU during 2009 and 2010 were 4.4% and 10%, respectively, due to the consumption of contaminated fruits, vegetables and juices, and their end products (EFSA/ECDC, 2012). Another major outbreak was reported in Germany in 2011 where more than 4000 people were affected and 50 deaths occurred due to the intake of contaminated aromatic fenugreek seeds while L. monocytogenes outbreak occurred in the

28  Chapter 2 United States where 30 deaths including more than 135 affected people were reported due to the intake of contaminated melons. Outbreaks have a highly significant impact on the health department and on the economic status of a country. In fresh produce food supply chains, the safety issues must be addressed properly, for example, antimicrobial resistance, pesticide residues, and GM organisms (Magnuson et al., 2011; Domingo and Bordonaba, 2011).

2.3.2  Pesticide Residues Pesticides are biological and chemical agents that are used for the protection of crops from weeds, infection, and insects. Usually pesticides are used in wheat, rice, fruit, canola, and vegetables and nonfood crops like cotton, flowers, and grasses. The environmental status and the health of people depend on the pesticides used. Pesticide exposure is associated with an increased risk of cancer. Approximately, worldwide 2.5 MT of pesticides are used annually and the use is ever increasing.. In Pakistan, the same trend has been reported. Recently, approximately 3 million have been suffering from pesticides and 200,000 die annually throughout the world, with the majority belonging to developing countries (Tariq et al., 2007). There is an intensive use of agrochemicals to increase the production of food to fulfill the requirements of the communities. Pesticides and chemical fertilizers are two major group of agrochemicals. The worldwide use of chemical fertilizers has extremely increased and it is responsible for the “Green Revolution.” Maximum food production can be achieved from the same surface area with use of mineral fertilizers such as potassium, phosphorus, and nitrogen (Carvalho, 2006). The huge use of mineral fertilizers increases the production of food, but it seriously contaminates the aquifers, specially nitrates, decrease the quality of water and cause problems for human consumption (Camargo and Alonso, 2006). Other types of fertilizers, for example, phosphate and superphosphate, produce phosphoric acid and Phosphorite that rise the environmental contamination with heavy metals, such as arsenic, uranium, and cadmium. Additionally, excessive use of such fertilizers caused eutrophication problems to water bodies in the EU, Malaysia, Thailand, and Brazil (Carvalho, 2006). Pesticides use including fungicides, herbicides, insecticides, etc., significantly increased the yield and reduced the losses during and before harvesting of maize, vegetable, corn, cotton, and protect the crop from various diseases, but these not only kill the pests, but can also affect directly and indirectly human health (Munawar et al., 2013). Pesticides are intentionally used to control the pests, but on the other hand, they cause toxic effects on other nontarget species. Pesticide residues contaminate the water and soil, cause destruction of parasites, predators and penetrate the food chain and finally are ingested by human beings along with water and food stuffs. Pests and insects are developing resistance against pesticides. So, the chemical industries continuously manufacture new chemicals. In the EU, more than 800 chemicals have been

Benefits of Contamination Control on Consumers’ Health  29 registered as pesticides. At present, in Pakistan there are more than 108 types of insecticides that are being used such as fungicides (30 types), weedicides (39 types), acaricides (5 types), and rodenticides (6 types). A high-risk rate of pesticide poisoning of about 60%–70% was observed due to the direct exposure of pesticides in female cotton pickers. Pesticides must be applied in a calculated manner to not exceed the dose. The overdose of residues cause illness to humans such as hormonal imbalance and various dysfunctions (Munawar et al., 2013). Pesticides and their residues cause cancer, Parkinsonism and heart diseases. These residues can transfer into the food chain through animal feeding such as consumption of pesticidecontaminated foodstuff. After digestion, pesticides have an affinity for lipids that are absorbed into the intestine and then into the blood circulation. Highly fat-soluble pesticides are concentrated in tissues with high lipid containing tissues such as the brain, liver, adipose, and the kidney (Muhammad et al., 2012).

2.3.3  Antibiotic Residues Antibiotics are also known antibacterials prepared by specific microorganisms and they prevent the growth or destroy the microorganisms but have no effect on the viruses. Antibiotics are commonly used in dairy, poultry, honey-processing industries and in livestock management due to their availability and cost effectiveness. Veterinary medicines are used for nutritive, prophylactic, therapeutic, and metaphylactic purposes. Abnormal use of these practices such as overuse of antibiotics and lack of understanding the usage of drug is a serious dilemma. The presence of antibiotics in food causes potential hazard on human health including allergic reactions, gastrointestinal disturbance, tissue damage, neurological disorders, and hypersensitivity. Antibiotics also change the properties of starter cultures in food industry and increase the economic damage (Babapour et al., 2012). The classification of antibiotics is narrow but their use is broad spectrum, depending on the species of bacteria on which they act. They fall into four categories: those that inhibit the nucleic acid synthesis, damage function of the cell membrane, inhibit the protein synthesis, and those that inhibit the cell wall synthesis. The antibiotics are transferred into an animal by injections, orally through water or food. The antibiotic residues also appear in animal origin foods such as meat, eggs, and milk (Babapour et al., 2012). Hence consuming such food can cause problems if no proper processing of the food is done. Penicillin is the first antibiotic discovered; it has the ability to prevent mold and bacterial growth. The antibacterial drugs are used for the prevention of diseases in animals. They give preventive measures against pathogenic microorganisms, but weaken the immune system and dilute blood. The side effects of antibiotics depend on microbial contamination or infection, individual patient immunity, and the dose consumed (National Health Service (NHS), 2016). In the early stage of life, exposure of antibiotics increases the body mass in humans which

30  Chapter 2 causes obesity (Ray, 2012). During antibiotic therapy, the target bacteria develop resistance in evolutionary processes against a particular antibiotic drug. Antibiotics, such as erythromycin and penicillin, have a high ability to resist bacterial growth, but are less effective if the bacterial strain develops resistance. In cattle, mainly antibiotic drugs are used as a growth promoter, but not widely used in sheep (Babapour et al., 2012).

2.3.4 Mycotoxins Mycotoxins are natural toxic compounds produced by fungal species; if high levels of contamination are present in food, they cause health hazards and even death in humans and animals. These substances are toxic and contaminate the agricultural products. These toxins are metabolized inside animal tissues and lately appear in eggs of poultry and milk of livestock after eating such infected feed. The Fusarium, Aspergillus, and Pencillium species can produce several different mycotoxins that cause a toxic effect on human health (examples are Aflatoxin, Ochratoxins, Zeralenone, Trichothecenes, and Fumonisins). Globally awareness has been created by virtue of technology and decontamination methods of mycotoxins; hence, regulatory agencies are performing an excellent role to cope with the adverse situation of mycotoxicosis (Milicevic et al., 2015). The production of mycotoxins is dependent on the physical, biological, and chemical factors. Temperature, moisture, mechanical damage, and relative humidity are considered the physical factors, and fungicides, gasses, and pesticides are categorized as the chemical factors, while the biological factors are stress, spore load, plant variety, and insects. These aforementioned factors affect the production of mycotoxins. The biological factors are further categorized, including the strain specificity, instability, and variation of toxic properties. Some species of molds are capable to produce more than one mycotoxins and some mycotoxins are produced by more than one type of fungi (Fung and Clark, 2004). The mycotoxins are noncontagious, nontraceable, noninfectious, and nontransferable to microbes other than fungi in humans or animals. The mycotoxin poisoning is encountered in different environments. These can be in agriculture commodities or in the livestock feed. During preharvest and postharvest, a huge range is contaminated with mycotoxins. To maintain a high quality of food and feed, it is critical to have surveillance for mycotoxins (Škrbić et al., 2012, 2014, 2015). 2.3.4.1 Aflatoxins Difuranocomarin are derivatives of aflatoxins produced by polyketide pathways by many strains of Aspergillus parasiticus, Aspergillus flavus, and Aspergillus nomius, which contaminate the agri-commodities. They have carcinogenic, teratogenic, and mutogenic effects during testing in the laboratory subjects. The major target organ is the liver for aflatoxins carcinogenicity and toxicity. The metabolism and products formed depend on species susceptibility to aflatoxin (Milicevic et al., 2015).

Benefits of Contamination Control on Consumers’ Health  31 2.3.4.2 Ochratoxin Aspergillus and Penicillium are the genera of fungi that produce ochratoxins. The kidneys are the major target organ and cause porcine nephropathy during model feed trial. Endemic nephropathy, kidney tumors, and chronic interstitial nephropathy are noticeably human disorders related with ochratoxin. The consumption of pork is a source of ochratoxin intoxication in humans (Milićević et al., 2011). 2.3.4.3 Zearalenone Zearalenone is a phenolic resorcyclic acid lactone, produced by Fusarium. It is a nonsteroidal estrogen and possess significant estrogenic activity in humans and animals caused by Dand E-zearalenol, corresponding to hypothalamic, hepatic, mammary, and uterine estrogen receptors who have affinities for binding with ZEA. Poultry are much tolerant, but pigs are too much sensitive (Milicevic et al., 2015). 2.3.4.4 Fumonisin Fusarium produce fumonisins; among the 12 compounds in a group of fumonisins, B1 is most toxic. They have the ability to disrupt the metabolism of sphingolipids by inhibiting the enzyme ceramide synthase. This enzyme is responsible for acylation of sphingosine and sphinganine. It causes porcine pulmonary edema and leukoencephalomalacia and cancer in humans in regions like China and Southern Africa (Milicevic et al., 2015). To decrease the mycotoxin contamination level and to manage the stress of plant proper fertilization, weed control, proper crop rotation, and necessary irrigation are necessary. Prevention of the fungal growth favoring conditions and toxin production are needed to control these factors, for example, water activity, grain condition, microbial interaction, temperature, gas composition, and presence of biological and chemical preservatives (Milicevic et al., 2015).

2.4  Contamination and Health 2.4.1  Nervous System Nervous system is a significant part of the living body that coordinates with involuntary and voluntary action and provides a way of communication through transmitting signals between different parts of the body and CNS. Central and peripheral are the major parts of nervous system. Central nervous system is made of the spinal cord and the brain. In the peripheral nervous system, there are fiber-like structures that branch off the spinal cord and extend to all parts of the body. Brevetoxins are natural neurotoxins that are produced by Ptychodiscus brevis and Gymnodinium breve, which cause illness and even death in seabirds, marine mammals, and fish (Fleming et al., 2011).

32  Chapter 2 Brevetoxins are odorless, acid stable, tasteless, and heat stable. So, it not easy to detect and could not be removed during the normal cooking procedure of foods (Fleming et al., 2011). The brevetoxins exposure in humans occurred through the intake of contaminated foods, resulting in neurotoxin poisoning that causes serious gastrointestinal and neurological chronic diseases. Recent studies have shown that a large number of dolphins died due to the consumption of brevetoxin-contaminated food even with no active form of toxins (Kirkpatrick et al., 2010). Human listeriosis, a lethal foodborne infection, is caused by L. monocytogenes. The vegetables can be contaminated through antibiotic residues and soil. The contaminated food eaten by animals is transferred into the food chain such as dairy and meat products. It is a much severe infection and cause mortality up to 30%. It has the ability to cross into the intestinal barrier and then crosses the maternofetal barrier, and have capacity to hole into the blood–brain barrier and infect the center nervous system. Streptococcus pneumoniae, Haemophilus influenza, and Neisseria meningitides have the ability to cause parenchymal and meningitis brain infection. The highest mortality rate of L. monocytogenes is about 22% compared with other bacterial meningitides that has been reported (Lecuit, 2007).

2.4.2  Immune System Immune system is the defense system of the human body against the pathogenic microbes and toxin in the environment. Innate immunity and adaptive immunity are the major types of immune system. Innate immunity includes response against the chemical, physical, and microbiological barriers, but more usually include the element of immune system such as monocytes, neutrophils, macrophages, and complementary compounds, with immediate host defense. The adaptive immunity develop against the action of pathogenic microflora though antigen-specific reaction such as T-lymphoctyes and B-lymphoctyes. The adaptive response is specific but takes several days or weeks to develop. In the event of consumption of contaminated feed and food, the intestinal epithelial cell layer is the first barrier against the foreign toxin, antigens, and pathogens. This layer provides both innate and adaptive immunity. The release of histamine and cell degranulation can affect the immune system. The cellular effect may occur due to the entry of toxins into the respiratory tract. The chemokines are members of the cytokine family that have an important role in the migration of leukocytes. The chemokines are produced by most cells and are stimulated when the receptors are found on all leukocytes. The cytokine production increase the antibody level due to the brevertoxin exposure response. The apoptosis process of brevetoxin immunotoxicity based on interleukin-1 enzyme in lymphocytes and macrophages occurs by consuming food commodities infected with the aforesaid toxins (Fleming et al., 2011).

Benefits of Contamination Control on Consumers’ Health  33 The immune system is a major target for the development of treatment strategies, in particular to improve the management of infections, tumors, and autoimmune disease resistance as compared with conventional therapies. Approaches include immunomodulation with cytokines or their antagonists, therapeutic vaccination with designer adjuvants to drive specified types of immune responses, and regulation of cell function and survival by manipulation of coreceptor signaling molecules. The immune system is easily accessible through stem cells in the bone marrow. Bone marrow is present inside the long bone of arms and legs, vertebrae, and pelvic bones of the body. It is made of red marrow, which produces red and white blood cells and platelets, and yellow marrow, which contains fat and connective tissues and produce some white blood cells. The spleen filters the blood by removing the old or damaged blood cells and platelets and help the immune system by destroying bacteria and other foreign substances. White blood cells are made in the bone marrow and protect the body against infection. If an infection develops, white blood cells attack and destroy the bacteria, viruses, or other pathogenic organisms. The possibilities of manipulation through gene therapy has raised with the successful integration of the adenosine deaminase gene into the cells of children with severe combined immunodeficiency.

2.4.3  Reproductive System The awareness of reproductive health has been growing in the last few decades, which is affected by different chemical substances. Clinicians, scientists, and patients have concerns with the recent identified trends in reproduction and fertility. Reproductive disorders such as subfertility, delayed puberty, miscarriage, impaired fetal growth, and anatomical anomalies are caused by toxicants. Toxicants may have a slow impact at first but latter they may become drastic. Moreover, conception, gestation, and fetus development are affected owing to the reproductive toxicants, so awareness is very important to save oneself from such adverse consequences. Harmful substances may come from air, dust, soil, water, and food. These substances are biologically taken up by our body and affect the liver, heart, stomach, gonads, and all internal organs of our body. Some are fat soluble and become part of the body after residing in adipose tissue; they may live there for long periods of time and cause damage when feasible. For instance, lead (Pb) stays in bones for more than 10 years. Pb has extended half-life and cause damage to body on exposure to tissues or organs. Some chemicals disrupt the endocrine chemicals and have adverse effects on the pituitary, thyroid, and hormone-producing glands. Mechanism involved in the damage of reproductive health includes killing of cells, oocytes, sperms, or duct glands. Endocrinedisrupting chemicals may have a direct or indirect effect. Some chemicals exert their effect by damaging the DNA and alter the normal signaling causing birth defects and fetal development with anatomical disorders. Some also cause gene mutations which affect the genotype as well as the phenotype of an individual. Some like benzene and diethylstilbestrol have mutagenic effects. They also cause epigenetic effects on the way how a gene performs. Food if contains reproductive toxins affect the mechanism described above.

34  Chapter 2

2.4.4 Carcinogen A carcinogen is any substance, radiation, or radionuclide, that cause cancer due to the change in the genome or alter the cellular metabolic processes (Arya et al., 2011). These substances exert their effect if the food treated with overdose radiations, cancer-causing chemicals, and processing methods, for example, frying of potatoes produces acrylamides which are carcinogenic. Studies have shown that many carcinogenic chemicals undergo conversion by metabolic pathways into DNA-reactive intermediates and some carcinogenic compounds do not bind to the DNA and they are not mutagenic; however, they cause carcinogenic hazards on animal models and in humans. When carcinogenic chemicals are excited by cellular processes, metabolic processes, their retention or excretion by the cells start to occur. In the case of production inside the cell, the carcinogenic product affects the function of the gene directly or indirectly including DNA repair, cell-cycle control, and cell apoptosis. Sometimes carcinogens can be genotoxic resulting in fusion, addition of new components into the DNA, deletion, chromosome breakage, and nondisjunction. Some carcinogens are nongenotoxic and such they induce immunosuppression, inflammation, formation of reactive O2 species, cause genetic silencing, and activation of receptors. Both genotoxic and nongenotoxic mechanisms alter the transduction process and finally mutability, causing loss of proliferation control, resistance to breakdown and genomic instability can occur (Arya et al., 2011). Malignant tumorigenesis is a multistage process of tumor-forming cells from normal cells (Dholakia et al., 2011). Epidemiological studies demonstrated the occurrence of cancer development between the population groups due to the lifestyle and habits of that population. It is estimated by the exposure to environmental carcinogens such as aromatic amines, PAH, amino azo dyes, etc. which may contribute to human cancers (Arya et al., 2011) (Table 2.2).

2.5 Conclusion Microorganisms extract energy from foods by metabolizing it, they get into foods and deplete their nutrients. Microbes spoil foods and produce bad smell, taste, and aroma. Spoiled foods are unfit for consumption and should be discarded. Fungal spores hover in air everywhere and on residing on foods their hyphae penetrate and produce enzymes that damage food structure and function. Fungal sporangia on foods can produce numerous spores which can repeat the cycle again and infect other produce. To cope with fungal contamination, various decontamination techniques are on the way. Hence, various preservation techniques such as freezing, pasteurization, refrigeration, and dry sterilization are being used. The misuse of antibiotics creates resistance in microbes in the flesh of chicken. On consuming antibiotictreated food, the human body is also affected and develops resistance against antibiotics and becomes vulnerable to various pathogenic diseases. Chicken is consumed widely in various countries and so in order to save human beings from ailments the FDA must take steps and approve safe antibiotics for long-term use. Pesticides are also being considered a health

Benefits of Contamination Control on Consumers’ Health  35 Table 2.2: Carcinogen exposure and cancer risk Group

Compound

Affected Organs/Cancer Type

Polycylic aromatic hydrocarbon Aromatic amines/amides

Benzo(a)pyrene Polychlorinated biphenyls 2-Acetylaminofluroene 4-Aminobiphenyl 2-Naphthylamine o-Aminoazotoluene N, N-dimethyl-4-aminoazobenzene N-Nitrosodimethylamine Trichloroethylene

Skin, lungs, stomach Liver skin Liver, bladder Bladder Bladder Liver, lungs, bladder Lungs, liver Liver, lungs, kidneys Experimental results showed liver, kidneys, and lung cancer. Liver Lung, mesothelioma Skin, lungs, liver Lungs, prostate, kidney Lungs, nasal cavity

Aminoazo dyes N-nitroso compounds Halogenated compound Natural carcinogen Metals

Aflatoxin b1 Asbestos Aresenic Cadmium Nickel

Reproduced from Arya, A., Arya, S., Arya, M., 2011. Chemical carcinogen and cancer risk: an overview. J. Chem. Pharm. Res. 3, 621–631.

threat especially to low-age groups if they exceed the prescribed limits. These chemical contaminants can cause cancer and various respiratory diseases. Legislation is compulsory for all such dangerous chemical additives because some of them persist in foods and in the environment long after its use. We should be aware of the safe usage levels of such chemicals to treat effectively the sick animals or plants without exceeding the safe values and should safeguard human beings. Moreover, neurotoxins, reproductive toxins, fungal toxins, and related toxicants must be taken into consideration while selecting and processing foods.

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Benefits of Contamination Control on Consumers’ Health  37 Islami, F., Pourshams, A., Nasrollahzadeh, D., Kamangar, F., Fahimi, S., Shakeri, R., Abedi-Ardekani, B., Merat, S., Vahedi, H., Semnani, S., 2009. Tea drinking habits and oesophageal cancer in a high risk area in northern Iran: population based case-control study. Br. Med. J. 338, b929. Jacxsens, L., Uyttendaele, M., Devlieghere, F., Rovira, J., Gomez, S.O., Luning, P., 2010. Food safety performance indicators to benchmark food safety output of food safety management systems. Int. J. Food Microbiol. 141, S180–S187. Jay, M.T., Cooley, M., Carychao, D., Wiscomb, G.W., Sweitzer, R.A., Crawford-Miksza, L., Farrar, J.A., Lau, D.K., O’connell, J., Millington, A., 2007. Escherichia coli O157: H7 in feral swine near spinach fields and cattle, central California coast. Emerg. Infect. Dis. 13, 1908. Khalil, R.K., Frank, J.F., 2010. Behavior of Escherichia coli O157: H7 on damaged leaves of spinach, lettuce, cilantro, and parsley stored at abusive temperatures. J. Food Prot. 73, 212–220. Kirkpatrick, B., Bean, J.A., Fleming, L.E., Kirkpatrick, G., Grief, L., Nierenberg, K., Reich, A., Watkins, S., Naar, J., 2010. Gastrointestinal emergency room admissions and Florida red tide blooms. Harmful Algae 9, 82–86. Kitinoja, L., Saran, S., Roy, S.K., Kader, A.A., 2011. Postharvest technology for developing countries: challenges and opportunities in research, outreach and advocacy. J. Sci. Food Agric. 91, 597–603. Lake, I.R., Hooper, L., Abdelhamid, A., Bentham, G., Boxall, A.B., Draper, A., Fairweather-Tait, S., Hulme, M., Hunter, P.R., Nichols, G., 2012. Climate change and food security: health impacts in developed countries. Environ. Health Perspect. 120, 1520. Lecuit, M., 2007. Human listeriosis and animal models. Microb. Infect. 9, 1216–1225. Leygonie, C., Britz, T.J., Hoffman, L.C., 2012. Impact of freezing and thawing on the quality of meat. Meat Sci. 91, 93–98. Li, H., Tajkarimi, M., Osburn, B.I., 2008. Impact of vacuum cooling on Escherichia coli O157: H7 infiltration into lettuce tissue. Appl. Environ. Microbiol. 74, 3138–3142. Linscott, A.J., 2011. Food-borne illnesses. Clin. Microbiol. Newsl. 33, 41–45. Liu, C., Hofstra, N., Franz, E., 2013. Impacts of climate change on the microbial safety of pre-harvest leafy green vegetables as indicated by Escherichia coli O157 and Salmonella spp. Int. J. Food Microbiol. 163, 119–128. Magnuson, B.A., Jonaitis, T.S., Card, J.W., 2011. A brief review of the occurrence, use, and safety of food-related nanomaterials. J. Food Sci. 76, R126–R133. Matthews, K.R., 2013. Sources of enteric pathogen contamination of fruits and vegetables: future directions of research. Stewart Postharvest Rev. 9, 1–5. Matthews, K.R., Sapers, G.M., Gerba, C.P., 2014. The Produce Contamination Problem: Causes and Solutions. Academic Press, San Diego, CA. Milićević, D., Grubić, M., Radičević, T., Stefanović, S., Janković, S., Vranić, V., 2011. Ochratoxin a residue in broiler tissues-risk assessment. Tehnologija mesa 52, 268–275. Milicevic, D., Nesic, K., Jaksic, S., 2015. Mycotoxin contamination of the food supply chain-implications for one health programme. Procedia Food Sci. 5, 187–190. Mishra, L.K., 2013. Role of microorganisms in food. In: Recent Advances in Microbiology, Vol. 1. Nova Publishers, New York, pp. 215–260. Montville, T.J., Dengrove, R., De Siano, T., Bonnet, M., Schaffner, D.W., 2005. Thermal resistance of spores from virulent strains of bacillus anthracis and potential surrogates. J. Food Prot. 68, 2362–2366. Muhammad, F., Javed, I., Akhtar, M., Awais, M.M., Saleemi, M.K., Anwar, M.I., 2012. Quantitative structure activity relationship and risk analysis of some pesticides in the cattle milk. Pak. Vet. J. 32, 589–592. Munawar, A., Hameed, S.W., Sarwar, M., Wasim, M., Hashmi, A.S., Imran, M., 2013. Identification of pesticide residues in different vegetables collected from market of Lahore, Pakistan. J. Agro Aliment. Process. Technol. 19, 392–398. National Health Service (NHS), 2016. Antibiotics-Side Effects by the UK. Crown copyright. Nerín, C., Aznar, M., Carrizo, D., 2016. Food contamination during food process. Trends Food Sci. Technol. 48, 63–68. Oliveira, M., Usall, J., Solsona, C., Alegre, I., Viñas, I., Abadias, M., 2010. Effects of packaging type and storage temperature on the growth of foodborne pathogens on shredded ‘Romaine’lettuce. Food Microbiol. 27, 375–380.

38  Chapter 2 Park, S., Szonyi, B., Gautam, R., Nightingale, K., Anciso, J., Ivanek, R., 2012. Risk factors for microbial contamination in fruits and vegetables at the preharvest level: a systematic review. J. Food Prot. 75, 2055–2081. Pérez-Rodríguez, F., Saiz-Abajo, M.J., Garcia-Gimeno, R.M., Moreno, A., González, D., Vitas, A.I., 2014. Quantitative assessment of the salmonella distribution on fresh-cut leafy vegetables due to crosscontamination occurred in an industrial process simulated at laboratory scale. Int. J. Food Microbiol. 184, 86–91. Ray, K., 2012. Gut microbiota: adding weight to the microbiota’s role in obesity—exposure to antibiotics early in life can lead to increased adiposity. Nat. Rev. Endocrinol. 8, 623. Sant’ana, A.S., Barbosa, M.S., Destro, M.T., Landgraf, M., Franco, B.D., 2012. Growth potential of Salmonella spp. and Listeria monocytogenes in nine types of ready-to-eat vegetables stored at variable temperature conditions during shelf-life. Int. J. Food Microbiol. 157, 52–58. Scharff, R.L., 2010. Health-Related Costs From Foodborne Illness in the United States. The Produce Safety Project at Georgetown University. www.producesafetyproject.org. Sharma, M., Lakshman, S., Ferguson, S., Ingram, D.T., Luo, Y., Patel, J., 2011. Effect of modified atmosphere packaging on the persistence and expression of virulence factors of Escherichia coli O157: H7 on shredded iceberg lettuce. J. Food Prot. 74, 718–726. Škrbić, B., Živančev, J., Đurišić-Mladenović, N., Godula, M., 2012. Principal mycotoxins in wheat flour from the Serbian market: levels and assessment of the exposure by wheat-based products. Food Control 25, 389–396. Škrbić, B., Živančev, J., Antić, I., Godula, M., 2014. Levels of aflatoxin M1 in different types of milk collected in Serbia: assessment of human and animal exposure. Food Control 40, 113–119. Škrbić, B., Antić, I., Živančev, J., 2015. Presence of aflatoxin M1 in white and hard cheese samples from Serbia. Food Control 50, 111–117. Sneed, J., Phebus, R., Duncan-Goldsmith, D., Milke, D., Sauer, K., Roberts, K.R., Johnson, D., 2015. Consumer food handling practices lead to cross-contamination. Food Prot. Trends 35, 36–48. Snyder, S.A., 2014. Emerging chemical contaminants. J. Am. Water Works Assoc. 106, 38–52. Stenfors Arnesen, L.P., Fagerlund, A., Granum, P.E., 2008. From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol. Rev. 32, 579–606. Tager, J., 2014. Nanomaterials in food packaging: FSANZ fails consumers again [online]. Chain Reaction No. 122, 16–17. Available at: https://search.informit.com.au/documentSummary;dn=734372479368382;res=IELHSS, ISSN: 0312-1372 [accessed on 20 Feb. 18]. Taormina, P.J., Beuchat, L.R., Erickson, M.C., Ma, L., Zhang, G., Doyle, M.P., 2009. Transfer of Escherichia coli O157: H7 to iceberg lettuce via simulated field coring. J. Food Prot. 72, 465–472. Tariq, M.I., Afzal, S., Hussain, I., Sultana, N., 2007. Pesticides exposure in Pakistan: a review. Environ. Int. 33, 1107–1122. Todd, E., Greig, J., 2015. Viruses of foodborne origin: a review. Virus Adapt. Treat. 7, 25–45. Wei, J., Kniel, K.E., 2010. Pre-harvest viral contamination of crops originating from fecal matter. Food Environ. Virol. 2, 195–206. West, D.F., Meek, J.P., 2006. Nutrition, Food, and Fitness. Goodheart-Willcox, Tinley Park, IL. World Health Organization, 2014. Safety Evaluation of Certain Food Additives and Contaminants. vol. 68. World Health Organization, Geneva, Switzerland. Zealand, A.N., 2004. Food Standards Australia New Zealand.

Further Reading Baluka, S.A., Miller, R., Kaneene, J.B., 2015. Hygiene practices and food contamination in managed food service facilities in Uganda. Afr. J. Food Sci. 9, 31–42.

CHAPTE R 3

Preemptive and Proactive Strategies for Food Control and Biosecurity Ali T. Khalil⁎, Irum Iqrar†, Samina Bashir†, Muhammad Ali†, Ali H. Khalil‡, Zabta K. Shinwari⁎

*Qarshi University, Lahore, Pakistan †Quaid-i-Azam University, Islamabad, Pakistan ‡University of Engineering and Technology, Peshawar, Pakistan

3.1 Introduction The basic dogmas of thriving society are safety and security. Life is greatly influenced by notorious accidents; however, efficient and well-defined tactics can be assumed to decrease the risk of massive losses. Like blasting and killing, attacks on food supplies do not smack terror in our hearts, but they should. The supply chain of the food industry has many points of susceptibility. Food contamination is the presence of harmful entities such as chemicals or other microbes which can cause consumer illness as well as degrade the quality of food. Providing a sustainable, safe, and secure food supply has been a challenging task which requires continuous monitoring of the food quality and quantity from production to consumption. Without an efficacious and productive detectable system, the food supply chain is subjected to contamination. The economies of many countries are dependent on agriculture, and therefore the agriculture sector is an attractive target for deliberate attacks. All the downstream food-related industries can be compromised after such attacks. Since the food supply chain has a global nature, there are numerous target points for deliberately contaminating the food by exotic pathogens (Gullino et al., 2008; Kingsolver et al., 1983). Agricultural resources comprise the foundation of producing food. With an increase in population, climatic changes, globalization of food business, biological weapons, and biocrime could represent major hurdles to the sustainable production of food (Stack, 2008). Lack of diagnostic infrastructure, global strategies, and a lack of awareness about food biosecurity represent critical issues. The current population of the world is above 7 billion. Among the major issues faced by governments of the present day is the widespread food insecurity for the increasing population. One out of nine people on the globe is undernourished, which leads to the death of 3.1 million children. With the decrease in farm lands due to urbanization and environmental changes leading to calamities like floods, our Food Safety and Preservation. https://doi.org/10.1016/B978-0-12-814956-0.00003-2 © 2018 Elsevier Inc. All rights reserved.

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40  Chapter 3 food supplies are stressed. There is a great need to review and potentially reform the global agriculture and food production system to meet the needs of people in the future. Agricultural and food industries resources make a substantial contribution to the economy of Pakistan. Agriculture contributes to about 24% of the overall GDP (Pakistan Bureau of Statistics, n.d.), and absorbs nearly 45% of the human resources. About 62% of the human resources are estimated to be linked to agriculture directly or indirectly. Livestock has been a massive contributor (53.2%) to the agriculture setup in the form of fisheries, hatcheries, and poultries (Shinwari et al., 2014). Dairy farming is a popular business making Pakistan the third largest milk producing country. It contributes 11.7% to the GDP. Dairy farming has been the source of living for approximately 40 million people in Pakistan (The Nation, 2013). Meat represents a widely consumed food, with the consumption of poultry and beef on the rise over the past two decades. Over a time frame of just 11 years, the consumption of poultry has risen by 239%, from 322 million tons to 767 million tons from 2000 to 2010–2011 (Ahmad, 2011). Farm animals and crops are the potential targets for agricultural bioterrorism while other targets include items which are in the processing stage or distribution stage, processed food at the retail or wholesale level, and agricultural facilities such as food storage and processing facilities, transportation elements, food outlets, and research labs (Parker, 2002).

3.2  Basic Concepts “Biosecurity” is a relatively broad term that covers agricultural and food security, environmental security, health security, plant pathogens, zoonosis, pests, GMOs and LMOs. It can be said that biosecurity owes the 4 Ts (Trade, Transportation, Tourism, and Travel) (Clevestig, 2009; Waage and Mumford, 2008). “Biosecurity is the management of risks to the economy, the environment, and the community, of pests and diseases entering, emerging, establishing or spreading.” Biosecurity is a holistic term that includes policies and regulations to protect humans, food, agriculture, and the environment from potential biological threats intended to harm innovations, standards, and practices that are utilized to secure pathogens, poisons, and delicate advancements from unapproved access, abuse, theft, or deliberate discharge. Biosecurity is to make life safe. Some applications of biosecurity seem worldly with wide results (like inhibiting the motion of mouth and foot viruses at farms using disinfectants). Whatever the practice is, all of the instances involve monitoring, regulation, and/or inhibiting the movement of different types of life. Sometimes it focuses on plants or animals, and sometimes on microbes. Sometimes the biosecurity alarms are raised due to lab scale accidents while sometimes they are raised due to the intentional misuse of microorganisms. Sometimes dealing with a life to make it safe seems direct or simple (like the farm’s sheep and cattle), but at other times many complex questions are hidden in this visible straightforwardness. Briefly, biosecurity is as complex as life itself, and certain problems are released by its applications.

Preemptive and Proactive Strategies for Food Control and Biosecurity  41

3.3  Food Biosecurity Risks associated with food contamination comprise potential infection (e.g., bioterrorism) and dispersion of disease because of the growing distance of transportation of food and animals. Risk of contamination with pathogenic bacteria increases with an increase in the time span of transport and lairage of cattle and poultry. All food processing includes the component of risk and it is difficult to fortify the risk control of food safety. The trend of eating food in restaurants or food in the streets is growing day by day. To measure the influence of foodborne diseases, it is necessary to know that some people, such as teenagers, old men, pregnant women, the immune deficient, those susceptible to serious illness, and those who go through chemical treatment, might be more susceptible to serious illness in contrast to normal people. Predictions show that the number of such type of people is increasing. The lifestyle of inhabitants in the developing world is noxiously evolving due to existing food safety issues. The most well-known definition of “food security” is “access by all individuals at all times to enough and suitable food to give the vitality and supplements expected to keep up a dynamic and sound life.” “Food biosecurity” is the concept of food protection from biological hazards. Food security deals with the proactive precautionary measures against deliberate or accidental contamination of food that can cause harm and disruption. Eventually, it may lead to severe health care issues and to an economic outcry. Such events may be planned at different levels of the food chain by a violent group, a lone “copycat” person, or criminal activity. Such malicious activities can be focused on food items, production, and transportation processes. The aim of food and agricultural biosecurity is to alleviate the chances of introduction of exotic pests, pathogens, or their derived toxins in the food chain or in the environment. These biohazards have potential harmful repercussions on the environment, economy, and community (including people, plants, and animals). Effective biosecurity measures are intended to establish precautionary measures and an effective response mechanism for intentional or accidental events. Proper biosecurity protocols are intended to effectively manage, contain, or suppress infectious disease.

3.4  Food Terrorism (or Food Bioterrorism) Food terrorism can be defined as spiteful intentional contamination or corruption of food by using biological or chemical agents. A few of these agents (biological or chemical) have an instantaneous effect and are odorless, insipid, and hard to find. Since few of them are hard to detect with the present technologies, the chances of terrorists developing and utilizing biological or chemical agents for the contamination of food or water are extremely high.

42  Chapter 3 According to the World Health Organization (WHO) food terrorism can be defined as “an act of contaminating food intentionally for human use with chemical, biological or radio nuclear agents for the aim of inflicting harm or death to populations and/or breaching social, political, or economic stability.” Administrative frameworks are confronting various new and proceeding with sustenance security challenges. Conspicuously, administrative powers are addressing new potential foodborne dangers (for instance, Bovine Spongiform Encephalopathy (BSE) and hereditarily changed life forms), while looking for ways to enhance control on other food related microbial contaminants like Salmonella and E. coli. Further, there is developing political pressure for expanded controls as an instrument to bolster consumer trust in the security of the food supply taking after various “sustenance alarms.” In the meantime, food safety directions are progressively observed suspiciously from a monetary point of view, persuading for “productive” directions, especially for controls, taking into account execution criteria or, on the other hand, data procurement. Food sustenance protection controls, in any case, keep on concentrating overwhelmingly on procedure-based prerequisites. Comparable pressure has been created to guarantee that item obligation frameworks give proficient motivators to food producers, processors, and merchants to convey results of satisfactory protection. The study of disease transmission of foodborne infections is quickly evolving as recently perceived pathogens rise and highly perceived pathogens get connected to new food vehicles. Thus, there is a need to look at food sustenance instruction projects to guarantee that messages are focused at lessening the danger of the most pervasive and/or significant reasons for foodborne disease.

3.5  Biological Weapons Threats to Food and Agriculture: A Brief History The basic concept of bioterrorism is “the deliberate use of biological and chemical agents to inflict harm” Bioterrorism is a global issue. Since foods are traded globally, bioterrorism agents can easily disperse over the globe. Owing to these facts, international cooperation is needed to make an efficient system to contest bioterrorism. Biological weapons have been used for malicious purposes from ancient times to the present day, and have been executed by nonstate actors, criminals, and state enemies (Dudley and Woodford, 2002). The broad range of bioterrorists includes state enemies, state sponsored or military ambush, and individuals, among others (Zilinskas and Carus, 2000). Historically, plants and microorganisms or their derived toxins, contaminated blankets, animal carcasses, and infected human corpses have been used for bioterrorism. The work on bioterrorism has been for a long time ago; hence, it is not a new task (Hobbs, 2006; Kutz, 2013; Luning and Marcelis, 2009). Food and agricultural industries are particularly prone to bioterrorist

Preemptive and Proactive Strategies for Food Control and Biosecurity  43 activities, and although these threats may lack shock value, the economic consequences are immense. In sixth century BC, ergots were utilized by Assyrians to contaminate the wells of drinking water of their foes. Throughout the Krissa attack, flam cabbage was developed by Solon of Athens to defile the water supply. In 1996, research facility laborers at a vast restorative focus in Dallas, Texas, were welcomed through email to have muffins and doughnuts in the lunchroom. Eight people developed loose bowels and showed positive results for Shigella dysenteriae. One of the research facility laborers was the culprit. The FMD outbreak in 1997 led to a loss of $6.9 billion USD in addition to the loss of 50,000 jobs in Chinese, Taipei. Following the eradication of FMD, an additional $15 billion USD were spent on decontamination (Pearson, 2000). One million pigs were slaughtered in 1999 as a result of Nipah virus infection that lasted for about 3 months in Southeast Asia. In England, 3,900,000 animals were slaughtered, leading to a loss of $2 billion USD/day because of the notorious FMD outbreak in 2001 (Dudley and Woodford, 2002). These were accidental events which caused massive losses. A bioterrorist event can also have similar consequences. The food infrastructure is also prone to bioterrorist attacks. Some of the documented examples include the widespread food poisoning by the Rajneesh cult in Oregon, USA, in 1984. There were also attempts carried out by the Japanese-based “Aum Shinrikyo” organization in the 1990s to spread anthrax and botulinum toxin (Parker, 2002). There are many more examples of how microorganisms can be a source of economic damages, already reviewed by (Shinwari et al., 2014). Pakistan is the sixth most populous country in the world and it will be a challenge to feed the nation with continuously reducing arable lands and water scarcity. Costly fertilizers and other expensive agrochemicals pose a threat to production of food in sufficient quantities. In the wake of climatic changes, earthquakes, and recurrent floods, agricultural production will be a problem in the coming years.

3.6  Lack of Food Security and Safety Measures Besides the health and environmental issues, a bioterrorist event may lead to severe economic damage on the national and international levels. There are a number of interrelated repercussions. For example, the foremost consequence will be the immediate halt in the food production or at processing facilities until the time when the stocks are decontaminated. Depending on the scale of the devastation, there will be diagnostic and treatment services needed to stop the spread of the disease. Proper trainers must be hired to effectively dispose of the contaminated stocks. Overall, export markets will be lost and for a specified time restrictions will likely be in place. Loss of consumer confidence in the product or affected industry is another major issue. Another concern will be the changes in pricing. Let us assume a hypothetical scenario by considering two industries (A and B) of similar production nature.

44  Chapter 3 Let industry “A” be the one which has faced the event and is now under restrictions for a specified period. Such a situation will lead to the substitution effect in economic terms which means that eventually if products from industry A are not available, there will be an increase in demand for a similar product produced by industry B. Increasing demands can create scarcity of the product, which can lead to the increased price of the product. In addition, some government funds will be directed for the compensation of affected people. A recent example of the lapse in food safety was the presence of Salmonella in peanut butter in America, which led to the biggest recall of products in the history of United States that includes more than 200 downstream food manufacturers (Hobbs, 2006). Crop-related infections are difficult to monitor as they are grown over large areas making it impossible to detect a diseased plant. Usually plants are observed to be infected only after the disease has spread. Efforts are required on a large scale to reduce the time of discovery. Even after it is established that the disease is present, the samples may still needed to be transported for specific diagnosis, which delays the response. Building the technical capacity to deal with these issues is therefore critical. Often the labs are understaffed or lack an expert or equipment (Wheelis et al., 2002). Zoopathological and phytopathological labs should be built with highly sensitive equipment (Shinwari et al., 2014). International trade and globalization of food and agricultural markets have also provided ways of pathogen introduction. For example, the FMD outbreak in Japan occurred because of straw imported from China that is used as bedding for cattle (Matsubara, 2000). National animal and plant well-being frameworks attempt to stop the presentations of new vermin or maladies. Wherever this fizzles, obliteration is a plausibility if communities of the presented species are still relatively small and regional. In case this is not productive, the possible choice could likewise be to stifle populaces on a long haul premise to lessen the sway. The estimation of long haul control of buildup and infections is occasionally delivered by just agrarian makers. Through the spread of the possible new vermin menace, governments ought to organize where to give money in counteractive action, obliteration, and management. This has resulted in the intergovernmental biosecurity systems worldwide by taking in to account various pests as well as pathogens. For plants, these incorporate the IPPC (International Plant Protection Convention), conducted by the FAO (Food and Agriculture Organization) of the United Nations, related territorial plant insurance associations, and several particular provincial understandings. For creatures, they incorporate the FAO and OIE (Organization International des Epizooties). The regular livestocks of the provincial zones of Pakistan suffered enormously from different diseases. About 500 camels were tainted by a “puzzling ailment” in Noorpur Thal (District Khushab-Punjab) and connecting areas in May 2015. The foot-and-mouth disease is very common in the rural areas of Pakistan. The reemergence of FMD disease in cattle since mid2010 in a few villages of District Sargodha and Mandi-Bahudin, Punjab was also reported.

Preemptive and Proactive Strategies for Food Control and Biosecurity  45 Likewise, the Pakistani agriculture sector, especially the mango and citrus growing areas, has suffered from diseases affecting the plants and trees. The citrus greening, instigated by the microscopic organisms Liberibacter asiaticus is a major issue for citrus growers in the region (The infection was initially reported in China more than 20 years back, and has been spreading to citrus in locales in various mainlands.) In recent years, the mango growers’ economy was harshly affected especially in District Multan and Sind province. The iteration of the diseases is due to the poor management of biosecurity in Pakistan. The food supply chain has seen an expanding pattern in the economic process of food sourcing and therefore progressively intricate supply chains. Expanding globalization of food exchange implies that an incapability to react to a food crisis could have critical outcomes on the haleness and trade in numerous countries. Governments likewise have a part in encouraging deterrent food security through both deliberate and administrative components.

3.7  Food Safety Management and Control Producing a safe product is not a simple process and needs genuine controls across the food production to consumption chain. Everyone (managers, engineers, chemists, microbiologists, food technologists, etc.) has to play their part in order to maintain the quality and safety of food from farm to fork. Food safety engineers should apply the engineering principles integrated with biology and chemistry to ensure a sustainable and healthy food supply. Some of the intervention technologies have been found very useful in increasing the safety and quality of food. Technologies like Pulsed Electric Field Processing (PEFP) and Highpressure Processing (HPP) have replaced the traditional thermal means of decontamination of food products that usually cause chemical as well as physical changes in food (Kutz, 2013). Quality management can contribute significantly to the safety and sustainability of food production systems. Agri-food processing industries can use a quality management system (QMS) to direct the implementation of policies that underlies the safety and sustainability of the product. QMS includes the formation of the organization, processes, responsibilities, and procedures that are intended toward food safety and quality (Luning and Marcelis, 2009). •

Food items must be free of biological as well as chemical hazards if safe nutrition is to be provided to human beings. Some of the biological hazards such as pathogenic microbes can become increasingly possible under environmental and climate changes as microbial growth is favored by high humidity and temperature. Possible contaminations may occur across the food supply chains, and there must be wide-ranging monitoring of foodborne diseases (Gustafson et al., 2016). Some of the managerial steps can decrease the possible contamination of flocks or herds, and food or water from an infectious agent (https:// www.sdstate.edu/sdces/fcs/upload/FoodBiosecurity_PPT.pdf). Routine practices involved to train first responders are summarized in Figs. 3.1 and 3.2.

46  Chapter 3

Screening

Quarantine or isolation of newly purchased or returning animals

Testing

Monitoring or evaluation system

Fig. 3.1 Series of management steps.

Microbial food safety & antimicrobial systems

Risk assessment, including microbial & chemical hazards Food Packaging technology & materials in contact with foods

Mycotoxins

Hazard analysis, HACCP & food safety objectives

Good manufacturing practices

Quality assurance

Codes of practice, legislation & international harmonization

Education, training & research needs

Food process systems design & control

Rapid methods of analysis & detection, including sensor technology

Consumer issues

Fig. 3.2 Food control covers the areas which are related to food process control or to food safety of human food.

3.7.1  Food Control: A Shared Responsibility When seeking to establish, appraise, strengthen, or otherwise revise food control systems, national authorities must take into consideration a number of principles and values that underpin food control activities, including the following:

Preemptive and Proactive Strategies for Food Control and Biosecurity  47 • • • • • • •

maximize the risk reduction by applying the principle of prevention as fully as possible throughout the food chain; address the farm-to-table continuum; establish emergency procedures for controlling particular hazards (e.g., recall of products); develop science-based food control strategies; identify priorities based on risk analysis and efficacy in risk management; establish integrated, holistic initiatives which target risks and impact on economic well-being; recognize that food safety and security are a shared responsibility which needs healthy interaction between all of the stakeholders.

Certain basic principles and associated issues are discussed later. 3.7.1.1  Integrating the notion of farm to table Keeping in mind the end goal of shopper confidence in the safety of the food, it is important that well-being and product quality be considered along with nourishment from creation to consumption. The requirement is an extensive and incorporated farm-to-table methodology in which the maker, transporter, processor, merchant, and purchaser all assume a dynamic part in confirming the nourishment, well-being, and quality of the product. The goal of decreased biological risk can be attained most viably by anticipating risks all through the creation, handling, and showcasing chain. In some cases, it is hard to mastermind adequate security to the customer by just examining and dissecting the final product. The presentation of preventive measures at all phases of the nourishment creation and conveyance chain, instead of just assessment and dismissal at the last stage, bodes well on the grounds that inadmissible items can be recognized early in the chain. The more financial and viable technique is to oblige nourishment makers and administrators with an essential role in sustenance security and quality. An all-around organized, preventive approach that controls procedures is the favored strategy for enhancing sustenance security and quality. Sustenance perils and quality misfortune may happen at an assortment of focuses in the natural way of life, and it is troublesome and costly to test for their potential. Numerous food-related risks can be managed along the production chain with the help of standard practices such as good manufacturing practices (GMP), good agricultural practices (GAP), and good hygienic practices (GHP). The Hazard Analysis Critical Control Points (HACCP) is a deterrent approach that can be linked to every step, i.e., from manufacturing of food items to there supply. A standardized procedure for HACCP has already been documented by the Codex Committee on Food Hygiene that gives an important base for identifying and controlling of the food-related hazards (Food and Agriculture Organization of the United Nations,

48  Chapter 3 2003). Administration should perceive the utility of HACCP methods by the nourishment business as a central instrument for increasing the food safety and security. Government controllers are then in charge of examining the execution of the nourishment framework through observation and reconnaissance exercises and for upholding lawful and administrative prerequisites. Hazard control contrasts from survey-based controls in that it must be science based and be created from a game plan of nourishment well-being objectives. An operational danger administration point on an auxiliary, national, or nearby scale ought to address arranged nourishment sullying. The potential culprits of bioterrorism and the agents that they could utilize ought to be resolved so that powerful hazard controls can be executed. 3.7.1.2  Risk analysis Hazard investigation is settled in for compound dangers, WHO and FAO are presently incorporating the mastery and experience set up from danger examination of concoction dangers to that of microbiological perils (Zoonoses, n.d.). All food handling includes a component of risk and it is basic to guarantee viable danger administration of viand protection. Hazard valuation is the exploration of comprehension perils, the probability of their event, and the outcomes on the off chance that they do happen. Hazard control is the system of recognition and evaluation of different risks in the manufacturing, processing and supply of food. Hazard information is characterized by “the intuitive trade of data and opinions concerning hazard among risk assessors, risk managers, consumers, and other invested individuals.” Hazard investigation is characterized as a procedure consisting of three segments: •

•

•

Risk evaluation include: (1) hazard recognizable proof (2) hazard portrayal (3) exposure evaluation (4) risk portrayal Risk administration—the procedure, unmistakable from danger appraisal, of measuring arrangement choices, in interview with all invested individuals, considering hazard evaluation and different variables identified with the well-being of customers and for the upgradation of reasonable exchange practices, and selecting proper control and avoidance conceivable outcomes. Risk correspondence—the teaming up of feelings and data all through the danger investigation process concerning dangers and perils, hazard-related variables and danger experiences, among danger administrators, hazard assessors, industry, customers, the scholarly group.

Preemptive and Proactive Strategies for Food Control and Biosecurity  49 3.7.1.3 Transparency A sustenance control framework must be built up and executed in a straightforward way. Sustenance control powers ought to likewise inspect the way in which they exchange nourishment security data to people in general. As needs be, it is imperative that all basic leadership procedures are straightforward, permit all partners in the natural way of life to make successful commitments, and clarify the premise for all choices. This will empower collaboration from all concerned gatherings and enhance the adequacy and rate of consistence (Zoonoses, n.d.). The certification of purchasers in the well-being and nature of the sustenance supply relies on their familiarity with the dependability and adequacy of nourishment control operations and exercises. This may take the type of experimental sentiment on nourishment well-being matters, reviews of appraisal action, and discoveries on sustenance embroiled in foodborne sicknesses, nourishment harming scenes, or gross defilement. This could be considered as a piece of danger correspondence to encourage purchasers to better comprehend the dangers and their obligations regarding minimalizing the effect of foodborne risks (Food and Agriculture Organization of the United Nations, 2003; Zoonoses, n.d.). 3.7.1.4  Regulatory impact assessment Whenever arranging and executing nourishment control measures, thought must be given to the administrative expenses (staff, assets, and money-related claims) to the sustenance business, as these expenses are at last passed onto customers. The imperative inquiries are: What is the most very much sorted out administration alternative? Do the advantages of control legitimize the expenses? Send out examination frameworks that are intended to guarantee the well-being and nature of sent out sustenance, will ensure universal markets, produce business, and secure returns (Zoonoses, n.d.). Creature and plant well-being measures enhance agrarian efficiency (Waage and Mumford, 2008). Interestingly, nourishment security is a fundamental general well-being objective and may force costs on makers, yet interests in sustenance well-being may not be promptly compensated in the commercial center. Regulatory Impact Assessment (RIA) is of expanding significance in deciding needs and help sustenance control organizations in altering or reconsidering their procedures to accomplish the most useful impact. They are, be that as it may, hard to do. Two methodologies have been recommended for deciding cost/advantage of administrative measures in sustenance well-being: • •

Cost of ailment taking care of lifetime therapeutic expenses and lost efficiency. Theoretical models can be produced to gauge ability to pay (WTP) for lessened danger of grimness and mortality.

50  Chapter 3 Both methodologies require significant information for elucidation. COI evaluations are maybe less demanding for arrangement producers to comprehend and have been generally used to legitimize measures for nourishment control, despite the fact that they do not quantify the full estimation of danger decrease. As anyone might expect, it is less demanding to perform an RIA for a fare review intercession, than for an administrative approach which accomplishes a general well-being result. Various components of the operational risk management are indicated in Fig. 3.3.

Supervise and review Implement risk control Make control decisions Analyse risk control measures Assess the risk

Identify the hazards

Fig. 3.3 Steps of operational risk management. extensive framework to determine the burden of food-related infections.

In order to evaluate the burden of disease estimation in a specific area, WHO is internationally renowned for years for playing crucial role in: (a) Developing the capacity of public health leadership capacity (b) Collecting health information according to international standards (c) Assembling expertise and knowledge of organizations as well as individuals together to have a role in estimating the burden of foodborne disease for development of a food safety policy (Food and Agriculture Organization of the United Nations, 2003) There are no current data on the comprehensive levels and magnitude of foodborne diseases. In order to get a clear picture of foodborne diseases, the causes (parasitic, microbial, biological, and chemical toxins) need to be addressed using a multidisciplinary approach to get meaningful and integrated results.

Preemptive and Proactive Strategies for Food Control and Biosecurity  51

3.8  Strategic Plans for Protecting Food Supplies • • •

Generation of baseline and trend data on foodborne diseases, which will reinforce the capacity of countries Encouraging the stakeholders to streamline the food safety policies and standards and utilize cost-effectiveness of interventions estimation analysis Setting a priority list of agents of concerns for food safety (chemical or biological)

3.9  Responding to the Food-Related Health Crises For outbreak detection, assessment, and response, there is still a lack of essential investigation aptitude. In addition, owing to a lack of communication among veterinary, agriculture, and food sectors, foodborne disease occurrences go undetected. The WHO, along with its associates, has created a number of tools and networks to address these gaps. Global Foodborne Infections Network (GFN) was initiated to improve the regional and national prevention, investigation, assessment, and surveillance for controlling the foodborne pathogens. This network enhanced the surveillance capability of labs and increased the national and international communication and collaboration among epidemiologists and microbiologists across many disciplines. Targeted, needs-based capacity building efforts are key for strengthening this network to further improve the connected response mechanisms. On the basis of robust assessment, early warning to inform action and encourage timely communication is another important aspect of addressing health threats. The Global Early Warning System (GLEWS) for transboundary animal diseases, including zoonosis, was a joint project by the FAO, WHO, and OIE in order to respond to threats like Severe Acute Respiratory Syndrome (SARS) and Avian influenza virus (H5N1). GLEWS involves a multidisciplinary and international partnership for in time identification and calculation of health-related risks at the human-animal-ecosystem interface.

3.10  Food Safety Management Food safety administration programs need to confront the counteractive action, discovery, and control of food harm. The improvement of these projects will incorporate the nitty-gritty danger investigation to distinguish potential risks and the probability and seriousness of their existence. The potential culprits of intentional food defilement or food terrorism which should be considered in the danger administration approach incorporate the workforce. The one that links with the association that desire to defile the aliment origin; who desire to ingress the aliment origin inside an office either by deviousness, through constrained passage, or different manners; and the individuals who plan to make outside assaults from outside the

52  Chapter 3 office. The potential operators, which should be tended to inside the danger administration approach, are those that could prompt either a restricted or a boundless food security event and include: Coherently nonirresistible or irresistible pathogenic microorganisms, including viruses, bacteria, microbiological poisons, algae, protozoa, algae, parasites, worms, and insects which could be conveyed in the type of solids, liquids, or aerosols; synthetic compounds which could be conveyed as airborne beads, liquids, aerosols, or solids (eliminating conflicting specialists, i.e., manufactured and natural poisons including pesticides, rodenticides, heavy metals, cleaning chemicals, dangerous chemicals); Physical (including bone silvers, clay, glass splinters, metals, wood etc.) which can enter to the supply chain at any phase; Radiological (radioactive components capable for bringing about damage); Prions; and allergens including grains containing scavengers, milk, gluten, eggs and related items, nuts, soybeans, fish, sesame seeds, mustard, and celery. The strategies for exposure of these operators include identification apparatus (physical and radiological tainting), chemical examination (chemical pollution), and microbiological examination (organic tainting). These agents all have the potential to be utilized as a part of an occurrence of bioterrorism. It is necessary to train and aggravate consciousness between viands managers with respect to protected viand managing drills.

3.11  Improved Organizational Structures Can Enhance Food Control In order to strengthen the food control systems, a better organizational model with improved collaboration and coordination can be very effective. Collaboration, coordination, and integration of organization remain pivotal across the farm to table. The operational levels may include (Fig. 3.4): Such systems have the following advantages: • • • •

• • •

Uniformity in applying the risk control measures across the food chain, from production to consumption Consistent delivery in the food control system No interference in the routine investigation and implementation roles of other food control departments to make them politically more acceptable Separate risk assessment and risk management functions, resulting in objective consumer protection measures with resultant confidence among domestic consumers and credibility with foreign buyers A well-prepared and -informed population about international standards of food control Accountability in implementation and transparent decision-making are encouraged Long-term cost-effective influence

Preemptive and Proactive Strategies for Food Control and Biosecurity  53

Fig. 3.4 Different level of an integrated food control system.

An integrated Food Control Agency should have the mandate to move resources to highpriority areas, to discuss important sources of risk, and should address the entire food chain from farm to table. Such an agency establishment should not involve day-to-day food inspection responsibilities. These responsibilities should continue to lie with existing agencies at state/provincial, local, and national levels. The role of private analytical, inspection, and certification services, particularly for export trade, should be considered by the agency (Zoonoses, n.d.).

3.12  Funding Food Control Systems It is necessary for a country where food safety is managed by various state departments and agencies to discuss and revise the funding structure to ensure the continuity of funds. It will confirm continuity of funds and resources. Full assurance by the government is needed for this purpose to establish necessary structures and develop strategies to provide the optimal level of consumer protection. The resources and funds which can be used for restructuring and improving the systems for food control will be distributed by the national government.

54  Chapter 3 Over the course of time, the tendency toward public sector funding is little and the government needs to prioritize its funding arrangements with sufficient allocation for food safety and security, though with resource limitation securing enough resources may be a difficult task. Many nations practice cost recovery. This should be managed appropriately as it will be ultimately passed onto consumers in the form of some indirect taxation on items of food. Such taxation can affect the poor segments of the society. Private sector services can be hired to carry out specific food-related checks such as examining the quality of food, surveillance, and inspection. Some of the challenges are summarized in Fig. 3.5.

Alleviation of hunger and poverty

Policy reforms and governance

Major challenges and policy options and actions for food biosecurity

Farmermarket-value chainemployment linkage

Productivity, profitability, sustainability, and inclusiveness

Climate change and risk management

Fig. 3.5 Major challenges and policy options and actions for food security.

3.13  Food Security Challenges for Pakistan Pakistan is an agricultural country. Agriculture fulfills the food and fiber requirements of the fast growing population of the country (Ahmad and Farooq, 2010). Its population is increasing rapidly and if it continues to increase at the present rate, it is expected that the population will double by 2050. Currently Pakistan is the sixth most populous country in the world; doubling its population by 2050 will make it the fourth most populous country in the

Preemptive and Proactive Strategies for Food Control and Biosecurity  55 world (Pakistan, Government of National Nutrition Programme. Ministry of Heath, 2010). Wheat is the major food crop of Pakistan. During 2010, wheat production has been increased about fivefold but Pakistan became just a marginal importer of wheat (Ahmad and Farooq, 2010). To narrow the gap between the food supply and demand chain, dedicated efforts are required to control population growth and to achieve advances in technology. An important portion of SDGs (2015–2030) focus on decreasing hunger, poverty, and food insecurity. These are also prerequisites for economic development. Furthermore, the developmental process of national economic growth and food security mutually interact and support each other (Timmer, 2004). A country is said to be not a food sovereign state if it fails to produce the required food and lacks resources to buy food from the international market for fulfilling its supply and demand gap (Pinstrup-Andersen, 2009). The development and usage of an aliment biosecurity or safeguard administration arrangement is one of the most ideal approaches to diminish the dangers of sustenance terrorism and its results. For example, preventive practices are implemented by many food service operations for chemical use and storage as compared to other areas of practices. It is believed that having MSDS (material safety data sheets) in place and knowing the perils brought on by the substances have driven operations to practice safe food management practices. Moreover, the preventive practices identified with nourishment managing involve obtaining aliment fixings from fair suppliers who have suitable licenses. This practice is liable to be set up with the end goal of value control and nourishment security measures that the greater part of the food service operations had formerly applied before nourishment terrorism turned into a critical issue in the food service business.

3.14  Future Concerns The availability of food specifically in the form of calories and proteins is dependent on agricultural production. An ample supply of food at reasonable prices is the cornerstone of the food security policy in Pakistan and in other regimes. Noteworthy progress has been made by Pakistan with respect to increasing food supplies (Ahmad and Farooq, 2010). The policy failures relevant to food safety and security can be of two types which induces uncertainty in food control, safety, and security. One is the rapid withdrawal of funds from the agriculture sector for developmental programs, and the other is the failure in the food supply system due to paying less attention to safety and security of food supplies and agriculture. Owing to these chaotic events, governments of developing countries and international donor agencies decreased investment in research and development and withdrew their support respectively (Zezza et al., 2007). An increase in rural poverty and reduction in agricultural productivity have been observed due to a lack of policy support without providing alternative solutions. Another important reason for the massive reduction in accessing food is lack of awareness of essentially interlinked sectors. This contributes to the poverty-food insecurity

56  Chapter 3 helix. Apart from a few food security programs at the regional level, Pakistan has never had a national food policy (Mittal and Sethi, 2009).

3.15  Preventive Measures and Readiness Common protection frameworks which react to a scope of crises shape a noteworthy segment of national reaction components. Reaction systems are generally known as danger controls, which ought to be tended to in a case of conscious sustenance defilement. • • • • • • • • •

Preventive measures, for example, an HACCP administration arrangement involving instruments for the purpose of recognition as well as detection of harmful operators. The manufacturing as well as the distribution process of food should be designed in such a way to minimize any risk of contamination from production chain to the supply chain. Health experts monitoring the side effects in people brought on by potential agents including FAD and reporting systems to distinguish patterns instantly. New techniques for the rapid detection of food contaminants should be developed. Viable antibodies, therapeutics as well as chemoprophylaxis being promptly accessible. Various awareness raising and training programs in maintaining stringent biosecurity and biosafety standards in food industries will be fruitful. Rapid and meaningful ways of communication among the various stakeholders in case of any food safety or security event will be helpful to confine the large scale damage. Ejection of corpses and perhaps the human cadaver. Protecting the sustenance and water production network.

3.16  Evaluating Weakness The WHO (2002) proposes that weakness ought to be surveyed on the premise of “the political, social, experimental, and financial situations of a nation to gauge the degree of the risk and to set needs for assets.” The WHO further notes that powerlessness ought to be surveyed “as a multidisciplinary movement, with contribution from legitimate, insight, therapeutic, investigative, monetary, and political areas.” On a national level, vulnerability might be surveyed on the premise of: • • • • • • • •

the viability of the nation’s sustenance well-being administration foundation and current reconnaissance systems accessibility of the probable diet sullying doer inspiration for culprits of food terrorism capacity of the doer to taint lump created aliment and increase far reaching circulation capability of human-to-human transmission of the agents ability for a compelling crisis reaction probability of the danger to the viands production network, animal health, and welfare transport sustenance exchange, traversing, and people well-being

Preemptive and Proactive Strategies for Food Control and Biosecurity  57

3.17 Conclusion Keeping the food supply safe from biological or chemical risks requires teamwork that involves participation from federal and local governments, as well as the private sector. New and updated food standards are specifically required by national governments to address issues related to food security objectives. Implementing such standards would allow for a food chain that is greatly controlled and supplied with appropriate data on contaminants, hazards, and risk management strategies. Biosecurity standards need to be adopted by a country to the greatest extent possible. Solid scientific advice must be used as a base for developing and implementing biosecurity standards. It is also critical to build risk assessment competence in the country or region. Risk assessment will confirm that standards are reformed to the prevailing conditions and are capable of delivering a maximal level of public health protection when implemented appropriately.

Acknowledgments The authors thank Dr. Hillary Carter of George Washington University for reviewing the chapter.

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CHAPTE R 4

Validation of Analytical Methods for the Assessment of Hazards in Food Sevinc Kurbanoglu, Bengi Uslu, Sibel A. Ozkan Ankara University, Ankara, Turkey

4.1 Introduction Including farmers, manufacturers, processors, distributors, retailers, restaurants, food service establishments, and customers, today’s food industry is a composite system of producers and users. The manufacture of “safe food” is the responsibility of food companies. Companies should meet the regulatory requirements. In order to ensure a safe food supply, the US Department of Agriculture (USDA) and the Food and Drug Administration (FDA) mainly regulate the food industry. Among all problems faced in food analyses, shelf life is a vital one. Shelf life mainly results from products showing degradation because of microbial degeneration. Moreover, chemical contaminants can cause degradation, such as antibiotic drug residues in fish, sulfites in apricots, pesticides residues, etc. There exist different types of validation such as analytical method validation, equipment validation, process validation, and cleaning validation (Werner et al., 2006; Barwick and Ellison, 2005; Feldsine et al., 2002; Christodoulakis and Satchell, 2008; Christian, 2004; Chandra, 2001; Crowter, 2001). In this chapter, analytical method validation for food analysis is the main concern. From this point of view, analytical methods have a vital role in following the quality and safety of the final product. The quality and safety of the final product are followed by analytical methods that are used in the processing of food. Many judgments are found on the results of quantitative analyses, and it is important to be aware of the quality of the results when analytical methods are used. There exist some comities and guidelines, judge for these criteria such as the Hazard Analysis and Critical Control Point Principles and Application Guidelines (Safefood, 2011, 2013), Good Manufacturing Practices (GMPs), Food and Drug Administration (FDA, 1983, 1987, 2000, 2015), and the Codex Alimentarius Commission (CAC, 1994). According to CAC, a method of analysis to be included in a Codex commodity standard, certain method performance information should be available. There is a continuing need for reliable analytical methods in all areas of food quality and safety, in determining chemical and biological hazard concords with national regulations and international Food Safety and Preservation. https://doi.org/10.1016/B978-0-12-814956-0.00004-4 © 2018 Elsevier Inc. All rights reserved.

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60  Chapter 4 requirements (Bean et al., 2012; Hubert et al., 2004, 2007a, 2007b; Horwitz, 1982, 1995; Huber, 1999; Branch, 2005; Bruce et al., 1998; Winslow and Meyer, 1997; Westgard, 1994; Westgard et al., 1999; Werner et al., 2006; Clute, 2009; Hey, 1998). The term “Validation” is the key factor in controlling the reliability of a method. Validation of a method can be achieved in three steps: ✓ identification of suitable and required validation parameters, ✓ design of experiments for parameter evaluation, and ✓ determination of acceptance standards. The reliability of a method is determined by validation results, where specificity, accuracy, precision, limit of detection (LOD) and determination, and sensitivity and applicability are reported. Validated analytical methods play a major role in achieving the quality and safety of the final product (Armbruster et al., 1994; Asuero et al., 2006; Huber, 1999; Bakshi and Singh, 2002; Bliesner, 2006; Braggio et al., 1996; ICH-Q2B, 1996; ICH-Q2A, 2005; FDA, 1983, 2000; Werner et al., 2006; Van Loco et al., 2002; Ozkan, 2012).

4.2  Analytical Quality Control Related to Different Committee and Guidelines Quality is indicated by some analytical parameters for the system, substances, tools, and outputs that can be found in chemical or biochemical measurements (Rosing et al., 2000). In order to obtain reliable and accurate data, analytical processes should be controlled, compared, and validated by using some analytical measurements. Analytical method validation is an essential subject in the food industry for quality, development, and registration, since human health is the concern (Nilsen, 1996; Peters and Maurer, 2002; Pico, 2015; Prichard and Barwick, 2007). Basically, analytical method validation is used to justify that the intended method is reliable and achieves what is claimed. All analytical methods should be validated or revalidated if any changes in the method, even in the quality of reagents, chemicals, and experimental conditions, occur. To obtain reliable experiments, one should report validation report related to developed analytical method including specificity, accuracy, precision, LOD and determination, and sensitivity and applicability. In all areas of food quality and safety, use of national regulations and international requirements must be ensured (Freiser and Nancollas, 1987; Green, 1996; Hajicostas, 2003; Hibbert, 2007; Gumustas et al., 2013; Gumustas and Özkan, 2011). Method validation, is a process by which a laboratory authorizes by examination, presented as regulatory requirements in February 1987, by The USFDA. FDA, which is a scientific regulatory agency responsible for the safety of the nationally produced and imported

Validation of Analytical Methods for the Assessment of Hazards in Food  61 foods, cosmetics, drugs, biologics, medical devices, and radiological products, distributed a document entitled “Guideline for Submitting Samples and Analytical Data for Methods Validation” (FDA, 1987, 1983, 2000). This guideline is proposed to support applicants in submitting samples and analytical data to the FDA for methods evaluation. Moreover, a memorandum was published by the FDA Food and Veterinary Medicine Science and Research Steering Committee called “Guidelines for the Validation of Analytical Methods for the Detection of Microbial Pathogens in Foods and Feeds” (FDA, 1987, 1983, 2000; ICH Harmonised Tripartite Guideline, 1994, 2005; Bean et al., 2012). Owing to various food safety issues, including microbiological, foreign material, labeling, shelf life, chemical contamination, improper processing, and packaging issues, the FDA spread “recalls” which are classified as classes I, II, and II related to the use of or exposure to a violative product. If the violative product will cause serious adverse health consequences or death, it is in class I. If a violative product may cause temporary or medically reversible adverse health consequences or serious adverse health consequences, it is categorized as class II. In class III, a violative product is not likely to cause adverse health consequences. In all these classes, generally voluntary and in some extreme cases legal action was required (FDA, 1987, 1983, 2000; ICH Harmonised Tripartite Guideline, 1994, 2005; Bean et al., 2012). In the development of a new analytical procedure, initially, analytical instrumentation and methodology should be selected based on the intended purpose and scope of the analytical method. After developing the new analytical method, some parameters that may be evaluated during method development such as specificity, linearity, range, LOD and limit of quantitation (LOQ), accuracy, and precision should be reported. Validation of an analytical method offers objective evidence that the individual requirements for specific uses are satisfied (Armbruster et al., 1994; Asuero et al., 2006; Bakshi and Singh, 2002; Bliesner, 2006; Bouabidi et al., 2012, 2010). Method validation should be assessed in the following situations: ✓ early routine testing of a developed method, ✓ method transfer between laboratories, and ✓ changes in the developed method such as buffer, pH, etc. There are different types of method validation: Full validation, Partial validation, and Cross validation. ✓ Full validation is necessary when a new method is developed for a new compound, its metabolites, or impurities. ✓ Partial validation can be performed when a part of the method is changed, such as species within the same matrix, or vice versa. Moreover, if a parameter, procedure, or methodology in the developed method is changed, partial validation should be performed.

62  Chapter 4 ✓ Cross validation is achieved when two or more analytical methods are used to produce data within the same analyses. It is performed when a measurement is conducted in different laboratories (Konieczka, 2007; Krull and Swartz, 1999; Reichenbächer and Einax, 2011). According to the FDA “Food and Veterinary Medicine Science and Research Steering Committee,” there exist different levels related to their usages for the Detection of Microbial Pathogens in Foods and Feeds. At the first level, emergency usage is important. It is the lowest level where all the work is conducted in one or more laboratories. At this level, validation is done for the methods that are already developed or modified, and then a threat is determined to food safety or public health. Therefore, the rapid development and deployment of a method is needed. Sensitivity and specificity are the main concern related to this level. This level is achieved when there is an emergency in the analyses part and once the crisis has passed, it should be determined whether further validation levels should be performed, useful, or warranted (FDA, 1987, 1983, 2000; ICH Harmonised Tripartite Guideline, 1994, 2005; Bean et al., 2012; Swartz and Krull, 1997). For “Non-emergency Use Methods,” there exist other levels called the Single-laboratory Validation, Independent Laboratory Validation, and Collaborative Validation Study. In the level Single-laboratory Validation, the originating lab is the main concern where all the complete initial studies are performed. This level is the first stage for the use of the developed method in routine analyses. The second level is called “Independent Laboratory Validation.” At this level, as it is hidden in its name, another laboratory finds a place in the validation procedures, using the developed method in the originate laboratory. At the last level of validation, which is called the “Collaborative Validation Study,” an inter-laboratory participated in the validation. Reproducibility is calculated between laboratories and methods; therefore, by this level, the method is proven to be successfully developed as it is performed in another laboratory than the originating laboratory (FDA, 2015). According to the US Department of Health and Human Services FDA, Guidance for Industry, Analytical Procedures and Methods Validation: Chemistry, Manufacturing, and Controls Documentation, there are three types of analytical procedures: regulatory analytical procedure, alternative analytical procedure, and stability-indicating assay. The regulatory analytical procedure is used for the analyte that is defined. Alternative analytical procedure is used instead of the regulatory analytical procedure. The changes of the substances related with time in the related properties are followed by the stabilityindicating assay. These three methods can be used in food and drug analyses (FDA, 1987, 1983, 2000; ). Moreover, there exist standard operating procedures (SOP) related to “Method Development, Validation, and Implementation,” which is generated by the FDA. These SOPs illustrate the procedures systematically for the approval of “Foods and Veterinary Medicine Program”

Validation of Analytical Methods for the Assessment of Hazards in Food  63 analytical method development proposals for implementation in USFDA laboratories. The arms of the Foods and Veterinary Medicine, “Chemistry Research Coordinating Group,” and the Microbiology Research Coordinating Group are responsible for the SOPs. In these SOPS, one can find information about Identification and Prioritization of Method Development and Validation Needs, Development, Exploratory Investigations, Cross Center Collaborations, Implementation of Validated Methods, Implementation of Multi-Laboratory Validated Methods, Review, and Approval of the Annual Methods Development Plans and Reporting (FDA, 1987, 1983, 2000). For development of globally accepted standards, “AOAC International” has the priority. AOAC works on volunteer base and with industry partners, and this society develops analytical methods for a broad spectrum of safety interests including: foods and beverages, dietary supplements, infant formula, feeds fertilizers, etc. They distributed guidelines and appendixes, such as “Guidelines for Single-laboratory Validation of Chemical Methods for Dietary Supplements and Botanicals,” “Appendix D: Guidelines for Collaborative Study Procedures To Validate Characteristics of a Method of Analysis,” “Appendix J: AOAC International Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces,” “Appendix M: Validation Procedures for Quantitative Food Allergen ELISA Methods: Community Guidance and Best Practices” for the industry and academy (Feldsine et al., 2002; FDA, 2015). In Appendix J, all the parameters related to validation, qualitative methods—technical protocol for validation, quantitative methods—technical protocol for validation, confirmatory identification methods, and safety regulations are stated. In Appendix D, Minimum Criteria for Quantitative and for Qualitative Analyses, Design of the Collaborative Study in terms of Preparation of Materials for Collaborative Studies, and Submission of Test Samples and results are well stated ( FDA, 1987, 1983, 2000). “Hazard Analysis Critical Control Point” (HACCP) systems have shown that to build an effective “Food Safety Management System,” it is vital to have validation and verification to valuate regarding with the “Food Safety Modernization Act” regulations. In many countries, it is mandatory for food manufacturers to work in accordance with Codex principles of HACCP. HACCP has principles such as hazard analysis, critical control points, critical limits, monitoring, corrective actions, verification, validation, and record keeping. “HACCP” principles were first accepted by the industry and then included into US and international regulatory schemes. To determine if the plan is adequate to control hazards and to verify that the HACCP system is operating according to the plan are the main objectives of “HACCP” principles (CAC, 2003, 2008; Alli, 2016; HACCP, 1998). Food safety is related directly to the harmful substances that can be found in the food, called food safety hazards. These hazards can be biological hazards such as pathogenic

64  Chapter 4 bacteria, viruses, parasites; chemical hazards such as permitted food additives, naturally occurring harmful compounds, unavoidable contaminants, agricultural residues, industrial contaminants, chemical residues, prohibited chemicals, food allergens; physical hazards such as broken glass, plastic, metal pieces, wood pieces, stones, personal articles. These hazards can be controlled in several ways. For biological hazards: ✓ ✓ ✓ ✓

thermal processing to eliminate pathogens frozen storage to prevent pathogens use of preservatives to prevent pathogens testing for the presence of pathogens

For chemical hazards: ✓ formulation control of regulated food additives ✓ testing for the presence of antibiotics ✓ testing for the presence of pesticide residues For physical hazards: ✓ filtering or screening to remove foreign objects ✓ detection and removal of metal contaminants These are the known food safety hazards; however, there are several other specific food safety concerns. The safety of these foods, like all other foods, is covered by food laws and regulations (Alli, 2016). Another committee, the CAC, requires method performance information including specificity, accuracy, precision (repeatability, reproducibility), LOD, sensitivity, applicability, and practicability, as appropriate. Extensive collaborative studies do not need to be performed according to the CAC. To be validated according to Codex Commodity Standards, a method should require performance review testing with related validation parameters such as specificity, accuracy, precision (repeatability, reproducibility), LOD, sensitivity, applicability, and practicability. From these points of view, the ideal validated method is one that has advancement in terms of the design, conduct, and interpretation of method performance studies within a collaborative study in agreement with international harmonized protocols including at least five test materials with the participation of eight laboratories. This ideal case is not practical, not economical, and hence it is not needed for all analytical methods used for food control purposes such as low-level contaminants in foods, veterinary drugs, and pesticide residues.

Validation of Analytical Methods for the Assessment of Hazards in Food  65 For routine food control analyses there are many methods available depending on the requirements of the public at the national level. These analyses are chosen on the basis of their performance characteristics and applicabilities. The “Codex Committee on Methods of Analysis and Sampling” (CCMAS), “Codex Committee on Pesticide Residues Committees,” and “Codex Committee on Residues of Veterinary Drugs in Food” (CCRVDF) hold power to discuss about the validation of analytical methods used in food control for Codex purposes. All three Committees need to provide expert advice and guidance in food control areas. “Consultation on Validation of Analytical Methods for Food Control” was created for this purpose.

4.3  Validation Criteria and Its Parameters Method validation proves the scientific qualification of the analytical methods and it is reported with a validation report. In past years, a new strategy was proposed called “food fingerprinting approaches” (Riedl et al., 2015). By this approach, it is aimed to capture as many compounds or features as technically possible to gain a complete vision into the composition of the sample. The approach is very beneficial in the: ✓ discrimination of different origins or species varieties and ✓ identification of contaminants and unknowns. Food fingerprinting approaches can be used for a wide range of food commodities such as milk, oil, alcoholic beverages, and also fish, meat, fruit, vegetables, and so on. This action was taken by European research projects such as QSAFFE and FOODINTEGRITY dealing with the harmonization of authenticity testing (EFSA, 2012, EFSA (European Food Safety Authority) and ECDC (European Centre for Disease Prevention and Control), 2013, EFSA (European Food Safety Authority) and ECDC (European Centre for Disease Prevention and Control), 2014) Fig. 4.1, divided into four procedure phases: data preparation with preprocessing and pretreatment (upper part), internal validation for model generation and optimization (middle box), external validation for model testing (left box), and system challenge for (long-term) stability testing of the model (right box). The scheme covers test set objects retrieved independently from the training set objects as well as selected from the total samples set by a certain split mode. For model interpretation, in addition to the applied methods (white boxes), model input information such as quality and quantity of n, m, i, and j as well as performance parameters and criteria (dark gray boxes) should be reported. With the purpose of ensuring food consumer protection as well as to avoid barriers to trade and unnecessary duplications of laboratory tests and to gain mutual recognition, it is very important to guarantee the quality of laboratories and results of analyses related to food. For this purpose, the EC Council and the Commission have introduced requirements

66  Chapter 4

Non-targeted food fingerprinting approach

Method development

Application

Optimization

Sampling

Quality assurance measures

Validation

Instrumental Sample Data preparation chemical preparation analysis

Model generation

Model validation

System challenge

Method Validation

Quality control samples Interlaboratory comparison etc.

Fingerprinting specific steps in the workflow

(A)

Raw data (n objects)

Raw data ( j new cases)

Preprocessing/pretreatment

Processed data (n x m matrix)

External model validation

Processed data ( j x m matrix)

Split data

Internal model validation

Test set (i objects)

System challenge

Training set (n - i objects)

Model generation Data set refinement e.g., by remove outliers, noise reduction, feature selection

Model optimisation

No!

by select number of components

Performance results

ok? Yes! Model testing

Final model

Model testing

Performance results

compare

Performance results

(B) Fig. 4.1 See the legend on next page

Validation of Analytical Methods for the Assessment of Hazards in Food  67 related with accuracy data, validation test data and quality assurance that measures for official laboratories. Q2A, Text on Validation of Analytical procedures (March 1995), Q2B, Validation of Analytical Procedures: Methodology (May 1997) address almost all of the validation parameters. If only qualitative methods will be developed, selectivity, detection limit, precision, and stability of the raw material should be studied. In quantitative methods, selectivity, linearity and range, detection limit and quantitation limit, precision, accuracy, and applicability validation parameters should be given in the validation report. With the least number of analyses (five repeat), the validation tests should be realized. Validation can supply that, the method can detect and identify an analyte within reported sensitivity, specificity, accuracy, trueness, reproducibility, ruggedness and precision. One can ensure that results are meaningful and appropriate to make a judgment (Krull and Swartz, 1999; Kuselman and Sherman, 1999; Lazar, 2006). Validation is characterized by the following parameters: ✓ Selectivity ✓ Linearity ✓ Range ✓ Detection limit ✓ Quantification limit ✓ Accuracy ✓ Precision • Reproducibility • Intermediate precision • Repeatability ✓ Robustness

4.3.1 Selectivity Generally, selectivity is the first studied validation parameter of the methods, which gives an idea of the reliability of the method. ICH defines specificity as “the ability to assess unequivocally the analyte in the presence of components which may be expected to be present.” It is the ability to clearly identify/quantify the analytes in the presence of other

Fig. 4.1 (A) Schematic overview of optimization and validation steps for method development and quality assurance measures for application of nontargeted fingerprinting approaches. Critical workflow steps that are related explicitly to the evaluation of multivariate data are indicated by the gray box. (B) Validation scheme for multivariate models dealing with fingerprinting data. Reprinted with permission from Riedl, J., Esslinger, S., Fauhl-Hassek, C., 2015. Review of validation and reporting of non-targeted fingerprinting approaches for food authentication. Anal. Chim. Acta 885, 17–32.

68  Chapter 4 component that can interfere either endogenously or exogenously. Typically, this might be impurities, degradants, matrix, etc. The United States Pharmacopoeia (USP) refers to the same definition but also comments that other reputable authorities such as IUPAC and AOAC use the term “selectivity” for the same meaning. To illustrate, in residue analysis, there can be substances which give a response similar to the residue being measured (USP, 1994; ICH Q2B, 1996; ICH Q2A, 2005; Riley and Rosanske, 1996; Branch, 2005; Buick et al., 1990; Bruce et al., 1998; Chan et al., 2004; Chambers et al., 2005; Christodoulakis and Satchell, 2008; de Bievre and Günzler, 2005). Selectivity is important in this kind of residue analysis. For the complex matrixes such as milk, wine, etc., this validation parameter is crucial. If the analyte can distinguish from other chemicals, the method can be called “selective.” Selective methods can differentiate between analytes and matrix components, impurities, degradants, isomers, etc. Selectivity studies should evaluate interferences that may come from the nature of the matrix. In food analysis, matrix effect is crucial, since it can be changed very easily from sample to sample. The matrix composition should be well defined, and sample preparation should be optimized to minimize the effects from matrix components. The lack of matrix effect that can come from interferences should be demonstrated by the analysis of at least five independent sources of the control matrix (Branch, 2005; Buick et al., 1990; Bruce et al., 1998; Chan et al., 2004; Chambers et al., 2005; Christodoulakis and Satchell, 2008; de Bievre and Günzler, 2005). For the studies of specificity, the potential compounds that can interfere should be examined that can effect the analyte response. Specificity studies are very difficult since it is not always possible to prove that a single analytical procedure is specific for a particular analyte. In this case, a combination of two or more analytical procedures can be combined together to overcome this problem, to achieve the necessary level of discrimination. If impurities or degradants are available in the matrix, the method should be applied to the pure impurity or degradant samples, and it should be proven that in the presence of impurities, the reference samples can still be determined with the developed method. If impurities are not available, impurity profiles should be compared. If degradation studies are performed, degradation peaks need to be resolved from the reference sample peaks. If the matrix composition is unidentified, the standard addition method can be used. The slopes of the calibration graphs should be associated for the identification of the matrix composition effect. Similar slopes means that there is no matrix effect on the results (Branch, 2005; Buick et al., 1990; Bruce et al., 1998; Chan et al., 2004; Chambers et al., 2005; Christodoulakis and Satchell, 2008; de Bievre and Günzler, 2005). Stability indicating methods can also be developed for the measurement of selectivity. The forced stress degradation in mild and hard ways can be performed to the analyte and the analyte after stress conditions should be realized in the presence of degradation products (Ruberg and Stegeman, 1991). The ICH guideline Q1A can be followed for further conditions (ICH Q2B, 1996; ICH Q2A, 2005; FDA, 1983, 2000; Werner et al., 2006; Van Loco et al., 2002).

Validation of Analytical Methods for the Assessment of Hazards in Food  69 Stress conditions can be hard and weak according to the ICH criteria. The forced (stress) degradation studies can be realized for obtaining the stability parameters. Stress conditions can be obtained by exposing the sample to different treatments such as acid, base, heat, oxidation, and UV light treatment with different time limits and concentrations that show the hard or mild condition. To demonstrate specificity, forced degradation or stress testing can be achieved and the method can be called stability indicating methods. Stability indicating methods are defined as fully validated methods that are free from potential interferences (Table 4.1) (ICH Q2B, 1996; ICH Q2A, 2005; FDA, 2000, 1983; Werner et al., 2006; Van Loco et al., 2002; Taylor and Shivji, 1987). Table 4.1: Conditions of the stress-degradation studies Stress-Degradation Study

Conditions

Acid hydrolysis Base hydrolysis Neutral hydrolysis Oxidative conditions Photolytic degradation Temperature Humidity

0.1 N HCl 0.1 N NaOH Phosphate buffer at pH 7.0 Between 3% and 30% H2O2 or atmospheric O2 UV, Fluorescent, or white lamb Under selected temperatures such as 75°C Under >75% humidity

Hydrolytic Degradation can be studied by exposing the compound in 0.1 N HCl and/or 0.1 N NaOH and/or phosphate buffer at pH 7.0, separately for about 3 to 8 h. Oxidative Degradation can be achieved by exposing the compound H2O2 with different % such as between 3% and 30% concentration range and time about 3 to 8 h. Photolytic Degradation can be realized by exposing the compound to light using UV, fluorescent, and/or white lamps or their combination. For this type of degradation studies, Xenon and methyl halide lamps can also be used. Heat Degradation can be achieved by exposing the compound to the temperature value that should increase as 10°C above the accelerated temperatures such as from 65°C to 75°C. Humidity studies can also be performed as they can be applied at or >75% humidity values where appropriate (ICH Q2B, 1996; ICH Q2A, 2005; FDA, 1983, 2000, Werner et al., 2006; Van Loco et al., 2002; Taylor and Shivji, 1987). Every step for the stability indicating method should be performed depending on whether the compound is degrading in hard or mild conditions and the results should be compared with the blank solutions stored under normal conditions (Branch, 2005; Buick et al., 1990; Bruce et al., 1998; Chan et al., 2004; Chambers et al., 2005; de Bievre and Günzler, 2005). In practice, a test mixture is prepared that contains the analyte and all potential sample components. The result is compared with the response of the pure analyte. In the test mixtures, components can come from synthesis intermediates, excipients, and degradation products. Generation of degradation products can be accelerated by putting the sample

70  Chapter 4 under stress conditions, such as elevated temperature, oxidation, humidity, or light. The target compound response by using the analytical method should also be evaluated for purity (Branch, 2005; Buick et al., 1990; Bruce et al., 1998; Chan et al., 2004; Chambers et al., 2005; Christodoulakis and Satchell, 2008; de Bievre and Günzler, 2005).

4.3.2  Linearity and Range ICH defines the linearity of an analytical procedure as its ability (within a given range) to obtain test results that are directly proportional to the concentration of the analyte in the sample. Generally, methods are described as linear when there is a directly proportional relationship between the method response and concentration of the analyte in the matrix over the range of analyte concentrations in the working range. The working range is predefined by the purpose of the method and may reflect only a part of the full linear range. Linearity may be demonstrated directly on the test substance (by dilution of a standard stock solution) or by separately weighing synthetic mixtures of the test product components (ICH Q2B, 1996; ICH Q2A, 2005; Van Loco et al., 2002; Horwitz, 1995; Huber, 1999, 2004; Draper and Smith, 1996; Feinberg and de la Rochette, 1997; Feinberg, 1996; Funk et al., 2007; Garfield, 1985; González et al., 2006). For linear ranges, the deviations should be equally distributed between positive and negative values. Another approach is to divide signal data by their respective concentrations, yielding the relative responses. A graph is plotted with the relative responses on the y-axis and the corresponding concentrations on the x-axis, on a log scale. The obtained line should be horizontal over the full linear range. Linearity is determined by a series of five to six repetitions of five or more standard dilutions whose concentrations span 50%–150% of the expected concentration range. If there is a linear relationship evaluated by visual inspection, the test results should be evaluated by appropriate statistical methods. The response should be directly proportional to the concentrations of the analytes or proportional by means of a well-defined mathematical calculation (Dadgar and Burnett, 1995; Dadgar et al., 1995; Branch, 2005; Buick et al., 1990; Bruce et al., 1998; Chan et al., 2004; Chambers et al., 2005; de Bievre and Günzler, 2005). The linearity refers to the proportion between the concentration values of the validation standards and the analytical results of the developed method. The relation can be given as y = mx + n where n is the intercept of the line with the y-axis, and m is the slope (tangent) (Draper and Smith, 1996; Funk et al., 2007; Garfield, 1985; González et al., 2006). A linear relationship should be evaluated across the range. The following parameters should be calculated and reported: • • • •

correlation coefficient, y-intercept, RSD or SE of the y-intercept, slope of regression line,

Validation of Analytical Methods for the Assessment of Hazards in Food  71 • •

RSD or SE of the slope of the regression line, and residual sum of squares.

Ideally, the intercept is zero, meaning that when there is no analyte, there is also no response, but analytically this is not the case due to interferences, noise, contaminations, and other sources of bias (de Castro et al., 2005; Feinberg and de la Rochette, 1997; Feinberg, 1996; Funk et al., 2007; Meier and Zünd, 2000). The slope shows how sensitive is the method; the sharper the slope more sensitive the method means that the developed method is strong. Sensitivity is a measure of the determination coefficient “r2,” and the coefficient of correlation “r” should also be given. On the other hand, range is the interval of concentrations which have acceptable precision, accuracy, and linearity. A high correlation coefficient (r) of 0.99 is often used as a criterion of linearity. However, this is not sufficient to prove that a linear relationship exists, and a method with a coefficient of determination of 5 replicates (ICH Q2B, 1996; ICH Q2A, 2005; FDA, 1983, 2000; Werner et al., 2006; Van Loco et al., 2002). k¢ =

Resolution (Rs) is another system suitability test parameter showing how well the separation is. It can be calculated from the following equation where Rs is the resolution, W represents the width of the peaks, and tR is the retention time. This system suitability test parameter is very beneficial in that there exists an interference peak. Generally, Rs should be higher than 2 between two peaks (ICH Q2B, 1996; ICH Q2A, 2005; FDA, 1983, 2000; Werner et al., 2006; Van Loco et al., 2002). 2 ét R - t RI ùû RS = ë II WI + WII Tailing factor (T) is also one of the system suitability test parameters showing the peak quality. It can be calculated by the following equation where A and B are shown in Fig. 4.7. A T value lower than 2 or equal to 2 is acceptable (ICH Q2B, 1996; ICH Q2A, 2005; FDA, 1983, 2000; Werner et al., 2006; Van Loco et al., 2002). A+ B 2A

Detector response

T=

10% of Peak height

A

B 5 % of Peak height

Time

Fig. 4.7 Tailing parameter representation from system suitability test parameters.

Theoretical plate number (N) is the measure of the efficiency of the used column. Generally, higher than 2000 value is required to have well separated and eluated peaks (ICH Q2B, 1996; ICH Q2A, 2005; FDA, 1983, 2000; Werner et al., 2006; Van Loco et al., 2002).

Validation of Analytical Methods for the Assessment of Hazards in Food  85 2

ét ù N = 16 ê R ú ëW û At least three of the system suitability test parameters should be satisfied. If all can be satisfied, the method can be more accurate, precise, and reliable (ICH Q2B, 1996; ICH Q2A, 2005; FDA, 1983, 2000; Werner et al., 2006; Van Loco et al., 2002).

4.4 Conclusion Test methods for materials and articles in contact with foodstuffs are required to determine the concentration of active material, residues of monomers in the materials themselves, or to determine the concentration of individual or groups of substances in food (or food simulants) which have migrated from the food contact materials. There is a continuing need for reliable analytical methods for use in determining compliance with national regulations as well as international requirements in all areas of food quality and safety. The reliability of a method is determined by some form of validation procedure. Analytical method validation is the process of demonstrating that an analytical procedure is suitable for its intended purpose. The methodology and objective of the analytical procedures should be clearly defined and understood before initiating validation studies. This understanding is obtained from scientifically based method development and optimization studies. Method validation gives an idea of a method’s capabilities and limitations, which may be experienced in routine use while the method is in control in food assay. The validation of a specific method should be demonstrated in laboratory experiments using samples or standards that are similar to unknown samples analyzed routinely. Samples can be analyzed with well-validated methods, as long as the variability of precision and accuracy routinely falls within acceptable tolerance limits. If this is not the case, duplicate or even triplicate analyses should be performed. The proposed procedure assumes that the type of instrument has been selected and the method has been developed. It meets criteria such as ease of use; ability to be automated and to be controlled by computer systems; costs per analysis; sample throughput; turnaround time; and environmentally friendly health and safety requirements. Specific controls need to be applied to the method to verify that it remains in control, that is, performing in the way expected. During the validation stage, the method was largely applied to samples of known content. Once the method is in routine use, it is used for samples of unknown content. In the field of materials and articles in contact with food, numerous chemicals are used in the manufacturing processes and it is not possible to prepare standard test methods for all. Therefore, the concept of routine methods and reference methods should be superseded by a criteria approach, in which performance criteria and procedures for the validation of screening and confirmatory methods are defined.

86  Chapter 4

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Further Reading Code of Federal Regulations, n.d. Title 21, Food and Drugs, National Archives and Records Administration. Codex Alimentarius, n.d. FAO/WHO Food Standards, www.codexalimentarius.net/. FAO/WHO. 1997. ALINORM 97/23A. Report of the 21st Session. International Standard, n.d. ISO 9001, third ed., 2000–12–15, Quality Management Systems—Requirements, ISO 2000, Geneva.

CHAPTE R 5

The Detection of Pesticide in Foods Using Electrochemical Sensors Nurgul K. Bakirhan, Bengi Uslu, Sibel A. Ozkan Ankara University, Ankara, Turkey

Abbreviations Ab/glutaraldehyde/chitosan/GCE  antibody/glutaraldehyde/chitosan/glassy carbon electrode Ab-fG-SPE  antibody-functionalized graphene-screen-printed electrode AChE/[BSmim] HSO4-AuNPs-porous carbon/BDD  acetylcholinesterase/based on honeycomb-like hierarchically ion liquidsporous carbon composite-modified boron-doped diamond electrode AChE/Chit-PB-MWNTs-HGNs/Au  acetylcholinesterase/chitosan-prussian blue-multiwall carbon nanotubes-hollow gold nanospheres-modified gold electrode AChE/CoPC/SPE  acetylcholinesterase/cobalt (II) phthalocyanine/screen-printed electrode AChE–Au6–PDDA–PB/GCE  acetylcholinesterase-gold nanoparticles/poly (dimethyl diallyl ammonium chloride) protectedprussian blue/glassy carbon electrode AChE-MSF-PVA  acetylcholinesterase-mesocellular silica foam-poly (vinyl alcohol) AChE–SiSG–CPE  acetylcholinesterase-silica sol–gel-carbon paste electrode Atrazine/BSA/antiatrazine/GNPs/Au  atrazine/bovine serum albumin/antiatrazine/gold nanoparticles/gold electrode Au/AET/PAMAM/ATR-BSA/Ab-ATR  gold/2 amino ethane thiol/polyamido aminic dendrimers/atrazine and bovine serum albumine/antiatrazine monoclonal antibody Au-alfalfa sprout–SAMs  gold electrode modified with a self-assembled monolayer and immobilized alfalfa sprout AuNPs-chi-GNs/GCE  Au nanoparticles-chitosan/decorated graphene nanosheets-modified glassy carbon electrode BiFE  bismuth-film electrode CA  chronoamperometry CB/P [5] A  carbon black/pillar [5] arene CB/TC-0–Ag  carbon black/5,11,17,23-Tetra-tert-butyl-25,26,27,28-tetrakis-[1-(2′-hydroxyethyl)-N-(3″,4″dihydroxyphenyl) amidocarbonyl)-methoxy)-2,8,14,20-tetrathiacalix [4] arene in 1,3-alternate conformation-silver nanoparticles CdTeQDs@MIPs/Au  CdTe quantum dots dotted molecular imprinted polymers-modified gold electrode CHcych-PPO  polyphenol oxidase was obtained and immobilized in chitosan cross-linked with cyanuric chloride CHIT/CaONPs/[EMIM] [Otf]/Au  chitosan/calciumoxidenanoparticles/ionicliquid (1-ethyl-3methylimidazolium trifluoromethanesulfonate/goldelectrode CHIT/ZnONPs/[EMIM] [Otf]/AuE  chitosan/zinc oxidenanoparticles/ionicliquid (1-ethyl-3-methylimidazolium trifluoromethanesulfonate CNT–IL/MIP/GCE  carbonnanotubes-ionicliquid/molecularlyimprintedpolymer CoO/rGO/GCE  cobalt (II) oxide-decorated reduced graphene oxide/glassy carbon electrode Food Safety and Preservation. https://doi.org/10.1016/B978-0-12-814956-0.00005-6 © 2018 Elsevier Inc. All rights reserved.

91

92  Chapter 5 CoPc-fMWCNT/GCE  cobalt phthalocyanine functionalized multiwalled carbon nanotubes-modified glassy carbon electrode CoPC-SPCE  cobalt particles carbon-screen printed electrode CSV  cathodic stripping voltammetry CuE  copper electrode CuO NWs–SWCNTs/GCE  copper oxide nanowires-single-walled carbon nanotubes/glassy carbon electrode EC  electrochemiluminescence ƒ-SWCNT–β-CD/GCE  carboxylic acid-functionalized single-walled carbon nanotubes–β-cyclodextrin-modified glassy carbon electrode GA/AChE–IL-GR–Gel/GCE  glutaraldehyde/acetylcholinesterase—Ionic liquid functionalized graphene-gelatin/ glassy carbon electrode GBP-OPH/Au  gold-binding polypeptide (GBP) and an organophosphorus hydrolase (OPH) fusion protein on a gold electrode GC/MWCNT/PANI/AChE  acetylcholinesterase/polyaniline/multiwalled carbon nanotubes/core–shell-modified glassy carbon electrode Gelatin/Ab/GA/L-Cys/Au  gelatin/antibody/glutaraldehyde/l-cystein/gold electrode GN-AuNRs/GCE  graphene-gold nanorods/glassy carbon electrode GO-Hemin/CPE  graphene oxide-hemin complex/carbon paste electrode Graphene/CdSe@ZnS/AChE/ITO  Graphene/CdSe@ZnS quantum dots/acetylcholinesterase/Indium tin oxide Hg (Ag) FE  silver amalgam film electrode HMDE  hanging mercury drop electrode LACC/PB/GPE  laccase/prussian blue films/graphene doped carbon paste electrode Laccase(Glu)/AuNPs/AuE  laccase/gold nanoparticles/gold electrode LACC–TYR–AuNPs–CS/GPE  laccase–tyrosinase–AuNPs–chitosan/graphene doped carbon paste LDH/CMCD)4-PoPD/GCE  Mg–Al-layered double hydroxide/carboxymethyl-β-cyclodextrin/poly o-phenylenediamine/glassy carbon electrode m-GEC  magnetic graphite-epoxy composite MIP(aminothiophenol)/Au  molecularly imprinted polymer (aminothiophenol)/gold electrode MIP (Ppy)-SPCE  molecularly imprinted polymer (poly pyrrole)—screen-printed carbon electrode MIP/rGO@Au/GCE  molecularly imprinted polymer/reduced graphene oxide and gold nanoparticles/glassy carbon electrode MIP-CP  molecularly imprinted polymer-carbon paste MRLs  maximum residue levels mSiO2nanospheres/Ru (bpy)32+/Nafion/GCE  mesoporous SiO2nanospheres/Ru(bpy)32+/Nafion-modified glassy carbon electrode MWCNT–(PEI/DNA)2/OPH/AChE  multiwalled carbon nanotube-(polyethyleneimine/DNA)2/organophosphate hydrolase/acetylcholinesterase MWCNT/Pd-Ir/4TB [8] A/MIP/GCE  multiwalled carbon nanotube-supported Pd-Ir nanocomposite-tertbutylcalix [8] arene catalyst with methylene blue Nano-Au/SDBS/GCE  nanogold/sodium dodecylbenzene sulfonate nanoparticles-modified glassy carbon electrode NF/AChE-CS/NiONPs-CGR-NF  nafion/acetylcholinesterase—chitosan/nickel oxide nanoparticles-carboxylic graphene-nafion/glassy carbon electrode NF/AChE–CS/SnO2NPs–CGR–NF/GCE  nafion/acetylcholinesterase-chitosan/SnO2nanoparticles-carboxylic graphene-nafion/glassy carbon electrode OMC–Nafion/GCE  ordered mesoporous carbon—nafion-modified glassy carbon electrode OPs  organophosphates OCPs  organochlorine pesticides PEC  photo electrochemistry PMBI-MIP/Au  poly(2-mercaptobenzimidazole) molecularly imprinted polymer-modified gold electrode

The Detection of Pesticide in Foods Using Electrochemical Sensors  93 poly(TTBO)/AgNWs/BChE  poly(5,6-bis(octyloxy)-4,7-di(thieno[3][3,2-b]thiophen-2-yl) benzo[c] [1,2,5] oxoadiazole)/silver nanowires/butyrylcholinesterase Poly-o-AT/Au  p-aminothiophenol/gold electrode pSC6–Ag NPs/GCE  para-sulfonatocalix [6] arene-modified silver nanoparticles coated on glassy carbon electrode Pt–BMI·BF4-MMT  platinum nanoparticles-1-butyl-3-methylimidazolium tetrafluoroborate ionic liquid in montmorillonite PVA-AWP/Fe–Ni NP/AChE B394  azide-unit water pendant polyvinyl alcohol/Iron–Nickel Nanoparticle/ acetylcholinesterase B394 SiO2/MWCNT/GCE  mesoporous silica/multiwalled carbon nanotubes/glassy carbon electrode SiO2/MWCNTs/RuPc  hybrid silica/multiwalled partially oriented carbon nanotubes/ruthenium phthalocyanine SnO2 NPs–CGR–NF/GCE  tin oxide nanoparticles-carboxylic graphene and nafion-modified glassy carbon electrode SWCV  square-wave cathodic voltammetry TvL(composition)/MWCPE  TvL(composition)/multiwalled carbon paste electrode β-CD-rGO/GCE  β-cyclodextrin-graphene-modified glassy carbon electrode

5.1 Introduction Pesticides involving fungicides, herbicides, and insecticides are widely used in most food production to control pests (e.g., insects, fungi, rodents, worms, and herbs) that would otherwise destroy or reduce food production. In the area of agriculture, the usage of insecticides, herbicides, molluscicides, and fungicides has an increasing importance. Pesticides might be harmful as well as have beneficial effects. Pesticides may also be nonbiodegradable, so they persist in the environment and may accumulate in living organisms. Many pesticides are toxic property, so they can be dangerous to health. Some health problems can occur, such as bone marrow disorders, carcinogenicity, infertility, cytogenic effects, neurological diseases, and immunological and respiratory problems. Hence, food control is very important and necessary for living organisms. Pesticide can be found in chemicals, natural, or synthetic forms. They are used for the control of insects, fungi, bacteria, nematodes, rodents, weeds, and other pests. In the field of agriculture, production is affected by the usage of pesticides. Pesticides and their derived forms increase residues and contaminants which may find at environment, superficial and ground waters, in soil and in food products due to the solubility and toxicity properties. They can cause serious diseases in living organisms. They have organic structures that may contain electroactive parts. Hence, electrochemical methods can be used for their mechanistic behavior and quantifications. Many analytical methods have been applied for the analysis of pesticides such as fluorimetry, spectrophotometry, capillary electrophoresis, chromatography, mass spectroscopy (Anson and Wade, 1976; Shivhare and Gupta, 1991; Tomita et al., 1992; Jain et al., 1993; Eremin et al., 1996; Coly and Aaron, 1998a,b; Pico et al., 2003; Leandro et al., 2006; McGarvey, 1993; Castro et al., 2000; Corasaniti and Nistico, 1993). However, these methods require

94  Chapter 5 expensive equipments, have limited laboratories, and time-consuming experiments (Mulchandani et al., 2001). Also, an extraction or pretreatment process can be necessary in these methods. Electrochemical methods provide the elucidation of processes and mechanisms of redox reaction of pesticides and their residues. Moreover, the use of electrochemistry with hyphenated methods can give important information about the understanding of the degradation pathways of pesticides in various media. These methods are sensitive, selective, low in cost, and fast (Parham and Rahbar, 2010; Tapsoba et al. 2009; Tcheumi et al., 2010). Trace amounts of pesticides can be found in the air, in soil, and from environmental and food samples. Analytical methods sometimes have problems about the quantification of pesticides, residues, contaminants, and metabolites in different application fields: – chemicals (in pesticides) are too large, – these chemicals have a specificity in terms of reactivity, and – a pesticide matrix can include active ingredients. Therefore, electrochemical methods are needed for sensitive, fast, and selective determination of pesticides in various media. In this chapter, electrochemical methods will be discussed for pesticide analysis in food samples. The transducers used in pesticide analysis will be shown; an increasing interest in the new modified sensors in recent years will be described.

5.2 Pesticides Pesticides usage has benefits and risks. Crop protection, food preservation, and vector-borne diseases prevention can be possible with pesticides. They have many benefits such as prevention of malaria that kills a lot of children in a year and prevention of dengue, leishmaniasis, and Japanese encephalitis. Besides these beneficial effects, they have harmful risks such as toxicity and environmental results because of their mode of action for targeting systems or enzymes in pests that might be very similar to systems or enzymes in human beings. Therefore, they can affect human health and the ecosystem. In the environment, pesticides have common usage due to the killing of insects, rodents, weeds, and control of plants. Children are especially exposed to these pesticides. Against of adults, children can expose higher toxicity of pesticides (National Resource Council, 1993). Pesticides can be put in farm machines, so there is no need for many workers. Production of large quantities of food is possible. In other words, pesticide usage can increase yields and farm economy. Pesticides are classified as following chart (Fig. 5.1):

The Detection of Pesticide in Foods Using Electrochemical Sensors  95

Fig. 5.1 Classification of pesticides.

The usage ratio

The first usage of synthetic pesticides was in 1940. Consumption of pesticides has been growing over the years. 2.26 million tons of pesticide were consumed in 2001, that value correspond to 25% production of developing countries the world over. Herbicides and insecticides have widespread usage particularly in developing countries. The most used pesticides are shown in Fig. 5.2:

Herbicides

Insecticides

Fungicides

Others

Fig. 5.2 The most used pesticide types.

5.2.1  Pesticides Classification Related to Usage and Chemical Structure The World Health Organization (WHO) classified the pesticides based on their health risk, toxicity, estimation of median lethal dose (LD50). In toxicology, LD50 is the dose required to kill 50% of a tested population that is exposed to toxicity. If this dose decrease, toxicity can increase. The term toxicity has different levels like slightly toxic, moderately toxic, highly toxic, and extremely toxic (CICOPLAFEST 1998, WHO 2004). In Table 5.1, pesticide classification is presented according to the chemical structure.

96  Chapter 5

Table 5.1: Pesticide classification based on chemical structure Insecticides

Herbicides

Fungicides

Rodenticides

Fumigants

Insect Repellents

Pyrethroids

Bipyridyls

Thiocarbamates

Warfarines

Diethyltoluamide

Organophosphorus Carbamates Organochlorine Manganese compounds

Chlorophenoxy Glyphosate Acetanilides Triazines

Dithiocarbamates Cupric salts Tiabendazoles Triazoles Dicarboximides Dinitrophenoles Organotin compounds Miscellaneous

Indanodiones

Aluminum and zinc phosphide Methyl bromide Ethylene dibromide

The Detection of Pesticide in Foods Using Electrochemical Sensors  97 Pesticides are generally subclassified based on the chemical structure into four main groups: 5.2.1.1 Organochlorine Organochlorine pesticides (OCPs) are especially used in agriculture to protect cultivated plants. They affect the nervous system (1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane (DDT) is a well-known pesticide which has an extensive usage to prevent the spread of malaria, dengue, leishmaniasis, and Japanese encephalitis through the prevention of the growth of mosquitoes. Another most commonly used OCP is lindane. It is used to treat head lice in children’s hair (Benbrook, 2002). OCPs are persistent chemicals that can accumulate in food chain (Waliszewski et al., 2003, 2004). OCP residues are found in fatty foods of animal origin, such as meat, fish, eggs, and milk, and those of plant origin, such as sesame, corn, rice, oat, olives, vegetable oil, nuts, grapes, lettuce, and avocado, for preservation. About 60% ratio of organic vegetables have OCPs (Benbrook, 2002). 5.2.1.2 Organophosphates Organophosphorus pesticides (OPs) are esters derived from phosphoric acid and widely used pesticides to control insects and miticides on vegetables. OPs penetrate to humans by ingestion and contact. They have high efficiency and a broad spectrum of activity. The residues of OPs are found on vegetables such as vegetable crops, fruit trees, grains, cotton, sugarcane, lettuce, and cabbage. Vegetables must be controlled about amounts of OPs to protect human health (de Silva et al., 2006; Uygun et al., 2007; Darko and Akoto, 2008; Zhao and Zhao, 2009). OPs are effective on the nervous system via inhibiting acetyl cholinesterase enzyme (Sorgob and Vilanova, 2002). Its symptoms are headache, dizziness, nausea, loss of reflexes, convulsions, coma, and even death (Perry, 1974). 5.2.1.3 Carbamates Carbamates are esters derived from acids and carbamic acid. These pesticides have become very important in recent years due to their low mammalian toxicity, rapid disappearance, and broad spectrum activity (Saunders and Harper, 1994). However, they can inhibit acetylcholinesterase enzyme. Therefore, they are harmful to people. Carbamate pesticides are generally used as insecticides, herbicides, fungicides, and nematicides. OCPs and OPs are more persistent than carbamates. However, carbamylation of the acetylcholinesterase enzyme occurs very fast due to the covalent attachment of electrophilic groups of the carbamoyl part to enzyme. 5.2.1.4 Pyrethroids Pyrethroids are used as insecticides that have a similar structure to pyrethrins whose extract is derived from chrysanthemum flowers. They were obtained synthetically and are presently manufactured in around 100 different commercial products (Sorgob and Vilanova, 2002). Pyrethrins are slightly soluble in water. Pyrethroids are more toxic than

98  Chapter 5 pyrethrins. Pyrethroids and pyrethrins are primarily released to the air because of their use as insecticides. Generally, pyrethroids are sprayed on crops and flight insects. Owing to the sunlight and compounds in the atmosphere, many pyrethroids can be rapidly degraded. If the atmosphere has rain or snow, they can be removed from the air before degradation. Pyrethroids can be absorbed into the body by eating contaminated foods, not with touch. These insecticides affect the central nervous system, causing changes in the dynamics of the Na+ channels in the membrane of the nerve cell. Perry (1974) explained that this effect can cause neuronal hyperexcitation. 5.2.1.5 Others Other pesticides are ordered as triazine herbicides, amides, nitro compounds, ethylene dibromide, phtalamide, bipyridyl compounds, nitro compounds, sulfur containing compounds, urea, hormon, copper, or mercury.

5.2.2  Pesticides and Human Health Pesticide usage has increased production in agriculture. Therefore, pesticide concentration has also increased in food and the ecosystem with accompanying bad results on human health. There are so many poisonings from pesticides every year (Richter, 2002). Chronic problems especially have occurred such as cancer, diabetes, genetic disorders, neurological effects, as well as respiratory and fetal diseases besides headaches, dizziness, fatigue, and short-term impacts. These problems are different in degree due to the exposure time and type. Chronic risk effects might occur after years if exposed to pesticides in the environment. Pesticides are also affecting people to a different degree in relation to those directly exposed, less directly exposed, and consumers. Acute problems can occur such as skin, nerve, and eye irritation. The central nervous system of children is not fully formed until age 12, so it is very important to expose pesticides. Pesticides can affect the human brain. Owing to the exposure to pesticides, children’s immune systems and nervous systems can be more affected. In other words, children have absorbed pesticides more than adults and they can absorb these hazardous materials through their lungs, intestinal tracts, water, air, and food.

5.2.3  Pesticides and the Environment Pesticide usage can affect the living organisms (beneficial insect species, soil microorganisms, and worms) in the environment. Some pesticides can accumulate on the vegetables, soil, water, and air. They can decrease the population of pests but can cause soil problems. Plant root and immune systems can become weak. Essential plant nutrients can reduce such as nitrogen and phosphorus.

The Detection of Pesticide in Foods Using Electrochemical Sensors  99 Of the pesticides used in the soil, some parts of them go to the air or waters with emission, volatilization, or drifting ways. From the air and waters, they can deposit on the soils, plants, animals, and people. Some pesticides which are used in the environment can be very persistent. They join the food chain so that humans and especially babies are at the top of the food chain (Fig. 5.3). Humans, birds, and mammals

Soil and surface water

Granules, seeds Drinking water

Crops

Ground water and food product

Air

Sea animals

Farm animals

Sprayed pesticides

Fig. 5.3 Possible ways of pesticide contamination in the food chain. Reproduced with permission from Kumar, P., Kim, K.-H, Deep, A., 2015. Recent advancements in sensing techniques based on functional materials for organophosphate pesticides. Biosens. Bioelectron. 70, 469–481.

Persistent organic pollutants (POPs) or persistent toxic substances (PTSs) are OCPs such as aldrin, endrin, hexachlorobenzene, toxaphene, chlordane, DDT, heptachlor, and mirex. They are lipophilic compounds which are low in solubility, resistant to environmental breakdown, and accumulate in adipose tissue and marine mammals (Kutz et al., 1991). Persistent OCPs have been banned in agricultural use and domestic uses in Europe, North America, and South America at the Stockholm Convention in 1992. However, DDT has been still used in some developing countries to control malaria.

5.2.4  Maximum Levels of Pesticide Residues in Food Maximum residue levels (MRLs) are the highest levels of pesticide residues on foods or feed crops. They are passed to humans in direct or indirect ways. Therefore, the European Union has defined legislative rules about approved active substances used in plant protection products and pesticide residues in foods. Active substance is defined as the active component

100  Chapter 5 by European Union in plant protection product to against pesticides or plant diseases. The European Union controls the foods or products before they are sold. If the substances are found in foods, they must be safe for humans, animals, and the environment.

5.2.5  Who Monitors Pesticides? Environmental Protection Agency (EPA): EPA is responsible for two groups. First, it regulates the pesticides which are used by farmers. Its second responsibility is the setting up of MRLs of pesticides in foods marketed in America. Food and Drug Administration (FDA): The FDA is responsible for establishing the laws related to crops and agricultural processes. US Department of Agriculture (USDA): The USDA is responsible for evaluating and establishing the regulations related to the pesticides though analytical studies.

5.3  Overview to Electroanalytical Studies on Pesticides In pesticide analysis, developed electroanalytical methods are very important. The proper electroanalytical method for a pesticide analysis depends on the selected method, its optimization, and proper working electrode. The reaction of interest occurs on the working electrode as a transducer. The proper electrode material depends on the possible redox reaction of the analyte and working potential range.

5.3.1  Electrochemical Behaviors of Pesticides 5.3.1.1  Organochloride pesticides Electrochemical studies of organochloride pesticides depend on removal of the chlorine atom (Carrai et al., 1992; Garcia et al., 1991). Dieldrin, endosulfan, heptachlor, and endosulfan sulfate are cycloalkene-containing organochloride pesticides in which electrochemical behaviors have been reported (Fig. 5.4) (Garcia et al., 1991; Reviejo et al., 1992a,b,c, 1993).

Cl R1

Cl Cl Cl

Cl R2

Cl

Fig. 5.4 The general structures of organochlorine pesticides.

The Detection of Pesticide in Foods Using Electrochemical Sensors  101 Generally, they are studied in micellar solutions due to the low solubility properties in water. Hence, surfactants usage is common to obtain a better peak response. p,p′-DDT and dieldrin have a similar electrochemical behavior. Two pesticides can be determined at different potentials by adding Fe (II). Dieldrin can form a metallic complex with Fe (II). According to the literature survey, the determinations of organochloride pesticides have been performed with DPV and AdSV methods. 5.3.1.2  Organophosphate pesticides Organophosphate pesticides can be divided into six groups (Fig. 5.5). These pesticides have −PO, −PS−, and −S−P [groups (c), (d), (e), and (f)] structures. −PS− and −S−P bonds show strong adsorption responses which allow the determination of low concentrations. O

O

O R

R P

O

R P

R

P

R

R

R

R

(A)

(B)

(C)

S P

O

R P

R

R

S

S R

R

S

S

P

R

R

R

R

(D)

(E)

(F)

R

Fig. 5.5 Chemical structures of six organophosphate pesticide groups.

Dichlorvos, chlorfenvinphos, dicrotophos, and crotoxyphos are also examples of organophosphate pesticides (Fig. 5.6) which have CC bonds (Subbalakskmamma and Reddy, 1994; Sreedhar et al., 1997a,b). These compounds are studied in a broad pH range 2.0–12.0 and each compound can give a well-defined reduction response (Subbalakskmamma and Reddy, 1994).

H3CO

O P

O

H3CO

Cl C

H

C Cl

Fig. 5.6 Chemical structure of organophosphate pesticide groups which have C=C bond.

102  Chapter 5 5.3.1.3  Carbamate and thiocarbamate pesticides Electrochemical behavior of carbamate pesticides is studied generally with polarography. Metal carbamate complexes are formed and used as pesticides (Halls et al., 1968). Aldicarb, carbofuran, carbaryl, ethienocarb, fenobucarb, oxamyl, and methomyl are some insecticides in carbamate groups. Carbaryl is a well-known insecticide in this family. Its chemical structure is shown in Fig. 5.7.

Fig. 5.7 Chemical structure of carbaryl.

5.3.1.4  Triazine pesticides Triazines and their derivatives are used as herbicides in agriculture. According to the literature, triazines have two groups, s-triazine and asymmetrical triazine (Fig. 5.8). s-Triazine has an aromatic heterocyclic structure. Reduction reaction occurs on the −CN− bond of the heterocyclic ring (Ignjatovic et al., 1993; Pedrero et al., 1993; Stastny et al., 1982). The reduction process is begun with protonation of nitrogen. For asymmetrical triazines, the reduction reaction depends on the structure. For example, guthion (azinphos-methyl) which has an asymmetrical triazine structure, reduction occurs at the −NN− bond of the ring (Méndez et al., 1988). However, metamitron reduction occurs on −CN− and N−NH2 (Olmedo et al., 1994). Analysis of triazines generally needs pretreatment steps with organic solvents. They have solubility problems in water, so electrochemical methods require alternative conditions. As an example of this situation, simazine was studied with polarography in water–oil R1 N R2HN

O N

N N

s-Triazine

S

NHR3

N

CH2

S

N

P

OCH3 OCH3

Asymmetric triazine

Fig. 5.8 s-Triazine and asymmetric triazine structures (R1 = Cl; OCH3; SCH3, R2, and R3 = Alkyl groups).

The Detection of Pesticide in Foods Using Electrochemical Sensors  103 and micellar solutions (Gálvez et al., 1993). The most used electrochemical methods are differential pulse voltammetry and adsorptive stripping voltammetry (AdSV) for analysis of triazine pesticides. 5.3.1.5 Nitropesticides Nitropesticides have very toxic properties, so their accurate, reliable, and sensitive analysis is important. Nitropesticide reduction reaction occurs in nitro groups with consequent formation of hydroxylamines (Southwick et al., 1976; Benadikova et al., 1983). Nitropesticides are divided into four groups such as nitroorganophosphates, nitrophenol derivatives, dinitroaniline derivatives, and nitrorganochlorides. Electrochemical studies can also achieve determination of metabolites. For example, parathion is a nitroorganophosphate pesticide; it has two metabolites, paraoxon and p-nitrophenol (Bowen and Eduards, 1950). They have different reduction potentials so that they do not interfere with each other and simultaneous analysis can be possible (Smyth and Osteryoung, 1978). Like triazine pesticides, they have adsorption behavior on mercury electrode and need AdSV for the determination of this group of pesticides. 5.3.1.6 Sulfonylureas Sulfonylureas are less dangerous pesticides which include low toxicity. They have three distinct parts: – aryl group – sulfonylurea bridge – nitrogen containing heterocycle. Chlorsulfuron is a well-known example of the family of sulfonylurea pesticides. Its chemical structure is shown as Fig. 5.9. CH3 Cl SO2NHCONH

N

N N

OCH3

Fig. 5.9 The chemical structure of sulfonylurea pesticide, chlorsulfuron.

To date, the literature has a few works based on sulfonylurea determination with electrochemical methods. DPV is the most commonly used method for these groups. Chlorsulfuron, metsulfuron-methyl, and chlorimuron-ethyl were analyzed by DPV (Concialini et al., 1989). pH is very important for the sulfonylurea pesticide electrochemical behavior. Generally, the best redox peak current was obtained at pH 2.5.

104  Chapter 5 5.3.1.7  Bipyridinium pesticides Bipyridinium pesticides are known as viologens which are derivatives of 4,4′-bipyridyl (IUPAC, 1997). They are used as herbicides. They can be easily reduced to the radical mono cation (Bird and Kuhn, 1981; Elofson and Edsberg, 1957; Pospisil et al., 1975; Walcarius and Lamberts, 1996). Their chemical structure is presented in Fig. 5.10. R-X N

X R N

N

X R N

N R X

e

N R X

N R X

R N

O2

R N

N R X

e

R N

N R

In the literature for electrochemical behavior of viologen, two peaks were observed, expressed that transferring of two electron and reduction of the cations. In bipyridinium pesticide groups, not all pesticides are electroactive. Paraquat and diquat are the commonly used herbicides in the bipyridinium pesticides groups.

R

+N

N+

R

Fig. 5.10 The chemical structure of bipyridinium pesticide.

5.3.2  The Electroanalytical Methods for Assay of Pesticides on Foods Electroanalytical methods depend on measuring changes in current with applied potential. Current is the change in charge with a function of time. I = dQ / dt According to Faraday’s law (equation is given below), measuring charge is proportional to the amount of analyte undergoing a gain or loss of electrons. Q=nFe where Q is the total charge in coulombs unit, n is the number of moles of an analyte undergoing redox reaction,

The Detection of Pesticide in Foods Using Electrochemical Sensors  105 F is Faraday's constant (96,487 C/mol), and e is the number of electrons per molecule gained or lost Hence, current response with time shows information about changes in the concentration of the analyte. 5.3.2.1  Cyclic voltammetry Among the variety of electrochemical methods which are available for pesticide analysis, voltammetry, that is, the recording of the current, as a function of applied potential, has become the most popular and common. Cyclic voltammetry (CV) is one of the potential sweep methods. It involves the most commonly used diagnostic tool for studying the electrode process (Smyth and Vas, 1992; Kissinger and Heineman, 1996; Brett and Oliveira-Brett, 1998; Wang, 2006). CV can show information about the electrochemical reactions and mechanism. It can also provide formal potentials, kinetics of electrochemical reactions on the electrode surface. It offers a rapid location of redox potentials and evaluation of the effect of pH and media on the redox reaction (Wang, 2006). The important parameters in the CV method are the magnitudes of the peak currents, Ipa and Ipc; the peak potentials at which peaks occur, Epa and Epc, and the half peak potential (E1/2). According to the parameters and relationship between them, provide an explanation about reversibility, irreversibility, or quasi-reversibility. Fig. 5.1 shows the usage of CV in pesticide analysis. Methylparathion was studied in 0.1 M acetate buffer (pH 5) with the CV method on montmorillonite and the gemini surfactant-modified glassy carbon electrode (Tcheumi et al., 2010). According to the cyclic voltammogram, methylparathion gave two redox systems, reversible and irreversible (Epc2 = −0.60 V). In the reversible part, methyl parathion showed an adsorption process on the modified electrode. Epc2 generated the reversible system because, after Epc2 formation, Epa1, Epc1 formed. Methylparathion was studied again on a different electrode (Nafion-coated GCE) (Zen et al., 1999). The same behavior was observed in this study. An irreversible system was obtained due to the reduction of the nitro group, after formation of a cathodic peak reversible couple was observed (Zen et al., 1999, Sbaï et al., 2007). 5.3.2.2  Pulse voltammetric methods Normal pulse voltammetric method (NPV) was studied using dropping mercury electrode (DME) by Barker and Gardner (1960). It was selected because of the minimizing effects of DME charging current. Hence, a more sensitive application could be done (Bockris and Khan, 1993; Gileadi et al., 1975; Bard and Faulkner, 2001; Brett and Oliveira-Brett, 1993).

106  Chapter 5 NPV is more sensitive than linear sweep voltammetry. NPV responses are not related to the electron transfer rate, so it is the proper method for the determination of diffusion coefficients of electroactive species. 10−6 M concentration levels can be performed by this method. The stirring procedure is needed in irreversible systems to remove depletion effects (Oldham and Parry, 1996). The Differential pulse voltammetry (DPV) principle depends on the waveform that consists of pulses of constant amplitude superimposed on a staircase waveform. In this method, charging current can be ignored against faradaic current and their ratio (faradaic current/ charging current) is obtained as large. Hence, the DPV method is the most sensitive one in electrochemistry. Barker and Gardner (1960) proposed the DPV method for the DME to obtain a lower limit of detection values of analytes. For the pulse voltammetric methods, there are important parameters which are affected from responses of analyte. They are ordered as pulse amplitude, pulse width, and the sampling period. The obtained peak current height is directly proportional to the analyte concentration. The great superiority of DPV is the minimization of charging current owing to calculating the difference in current at two potentials differing by less than 100 mV, while in NPV the charging current increases as Ep increases. The increasing values of the ratio faradaic current/ charging current mean that lower LOD values (10−8 M) can be reached. Owing to its sensitive property, it is used in trace analysis, drug analysis, and pesticide analysis. Differential pulse polarography is another pulse method. In DPP method principle is based on the linearly increasing voltage that superimposed during the lifetime of a single drop by an increase of voltage of small amplitude. This increasing voltage is applied toward the end of the drop-time. The difference of current between before and during the voltage step is measured and plotted as a function of the applied voltage. This method has the disadvantage about the application part. DPP is working on just reversible systems where the equilibrium between the oxidized and reduced forms at the electrode surface. 10−7 M concentration levels can be obtained with DPP. Although mercury is highly toxic, DPP is used frequently in analytical laboratories (especially pesticide analysis) due to their so many functional groups of pesticide and mercury electrode provides the adsorption of many organic and inorganic compounds. According to the one study (Sreedhar et al., 1997a,b) in the literature, DPP was used for determination of three fungicides which have the carbonyl group. Methyl parathion was studied in water and soil samples with DPP (Castanho et al., 2003). As a dinitro pesticide, fluazinam was determined from environmental samples by Balaji et al. (2010a). The same group (Balaji et al., 2010b) was studied on fluochlorin with DPP in grains, soils, and spiked water samples.

The Detection of Pesticide in Foods Using Electrochemical Sensors  107 Square wave voltammetry is one of the efficient, fast, and major voltammetric methods in electrochemical studies. Its principle depends on a potential, consisting of symmetrical square wave pulses superimposed on a staircase wave form that is applied to the working electrode. During each square wave cycle, the current is sampled twice, just before the end of each forward and each backward pulse followed by subtraction of the currents; a plot is made of the forward (If), backward (Ib), and the total net (It = If−Ib) currents versus the staircase potential. This method has three types, Barker, Kalousek, and Osteryoung. The Osteryoung-type SWV is widespread in electrochemical analysis (Osteryoung and Osteryoung, 1985; Osteryoung and O'Dea, 1986). SWV measurement can be taken with solid electrodes or on static mercury drop electrodes. 10−8 M detection limits can be obtained with this method. SWV is also more sensitive than DPV (about four times) and kinetic mechanism is so fast. Reversible and irreversible systems can be studied with SWV. In the literature, there have been published studies on pesticide determination with SWV. As a toxic herbicide, paraquat was studied by El Mhammedi et al. (2007) with a modified carbon paste electrode. Organophosphate pesticides have been studied with SWV (Liu and Lin, 2005; Parham and Rahbar, 2010; Zen et al., 1996a,b; Garrido et al., 1999). Nowadays, there are some studies on stripping SWV combined with solid phase extraction (Gong et al., 2009, 2010). SWV provides a fast, simple, and sensitive analysis of pesticides. 5.3.2.3  Stripping methods CV, LSV methods cannot applied to sensitive analysis due to their obtained detection limits about 10−5 M. DPV and SWV methods are chosen for 10−7–10−8 M values. The preconcentration step is very important for sensitivity. Detection limits can be lowered to 10−10–10−12 M by stripping methods. If the preconcentration step is used, this method is called the stripping method. The stripping method is divided into four subgroups: Anodic, cathodic, adsorptive voltammetry, and potentiometric stripping. The advantages of stripping voltammetric methods are: a. b. c. d. e. f.

very low detection limits (about 10−10–10−12 M), high precision and accuracy, online and in situ measurements, no effect from the matrix, low cost equipment, relative simplicity and rapidity.

Stripping analysis has special importance in pesticide analysis. Electroactive molecules are deposited on solid electrodes or liquid mercury electrodes before measurement (Bockris and Khan, 1993; Gileadi et al., 1975; Bard and Faulkner, 2001; Brett and Oliveira-Brett, 1993).

108  Chapter 5 The preconcentration step can be done by forming an amalgam or complex with the particular analyte and the electrode material or by adsorbing the analyte on the electrode surface (Wang, 2006; Koryta et al., 1993; Zoski, 2007; Brett and Oliveira-Brett, 2003; Kellner et al., 2004; Harvey, 2000; Hart, 1990). Stripping voltammetry has the following steps: a. Deposition or accumulation of analyte on the electrode surface at open circuit or constant potential b. Equilibration time (max. 30 s) c. Measurement (determination occurs by redox reaction of the analyte) Deposition potential and time should be optimized to obtain a more sensitive result and wellpeak shape. In pesticide analysis, DPV and SWV methods are the mostly used methods due to the obtain very low detection limits of organic and inorganic analytes (Bagotsky, 2005; Wang, 2006). Simultaneous analysis of compounds can be performed with these methods due to the improved resolution between peak potentials of compounds (Wang, 2006). For instance, dinitrophenolic herbicides and neonicotinoid insecticides were studied by adsorptive stripping differential pulse voltammetry (Sreedhar et al., 2003). Anodic stripping voltammetry (ASV) is the most widely used stripping method. It involves a preconcentration step and a determination step. In the preconcentration step, metal ion reduces to a metallic form on the electrode surface. In the determination step, metal is reoxidized from the electrode surface via a positive potential. Cathodic stripping voltammetry (CSV) has a different mechanism from that in ASV. The deposition step is performed by oxidation to a compound of the metal and stripping by reduction. This method has been used for the detection of inorganic ions such as iodide, bromide, and chloride, as well as sulfide ions and pesticides (Florence, 1979; Moore and Gaylor, 1977; Palecek, 1980; Palecek et al., 1982). Adsorptive stripping voltammetry (AdSV) has three steps: a. accumulation step by adsorption b. equilibration step (max. 30 s) c. stripping step In this method, in the accumulation step, a complexing or chelating agent is used to enable the formation of an adsorbable metal complex or chelate in solution in an optimized pH medium. Abrasive stripping voltammetry (AbSV) is used for electroactive all solid substances (Scholz et al., 1989, 1990, 1991; Scholz and Lange, 1990). A trace amount of a solid substance is transferred onto the electrode surface by abrasion. CV, DPV, or SWV can be selected for the electrochemical stripping step where the abraded substance is

The Detection of Pesticide in Foods Using Electrochemical Sensors  109 electrochemically stripped step where the abraded substance is electrochemically stripped and measured. The advantages of the AbSV method are: – – – –

it can be applied to all solid compounds it can elucidate the very complex condition on the surface electrode. very small amounts of compounds can be studied by this method. it provides voltammetric studies with insoluble compounds in water or other important solvents. – minerals, high-temperature superconductors, metal chelate complexes, and organometallics can be studied by AbSV. In the literature, chlorothion pesticide was studied by AbSV on cucumber and lettuce. AbSV could provide a rapid solid-state voltammetric procedure for the screening of chlorothion. 5.3.2.4 Amperometry In pesticide analysis methods, amperometry is one of the common selected methods. According to this method, a constant potential is applied and the change in current is measured with a function of time. In this method, the superiority is the limited time resolution. However, this method is not selective about chemicals. 5.3.2.5 Chronoamperometry Chronoamperometry (CA) is one of the most used electrochemical methods. It is a kind of square wave pulsed method. Electrochemical information of the analyte is limited; it can be obtained from the ratio of the peak oxidation current versus the peak reduction current. The disadvantage of CA is that it generates high charging currents, which in this case decays exponentially with time. Diffusion coefficient, kinetic and redox mechanism can be investigated by this method. According to CA, the analyte current response changes with time. The diffusion coefficient and electrode area can be obtained with the Cottrell equation. i= where n is the number of electrons, F is Faraday’s constant, D is the diffusion coefficient, Cb is the bulk concentration, and A is the electrode area.

nFAD1/ 2C b

(p t )

1/ 2

110  Chapter 5 5.3.2.6  Electrochemical impedance spectroscopy Electrochemical impedance spectroscopy (EIS) is a sensitive method which monitors the response of the studied system to the application of a periodic small amplitude AC signal. EIS is a sensitive method which monitors the response of the studied system to the application of a periodic small amplitude AC signal. Measurements are carried out at different frequencies. Analysis of the system response contains information about the interface, its structure, and occurring reactions. This technique does not lead to the identification of the chemical bonds or of the intermediates; however, information on the reaction rates occurring at the electrochemical interface can be obtained and provide characterization of the intermediates (Gabrieli, 1995). The EIS method can provide sensor characterization and development of new surfaces (Rohrbach et al., 2012; Tran et al., 2011). This method is very interesting for the biosensor fabrication (Radi et al., 2005). EIS is a sensitive method with rapid detection of analytes. Its disadvantageous part is the low selectivity (Bogomolova et al., 2009). However, this problem can be removed by modifying the electrode surfaces with nanoparticles or polymers.

5.3.3  Electrode Types in Pesticide Analysis 5.3.3.1  Dropping mercury electrode Mercury as an electrode material has positive and negative properties in the literature. It has a narrow potential range and is very toxic. However, it is still used especially in stripping analysis. For environmental studies, usually a mercury thin film electrode is selected. A hanging mercury electrode is often used for adsorptive stripping analyses of pesticides. 5.3.3.2  Metal electrodes Although the first electrochemical studies began with a dropping mercury electrode, recent studies used metal electrodes due to their high conductivity, low background current, and easy applicability in stationary or flow-through systems. In these days, metal electrodes have gained popularity because of their applicability to anodic oxidations. However, their important disadvantage is fouling of the surface. Although the first used metal electrode was silver (Ag) (Adams, 1969; Wang, 2006; Smyth and Vas, 1992), the most used metal electrode materials are platinum (Pt) and gold (Au). The Ag electrode has an extremely narrow potential window, which limited electrochemical studies. Pd, Bi, Rh, Cu, Ru, Ni, Cd, Sn, and In have also been used for specific electroanalytical applications. For example, Cu, Ni, and Ag electrodes can be proper for carbohydrates and amino acid studies. 5.3.3.3  Carbon-based electrodes Carbon is of different types depending on the bonding structures and properties. Pyrolytic carbon, vitreous carbon, and carbon fiber have an sp2 atomic structure. Diamond, fullerens, etc. have an sp3 type of carbon.

The Detection of Pesticide in Foods Using Electrochemical Sensors  111 In these types of carbon, sp2 ones are very conductive, stabile, porous, and hard. Carbon-based electrodes are more favorite electrodes than dropping mercury electrodes and metal electrodes due to the following reasons; – wider potential range, – rich surface area, – inertness, – low cost, – low background current, – broad potential window, – suitable for various sensing and detection applications, – simple renewal electrode surface. There are different type carbons as electrode materials. One of the most famous is glassy carbon, and the others can be ordered as graphite, carbon paste, carbon films, fullerenes, pyrolytic graphite, carbon fiber, wax-impregnated graphite, carbon nanotubes, screen-printed carbon, and whiskers. They all differ in their agglomeration, particle size, and degree of graphitization. Of these carbon electrode types, glassy carbon is the most popular electrode material in the literature due to its physical and electrochemical properties (van der Linden and Dieker, 1980). It is the most widely used carbon electrode in pesticide analysis. It has also easy cleaning and polishing for electrode surface renewability and fast electron transfer kinetics. Carbon paste electrode is especially selected as a transducer for organophosphate compounds analyses. Organophosphate pesticides can be strongly adsorbed on the carbon paste surface and provide sensitive analysis (Lin and Lin, 2005). Carbon fiber electrodes are increasingly being used in electrochemical studies. Pyrolytic carbon films have advantages such as a fast electron transfer rate and easy electrode pretreatment (McFadden et al., 1990). Boron-doped diamond has chemical inertness and low electrode capacitance (Pedrosa et al., 2004). In recent studies, carbon nanotubes have shown good properties such as they can improve electron transfer reactions, minimizing fouling effect, gain electrocatalytic activity (Baughman et al., 1999; Banks et al., 2004). The wall-jet glassy carbon electrode has also been used for determination of organic pollutants (Manisankar et al., 2002). 5.3.3.4  Clay-film modified electrode Clay minerals are widely preferred as modifiers due to their well-defined layered structure, low cost, and flexible properties. They can also form conductive surfaces.

112  Chapter 5 Clay-modified electrodes can show adsorption depending on the holes and porous structures. Adsorption can occur on the external surfaces of the clay or at the edges of the clay sheets. Natural clay minerals are used for the modification of solid electrodes to determine pesticides. For example, montmorillonite clay-modified GCE was fabricated for sensitive analysis of endosulfan, o-chlorophenol, methylparathion, isoproturan, and malathion with square wave stripping voltammetry method (Manisankar et al., 2006). In the literature, a nontronite clay-coated screen printed carbon electrode was developed for determination of amitrole by flow injection analysis. Nontronite clay includes a high iron content and various compounds can be analyzed with it (Fitch, 1990; Carter and Bard, 1987; Zen and Lo, 1996; Zen et al., 1996a,b, 1998). In other examples, zirconia nanoparticles were used for modification and they could have analyzed organophosphate pesticides and nerve agents (Liu and Lin, 2005). 5.3.3.5  Polymer-coated electrodes Conducting polymers have been used in numerous application fields such as in batteries, electronics, ion-selective membranes, and electrochemical sensors (Thevenot et al., 2001; Gerard and Chaubey, 2002; Nishizawa et al., 1992; Barlett and Whitaker, 1987). They have excellent properties; – – – –

high conductivity due to the π-conjugation structure, good electrochemical reversibility, easily functionalized with many chemical groups, highly specific cavities.

Electrochemical sensors based on conducting polymers hold promise for an expanded array of applicable airborne, biological, and environmental analytes. Isoproturan, carbendazim, and methyl parathion were analyzed with polypyrrole modified GCE (Manisankar et al., 2005). In the literature, atrazine pesticide was studied by molecularly imprinted polymers (MIPs) (Berg et al., 1995; Luo et al., 2001; Piletsky et al., 1995; Matsui et al., 1995; Pardieu et al., 2009). MIPs are used for the developing pesticide sensor. These polymers are cross-linked or copolymer forms. MIPs have a high physical and chemical stability, short time of synthesis process, low cost, and are selective. They also have great potential for biorecognition species as enzymes or antibodies. The interaction between biomolecule and MIPs depends on electrostatic, hydrophobic, and steric matching interactions. Owing to these properties of MIPs, they can be considered as artificial/plastic antibodies (Algieri et al., 2014; Volkert and Haes, 2013; Rotariu et al., 2016). Electrochemical polymerization of monomers is used for MIPs synthesis. Target compound is inside of template. The target compound is removed and active sites are formed in the structure of the polymer. These active sites are complementary in shape, functional group, and

The Detection of Pesticide in Foods Using Electrochemical Sensors  113 size with the template molecule (Fig. 5.11). Mimicking the biological activity of antibodies, MIPs can bind the analyte in the molecularly imprinted sites. An appropriate transducer will convert this interaction in a measurable signal. However, poor signal transduction and a lack of stability and selectivity represent the main drawbacks of MIP sensors. In the literature, there have been different ways to amplify the response, sensitivity, and selectivity. Electropolymerization of p-aminothiophenol functionalized gold nanoparticles in the presence of the template molecule leads to an amplification of the analytical signal of the MIP sensors (Jiang et al., 2015). Aflatoxin B1 was determined with very low detection limits as 4 fM (Jiang et al., 2015). Bougrini et al. (2016) applied the same principle of MIP sensors and tetracycline was detected at 0.22 fM from a honey sample. Quinoxaline-2-carboxylic acid (QCA) is a veterinary drug and major metabolite of carbadox. It can be found in commercial pork meat products at trace level so that its detection is very difficult. An MIP sensor was developed and QCA was detected at 4.4 × 10−7 M by Yang et al. (2013). The developed sensor was applied in commercial pork products with good stability and reproducibility. A long-acting sulfonamide antibiotic, sulfadimethoxine as a veterinary drug, was determined from milk samples by an MIP sensor (Turco et al., 2015). The detection limit was obtained at 70 μM with good selectivity and reproducibility. O

O N H

O OH

H3C

O

Drop-coating

Remove tamplate

Rebind

GCE

GCE

GCE

GCE

Modified with rGO@Au

H2C

MIP film

Fig. 5.11 The fabrication of MIP film on rGO@Au electrode. Reproduced with permission from Tan, X., Hu, Q., Wu, J., Li, X., Li, P., Yu, H., Li, X., Lei, F., 2015. Electrochemical sensor based on molecularly imprinted polymer reduced grapheme oxide and gold nanoparticles modified electrode for detection of carbofuran. Sensor. Actuat. B-Chem. 220, 216–221.

114  Chapter 5 As an organophosphate pesticide, isocarbophos was determined with an MIP sensor [poly(ophenylenediamine-co-gallic acid-co-maminobenzoic acid)] by Yan et al. (2012). They applied the DPV method with a linear range from 7.50 × 10−8 to 5.00 × 10−5 M and detection limit 2 × 10−8 M. They applied the developed sensor in cabbage and cowpea real samples. 5.3.3.6  Biosensors for pesticide analysis Biosensors have been used for detection of pesticides. According to Fig. 5.12A, biosensors are presented as analytical devices. Biorecognition and transducer parts are combined for detection of the target analyte (Donald, 1993; Guilbault et al., 2004; Gopel et al., 1991). The biorecognition part can be an enzyme, DNA, antibody, or microorganism (Fig. 5.12B). Hence, the biosensor is based on a reaction catalyzed by macromolecules, which are present in their biological environment or have been isolated previously or manufactured.

Fig. 5.12 (A) Schematic representation of biosensor and (B) an example for biosensor fabrication for pesticide determination. Reproduced with permission from Zhang, Y., Arugula, M.A., Wales, M., Wild, J., Simonian, A.L., 2015. A novel layer-by-layer assembled multi-enzyme/CNT biosensor for discriminative detection between organophosphorus and non-organophosphorus pesticides. Biosens. Bioelectron. 67, 287–295.

The Detection of Pesticide in Foods Using Electrochemical Sensors  115 The transducer part of the sensor serves to transfer the signal from the output part of the recognition system to mostly the electrical part. The transducer part of a biosensor can be used as a detector, electrode, or sensor. However, a transducer includes all of them, so this confusion is removed. Generally, electrochemical transducers (potentiometry, voltammetry, amperometry, surface charge using field effect transistors [FETs], and conductometry) can measure the output signal from the biorecognition part. The biochemical recognition mechanism was considered in this section to classify the biosensors and the main types of biosensors used for food analysis are presented in Fig. 5.13. Substrate detection

Substrate

Oxidases

Peroxidases

Enzyme biosensors

O2 H2O2 H2O2 H2 O NAD+

Dehydrogenases

NADH

Electrochemical biosensors for food

Product

Substrate

Enzyme inhibition

Substrate

Enzyme

Inhibitor

Product

Affinity biosensors

MIP sensors Molecularly imprinted polymers

Fig. 5.13 Types of biosensors that are used in food analysis. Reproduced with permission from Rotariu, L., Lagarde, F., Jaffrezic-Renault, N., Bala, C., 2015. Electrochemical biosensors for fast detection of food contaminants— trends and perspective. Trends Anal. Chem. 79, 80–87. https://doi.org/10.1016/j.trac.2015.12.017.

Enzyme-based biosensors for pesticide analysis

Enzyme-based electrodes are a big part of the biosensors. Enzyme systems are used as bioreceptors. They can be organelles, whole cells, and tissues. Enzyme biosensors are divided into two parts; one depends on substrate detection and the other depends on enzyme inhibition principles.

116  Chapter 5 In substrate detection enzyme biosensors, oxidoreductases are commonly used for development of biosensors. Substrates are catalyzed by the enzymes and then formed products can be detected with a transducer. Biosensor selectivity depends on the enzyme and developed transducer. Biogenic amines are formed in food after the microbial decarboxylation of amino acids (Naila et al., 2010). In food analysis, recent studies are based on the determination of these biogenic amines. Poor selectivity of these enzymes were determined by different research groups with higher selectivity for a particular amine, such as putrescine oxidase extracted from Micrococcus rubens (Henao-Escobar et al., 2016) characterized by specificity to putresceine, or spermine oxidase an enzyme specific to spermine (Boffi et al., 2015). l-Lactic acid detection is an important experiment to ascertain freshness of foods such as tomato, infant food, etc. l-Lactate biosensors were developed in the literature with l-lactate oxidase (Rassaei et al., 2013). Xanthine is also an important biomarker to detect freshness of fish (Dervisevic et al., 2015). Peroxidase has been used to catalyze the oxidation of phenol compounds (Chekin et al., 2015; Raghu et al., 2013; Gurban et al., 2015; Gurban et al., 2011). Tyrosinase can be used to catalyze the oxidation of some small and large substrates such as tyramine (Apetrei and Apetrei, 2015) or bisphenol A (Kochana et al., 2015; Reza et al., 2015; Zehani et al., 2015). Nitrite reductase has been used in the food industry (Nikolelis et al., 2013). Nitrite-based biosensors are used as a preservative. Dehydrogenases as a subgroup of oxidoreductases are a huge class of enzymes based on the NAD+/NADH or NADP+/NADPH redox systems in realization of the electrons transfer from or to the substrate. For example, histamine dehydrogenase was used to fabricate a histamine biosensor (Henao-Escobar et al., 2016). l-Lactate dehydrogenase is also used to test l-lactate with much superiority for electrochemical detection of NADH (Rassaei et al., 2013). Hydrolase enzymes are a very small group of electrochemical biosensors. β-Lactamase is the enzyme which is responsible for the resistance of the bacteria to some class of antibiotics. In recent years, there is an increased concern about the antibiotics in meat or milk products. For example, β-lactamase was used to detect penicillin G (Prado et al., 2015). Enzyme inhibition-based biosensors are commonly used for detection of pesticides. Acetylcholine esterase (AChE) and butyrylcholineesterase (BChE) are the most used enzymes (Amine et al., 2016). The principle depends on measuring the enzyme activity in the absence and presence of the inhibitor and the inhibition degree is correlated with the concentration

The Detection of Pesticide in Foods Using Electrochemical Sensors  117 of the inhibitor (Rotariu et al., 2012). In the literature, there are studies with other enzymes such as tyrosinase, alkaline phosphatase, or organophosphate hydrolase as bioreceptors for pesticide detection (Amine et al., 2016). These biosensors lack poor selectivity for a specific compound that could be sometimes an advantage when this type of device is intended to be used as a screening method for the presence of a class of toxic compounds in the sample. An exception is shown by the methyl parathion hydrolase, which is specific to methyl parathion (Liu et al., 2014a,b). Puiu et al. (2012) have reported an approach about the use of AChE for detection of mycotoxins like aflatoxin B1. Attar et al. (2015) used peroxidase for detection of cyanide. Affinity biosensors for pesticide analysis

The main difference between the affinity (bio)sensors and enzyme biosensors is the absence of a biochemical transformation of the analyte after interaction with the bioreceptor. The interaction between the analyte and the affinity partner is reversible, the formation and the decomposition of the affinity complex being realized in mild conditions by modifying the physicochemical parameters like pH or ionic strength. Affinity biosensors are based on analytical recognition systems such as antigen–antibody, hormone–receptor, or interaction between DNA strands, which do not imply a chemical transformation of the target compound. In recent studies, new classes of recognition elements were reported. Among them, the aptamers, MIPs, or antibodies attracted the attention of researchers and was the subject of review (Tiwari and Turner, 2014). 5.3.3.7  Immunosensors for pesticide determination As shown in Fig. 5.14, immunosensors are based on the immunochemical reactions, that is, binding of the antigen (Ag) to a specific antibody (Ab). Nonspecific interactions are minimized when the Ab-Ag complexes are detected. Every Ag detection needs a special Ab production, isolation and purification. If the sensitivity wants to increase, generally enzyme labels couple to Ab or Ag. Hence, additional chemical synthesis steps are required. In the literature, there have been related articles which are used in pesticide detection (Xuesong et al., 2008; Ciumasu et al., 2005; Zacco et al., 2007; Tschmelak et al., 2005).

5.4  Applications of Electrochemical Pesticide Analysis on Foods The majority of the studies found in the literature have as their objective the quantitative measurement of pesticides in a wide variety of sample types and different analytical methods. Electrochemical methods are very popular and sensitive in this area due to the selectivity, sensitivity, and fast properties. Many kinds of nanomaterials have been widely used in

118  Chapter 5

(A)

(B) Transducer interface

Antibody

Labeled antibody

Linker layer

Antigen

Labeled antigen

Fig. 5.14 Schematic representation of immunosensors: (A) sandwich format and (B) competitive format. Reproduced with permission from Jiang, X., Li, D., Xu, X., Ying, Y., Li, Y., Ye, Z., Wang, J., 2008. Immunosensors for detection of pesticide residues. Biosens. Bioelectron. 23, 1577–1587.

different electrochemical sensing systems based on their attractive properties. In this chapter, our aim is present electrochemical methods and developed sensors for determination of pesticides in food samples. El-Moghazy et al. (2016) developed an ultrasensitive screen-printed biosensor with a genetically engineered AChE immobilized in an azide unit water pendant polyvinyl alcohol (PVA-AWP)/Fe–Ni alloy nanocomposite. The Fe–Ni alloy provided a catalytic effect by lowering the potential from 80 to 30 mV. The developed biosensor was used for reproducible and stabile phosmet detection in olive-oil samples with a 96% recovery rate. The detection limit was obtained as 0.1 nM. Mehta et al. (2016) determined parathion pesticide from tomato and carrot samples. They fabricated an immunosensor with a 2-aminobenzylamine functionalized graphene sheets modified screen printed electrode and antiparathion antibody (Fig. 5.15). The modification steps are given below: • • • •

drop casting of graphene suspension on SPE 2-aminobenzylamine is used for functionalization of graphene sheets immobilization of antiparathion antibodies sensing of parathion pesticide

The Detection of Pesticide in Foods Using Electrochemical Sensors  119

O

4

C

O

C NH

C

NH

O

NH

C NH

NH2

3

Z′′ (Ω)

2

NH2

O

1

Electrochemical impedance analysis Z′ (Ω)

Step 1: Drop-casting of graphene on carbon screen printed electrode Step 2: Electro-catalyzed amine (−NH2) functionalization of graphene with 2-aminobenzylamine Step 3: Immobilization of anti-parathion antibodies on −NH2 functionalized graphene SPE Step 4: Immunosensing of parathion with above sensor Antibody

Parathion

Nonspecific pesticides

Fig. 5.15 Schematic of the graphene-based screen-printed immunosensor for parathion detection. Reproduced with permission from Mehta, J., Vinayak, P., Tuteja, S.K., Chhabra, V.A., Bhardwaj, N., Paul, A.K., Kim, K-H., Deep, A., 2016. Graphene modified screen printed immunosensor for highly sensitive detection of parathion. Biosens. Bioelectron. 83, 339–346.

5.5  0.1–1000 ng/L linear Range and 52 pg/L Limit of Detection Were Obtained by Impedance Spectroscopy SiO2/MWCNTs/RuPc was developed by Canevari et al. (2016) for determination of organophosphate pesticide fenitrothion. The modified electrode showed well-defined peaks in the presence of fenitrothion in acetate buffer, pH 4.5, with a sensitivity of 0.0822 μA μM−1 mm−2 and a detection limit of 0.45 ppm. The electrocatalytic property of the developed electrode was compared with the SiO2/MWCNTs/GCE and bare GCE electrodes (Fig. 5.16). According to the DP voltammograms of three electrodes (SiO2/MWCNTs/RuPc/ GCE (Ered = −0.54 V), SiO2/MWCNTs/GCE (Ered = −0.58 V), and bare GCE (Ered = −0.55 V)), SiO2/MWCNTs/RuPc/GCE (Ered = −0.54 V) showed a better electrocatalytic effect with increasing current response of 1.66 × 10−5 M fenitrothion. Recent studies (2010–16) about organophosphate pesticide analysis from foods are presented in Table 5.2. Li et al. (2016) developed a novel supramolecular imprinted sensor for carbamate-based pesticide, carbofuran. An imprinted sensor was fabricated with an MWCNT/supported Pd-Ir

120  Chapter 5 –0.2

–0.2

–0.4

I / mA

–0.6 I / mA

–0.4

Absence 1.66 x 10–5 mol L–1

–0.8 –1.0

Absence 1.66 x 10–5 mol L–1

–0.6 –0.8 –1.0

–1.2

SiO2/MWCNTS/RuPc/GCE

SiO2/MWCNTS/GCE

–1.2

–1.4 –0.8

(A)

–0.6 –0.4 –0.2 0.0 E / V vs Ag / AgCl

0.2

0.4

–0.8

–0.6

(B)

–0.4 –0.2 0.0 E / V vs Ag / AgCl

0.2

0.4

–0.3 –0.4 Absence 1.66 x 10–5 mol L–1

I / mA

–0.5 –0.6 –0.7 –0.8

GCE bare

–0.9 –0.8

(C)

–0.6 –0.4 –0.2

0.0

0.2

0.4

E / V vs Ag / AgCl

Fig. 5.16 Electrocatalytic study of fenitrothion pesticide using the (A) SiO2/MWCNTs/RuPc/GCE electrode, (B) SiO2/MWCNTs/GCE electrode, and (C) GCE electrode using differential pulse voltammetry in a 0.1 M acetate buffer (pH 4.5) and a scan rate of 20 mV s−1. Reproduced with permission from Canevari, T.C., Prado, T.M., Cincotto, F.H., Machado, S.A.S., 2016. Immobilization of ruthenium phthalocyanine on silicacoated multi-wall partially oriented carbon nanotubes: Electrochemical detection of fenitrothion pesticide. Mater. Res. Bull. 76, 41–47.

nanocomposite catalyst (MWCNT/Pd-Ir) with methylene blue (MB). At the beginning of the modification of GCE, MWCNT/Pd-Ir composite nanoparticles were formed. At the second step, MB-doped o-phenylenediamine as the functional monomer and a supramolecular complex (4-tert-butylcalix [8] arene-CBF (4TB [8] A-CBF)) as the template were used (Fig. 5.17). CBF signal was controlled with a modified electrode and a developed sensor showed sensitive quantification of the agricultural pesticide carbofuran. Malaoxon, methyl-paraoxon, carbofuran, and aldicarb pesticides were determined with a acetylcholinesterase biosensor by Shamagsumova et al. (2015). Pillar [5] arene was used as electron mediator. This biosensor fabrication steps is shown in Fig. 5.18. The CA method was applied to determine these organophosphate and carbamate pesticides. 200 mV and

Table 5.2: Recent electrochemical studies (2010–16) on organophosphate pesticide in/on foods Pesticide

Class

Structure

Method

Transducer

LOD/LOQ

Matrix

References

Parathion

Insecticide

Ops

EIS

Ab-fG-SPE

0.18 pM

Mehta et al. (2016)

Phosmet

Insecticide

Ops

Amperometry

0.1 nM

Paraoxon

Insecticide

Ops

Amperometry

0.212 μM

Milk

Parathion

Insecticide

Ops

SWCV

PVA-AWP/Fe–Ni NP/ AChE B394 poly(TTBO)/AgNWs/ BChE BiFE

Tomato Carrot Olive oil

55.7 nM

Skimmed milk

Fenitrothion

Insecticide

Ops

DPV

1.62 μM

Orange juice

Paraoxon

Insecticide

Ops

CV

0.5 μM

Apple

Dichlorvos

Insecticide

Ops

DPV

0.30 pM

Lettuce

Wei and Wang (2015)

Carbaryl Monocrotophos

Insecticide Insecticide

Carbamate Ops

DPV

5.3 fM 0.46 fM

Tomato juice

Zheng et al. (2015)

Paraoxon Dichlorvos

Insecticide Insecticide

Ops

PEC

0.61 fM 2.5 pM

Apple

Li et al. (2015)

Chlorpyrifos-oxon

Insecticide

OPs

0.10 μM

Apple

Methyl parathion Acephatemet

Insecticide Insecticide

OPs OPs

AutoDip platform SWV EIS

3.11 nM 4.25 nM

Kiwi Cabbage Apple

Drechsel et al. (2015) Zhu et al. (2014) Gong et al. (2014)

Paraoxon Malaoxon Aldicarb Carbofuran Malathion

Insecticide Insecticide Insecticide Insecticide Insecticide

OPs OPs Carbamate Carbamate OPs

0.05 nM 0.1 nM 0.01 μM 0.1 nM 0.3 nM

Peanut Grape juice

Evtugyn et al. (2014)

Garlic

Huo et al. (2014)

DPV

GN-AuNRs/GCE LDH/ CMCD)4-PoPD/GCE CB/TC-0–AgNPs/ GCE

CuO NWs–SWCNTs/ GCE

Gerent and Spinelli (2016) Canevari et al. (2016) Zhang et al. (2015)

Continued

The Detection of Pesticide in Foods Using Electrochemical Sensors  121

CA

SiO2/MWCNTs/ RuPc/GCE MWCNT–(PEI/ DNA)2/OPH/AChE AChE/[BSmim] HSO4-AuNPs-porous carbon/BDD GA/AChE– IL-GR–Gel/ GCE Graphene/CdSe@ ZnS/ AChE/ITO Chip

El-Moghazy et al. (2016) Turan et al. (2016)

Pesticide

Class

Structure

Method

Transducer

LOD/LOQ

Matrix

References

Methyl parathion

Insecticide

OPs

DPV

ƒ-SWCNT–β-CD/ GCE

1.52 nM

Yao et al. (2014)

Malathion Acephate

Insecticide Insecticide

OPs OPs

DPV

AChE–SiSG–CPE

0.18 μM 0.24 μM

Methyl parathion Carbofuran

Insecticide Insecticide

OPs Carbamate

DPV

0.05 pM 0.5 pM

Malathion Chlorpyrifos Monocrotophos Chlorpyriphosoxon Ethyl paraoxon Malaoxon Monocrotophos

Insecticide Insecticide Insecticide Insecticide Insecticide Insecticide

OPs

Amperometry

OPs

Amperometry

NF/AChE–CS/ SnO2NPs–CGR–NF/ GCE AChE/Chit-PBMWNTs-HGNs/ Au AChE/CoPC/SPE

Onion Lettuce Rape Spinach Grape Apple Mango Orange Banana Tomato Rice Wheat Apple Cabbage

0.05 nM 0.05 nM 0.1 nM 5 pM 5 nM 0.5 nM

Cabbage Lettuce Leek Pakchoi Milk

Mishra et al. (2012)

Insecticide

OPs

Amperometry

3.6 pM

Garlic

Wu et al. (2011a,b)

Methyl parathion

Insecticide

OPs

SWV

AChE–Au6– PDDA–PB/ GCE AuNPs-chi-GNs/GCE

2.28 nM

Gong et al. (2011)

Chlorfenvinphos Monocrotophos Methyl parathion Paraoxon Methyl parathion

Insecticide Insecticide Insecticide Insecticide Insecticide

OPs OPs OPs OPs OPs

CA Amperometry SWV SWV DPV

CoPC-SPCE AChE-MSF-PVA nano-Au/SDBS/GCE MIP-CP pSC6-Ag NPs/GCE

– 0.2 nM 0.09 nM 1.0 nM 4.0 nM

Garlic Cabbage Tea Wheat Garlic Pear Cabbage Pear

Raghu et al. (2014)

Zhou et al. (2013)

Zhai et al. (2013)

Crew et al. (2011) Wu et al. (2011a,b) Li et al. (2011a,b) Alizadeh (2010) Bian et al. (2010)

122  Chapter 5

Table 5.2: Recent electrochemical studies (2010–16) on organophosphate pesticide in/on foods—cont’d

The Detection of Pesticide in Foods Using Electrochemical Sensors  123

Elution

Polymerization Modify

Re-adsorption

Deposition

GCE

MWCNT

Pd-Ir manoparticles 4-Tert-butylcalix [8] arene

Detection

Detection

Self-assembly

CBF current

CBF MB MIP

Catalytic current

Fig. 5.17 The procedure to construct a sensor and determine CBF. Reproduced with permission from Li, S., Yin, G., Wu, X., Liu, C., Luo, J., 2016. Supramolecular imprinted sensor for carbofuran detection based on a functionalized multiwalled carbon nanotube-supported Pd-Ir composite and methylene blue as catalyst. Electrochim. Acta. 188, 294–300.

180 s were optimum conditions to obtain the best responses. Detection limits were obtained for malaoxon, methyl-paraoxon, carbofuran, and aldicarb at 4 × 10−12, 5 × 10−9, 2 × 10−11, and 6 × 10−10 M, respectively. The AChE biosensor was tested in the organophosphate and carbamate pesticides spiked samples of peanut and beetroot.

Glassy carbon

Glassy carbon

Glassy carbon

OH

OH

90

Enzyme immobilization

OH OH

Pillar[5]arene

75 I, %

Pillar[5]arene deposition

OH

OH

AChE

OH OH

OH

AChE

AChE Carbon black

OH

60 45 30 1E-4

CH3 H3C N+ CH2-CH2-S C CH3 CH3

O

AChE

CH3COOH

CH3 H3C N+ CH2-CH2-SH

1E-3 0.01 0.1 [Malaoxon], µM

CB/P[5]A,-2e

1

−

Dimer

CH3

Fig. 5.18 Biosensor fabrication for malaoxon, methyl-paraoxon, carbofuran, and aldicarb pesticides determination. Reproduced with permission from Shamagsumova, R.V., Shurpik, D. N., Padnya, P. L., Stoikov, I. I., Evtugyn, G.A., 2015. Acetylcholinesterase biosensor for inhibitor measurements based on glassy carbon electrode modified with carbon black and pillar [5] arene. Talanta. 144, 559–568.

Recent studies (2010–16) about carbamate pesticide analysis from foods are presented in Table 5.3.

Pesticide

Class

Structure

Method

Transducer

LOD/LOQ

Matrix

References

Formetanate Carbofuran

Insecticide Insecticide

Carbamate Carbamate

SWV DPV

CoPc-fMWCNT/GCE MWCNT/Pd-Ir/4TB[8] A/MIP/GCE

0.097 μM 1.7 pM

Ribeiro et al. (2016) Li et al. (2016)

Carbofuran

Insecticide

Carbamate

DPV

MIP/rGO@Au/GCE

0.02 μM

Aldicarb Carbofuran Carbofuran

Insecticide Insecticide Insecticide

Carbamate

CA

CB/P[5]A

Carbamate

EIS

Gelatin/Ab/GA/L-Cys/ Au

0.02 nM 0.4 nM 0.45 nM

Carbendazim Carbofuran

Fungicide Insecticide

Carbamate Carbamate

SWV SWV

SiO2/MWCNT/GCE Biomimetic sensor

0.056 μM 9.0 nM

Oxfendazole

Anthelmintic

Carbamate

MIP(Ppy)-SPCE

Aldicarb Carbofuran Carbaryl Formetanate Propoxur Ziram Carbofuran carbaryl

Insecticide Insecticide Insecticide Insecticide Insecticide Fungicide Insecticide Insecticide

Carbamate

DPV SWV CA

Carbamate

SWV

LACC–TYR–AuNPs–CS/ GPE

Carbamate

DPV

CoO/rGO/GCE

0.025 μM 0.015 μM 0.01 μM 0.1 nM 0.02 μM 0.22 μM 0.19 μM 1.68 nM 0.034 μM 0.021 μM

Mango grape Cowpea Chinese cabbage Tomato Apple Cabbage Cucumber Peanuts Beetroot Tomato Cabbage Lettuce Orange juice Carrots Tomato Milk

Formetanate

Insecticide

Carbamate

SWV

Laccase(Glu)/AuNPs/AuE

0.095 μM

Carbofuran

Insecticide

Carbamate

SWV

GO-Hemin/CPE

9 nM

CB/TC-0–Ag

Tan et al. (2015) Shamagsumova et al. (2015) Liu et al. (2015a,b)

Razzino et al. (2015) Wong et al. (2014) Radi et al. (2014)

Peanut

Evtugyn et al. (2014)

Orange Tangerine Lemon

Oliveira et al. (2014)

Grapes Oranges Tomato Cabbages Mango Grape Carrots Tomatoes

Wang et al. (2014)

Ribeiro et al. (2014) Wong et al. (2014)

124  Chapter 5

Table 5.3: Recent electrochemical studies (2010–16) on carbamate pesticide in/on foods

Insecticide

Carbamate

Amperometry

SnO2 NPs–CGR–NF/ GCE Ab/glutaraldehyde/ chitosan/GCE LACC/PB/GPE

0.5 pM

Fenvalerate

Insecticide

Carbamate

EIS

Carbofuran Ziram Carbofuran

Insecticide Fungicide Insecticide

Carbamate

SWV

Carbamate

Amperometry

AChE/Chit-PBMWNTs-HGNs/ Au

Pirimicarb

Insecticide

Carbamate

SWV

TvL(composition)/MWCPE

0.18 μM

Carbofuran

Insecticide

Carbamate

Amperometry

0.5 pM

Carbaryl Methomyl Methomyl

Insecticide Insecticide Insecticide

Carbamate

CA

Carbamate

SWV

NF/AChE-CS/NiO NPs-CGR-NF GC/MWCNT/PANI/ AChE Pt–BMI·BF4-MMT

1.4 μM 0.95 μM 1.0 μM

Metolcarb

Insecticide

Carbamate

CA

poly-o-AT/Au

0.013 μM

m-tolyl methyl carbamate Thiodicarb

Insecticide

Carbamate

CA

0.013 μM

Insecticide

Carbamate

SWV

MIP(aminothiophenol)/ Au CHcych-PPO

Thiodicarb

Insecticide

Carbamate

SWV

Au-alfalfa sprout–SAM

0.58 μM

1.91 nM 0.1 μM 5.2 nM 2.5 nM

0.16 μM

Apple Cabbage Tea Tomato Potato Cabbage Lettuce Leek Pakchoi Tomato Lettuce Apple Cabbage Cabbage Broccoli Carrot Tomato Apple juice Cucumber Cabbage Apple juice Peach Grape Lettuce Potato Apple Strawberry

Zhou et al. (2013) Wang et al. (2013a,b) Oliveira et al. (2013a,b) Zhai et al. (2013)

Oliveira et al. (2013a,b) Yang et al. (2013) Cesarino et al. (2012) Zapp et al. (2011) Pan et al. (2011)

Pan et al. (2011) Lima et al. (2010)

Moccelini et al. (2010)

The Detection of Pesticide in Foods Using Electrochemical Sensors  125

Carbofuran

126  Chapter 5 Ribeiro et al. (2013) studied OCP endosulfan (Fig. 5.19) with SWV. HMDE was used as a working electrode and a quasi-reversible behavior was obtained for endosulfan in acidic media. The SWV method was optimized as the pulse potential frequency of 200 s−1, the amplitude of the pulse of 20 mV, and the height of the potential step of 4 mV (Fig. 5.19). Analytical parameters such as linearity range, detection and quantification limits, recovery values, precision, and accuracy were obtained. The developed method was applied in sugarcane and tomato samples. The recovery test showed that the proposed method is proper for quantification of endosulfan in the matrix. Reproducibility was also checked and stabilesensitive results were obtained.

Fig. 5.19 Molecular structure of endosulfan and SW voltammograms for 2.43 × 10−5 mol L−1 of endosulfan on the HMDE in the britton–robinson buffer, pH 4.0, with a scan potential ranging from −0.5 to −1.4 V, f = 200 s−1, a = 20 mV and ∆Es = 4 mV. Forward component (…), backward component (---), and resultant component (−−). Reproduced with permission from Ribeiro, F.W.P., Oliveira, T.M.B.F., Silva, F.L.F., Mendonça, G.L.F., Mello, P.H., Becker, H., Lima-Neto, P., Correia, A.N., Freire, V.N., 2013. Sensitive voltammetric responses and mechanistic insights into the determination of residue levels of endosulfan in fresh food stuffs and raw natural waters. Microchem. J. 110, 40–47.

Clothianidin insecticide was determined by AdsSWV using a silver amalgam film electrode (Hg (Ag) FE) (Brycht et al., 2013). The pretreatment of the Hg (Ag) FE by applying the appropriate conditioning potential is very important. It was used at −0.60 V in the Britton– Robinson buffer pH 9.0. The proposed AdsSWV method was optimized (Fig. 5.20). Two linear ranges were obtained as 6.0 × 10−7–7.0 × 10−6 M and 7.0 × 10−6–4.0 × 10−5 M. The lower

The Detection of Pesticide in Foods Using Electrochemical Sensors  127 O2N N H 3C

H2 C N H

S

CI

N H

-10 N

-30

-5

Ip (mA)

Ip (mA)

(b)

-20

-10 (a)

0

-0.50

-1.00

0 -1.50

Econd (V)

Fig. 5.20 SWV signals of 3.0 × 10−5 M Clothianidin recorded in the Britton-Robinson buffer pH 9.0 at (A) HMDE and (B) Hg (Ag) FE. Measurement parameters: conditioning potential Econd = −1.70 V, conditioning time tcond=15 s, frequency f = 100 Hz, amplitude Esw = 60 mV, and step potential ΔE = 7 mV. Reproduced with permission from Brycht, M., Skrzypek, S., Guzsvány, V., Berenji, J., 2013. Conditioning of renewable silver amalgam film electrode for the characterization of clothianidin and its determination in selected samples by adsorptive square-wave voltammetry. Talanta. 117, 242–249.

LOD value was obtained at 1.8 × 10−7 M. Repeatability, recovery, and interference tests were performed. Analytical application was applied with corn seeds samples. Melamine is the most studied material in triazine pesticides. It is especially used in white products such as milk and milk products. Liu et al. (2015a,b) developed a sensor for the determination of the toxic material melamine in milk samples. They fabricated a selective and sensitive molecularly imprinted electrochemical sensor. This sensor was fabricated with a carbon nanotube–ionic liquid composite (Fig. 5.21). In this sensor, nanomaterials formed imprinted cavities so that the selectivity and sensitivity were increased. Linear range was obtained as 0.4–9.2 μM with a detection limit of 0.11 μM. The detection of melamine compound in real samples was successfully applied with good recovery. Recent studies (2010–16) about other pesticide analysis from foods are presented in Table 5.4.

128  Chapter 5 Polymerization CNT-IL

Extraction Rebinding Polymerization

Melamine Polypyrroles

Fig. 5.21 The preparation process of CNT–IL/MIP and CNT–IL/NIP, and the comparison of electrochemical response of melamine at different modified electrodes. Reproduced with permission from Liu, B., Xiao, B., Cui, L., M. Wang., 2015a. Molecularly imprinted electrochemical sensor for the highly selective and sensitive determination of melamine. Mat. Sci. Eng. C-Biomim. 55, 457–461; Liu, L., Xu, D., Hu, Y., Liu, S., Wei, H., Zheng, J., Wang, G., Hu, X., Wang, C., 2015b. Construction of an impedimetric immunosensor for label-free detecting carbofuran residual in agricultural and environmental samples. Food Control. 53, 72–80.

5.6 Conclusion Pesticides are used in various steps of food crops cultivation and/or in agriculture. Some of them have persistent property and can be accumulate on people. Hence they can be hazardous for humans and the environment. Children are the most affected part of the chain, so the European Commission set maximum residue limits at 0.01 mg/kg for any pesticide residue. Several electrochemical approaches developed for the analyses of pesticides in food samples were discussed in this chapter. It is encouraging that electroanalyses for food samples are conceptually very similar to those used for clinical or environmental analyses. The greatest difficulty in the analysis was the fouling of the electrode surface. Using proper electrode material, method optimization and modification of the surface can be a solution for this problem. Spending the minimum solvent amount is perhaps the most active area of green chemistry. In electrochemistry, usually small amounts of solutions are used. Aqueous media are also preferred for recent studies. Hence, electrochemical methods have superiorities based on green chemistry. In this chapter, several electrochemical approaches were developed for the analysis of different pesticide groups in food matrices. Biosensor-based sensors, especially cholinesterase, were used for the determination of organophosphate, carbamate, triazine, and pyrethroid types. MIPs and nanoparticles have been preferred for fabrication of transducers. To perform sensitive, reliable, fast control of low levels analysis of pesticides have to be done with instrumental methods. Modern electroanalytical techniques, especially voltammetric methods, provide a reliable, sensitive, selective, and reproducible analysis for the pesticide residues determination.

Table 5.4: Recent electrochemical studies (2010–16) on other pesticides in/on foods Class

Structure

Method

Transducer

LOD/LOQ

Matrix

References

Endosulfan

Insecticide

OCs

SWV

HMDE

0.30 nM

Paichongding

Insecticide

Neonicotinoid

CV

β-CD-rGO/GCE

0.11 μM

Sugar cane Tomato Grain

Paraquat

Herbicide

Amperometry

m-GEC

0.7 nM

Potato

Cypermethrin

Insecticide

Bipyridinium dichloride Pyrethroid

CV

Pt

4.8 nM

Tomato

Clothianidin

Insecticide

Pyrethroid

AdSSWV

Hg(Ag)FE

0.18 μM

Corn seeds

Fenvalerate

Insecticide

Pyrethroid

EIS

1.91 nM

Tea

S-Fenvalerate

Insecticide

Pyrethroid

Photoelectrochemistry

Glutaraldehyde/ chitosan/GCE CdTeQDs@MIPs/Au

3.2 nM

Melamine

Polymer

Triazine

DPV

0.096 pM

Melamine

Polymer

Triazine

SWV

CHIT/ZnONPs/ [EMIM][Otf]/AuE CNT–IL/NIP/GCE

Apples Pears Tomatoes Cucumbers Milk powder

Ribeiro et al. (2013) Zhang et al. (2016) Garcia-Febrero et al. (2013) Borah et al. (2016) Brycht et al. (2013) Wang et al. (2013a,b) Wang et al. (2013a,b)

0.11 μM

Milk

Melamine

Polymer

Triazine

DPV

0.96 fM

Milk

Melamine Atrazine

Polymer Herbicide

Triazine Triazine

DPV DPV

2.4 nM 0.074 nM

Milk Maize

Melamine

Polymer

Triazine

DPV

CHIT/CaONPs/ [EMIM][Otf]/Au OMC–Nafion/GCE Atrazine/BSA/antiatrazine/GNPs/ Au CuE

0.85 μM

Milk

Melamine

Polymer

Triazine

DPV

MIPs/GCE

0.068 μM

Milk Infant Formula Pet food

Rovina and Siddiquee (2016) Liu et al. (2015a,b) Rovina et al. (2015) Guo et al. (2014) Liu et al. (2014a,b) Araujo and Paixão (2014) Xu et al. (2014)

Continued

The Detection of Pesticide in Foods Using Electrochemical Sensors  129

Pesticide

130  Chapter 5

Table 5.4: Recent electrochemical studies (2010–16) on other pesticides in/on foods—cont’d Pesticide

Class

Structure

Method

Transducer

LOD/LOQ

Matrix

References

Atrazine

Herbicide

Triazine

CV

5.6 nM

Corn flakes

Melamine

Polymer

Triazine

EC

2.6 nM

Milk powder

Giannetto et al. (2014) Cao et al. (2013)

Melamine

Polymer

Triazine

EIS

Au/AET/PAMAM/ ATR-BSA/Ab-ATR mSiO2nanospheres/ Ru(bpy)32+/Nafion/ GCE PMBI-MIP/Au

3.0 nM

Cyanazine

Pesticide

Triazine

CSV

MIP-CP

3.2 nM

Propazine

Herbicide

Triazine

CSV

MIP-CP

0.001 μM

Melamine Melamine Melamine

Polymer Polymer Polymer

Triazine Triazine Triazine

DPV DPV DPV

MIP-CP MIP/GCE GCE

0.7 nM 0.36 μM 3.0 nM

Liquid milk Yogurt Milk powder Onion Tomato Lettuce Rice Onion Tomato Lettuce Rice Milk Milk Milk

Wu et al. (2012)

Gholivand et al. (2012)

Gholivand et al. (2012)

Li et al. (2011a,b) Liu et al. (2011) Cao et al. (2010)

The Detection of Pesticide in Foods Using Electrochemical Sensors  131

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140  Chapter 5 Wang, M.Y., Huang, J.R., Wang, M., Zhang, D.E., Chen, J., 2014. Electrochemical nonenzymatic sensor based on CoO decorated reduced graphene oxide for the simultaneous determination of carbofuran and carbaryl in fruits and vegetables. Food Chem. 151, 191–197. Wei, M., Wang, J., 2015. A novel acetylcholinesterase biosensor based on ionic liquids-AuNPs-porous carbon composite matrix for detection of organophosphate pesticides. Sensor. Actuat. B-Chem. 211, 290–296. WHO, 2004. The WHO Recommended Classification of Pesticides by Hazard and Guidelines to Classification: 2004. World Health Organization, Ginebra. Wong, A., Materon, E.M., Sotomayor, M.D.P.T., 2014. Development of a biomimetic sensor modified with hemin and grapheme oxide for monitoring of carbofuran in food. Electrochim. Acta. 146, 830–837. Wu, S., Lan, X., Zhao, W., Li, Y., Zhang, L., Wang, H., Han, M., Tao, S., 2011a. Controlled immobilization of acetylcholinesterase on improved hydrophobic gold nanoparticle/Prussian blue modified surface for ultratrace organophosphate pesticide detection. Biosens. Bioelectron. 27, 82–87. Wu, S., Zhang, L., Qi, L., Tao, S., Lan, X., Liu, Z., Meng, C., 2011b. Ultra-sensitive biosensor based on mesocellular silica foam for organophosphorous pesticide detection. Biosens. Bioelectron. 26, 2864–2869. Wu, B., Wang, Z., Zhao, D., Lu, X., 2012. A novel molecularly imprinted impedimetric sensor for melamine determination. Talanta. 101, 374–381. Xu, G., Zhang, H., Zhong, M., Zhang, T., Lu, X., Kan, X., 2014. Imprinted sol–gel electrochemical sensor for melamine direct recognition and detection. J. Electroanal. Chem. 713, 112–118. Xuesong, J., Dongyang, L., Xia, X., Yibin, Y., Yanbin, L., Zunzhong, Y., 2008. Immunosensors for detection of pesticide residues. Biosens. Bioelectron. 23, 1577–1587. Yan, X., Deng, J., Xu, J., Li, H., Wang, L., Chen, D., Xie, J., 2012. A novel electrochemical sensor for isocarbophos based on a glassy carbon electrode modified with electropolymerized molecularly imprinted terpolymer. Sensor. Actuat. B-Chem. 171-172, 1087–1094. Yang, Y., Fang, G., Liu, G., Pan, M., Wang, X., Kong, L., He, X., Wang, S., 2013. Electrochemical sensor based on molecularly imprinted polymer film via sol-gel technology and multi-walled carbon nanotubes-chitosan functional layer for sensitive determination of quinoxaline-2-carboxylic acid. Biosens. Bioelectron. 47, 475–481. Yao, Y., Zhang, L., Xu, J., Wang, X., Duan, X., Wen, Y., 2014. Rapid and sensitive stripping voltammetric analysis of methyl parathion in vegetable samples at carboxylic acid-functionalized SWCNTs–β-cyclodextrin modified electrode. J. Electroanal. Chem. 713, 1–8. Zacco, E., Galve, R., Marcob, M.P., Alegret, S., Pividori, M.I., 2007. Electrochemical biosensing of pesticide residues based onaffinity biocomposite platforms. Biosens. Bioelectron. 22, 1707–1715. Zapp, E., Brondani, D., Vieira, I.C., Scheeren, C.W., Dupont, J., Barbosa, A.M.J., Ferreira, V.S., 2011. Biomonitoring of methomyl pesticide by laccase inhibition on sensor containing platinum nanoparticles in ionic liquid phase supported in montmorillonite. Sensor. Actuat. B-Chem. 155, 331–339. Zehani, N., Fortgang, P., Saddek Lachgar, M., Baraket, A., Arab, M., Dzyadevych, S.V., Kherrat, R., JaffrezicRenault, N., 2015. Highly sensitive electrochemical biosensor for bisphenol A detection basedon a diazonium-functionalized boron-doped diamond electrode modified witha multi-walled carbon nanotubetyrosinase hybrid film. Biosens. Bioelectron. 74, 830–835. Zen, J.M., Lo, C.W., 1996. A glucose sensor made of an enzymatic clay-modified electrode and methyl viologen mediator. Anal. Chem. 68, 2635–2640. Zen, J.M., Jeng, S.H., Ji-Chen, H., 1996a. Determination of paraquat by square wave voltammetry at a perfluorosulfonated ionomer clay-modified electrode. Anal. Chem. 68, 498–502. Zen, J.M., Jeng, S.H., Chen, H.J., 1996b. Catalysis of the electroreduction of hydrogen peroxide by nontronite clay coatings on glassy carbon electrodes. J. Electroanal. Chem. 408, 157–163. Zen, J.M., Wang, W.M., Ilangovan, G., 1998. Adsorptive potentiometric stripping analysis of dopamine on claymodified electrode. Anal. Chim. Acta. 372, 315–321. Zen, J.M., Jou, J.J., Kumar, A.S., 1999. A sensitive voltammetric method for the determination of parathion insecticide. Anal. Chim. Acta. 396, 39–44.

The Detection of Pesticide in Foods Using Electrochemical Sensors  141 Zhai, C., Sun, X., Zhao, W., Gong, Z., Wang, X., 2013. Acetylcholinesterase biosensor based on chitosan/prussian blue/multiwall carbon nanotubes/hollow gold nanospheres nanocomposite film by one-stepelectrodeposition. Biosens. Bioelectron. 42, 124–130. Zhang, Y., Arugula, M.A., Wales, M., Wild, J., Simonian, A.L., 2015. A novel layer-by-layer assembled multienzyme/CNT biosensor for discriminative detection between organophosphorus and non-organophosphorus pesticides. Biosens. Bioelectron. 67, 287–295. Zhang, M., Zhao, H.T., Yang, X., Dong, A.J., Zhang, H., Wang, J., Liu, G.Y., Zhai, X.C., 2016. A simple and sensitive electrochemical sensor for new neonicotinoid insecticide Paichongding in grain samples based on β-cyclodextrin-graphene modified glassy carbon electrode. Sensor. Actuat. B-Chem. 229, 190–199. Zhao, J.H., Zhao, D.G., 2009. Transient expression of organophosphorus hydrolase to enhance the degrading activityof tomato fruit on coumaphos. J. Zhejiang Univ.-Sci. B (Biomed. & Biotechnol.) 10, 142–146. Zheng, Y., Liu, Z., Jing, Y., Li, J., Zhan, H., 2015. An acetylcholinesterase biosensor based on ionic liquid functionalized graphene–gelatin-modified electrode for sensitive detection of pesticides. Sensors and Act. B: Chem. 210, 389–397. Zhou, Q., Yang, L., Wang, G., Yang, Y., 2013. Acetylcholinesterase biosensor based on SnO2 nanoparticles– carboxylic graphene–nafion modified electrode for detection of pesticides. Biosens. Bioelectron. 49, 25–31. Zhu, W., Liu, W., Li, T., Yue, X., Liu, T., Zhang, W., Yu, S., Zhang, D., Wang, J., 2014. Facile green synthesis of graphene-Au nanorod nanoassembly for online extraction and sensitive stripping analysis of methyl parathion. Electrochim. Acta. 146, 419–428. Zoski, C.G. (Ed.), 2007. Handbook of Electrochemistry, first ed. Elsevier, Amsterdam.

Further Reading Francisco, W.P., Oliveira, R.T.M.B.F., Silva, F.L.F., Mendonça, G.L.F., Mello, P.H., Becker, H., Lima-Neto, P., Correia, A.N., Freire, V.N., 2013. Sensitive voltammetric responses and mechanistic insights into the determination of residue levels of endosulfan in fresh foodstuffs and raw natural waters. Microchem. J. 110, 40–47. Gholivand, M.B., Malekzadeh, G., 2012. Computational design and synthesis of a high selective molecularly imprinted polymer for voltammetric sensing of propazine in food samples. Talanta. 89, 513–520. Jiang, X., Li, D., Xu, X., Ying, Y., Li, Y., Ye, Z., Wang, J., 2008. Immunosensors for detection of pesticide residues. Biosens. Bioelectron. 23, 1577–1587. Kumar, P., Kim, K.-H., Deep, A., 2015. Recent advancements in sensing techniques based on functional materials for organophosphate pesticides. Biosens. Bioelectron. 70, 469–481.

CHAPTE R 6

Multiway Calibration Approaches for Quality Control of Food Samples Romina Brasca, Héctor C. Goicoechea, María J. Culzoni Universidad Nacional del Litoral-CONICET, Ciudad Universitaria, Santa Fe, Argentina

6.1  Scope of the Chapter Nowadays, the use of modern instruments in analytical laboratories is generating a large variety of second- and higher-order instrumental data, that is, instead of obtaining a scalar for each sample measurement as when, for example, one absorbance at one wavelength is registered, a matrix tensor (second-order data) or a cube tensor (third-order data) of data points is recorded for each sample under analysis. Interestingly, an enhancement in the analytical properties is obtained by processing the latter data, which have made multiway calibration a subject of high interest for the analytical community, producing a significant impact on the development of analytical methods, especially for the quantitation of analytes of interest in complex matrices, such as those found in environmental, biological, and food samples, among others. Modeling second- and higher-order data allows one to exploit the remarkable and wellknown second-order advantage (Escandar et al., 2014). This means that an analytical method developed employing proper second- or higher-order data modeling can quantitate analytes of interest in complex systems even in the presence of unmodeled substances (i.e., potential interferences). This means that no physical separation is required for quantitation giving raise to methods consuming less laboratory effort, for example, time, money, and complex instrumentation, among others. In addition, second and higher-order calibration methods become an attractive choice in the field of quality control laboratories due to the improvement in sensitivity and selectivity. The objective of the present chapter is to revise the most recent practical analytical applications in quality control of food samples of higher-order data generation with instrumental techniques and data processing with proper algorithms such as parallel factor analysis (PARAFAC) (Bro, 1997), multivariate curve resolution with alternating least squares (MCR-ALS) (De Juan and Tauler, 2001), unfolded partial least-squares/residual bilinearization (U-PLS/RBL) (Wold et al., 1987), and alternating trilinear decomposition (ATLD) (Wu et al., 1998). Food Safety and Preservation. https://doi.org/10.1016/B978-0-12-814956-0.00006-8 © 2018 Elsevier Inc. All rights reserved.

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144  Chapter 6

6.2  Second- and Higher-Order Data Generation Second- and higher-order data generation usually includes fluorescence, high-performance HPLC, and CE. Emission fluorescence matrix (EEFM) data can be obtained by registering emission spectra at several different excitation wavelengths. In order to generate an EEFM, a proper emission spectral range is usually selected, including the wavelengths where the sample constituents are known to emit. It should be noted that when recording fluorescence emission data, other signals may be detected by the instrument, as the Rayleigh and Raman dispersions, and the second-order harmonic of the Rayleigh dispersion. Several procedures were developed to cope with the above-mentioned dispersion phenomena, which make the data analysis considerably simpler (Eilers and Kroonenberg, 2014). Fig. 6.1 shows the contour plot corresponding to an EEFM recorded for a Sauvignon blanc wine sample in the excitation range from 245 to 341 nm with a resolution of 2 nm and emission range from 375 to 500 nm with a resolution of 5 nm. Finally, the excitation was restricted in order to eliminate the Rayleigh dispersion (Azcarate et al., 2015). Additionally, third-order data can be generated registering EEFMs (either fluorescence or phosphorescence) as a function of reaction time or decay time (Olivieri et al., 2004). It should be commented that only in a few reports have four-way data been recorded and used to develop analytical methodologies, a fact that could be attributed to lack of understanding of

Fig. 6.1 Contour plot corresponding to an EEFM recorded for a Sauvignon blanc wine sample.

Multiway Calibration Approaches for Quality Control of Food Samples  145 their analytical advantages. It should be considered that the practical acquisition of these data arrays is still difficult to implement (Escandar et al., 2014). HPLC and CE are two other analytical techniques which can provide second- and third-order data when they are coupled with a diode array detector (DAD) or a fast scanning fluorescence detector. Chromatographic (and electrophoretic) separations can become a difficult task when complex samples have to be analyzed. This is owing to the presence of constituents with similar retention times (or electrophoretic migrations). However, multiway calibration can become a useful choice for accurate analyte quantitation when complete separation is not accomplished, or new constituents are present in the sample being analyzed (Escandar et al., 2014). Fig. 6.2 shows a typical example of a second-order data matrix generated by CE-DAD corresponding to a Chardonnay wine sample (Azcarate et al., 2016). The working region corresponds to a CE-DAD matrix with size 397 elution times (from 1 to 120 s) × 107 wavelengths (from 195 to 301 nm). As can be appreciated, information regarding the full spectrum of each substance can help to better understand and model the system variability. The use of a proper algorithm allows the calibration with pure standard solutions and quantitation of the analyte even in the presence of substances with similar migration time (or retention time).

Fig. 6.2 CE-DAD matrix corresponding to a Chardonnay wine sample.

Finally, another interesting and simple way to obtain second-order data should be mentioned: kinetic-spectroscopic matrices and pH-spectral matrices. In both cases, a chemical reaction takes place in a sample in which the analyte changes its chemical composition with time or

146  Chapter 6 pH, respectively. Consequently, recording UV–visible absorption or emission fluorescence spectra as a function of time or pH can become an interesting way to generate second- or higher-order data. The latter can be done when the kinetics or the pH evolution is followed when registering EEFMs (Muñoz de la Peña et al., 2015a). The change should develop in a suitable time window, that is, in the order of minutes, which can be conveniently followed using a diode array spectrophotometer or a fast-scanning fluorimeter. Fig. 6.3 shows a pertinent example of the use of kinetic-spectroscopic matrices for the analysis of tartrazine in beverage samples (Schenone et al., 2013).

Fig. 6.3 Kinetic-spectroscopic matrix for the analysis of tartrazine in beverage samples.

6.3  Multiway Calibration Algorithms 6.3.1 PARAFAC The algorithm PARAFAC is based on the decomposition of a three-way array X, obtained by joining second-order data from the calibration and test samples according to the following Eq. (6.1): N X = å a n Ä bn Ä c n + E (6.1) n =1

where ⊗ indicates the Kronecker product, N is the total number of responsive components, the column vectors an, bn, and cn are usually collected into the score matrix A and the loading

Multiway Calibration Approaches for Quality Control of Food Samples  147 matrices B and C; and E is a residual error term of the same dimensions as X[(I + 1) × J × K]. I is the number of calibration samples, and hence the first dimension of X is (I + 1), since the test sample is also included in X. In the case of EEFM data, J and K are the number of digitized wavelengths. ALS minimization is carried out through decomposition (Smilde et al., 2005). A typical array built with five EEFMs recorded for four pure standard solutions and a test sample containing three components is shown in Fig. 6.4. As can be seen, the X array is decomposed into three matrices containing both excitation and emission loadings (B and C), and a matrix A containing the relative concentration of the analytes in each sample. The first four samples do not contain the unknown components (interferences). The implementation of PARAFAC requires fulfilling the condition of trilinearity in the data (see later).

Calibration samples + test sample

X

Decomposition in profiles of three components

A

B

C

Fig. 6.4 Typical array built with several EEFMs recorded for four pure standard solutions and a test sample containing three components.

The application of the PARAFAC model requires initializing and/or constraining the algorithm, defining the number of components, and recognizing specific components from the information provided by the model. Finally, the model should be calibrated in order to obtain absolute concentrations for a particular component in a test sample. The first step can be carried out with profiles obtained by several procedures including direct trilinear decomposition (DTLD) (Sanchez and Kowalski, 1990). The number of components (N) can be estimated by considering the internal PARAFAC parameter, known as core consistency (Bro, 1997). The exploration of the profiles contained in B and C allow the identification of the chemical constituents, and comparing them with those for a standard solution of the analyte of interest. Finally, absolute analyte concentrations are obtained after proper calibration, since only relative values (A) are provided by decomposing the three-way data array (see Fig. 6.4). Experimentally, this is done by preparing a data set of standards of known composition and regressing the first I elements of column A against known standard concentrations y of analyte n. For more details, consult Muñoz de la Peña et al. (2015b).

148  Chapter 6

6.3.2 MCR-ALS The augmented MCR-ALS algorithm requires to build an augmented matrix (instead of a cube tensor structure) arranging the matrices along the mode that is suspected of breaking the trilinear structure. The latter phenomenon appears when a matrix-tomatrix variation of profiles occurs along one of the directions. Thus, if a matrix has time × wavelength dimensions (like a chromatogram-absorbance matrix), a columnwise augmented matrix is created when the trilinearity loss is in the time mode. The bilinear decomposition of the augmented matrix D is performed according to the expression Eq. (6.2): D = CS + E

(6.2)

where the rows of D contain, in the case of chromatographic data registered with DAD, the absorption spectra measured as a function of time, the columns of C contain the time profiles of the compounds involved in the process, the columns of S contain their related spectra, and E is a matrix of residuals not fitted by the model. The matrices have the following dimensions: D (1 + I)J× K, C (1 + I) J × N, ST(N × K) and E (1 + I)J× K, being I = number of training samples, J = number of elution times, K = number of digitized wavelengths, and N = number of responsive components. Decomposition of D is achieved by iterative least-squares minimization of the norm of E. Of remarkable importance are the constraining conditions: nonnegativity in spectral profiles, unimodality and nonnegativity in concentration profiles, and closure relations between reagents and products of a given reaction, among others. One of the most important characteristics of MCR is that the profiles in the different C submatrices need not share a common shape, while the pure spectra of the compounds should be the same in all experiments. For this reason, chromatographic (or electrophoretic) runs performed at different times and hence showing different profiles (owing to time-shifts and warping) can be analyzed together, as long as the spectra of the compounds involved in the process remain invariant. Similar considerations can be made for variations in kinetics or pH-dependent experiments. Fig. 6.5 represents an array built with five HPLC-DAD matrices (time × wavelength) recorded for four pure standard solutions and one test sample containing three components (the standard and two interferences). The D column-wise augmented matrix is decomposed into two matrices: the augmented C matrix with three components (N = 3) containing the evolution during the chromatographic run of every component in each individual matrix (in the first four matrices, there is only a single substance, the analyte), and the ST matrix containing the spectra of each component. The area under the evolution curve for the analyte can be used to build the univariate calibration graph with a quantitative purpose. For more details, consult Muñoz de la Peña et al. (2015c).

Multiway Calibration Approaches for Quality Control of Food Samples  149

Calibration samples + test sample

Decomposition in profiles of spectra and concentration

D

C

ST

Fig. 6.5 Array built with five HPLC-DAD matrices (time × wavelength) recorded for four pure standard solutions and one test sample containing three components.

6.3.3 U-PLS/RBL The U-PLS/RBL algorithm comprises a first calibration step including information regarding concentrations. Interestingly, this step does not include data for the unknown sample (Wold et al., 1987). The I calibration data matrices Xcal,i (size J × K, where J and K are the number of channels in each dimension) are unfolded. Then, a usual U-PLS model is built using these data and the vector of calibration concentrations y (Ncal × 1, where Ncal is the number of calibrated analytes). The calibration procedure provides a set of loadings P and weight loadings W (both of size JK × A, A being the number of latent factors), as well as regression coefficients v (size A × 1). The acquisition of the parameter A is carried out following the well-known leave-one-out cross-validation technique. Finally, v can be employed to estimate the analyte concentration, when no unexpected interferences are present in the test sample: yu = t uT v

(6.3)

where tu is the test sample score, obtained by projection of the unfolded data for the test sample Xu onto the space of the A latent factors, according to Eq. (6.4):

(

t u = WTP

)

-1

W T vec ( X u )

(6.4)

In the case in which the sample contains unmodeled components, the sample scores given by Eq. (6.4) are not suitable for analyte prediction using Eq. (6.3). Thus, the residuals of the U-PLS prediction step will be abnormally large in comparison with the typical instrumental noise, which is easily assessed by replicate measurements (Muñoz de la Peña et al., 2015d). In the latter case, the situation can be handled by the RBL procedure, which consists in modeling of the interferent effects with singular value decomposition (SVD). The object is

150  Chapter 6 to minimize the norm of the residual vector eu, computed while fitting the sample data to the sum of the relevant contributions to the sample signal. For a single interferent: T vec ( X u ) = Pt u + vec ég int bint ( c int ) ù + e u (6.5) ë û where bint and cint are the left and right eigenvectors of Ep and gint is a scaling factor. Ep is the J × K matrix obtained after reshaping the JK × 1 ep vector. During this RBL procedure, P is kept constant at the calibration values and tu is varied until the norm is minimized, generally using a Gauss–Newton (GN) procedure. Then, the analyte concentrations are provided by Eq. (6.3), by introducing the final tu vector found by the RBL procedure. The number of unmodeled constituents Nunx can be assessed by comparing the final residuals su with the instrumental noise level. A plot of su computed for a trial number of components will show decreasing values, starting at sp when the number of components is equal to A (the number of latent variables used to describe the calibration data), until it stabilizes at a value compatible with the experimental noise, allowing to locate the correct number of components. It is important to comment that the U-PLS/RBL algorithm does not provide qualitative information about the components as the previously discussed algorithms. On the other hand, the use of latent variables becomes U-PLS/RBL, a powerful tool for quantitative purposes when the data present serious losses in the tri- and quadri-linearity property. Fig. 6.6 shows a general scheme of the U-PLS/RBL procedure. For more details, consult Muñoz de la Peña et al. (2015d). Analyte concentrations

RBL

U-PLS model

Prediction Fig. 6.6 General scheme of the U-PLS/RBL procedure.

6.3.4 ATLD The ATLD method, first developed by Wu et al. in 1996, is an iterative algorithm with similar characteristics to PARAFAC. It has been commonly used due to the advantages of being

Multiway Calibration Approaches for Quality Control of Food Samples  151 insensitive to excessive component number, fast convergence, and fully exploiting the second-order advantage. It is based on the alternating least-squares principle without any constraints, which is able to correlate the signals of spectra with the concentrations of the analytes. The ATLD algorithm alternately minimizes the following three objective functions to update the two normalized qualitative matrixes (A and B) and the corresponding relative concentrations matrix (C) by using the Moore–Penrose generalized inverse strategy which is based on SVD (Wu et al., 1998).

( )

(6.6)

( )

(6.7)

( )

(6.8)

s 1 = å k =1  X.. k - Adiag c ( k ) BT 2F K

s 2 = å i =1  X..i - Bdiag a (i ) C T 2F I

s 3 = å j =1  X.. j - Cdiag b( j ) A T 2F J

The authors postulate that different to other second-order calibration algorithms such as PARAFAC and MCR-ALS, which are based on the decomposition of a bilinear extended matrix model, ATLD adopts a simultaneous iteration strategy from the three directions; thus, the computation speed can be acquired quicker and the possibility of being trapped into colinear factors can be significantly decreased. Several algorithms employing a similar ideology than ATLD have been presented by the same author: (a) self-weighted alternating trilinear decomposition (SWATLD), which retains the merits of fast convergence and insensitivity to excessive factors used in calculation, and offers more satisfactory results when compared with those from ATLD under higher noise and collinear levels; and (b) alternating penalty trilinear decomposition (APTLD), which brings penalty functions into three objective functions, consequently transforming the process of target minimization into a constrained optimization problem. For more details, consult Muñoz de la Peña et al. (2015e).

6.3.5 Software The software freely available on the Internet to implement second- and higher-order calibration is in the form of MATLAB codes, including several useful graphical user interfaces (GUI) (Jaumot et al., 2005; Gemperline and Cash, 2003; Wu et al., 2009; Olivieri et al., 2012). Among the most popular GUI can be mentioned: (a) N-way toolbox (http:// www.models.life.ku.dk/nwaytoolbox/download) for PARAFAC, PARAFAC2, GRAM, DTLD, U-PLS, and N-PLS; (b) MCR graphical interface (http://www.mcrals.info/) for MCR-ALS; and (c) MVC2 and MVC3 graphical interfaces (http://www.iquir-conicet.gov.ar/ descargas/mvc2.rar) for PARAFAC, APTLD, SWATLD, BLLS/RBL, U-PLS/RBL, N-PLS/ RBL, and MCR-ALS.

152  Chapter 6

6.4  Analytical Applications In this section, several analytical applications of PARAFAC, MCR-ALS, U-PLS/RBL and ATLD, SWATLD, and APTLD to the quality control of food samples are described. A summary of each methodology can be found in Table 6.1.

6.4.1 PARAFAC Synthetic dyes can be added to drinks to enhance color intensity or impart a convenient colored appearance, but in certain quantities they are harmful to human health (Codex Alimentarius-FAO/WHO, 2016a; Silva et al., 2010). Therefore, the supervision of the content of synthetic dyes in this kind of highly consumed products is an indispensable task. In this way, three common food colorants, which exhibited acid–base properties, were simultaneously quantified in powdered soft drinks by generation of absorbance spectral-pH data matrices using HPLC and chemometric modeling by means of PARAFAC and bilinear least-squares–residual bilinearization (BLLS–RBL) (El-Sheikh and Al-Degs, 2013). PARAFAC showed modest performance for the quantitation of tartrazine, allura red, and sunset yellow, while BLLS/RBL clearly demonstrated a superior capability for handling rank deficiency and linear dependency problems involved in the equilibrium systems. The contamination of human food and animal feed with mycotoxins is a significant problem which can cause serious adverse health effects as well as economic losses. Therefore, sanitary and phytosanitary regulations and recommendations have been established to protect consumers around the world (European Comission Regulation, 2012; Codex AlimentariusFAO/WHO, 2016b). Ochratoxin A, which is produced by several species of Aspergillus and Penicillium (Creppy, 2002), was quantified in cereal samples using EEFM with the aim of developing a rapid, sensitive, and simple quantitation method. Satisfactory results were obtained when PARAFAC was applied to predict artificial and real sample concentrations of OTA in sorghum grains (Rodríguez et al., 2013). The use of fluoroquinolones as antibiotics in animals is a common practice for livestock producers and in aquaculture in order to prevent and control infectious diseases and also to promote growing (Sarmah et al., 2006; Du and Liu, 2012). Moreover, high volumes of fluoroquinolone antibiotics are consumed by humans, and then a portion of the active ingredients is excreted. Therefore, the presence of fluoroquinolones in the environment is not uncommon and is considered as a potential problem (Alcaráz et al., 2014). These kinds of “contaminants of emerging concern” have been found in groundwater, surface water (i.e., river, lake, sea), tap water, raw and treated wastewater (domestic and industrial), sediments, soil, and food (Snow et al., 2015; Ferrer and Thurman, 2013; Deblonde et al., 2011; Guo et al., 2016; Farré and Barceló, 2013). Very recently, wildlife contamination with fluoroquinolones from livestock carrion has been documented by Blanco et al. (2016). Owing

Table 6.1: List of samples, applications, second-order instrumental data, other applied algorithms and references for the application of PARAFAC, MCR-ALS, U-PLS/RBL and ATLD, SWATLD, and APTLD to the analysis of food samples Sample

Application

Instrumental Data

Other Applied Algorithms

References

PARAFAC UV spectral-pH matrices EEFM EEFM

BLLS/RBL

Sorghum Milk

Quantitation of tartrazine, allura red, and sunset yellow Quantitation of ochratoxin A Quantitation of danofloxacin

Vinegar Brandy Meat

Classification Classification Classification

PLS-DA and SVM PLS PCA

Honey

Classification

EEFM EEFM Front-face SFS matrices Front-face FS matrices

El-Sheikh and Al-Degs (2013) Rodríguez et al. (2013) Cañada-Cañada et al. (2009) Callejón et al. (2012) Markechová et al. (2014) Sahar et al. (2016)

PLS-DA

Lenhardt et al. (2015)

LC-DAD matrices

PCA

Salvatore et al. (2013)

LC-DAD matrices LC–MS matrices LC-DAD matrices

PCA D-UPLS PCA, PARAFAC, PLS-DA and NPLS-DA –

Pisano et al. (2014) Pisano et al. (2015) Silvestri et al. (2014)

— —

MCR-ALS Wine Wine Wine Wine Olive oil

Fish Food peels of plum and tomato Milk powder Drink

Quantitation of phenolic compounds. Classification Classification Classification Classification Quantitation of p-coumaric, caffeic, ferulic, 3,4-dihydroxyphenylacetic, vanillic, and 4-hydroxyphenilacetic acids Tryptamine, 2-phenylethylamine, putrescine, cadaverine and histamine Malathion

CE-DAD matrices

SERS

NMF-ALS and MCR-WALS

Albuquerque and Poppi (2015)

Starch, urea, whey powder, and melamine

NIR hyperspectral imaging matrices Kineticspectrophotometric matrices

—

Forchetti and Poppi (2016)

—

Schenone et al. (2013)

Tartrazine

LC-DAD matrices

Godoy-Caballero et al. (2013) Pinto et al. (2016)

Continued

Multiway Calibration Approaches for Quality Control of Food Samples  153

Drinks

Sample

Application

Instrumental Data

Drink Honey, jam, fruit juice, milk, milkbased food

Amaranth, sunset yellow FCF, and tartrazine Glucose, fructose, and lactose

LC-DAD matrices Kineticspectrophotometric matrices

Other Applied Algorithms

References

U-PLS/RBL U-PLS/RBL and N-PLS/RBL

Culzoni et al. (2009) Aimo et al. (2016)

PARAFAC and N-PLS/ RBL PARAFAC

Monago-Maraña et al. (2016) Alarcón et al. (2013)

LC-DAD matrices

—

Zhang et al. (2013)

EEFM LC-DAD matrices

— —

Zhu et al. (2008) Yua et al. (2011)

LC-DAD matrices LC-DAD matrices LC-DAD matrices UV spectral-pH matrices

— — PARAFAC PARAFAC

Tan et al. (2011) Yin et al. (2014) Zhang et al. (2007) Masoum et al. (2015)

U-PLS/RBL Paprika Extra virgin and sunflower oils

Quantitation of quercetin, myricentin, and kaemferol. Classification Quantitation of hydrocarbons benzo[a] anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene, benzo[g,h,i] peryleneandindeno[1,2,3-c,d]-pyrene

EEFM EEFM

ATLD, SWATLD, and APTLD Honey Honey Honey

Tea Tea Hot chili Saffron

Quantification of nine polyphenols. Classification Carbaryl, carbendazim, and 1-naphthol Ciprofloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, fleroxacin, lomefloxacin, marbofloxacin, ofloxacin, orbfloxacin, pefloxacin, and sarafloxacin Free aminoacids Gallic acid, caffeine, and six catechins Sudan dyes Quantitation of tartrazine and sunset yellow. Classification

154  Chapter 6

Table 6.1  List of samples, applications, second-order instrumental data, other applied algorithms and references for the application of PARAFAC, MCR-ALS, U-PLS/RBL and ATLD, SWATLD, and APTLD to the analysis of food samples—cont’d

Multiway Calibration Approaches for Quality Control of Food Samples  155 to the use of fluoroquinolones in farm animals, the supervision of related products for human consumption is a common task. In this direction, a new procedure for fast determination of danofloxacin (DANO) in commercial bovine milks was proposed by Cañada-Cañada et al. (2009). The method is based on the generation of EEFM and combines secondorder calibration and standard addition method. Firstly, a calibration curve was performed in deionized water and five samples were used to test the PARAFAC model, producing satisfactory results. In the subsequent quantitation of DANO in spiked milk samples, no good results were obtained. Therefore, PARAFAC standard addition was employed by adding increasing amounts of DANO to different milk samples that were previously spiked at three concentration levels. In this way, three calibration curves were performed for whole, low fat, and fat-free milk, respectively, and excellent results were obtained regarding mean recoveries, decision limits, and detection capabilities. The classification of wines according to the grape variety and the classification of vinegars according to their aging are important tools which can help producers and investors to have insights about the quality of the products, which is obviously connected with different prices. Therefore, classification techniques can be very useful to guarantee the authenticity of the products. In this way, multidimensional fluorescence measurements are done for the intact products, and subsequently, the interpretation and classification using chemometrics is carried out (Azcarate et al., 2015; Callejón et al., 2012).For example, EEM data sets were obtained from 39 different classes of Sherry vinegars from 14 different producers and they were evaluated by means of PARAFAC (Callejón et al., 2012). It was estimated that five fluorophores were present in the samples. With the help of the available literature, it was possible to make the corresponding chemical interpretation of the data retrieved by PARAFAC and to get an impression of the separation between the different vinegar classes. Subsequently, classification tools such as partial least-squares-discriminant analysis (PLS-DA) (Nocairi et al., 2005) and support vectors machine for classification (SVM) (Vapnik, 1995) were applied by using the scores obtained by PARAFAC. Both methods performed quite satisfactorily in classifying the different samples, but the ability of SVM to predict external samples was superior to that of PLS-DA. A model based on PARAFAC and PLS was also used for the determination of brandy adulteration with mixed wine spirit (Markechová et al., 2014). To detect adulteration of brandy samples, the authors developed a method based on the acquisition of EEFM and modeling with a previously published PARAFAC–PLS model (Surribas et al., 2006). It was possible to successfully determine the content of mixed wine spirit added to brandy samples at levels down to 1.9% (v/v). Regarding the use of classification techniques, Sahar et al. performed the monitoring of the changes produced in meat samples by the application of heat by front-face synchronous fluorescence spectroscopy (SFS) allowing the classification of the samples as a function of

156  Chapter 6 the cooking time and temperature (Sahar et al., 2016). PCA was used in order to discriminate the samples on the basis of the changes produced by cooking and PARAFAC revealed spectral information of the fluorophores present in the meat samples. The application of front-face fluorescence spectroscopy (FS) in combination with PCA and PARAFAC was also employed for the characterization and classification of honey (Lenhardt et al., 2015). Lenhardt et al. were able to distinguish between different honey types such as acacia, linden, sunflower, meadow mix, and fake honey. PARAFAC analysis, based on a six-component model, showed that mainly phenolic compounds and Maillard reaction products are responsible for the spectral differences observed among sample groups of different botanical origin. Moreover, the largest difference was detected between fake and natural honey samples. Therefore, a PLS-DA model constructed from PARAFAC scores was successfully applied to the classification of the different natural samples and the detection of fake honey samples.

6.4.2 MCR-ALS MCR-ALS in combination with classification algorithms such as PCA has been successfully applied in wine analyses in order to distinguish among wine samples of different varietal and geographical origin (Salvatore et al., 2013; Pisano et al., 2014). For example, wine samples were first analyzed by HPLC-DAD, and subsequently, chemometric techniques were applied using different strategies. In the case of the investigation of the phenolic compounds in certified Lambrusco wines (Salvatore et al., 2013), the individual data matrices (corresponding to each chromatographic run of a single sample) were organized appending one on top of each other to obtain the column-wise augmented multisets (formed by standards and samples). In this case, the use of local rank constraints in MCR-ALS was demonstrated to be mandatory for the correct resolution of the chromatographic peaks and quantitation of the phenolic compounds. Another strategy for the resolution of component chromatograms and spectra was described by Pisano et al. (2014) and involved the time alignment of the chromatograms using the correlation optimized shifting (Tomasi et al., 2004) synchronization algorithm as an additional step previous to the arrangement of the individual data matrices in a row-wise augmented data matrix. PCA was successful in discriminating wine varietals with only partial success to explore the wine samples by geographical origin. Moreover, MCR-ALS and discriminant unfolded partial least-squares (D-UPLS) (Wold et al., 1987) were used to process data from different red wines acquired by the HPLC–mass spectrometry system (LC–MS) with the objective of developing a classification method according to the botanical and geographical origin of wines using anthocyanins as markers (Pisano et al., 2015). The column-wise augmentation mode was used for MCRALS decomposition which rendered the chromatographic and mass spectral profiles for each component in a particular sample. After MCR resolution, the areas under the resolved

Multiway Calibration Approaches for Quality Control of Food Samples  157 chromatographic profiles for each sample, that is, the scores, were arranged into a matrix and submitted to PCA. Complementary second-order data were rearranged into vectors and D-UPLS was applied for discrimination purposes using the concept of variables importance in the projection (Mehmood et al., 2012). As a result, the samples were adequately discriminated according to varietal and/or geographical origin using both chemometric models, and also, it was possible to ascribe anthocyanin compounds as responsible for both types of discriminations. The data fusion approach, which consists in jointly analyzing samples using different analytical techniques, can be a useful tool for the recovery of chemical information especially when the a priori knowledge about the composition of the samples is not complete (Forshed et al., 2007; Vera et al., 2011). For example, the use of mid-level data fusion was proposed by Silvestri et al. (2014) as a very efficient tool for the characterization of different varieties of Lambrusco wine. The experimental data, which were arranged as two- and three-way arrays, were generated using three analytical techniques and three different data analysis algorithms such as PCA, PARAFAC, and MCR were used for data reduction and explorative analysis. In particular, PCA was used for 1H NMR data, PARAFAC for EEM data, and MCR for HPLC-DAD data. Then, the results of the explorative analysis were merged together and a classification was performed by means of PLS-DA. The performance of the data fusion methodology was compared with the performance of the single set of data and better results were obtained in the former cases. Thus, PLS-DA and NPLS-DA were built for the three separate data sets, respectively. Using the proposed approach, it was possible to characterize different Lambrusco samples and understand the correlation between data of different nature and dimensionality. MCR-ALS was also applied to CE-DAD data for the resolution and quantitation of six phenolic acids, that is, p-coumaric, caffeic, ferulic, 3,4-dihydroxyphenylacetic, vanillic, and 4-hydroxyphenilacetic acids, in virgin olive-oil samples (Godoy-Caballero et al., 2013). The individual data matrices were augmented in the temporal mode so that the time profiles may change from sample to sample, that is, data deviating from trilinearity. The method was successfully applied to resolve data sets of standard mixtures of the analytes prepared in organic solvent and in real virgin olive-oil samples as well. Time misalignment and rank deficiency were also present in the analysis of biogenic amines in fish by HPLC-DAD (Pinto et al., 2016). The proposed method for the quantitation of five amines, that is, tryptamine, 2-phenylethylamine, putre scine, cadaverine, and histamine, in fish samples is based on chemical reactions with dansyl chloride to obtain products which exhibited the same spectral profiles and partial separations in the LC system. Moreover, the chromatographic data also showed peak misalignment. These problems were conveniently handled by the use of the Interval Correlation Optimized Shifting algorithm (Tomasi et al., 2011) and spectral augmented MCR-ALS. The method required 3.5 min for the complete

158  Chapter 6 elution of the analytes which is performed under isocratic conditions and showed a good performance as compared to previous studies. Therefore, it can be a useful methodology for monitoring the presence of amines in foodstuffs, considering their potential effects on human health and food security. The detection of malathion, an insecticide widely used in agriculture, was satisfactorily accomplished by surface-enhanced Raman imaging spectroscopy (SERS) and MCRALS analysis in samples of tomato and plum peels (Albuquerque and Poppi, 2015). The SERS images formed hyperspectral data cubes which were unfolded, augmented, and preprocessedng. Finally, the pure spectra and distribution mapping of the analyzed surfaces were recovered with the aids of nonnegative matrix factorization (NMF) (Lee and Seung, 1999), MCR-ALS, and MCR with weighted alternating least square (MCR-WALS). For the tomato data, NMF-ALS and MCR-ALS showed excellent spectral recovery, whereas for the Damson plum data better results were obtained with MCR-WALS due to the existence of heteroscedastic noise. This methodology permitted detection below the maximum residue limit allowed for this pesticide, and therefore it can be implemented for in situ monitoring. The use of hyperspectral imaging in combination with MCR-ALS was also demonstrated for near-infrared (NIR) data with the objective of detecting adulterants such as starch, urea, whey powder, and melamine in milk powder samples (Forchetti and Poppi, 2016). It was possible to recover the adulterant spectral profiles providing its identification and to predict concentrations by using nonnegativity constraints for concentration and correlation criteria. MCR-ALS has been demonstrated to be an efficient tool for the determination of dyes in beverages by modeling of nonlinear kinetic-spectrophotometric data acquired by a stoppedflow system and spectroscopic data acquired by a fast HPLC-DAD methodology (Schenone et al., 2013; Culzoni et al., 2009). In the first case, the spectral changes during the reaction of tartrazine with potassium bromate were analyzed. The quantitation of tartrazine in the presence of other interfering dyes was possible by removing the contribution of unexpected components not included in the calibration set by MCR-ALS and applying a polynomial function to the nonlinear data (Schenone et al., 2013). In the second case, three synthetic dyes, that is, amaranth, sunset yellow FCF, and tartrazine, were analyzed in nonalcoholic beverages (Culzoni et al., 2009) using two second-order algorithms (MCR-ALS and U-PLS/ RBL). The U-PLS/RBL algorithm failed when modeling this kind of data due to the lack of trilinearity, but MCR-ALS showed a good performance leading to acceptable figures of merit. The quantitation of carbohydrates is very important in the food industry for quality controls and also for determining the nutritional content. Therefore, a second-order kineticspectrophotometric method was developed for the determination of reducing sugars such as glucose, fructose, and lactose in food samples (Aimo et al., 2016). The method involved the reaction with hexacyanoferrate in basic media and modeling of the generated data with MCRALS using the spectral augmentation mode to successfully resolve data linear dependence.

Multiway Calibration Approaches for Quality Control of Food Samples  159 The performance of MCR-ALS was satisfactory regarding the quantitation of fructose and glucose in real samples such as honeys, jams, fruit juices, milks, and milk-based foods, but nonsatisfactory results were obtained for lactose. Therefore, these three reducing sugars were simultaneously analyzed by means of U-PLS/RBL and N-PLS/RBL in validation samples and in test samples containing only active spectral interferents, and good results were obtained. Quantitation of lactose, glucose, and fructose in real milk samples was successfully achieved.

6.4.3 U-PLS/RBL Muñoz de la Peña and colleagues developed an analytical method able to quantitate and identify flavonoids in paprika samples by coupling spectrofluorimetry to secondorder chemometric tools. Firstly, the fluorescence features of the target flavonoids, that is, quercetin, myricetin, and kaempferol, were studied and optimized focalizing on both fluorescence intensity enhancement and compound stability, which were intensified in basic medium for the first time. These previous analyses were conceived not only for the development of an analytical method with quantitative purposes, but also considering the importance of precluding fraud in this kind of protected designation of origin (PDO) samples (Monago-Maraña et al., 2016). The excitation–emission fluorescence matrices were analyzed by means of PARAFAC in the first place in order to group the results according to their belonging or not to a Spanish PDO. Eventually, U-PLS/RBL and N-PLS/RBL were evaluated to quantitate quercetin and kaempferol in the same samples. While PARAFAC only provided quantitative results for quercetin and kaempferol together, U-PLS/RBL and N-PLS/RBL allowed their differentiation in synthetic samples, and their quantitation in paprika samples. The feasibility of simultaneously determining the US-EPA-polycyclic aromatic hydrocarbons benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h] anthracene, benzo[g,h,i]peryleneandindeno[1,2,3-c,d]-pyrene in edible oils, that is, extra olive and sunflower oils, by analyzing EEMs with U-PLS/RBL and PARAFAC has been explored by Alarcón et al. A pretreatment step involving microwave assisted liquid–liquid extraction and solid-phase extraction on silica should have been included in order to handle the inner filter effects and spectral overlapping due to the native compounds present in the complex matrices. Since the U-PLS/RBL algorithm provided the best results, they were compared to those obtained by HPLC-FLD. It should be mentioned that not only no significant differences between them were observed, but also similar LODs. Therefore, the proposed method constitutes a simple and faster analysis with less organic solvent requirements (Alarcón et al., 2013).

6.4.4  ATLD, SWATLD, and APTLD An isocratic HPLC-DAD method for the simultaneous determination of nine polyphenols in five kinds of honey samples has been developed by Zhang et al. (2013). The content of polyphenols in this kind of samples has been found to be highly correlated to their antioxidant

160  Chapter 6 properties (Gheldof et al., 2002). Owing to the strategy performed to reduce the analysis time, the analytes became highly overlapped between them and to the interferences inherent to the complex sample matrix. The procedure involved the modeling of second-order data by means of ATLD without performing a baseline correction, which is a common undesirable phenomenon in the analysis of real samples using HPLC-DAD. Instead of removing it previously to the ATLD resolution, the background drift was overcome by considering it as additional component(s) in the mathematical model. With the quantitative results obtained by ATLD, the relationship between the content of the polyphenols and the honey variety was also studied. Taking into account that the content of polyphenols significantly varies between different types of honeys, this parameter has been successfully used for their classification. The proposed method can be considered as an accurate alternative to the conventional zeroorder calibration method for rapid identification and quantitation of the analytes in complex matrices, regarding its advantages related to solvent and time saving, and reduction of both the cost per analysis and the environmental impact. Carbaryl (1-naphthol-N-methycarbamate) and carbendazim (methyl-2-benzimdazole carbamate) are highly used broad-spectrum pesticides that could become toxic for ecosystems and human health when used indiscriminately. Since carbaryl hydrolyses in water lead to the major degradation product 1-naphthol, the second-order advantage achieved by applying the SWATLD-based second-order calibration method to EEMs was exploited to simultaneously determine carbaryl, carbendazim, and 1-naphthol in honey samples. Although the analytical results for both carbendazim and 1-naphthol were satisfactory, those related to carbaryl should have been confirmed by applying the second-order standard addition method (SOSAM) based on SWATLD and high-performance HPLC–tandem mass spectrometry (HPLC–MS) due to either a strong matrix effect occurring for this analyte or the fact that the honey sample already contained the analyte, being the results consistent with this latter fact (Zhu et al., 2008). Another method illustrating the potentiality of ATLD to model second-order analytical data has been described by Yua et al. In this case, HPLC-DAD coupled to ATLD was employed to quantitate 12 quinolones in honey samples, that is, ciprofloxacin, danofloxacin, difloxacin, enoxacin, enrofloxacin, fleroxacin, lomefloxacin, marbofloxacin, ofloxacin, orbfloxacin, pefloxacin, and sarafloxacin. The multiresidue analysis of quinolones in food samples is of current interest, since their concentrations have been significantly increased in recent years leading to negative consequences for public health. The proposed method proved to be more effective and simpler in relation to the traditionally applied chromatographic methods (Yua et al., 2011). Tan and coworkers reported the application of APTLD to the simultaneous determination of the content of free amino acids in tea, which are related to the characteristic flavor and taste of tea, besides being indispensable nutritional elements, by HPLC-FLD. The second-order

Multiway Calibration Approaches for Quality Control of Food Samples  161 advantage allowed the accurate concentration prediction of the target amino acids immersed in a complex matrix, and provided resolved chromatographic and spectral profiles as well. This procedure constitutes an inexpensive alternative useful to quantitate the analytes in the presence of potential interferences and/or peak overlapping. Besides, it proved to be considerably faster than the automatic amino acid analysis widely used to profit from its excellent stabilization, since it can be accomplished in 20 min instead of 90–150 min, and the calculations are automatic (Tan et al., 2011). Also related to the analysis of tea samples, an HPLC-DAD/ATLD strategy was proposed for the simultaneous and fast determination of eight co-eluted compounds including gallic acid, caffeine, and six catechins in 10 kinds of Chinese teas within 8 min using a simple mobile phase. As previously mentioned (Gheldof et al., 2002), the baseline drift was also removed by considering it as additional factor(s) in the mathematical model. Results achieved with LC–MS/MS allowed concluding about the accuracy of the proposed method, which also proved to be sensitive, fast, universal, and suitable for the routine analysis of the target analytes in Chinese tea samples (Yin et al., 2014). Owing to the presence of artificial dye adulterants in spices and herbs, Zhang et al. developed an HPLC-DAD method for the determination of Sudan dyes in hot chili using three-way data (Zhang et al., 2007). Seven calibration samples together with ten spiked chili samples were conveniently analyzed with second-order calibration algorithms based on PARAFAC, ATLD, and SWATLD in order to get the accurate concentration of the analytes in the complex chili samples. In spite of SWATLD providing slightly better results than PARAFAC and ATLD, the three second-order calibration methods showed to be excellent tools for the determination of Sudan I and II in complex chili samples. A selective and nonseparative method using second-order spectrophotometric data assisted by chemometrics has been developed to distinguish between genuine and adulterated saffron samples. This spice, which can be used not only for dying and cooking, but also for medical purposes, is in the spotlight of the fraudulent manufacturers due to both its high economic value and restricted production. Since the addition of synthetic colorants to saffron is a way of adulteration, the simultaneous determination of tartrazine and sunset yellow was evaluated in real samples. The presence of unknown interferences was overcome by using the three-way chemometric methods APTLD, SWATLD, and PARAFAC for the analysis of three-dimensional absorbance spectra– pH gradient data. The high collinearity among the spectra of the colorants resulted in rank deficient data, which were overcome by subtracting the first pH spectrum from each spectrum at each pH before building the three-way array. The proposed method constitutes an alternative to classify saffron samples as genuine or fraudulent, which does not require a separation step and long analysis time such as the reported HPLC method (Masoum et al., 2015).

162  Chapter 6

6.5  Concluding Remarks The usefulness of chemometric tools to the simultaneous identification and quantitation of different kind of analytes immersed in complex food samples has been summarized in the present chapter. Many of the proposed strategies allowed not only determining the analytes of interest, but also inferring about sample authenticity through classification approaches, which is of vital importance to ensure public health. In this sense, the generation of higherorder data and their analysis by chemometric algorithms constitutes a valuable association to develop rapid, simple, inexpensive, and environmentally friendly methods to the quality control of food samples.

Acknowledgments The authors are grateful to Universidad Nacional del Litoral (Projects CAI + D 2011 No 11–11 and 11–7), CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas, Project PIP-2015 No 0111), and ANPCyT (Agencia Nacional de Promoción Científica y Tecnológica, Project PICT 2014–0347) for financial support.

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CHAPTE R 7

Biocontrol as an Efficient Tool for Food Control and Biosecurity Karen Nathiely Ramírez-Guzmán⁎, Cristian Torres-León⁎, Eduardo Martínez-Terrazas†, Reynaldo De la Cruz-Quiroz‡, Adriana C. Flores-Gallegos⁎, Raúl Rodríguez-Herrera⁎, Cristóbal N. Aguilar⁎ ⁎

Autonomous University of Coahuila, Saltillo, Mexico †Autonomous University of San Luis Potosí, Ciudad Valles, Mexico ‡Monterrey Institute of Technology, Monterrey, Mexico

7.1 Introduction Nowadays, agriculture has suffered a very considerable economic decrement due to its little production or bust promoted by diverse factors, among the most common being the illnesses and plagues (Galindo et al., 2015). The latter, called “natural enemies,” can be classified into three big groups: parasites, predatory, and pathogens (Badii and Abreu, 2006). The mechanisms or agents used to combat and protect the harvests of this detriment mostly belong to products of synthetic origin or chemistry (Brimner and Boland, 2003). However, in past years, the use of pesticides and herbicides has caused controversy since it has adverse effects on consumers, affecting their health, and also on the environment. Even these seem cheap, and in the long run, they cause environmental pollution and negative effects on food and human health due to the concentration of such compounds, prolonged periods of degradation required (Manandhar and Wright, 2015), carcinogenic potential and teratogenic effect (Droby et al., 2009), besides, that they can promote the generation of resistant organisms due to the permanence of such chemical compounds (Paredes et al., 2011). Thus, a reduction or elimination of the applications of synthetic pesticides in agriculture is highly desirable. This has led to the promotion of research of other control options, and for this reason, attention has been paid to control agents of biological origin (Vos et al., 2014). Biological control is the use of an organism to combat another organism (Mmbaga et al., 2008). This technology can have two approaches: (1) the use of native organisms in the system or (2) the incorporation of some that are unrelated to this, which are able to fight problematic pathogens of the crop in question (Sharma et al., 2009). This alternative goes in hand with the sustainable development and conservation of natural resources, its principle being the use of natural compounds (Yu et al., 2013). Research has focused on identifying new organisms with a clear potential to be Food Safety and Preservation. https://doi.org/10.1016/B978-0-12-814956-0.00007-X © 2018 Elsevier Inc. All rights reserved.

167

168  Chapter 7 part of a select group called “biological control agents” (BCAs) and their action mechanisms (Manandhar and Wright, 2015). This has already led to the exploitation of certain biological control organisms in a commercial context, and it is expected that the so-called “biopesticides market” increase in the coming years (Wyckhuys et al., 2013). This review aims to present basic aspects of Biological Control (CB), the beginning, types of agents as well as trends and future perspectives.

7.2  Food Control 7.2.1  Generalities and Definition Nowadays, the sufficiency of safe and quality food is one of the priorities of modern society. In fact, food, together with drinking water and energy resources, belongs to the strategic components for the protection of public health, stability, and economic development of society. However, food safety has become a major concern due to the increased incidence of foodborne diseases: in 2009, the World Health Organization (WHO) reported that more than 200 diseases could be spread by contaminated food or water, killing about 2.2 million people annually, of whom 1.9 million are children (WHO, 2009, 2010). Besides health injuries, economic losses caused by damage to food are estimated to be more than $35 billion a year, according to Forbes magazine (Steinhauserova and Borilova, 2015). Although the microbiological causes of food-borne diseases are often seen as one of the most critical risks, pesticide residues, veterinary drugs, chemicals that are leached from containers and products processing chemicals also pose significant risks. Thus, according to current knowledge, food safety means that the food does not contain pathogens or chemical contaminants in amounts which could induce an illness in a person, and that confirm the conditions given by food legislation. In contrast to food safety, this fact is more related to the quality of food, which means the food meets the typical standards characteristic of a particular product. In this sense, the Food and Agriculture Organization (FAO) of the United Nations has defined Food Control Management as “the mandatory regulatory activity of the enforcement of food laws and regulations by national or local authorities to provide consumer protection and ensure that all foods during production, handling, storage, processing, and distribution are safe, wholesome and fit for human consumption; conform to safety and quality requirements; and are honestly and accurately labelled as prescribed by law” (FAO/WHO, 2003). It can also be defined as “a continuous process of planning, organizing, monitoring, coordinating, and communicating, in an integrated way, a broad range of risk-based decisions and actions to ensure the safety and quality of domestically produced, imported and exported food for national consumers and export markets as appropriate” (FAO, 2006).

Biocontrol as an Efficient Tool for Food Control and Biosecurity  169

7.2.2  Food Risks Since food safety and quality are based on risk analysis with an integrated farm-to-table approach, FAO recommends the application of the Codex Alimentarius Commission (CAC) working principles which comprise three interlinked components: (1) risk assessment; (2) risk management; and (3) risk communication (Codex Alimentarius Commission, 2007). There are several systems in food production to prevent risks to human health. Some of them are compulsory for the producers and some are optional, depending on the requirements of the business and in addition to the food security, they also help to guarantee the quality of the food. For risk assessment of foods and food ingredients, it is desirable to identify possible toxic compounds also on the basis of their chemical structure and mechanism of action (Daneshian et al., 2013). Moreover, it is desirable to describe concentration-dependent effects, longterm low-concentration exposure effects as well as a proper risk-benefit analysis. The safety assessment of foods and ingredients is clearly much more complicated than for wellcharacterized nonfood substances, for example, industrial chemicals, plant protection products, or medicines. Food additives and flavorings may have more similarities to the aforementioned examples, but the majority of food ingredients, including GMOs, present a greater challenge, for example, because of the complexity of the food composition (Palafox-Carlos et al., 2011).

7.2.3  Regulation and Food Control According to the FAO (2006), food legislation is defined as “the complete body of legal texts that establish broad principles for food control in a country, and that governs all aspects of the production, handling, marketing and trade of food as a means to protect consumers against unsafe food and fraudulent practices.” Regulations governing food hygiene can be found in numerous early sources such as the Old Testament, and the writings of Confucius, Hinduism, and Islam with a vague conception of foodborne illness. Despite our expansion of knowledge nowadays, foodborne diseases are still a widespread health problem and an important cause of reduced economic productivity (WHO, 1992). Even demanding completely safe food is unrealistic, though it is possible to have food in which potential hazards have been reduced (Adams and Moss, 2000). With the aim to reduce those potential hazards, many systems have been created. In 2010, the Food and Agriculture Organization (FAO) of the UN, the World Organization for Animal Health (OIE), and the WHO signed ‘A Tripartite Concept Note’ on sharing responsibilities and coordinating global activities to address health risks at the animal-human-ecosystems interfaces. This global agreement is reflected, for example, in the EU legislative food safety framework with a number of initiatives, one of them being that flexible provisions were adopted in legislation to protect the diversity of EU agricultural production, as well as to serve consumers and the needs of small-scale producers (Radakovic, 2015).

170  Chapter 7 Effective national food control systems (NFCS) are seen as important. Guidance on the key components of NFCS has been defined by international bodies. In particular, the FAO and WHO have jointly published guidance on the strengthening of NFCS (FAO/WHO, 2003). In addition, more recently, the international CAC has adopted “Principles and Guidelines for National Food Control Systems” (Codex Alimentarius Commission, 2013). Assessing compliance with these documents can assist in the development of improved NFCS within a country and enables good practice developed in one country to be shared and adopted elsewhere. One of the most worldwide used systems at the level of the producers is HACCP (Hazard Analysis Critical Control Points) whose principle is to identify technological methods in the production process which can eliminate hazards (physical, chemical, or biological). Besides identifying critical control points, the producer has to establish their limits, methods for their monitoring, and corrective steps. Verification of the whole system is a very important step to confirm that the system is working properly and the hazards are effectively kept under control (Milios et al., 2014). In primary production and food processing, it is almost impossible to introduce HACCP, but food safety can be increased with Good Manufacturing Practice (GMP). This system operates by setting rules for processing, assuring that the risks of a harmful food occurrence are eliminated and, at the same time, the law is respected. The principles of GMP can be applied at each production stage and describe precisely the basic requirements for technological procedures, staff behavior, premises, etc. Those two systems, GMP and HACCP, are complementary (Steinhauserova and Borilova, 2015). Besides the HACCP system, there are other systems, including British Retail Consortium (BRC), Food Safety System Certification 22000, or International Food Standard (IFS). One of the methods for validation and verification of GMP and HACCP systems is the implementation of process hygiene criteria at slaughterhouses (EC No 2073/2005 on microbiological criteria for foodstuffs). The existing food safety tools, such as Hazard Analysis and Critical Control Point (HACCP), are less capable of addressing the need for identification, quantification, control, and management of these food safety risks (Sperber, 2005). Hence, the Food Safety Modernization Act (FSMA) was signed into law in the United States in 2011 to shift the existing approach to food safety focus from reactive to preventive control. This law provides the Food and Drug Administration (FDA) with increased authority to inspect food products and authorize mandatory recalls for contaminated products. The proposed FSMA rules are divided into four titles: (1) improving the capacity to prevent food safety problems, (2) improving the capacity to detect and respond to food safety problems, (3) improving the safety of imported food, and (4) miscellaneous provisions (e.g., employee protection and budget details). This law contains the requirements of Hazard Analysis and Risk-Based

Biocontrol as an Efficient Tool for Food Control and Biosecurity  171 Preventive Controls (HARPC) (Kheradia and Warriner, 2013), which necessitates a preventive food safety system for facilities handling/processing food or food ingredients (FDA, 2015). HARPC is a shift from the existing food safety management system (FSMS) because it mandates a logical pre-assessment of food safety hazards. This system will mainly benefit small businesses adopting preventive control (Center for Progressive Reform, 2013), because they have not been extensively experienced with prevalent FSMS and standards such as ISO 22000: 2005, SQF code (Safe Quality Food Institute), GFSI guidelines (Global Food Safety Initiative), or HACCP (FDA Federal Register, 2014), which form the basis for HARPC implementation in food businesses. While HACCP has proven to be very effective for the control of food safety (Arvanitoyannis and Traikou, 2005), it must be acknowledged that it is designed on the basis of known hazards and that potential future risks are not necessarily taken into account. The European Food Safety Authority (EFSA) defines an emerging risk to human, animal, and/or plant health as a health risk resulting from a newly identified hazard to which a significant exposure may occur or from an unexpected new or increased significant exposure and/or susceptibility to a known hazard (EFSA, 2007). This authority operates the Emerging Risks Exchange Network (EREN) as a system for identification of multifaceted emerging risks. Finally, another procedure to ensure nutritional quality of food throughout all stages of production, in order to define the impact on human health, is the Nutrient, Hazard Analysis, and Critical Control Point (NACCP) process, which was proposed to ensure nutritional quality of food throughout all stages of production. The NACCP process aims to evaluate and guarantee Total Quality Management (TQM) in the maintenance of high nutritional levels with a consequent positive impact on consumer’s health. This system is based on the principle that the food issue must be dealt with using a “holistic” approach, targeting both safety and nutritional aspects, and takes into account four general principles: (1) guarantee of health maintenance; (2) evaluate and assure the nutritional quality of food and TQM; (3) give correct information to the consumers; (4) ensure an ethical profit (Di Renzo et al., 2015).

7.3  Food Safety Food safety encompasses actions aimed at ensuring that all food is as safe as possible; this is a major public health concern worldwide (Shaheen et al., 2016), because of the increasing risk of contamination of food by bacteria (Husseina et al., 2016), viruses (Bostan and Mahmooda, 2010), parasites (Robertson, 2016), and chemicals (Kariathi et al., 2016). An estimated 600 million—almost 1 in 10 people in the world get sick and die as a result of diseases associated with contaminated food intake (WHO, 2007). Table 7.1 presents the major foodborne pathogens, diseases, and symptoms.

Cause

Agent

Disease

Signs and Symptoms

Bacteria

Salmonella

Salmonellosis

Campylobacter

Campylobacteriosis

Fever, headache, nausea, vomiting, abdominal pain and diarrhoea Diarrhoea, abdominal pain, fever, headache, nausea, and/or vomiting

Enterohaemorrhagic Escherichia coli

Haemolytic uraemic syndrome

Watery diarrhoea, abdominal cramping

Listeria

Listeriosis

Vibrio cholerae

Cholera

Diarrhea, fever, muscle aches, headache, stiff neck, confusion, loss of balance, and convulsions Watery diarrhea, occasional vomiting, and abdominal cramps

Norovirus

Gastroenteritis

Viruses

Hepatitis A

Parasites

Prions

Trematodes

Echinococcus spp.

Echinococcosis

Taenia solium

Taeniasis

Giardia

Giardiasis

Transmissible spongiform encephalopathy

Diarrhea, throwing up, nausea, stomach pain Fever, malaise, loss of appetite, diarrhoea, nausea, abdominal discomfort, dark-colored urine and jaundice Dysuria, hema turia, and uremia, fever, hepatomegaly, abdominal pain, and jaundice. Unspecific symptoms intestinal irritation, anemia, and indigestion occur Violent diarrhea, excess gas, stomach or abdominal cramps, upset stomach, and nausea Slow thinking, difficulty concentrating and memory loss

Foods

Reference

Poultry and other products of animal origin Raw milk, raw or (Husseina et al., undercooked poultry and 2016) drinking water (Karch et al., 2005) Unpasteurized milk, undercooked meat and fresh fruits and vegetables Soft cheeses, (Fox et al., 2015) unpasteurized milk and unpasteurized (Finkelstein, 1996) Seafood, raw fruit and vegetables, and other foods contaminated during preparation or storage Fruits and vegetables (Knight et al., 2013) Contaminated food or water

(Bostan and Mahmooda, 2010)

Fish and contaminated water

(Doughty, 1996)

Beef or contact with animals Pork or contact with animals Water or soil

(Zhenghuan et al., 2008) (García et al., 2003)

Bovine products, water or soil contaminated

(Castilla et al., 2005)

(Lindsay et al., 1997)

172  Chapter 7

Table 7.1: Major foodborne illnesses and causes

Biocontrol as an Efficient Tool for Food Control and Biosecurity  173

7.4  Chemical Substances 7.4.1  Naturally Occurring Toxins Mycotoxins are toxic compounds produced by different types of fungi belonging mainly to the Aspergillus, Penicillium, and Fusarium genera (Picó, 2016). There are five mycotoxins or groups of mycotoxins that occur quite often in food: deoxynivalenol/Nivalenol, zearalenone, ochratoxin, fumonisins, and aflatoxins (Tola et al., 2016). Staple foods such as maize or cereals may have mycotoxins (Hove et al., 2016), the adverse effects of these mycotoxins in humans, termed mycotoxicosis, can affect the immune system and cause cancer (Mostrom, 2016). Some mycotoxins have been further classified as mutagenic, carcinogenic, or teratogenic (Hove et al., 2016). Among the mycotoxins, aflatoxin B1 (AFB1) and ochratoxin A (OTA) are the most important (Ariane Vettorazzi, 2016), and the International Agency for Cancer Research (IARC), has included the first of these as one of the most dangerous aflatoxins, in its Classification of group 1, due to evidence of its carcinogenic effect in humans. OTA, which is commonly found in foods, has been included in IARC’s Group 2B classification because it is also a potential carcinogen in humans (Çağındı and Gürhayta, 2016). Given the reported danger, quality control is necessary to ensure the protection of persons against the potential hazards of mycotoxins.

7.4.2  Biological Control of Chemicals The use of fungicides, insecticides, and herbicides in the production of fruit is inevitable. For example, in the case of tomato, evaluations using Gas Chromatography-Mass Spectrometry have shown high levels of pesticides which pose a high risk to the health of consumers (Kariathi et al., 2016).

7.4.3  Heavy Metals Heavy metals are ubiquitous in the environment; these can contaminate food through the soil, air, and water (Ahmed et al., 2015). Some heavy metals are essential for either plant or human nutrition, while other micronutrients (e.g., Cu, Cr, and Ni) might be toxic at elevated concentrations (McLaughlin, 2013); however, other metals such as As, Cd, and Pb might also inadvertently enter the food chain and pose risks to humans and animals (Islam et al., 2015). Heavy metals can be harmful because their potential to accumulate in different parts of the body (e.g., in lipids and the gastrointestinal system), as well as in the body of heavy metals, has adverse effects such as: impaired kidney function, poor reproductive capacity, liver damage, skin and bladder cancer, and even death (Wei et al., 2014; Chowdhury et al., 2016). Since food consumption is the main pathway (accounting for >90%) for human exposure to heavy metals, compared with air or skin contact (Saha et al., 2016), control agencies must perform an exhaustive control of effluent water as well as food marketed to guarantee levels of heavy metals that do not pose a risk to consumer health.

174  Chapter 7

7.4.4  Regulatory and Socioeconomic Aspects The food chain (producer, processor, retailer, and consumer) is critical to ensure the delivery of safe food. Advances in science and food technology are aimed at minimizing the risk of a socially acceptable manner (Desmarcheliera and Szabob, 2008). In recent years, quality assurance systems are becoming more stringent, in response to the food security problems recorded. On every continent, there have been serious outbreaks of foodborne illness. For example, in 2011, enterohemorrhagic Escherichia coli outbreak in Germany that affected eight countries and claimed the lives of 53 people showed up. Parallel to global standards and manufacturing control (ISO 22000, Hazard Analysis and Critical Control Points and Codex Alimentarius), the WHO developed a strategy to reduce the burden of disease which meets five keys to safe food preparation: (1) keep clean; (2) separating raw and cooked foods; (3) cook thoroughly; (4) keep food at safe temperatures; and (5) use safe water and raw materials (WHO, 2007); these are used to train food handlers and consumers.

7.5  Biosecurity of Biocontrol Agents Recently, use of biocontrol agents (BCA) for disease and pest control has been the focus of research and implementation in developed and developing countries because they play an important role in agricultural ecosystems maintaining their structure and functionality and reducing the need for synthetic pesticides. Hoeschle-Zeledon et al. (2013) stated that BCAs are an important component of integrated pest management (IPM). Loss of biocontrol by natural enemies of pests results in an increase in the employment of synthetic pesticides which will impact on crops profitability (Skevas et al., 2014). Use of synthetic pesticides in modern agriculture has been associated with risks to human health. Fungicides were reported as representing 60% of oncogenic risk among all pesticides (National Research Council (US), 1987). In the same document, NRC also declared that ‘As a class, fungicides present special difficulties because nine oncogenic compounds account for about 90% of all fungicide sales.’ Later the same organization reported that children’s vulnerability increased to synthetic pesticides (National Research Council (US), 1987). In spite of the numerous advantages of BCA to pest control, uncertainty about the risk of unanticipated effects on nontarget organisms is a major concern about the commercial use of BCAs (Skevas et al., 2014). For these reasons, it is important to contemplate diverse biosecurity issues in the commercial use of BCA for pest control. FAO (2003) stated that “Biosecurity is a strategic and integrated approach that encompasses the policy and regulatory frameworks (including instruments and activities) that analyze and manage risks in the sectors of food safety, animal life and health, and plant life and health,

Biocontrol as an Efficient Tool for Food Control and Biosecurity  175 including associated environmental risk.” Wisniewski et al. (2007) reported the characteristics of a BCA. Some of these characteristics are related to biosecurity. Among them, the BCA should be genetically stable; it should not produce metabolites that are deleterious to human health or be associated with infections in humans; it should be nonpathogenic to the host commodity. Biosecurity is a holistic concept that covers the introduction of plant and animal pests, introduction of genetically modified organisms (GMOs) and their products, and the introduction and management of invasive alien species and genotypes which will have direct relevance for agriculture sustainability, food safety, environment protection, and human health (FAO, 2003). Biosecurity of some BCA has been tested. Jing-hua et al. (2006) evaluated some biosecurity aspects of the use of Trichoderma viridis T23 on the microbial ecology of Rhizosphere and the influence on the growth of melon, as well as toxicity in mice and white rabbits. These authors did not find significant effects of this BCA on the number of fungi, bacteria, and actinomyces in the rhizosphere soil. T. viridae had no harmful effect on fish growth. On the other hand, this fungal strain had low toxicity on rats in both stomach and skin toxicity experiments and no allergic reaction to rabbit skin and mucous membrane of eyes, while Jing et al. (2009) reported that the LD50 of the big mouse was more than 5000 mg/kg when the Bacillus subtilis strain B29 was supplied through its mouth and skin.

7.5.1  Regulations on Biosecurity of BCA There are international regulations such as the FAO Code of Conduct for the Import and Release of Exotic Biological Control Agents (FAO, 2006) and the International Standards for Phytosanitary Measures No. 3, Guidelines for the Export, Shipment, Import and Release of Biological Control Agents and other Beneficial Organisms (ISPM-3 2005). The last regulation provides phytosanitary measures, as well as recommended guidelines for safe export, shipment, import, and safe usage of BCAs. Existing standards in pest risk analysis (PRA), such as—ISPM No. 2, Framework for PRA (ISPM-2 2007) and ISPM No. 11, PRA for quarantine pests, including environmental risk analysis and living modified organisms (ISPM-11 2004) are a basic regulation to be performed for risk assessments (HoeschleZeledon et al., 2013). Other international regulations that may apply to commercial use of BCA are The International Standards for Phytosanitary Measures No. 3, and Guidelines for the Export, Shipment, Import and Release of Biological Control Agents and other Beneficial Organisms (ISPM-3 2005) established by The International Plant Protection Convention (IPPC) (Woo et al., 2014). The European and Mediterranean Plant Protection Organization (EPPO) has released some regulations that may apply to BCA;one of them is the EPPO Standard PM 6/1, First import of exotic BCAs for research under contained conditions. Another one is the PRA in accordance with ISPM No. 2 (Framework for PRA) and ISPM No. 11 (PRA for quarantine

176  Chapter 7 pests including analysis of environmental risks and living modified organisms). In the European Union, all plant protection products are regulated under EU Regulation 1107/2009/ EC (EU 2009a) and Regulation 1107/2009/EC (EU 2009a) and Directive 2009/128/EC (EU 2009b), while BCA products in Africa are regulated by the Inter-African Phytosanitary Council (IAPSC) (Hoeschle-Zeledon et al., 2013). Some countries have established specific regulations. In Kenya, BCA are considered as biopesticides and are regulated under the control of the Pest Control Products Board (PCPB). In Brazil, authorization for marketing of pest control products is regulated by the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA), the National Health Surveillance Agency (ANVISA), and the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA) (Woo et al., 2014). In India, BCA should follow the Insecticide Act of 1968 and are regulated by the Central Insecticides Board (CIB) of the Ministry of Agriculture. In Canada, registration of BCA is under the Guidelines for the registration of microbial pest control agents and products (DIR2001-02), while, Germany has regulations for non-native species used for pest control and is under the Federal Environment Protection Law (Hoeschle-Zeledon et al., 2013). In the United States of America, pesticides are regulated by the Environmental Protection Agency (EPA) where there is a special area that regulates BCA (Scoles et al., 2015). The US Code § 7712—Regulation of movement of plants, plant products, biological control organisms, noxious weeds, articles, and means of conveyance (Pub. L. 106–224, title IV, § 412, June 20, 2000, 114 Stat. 441.) was established in USA, which indicates the main regulations for biological control organisms. The Agriculture Secretary may publish a list of organisms which are allowed to be moved in interstate commerce; this list distinguishes between indigenous, non-indigenous, newly introduced, and commercially raised organisms and any person may ask to add or remove any organism from the list.

7.5.2  Hazard Identification and Risk Assessment to Human Health and the Environment The assessment of ecological, environmental, and health risks is limited, mainly due to a lack of information. As defined by the Environmental Protection Agency of the United States, “ecological risk assessment is a process used to systematically evaluate and organize data, information, assumptions and uncertainties in order to help understand and predict relationships between stressors and ecological effects in a way that is useful for decision-making” (Ives and Schellhorn, 2011). Before making a regulatory decision about a pesticide, EPA suggests the requirement of data on a range of subjects to ensure that the product meets Federal safety standards. For all pesticide products, including genetically engineered pesticides, EPA requires testing of product composition and chemical properties, human health effects, environmental effects on

Biocontrol as an Efficient Tool for Food Control and Biosecurity  177 nontarget pests, and the fate of the pesticide in the environment. Where appropriate, EPA also examines a pesticide’s potential to trigger allergenic responses. Specifically for genetically engineered pesticides, EPA routinely examines the following types of information and data: • • • • • • •

Identification of new genetic material and all new proteins. Mammalian toxicity testing of all new proteins. Comparison of new proteins to known toxins and allergens. Toxicity testing on birds, fish, earthworms, and representative insects such as bees, ladybird beetles, and lacewings. Toxicity testing on insects related to target insect pests. Length of time required for the new proteins to degrade in the environment. Toxicity testing will be conducted with a range of doses and concentrations 10–100 times higher than those expected in environmental conditions. EPA also consults the literature and other sources of supporting information related to any aspect of the proteins and the organisms from which they are derived.

7.5.3  Personal Protection in the Food Industry: An Overview In 2006, EPA issued a very strict rule about all pesticide research using human subjects. These rules need to be considered before we use the pesticides. There are strong standards and high ethical protections for adults who participate in intentional exposure studies for pesticides. EPA’s rules make it clear that all pregnant women, all nursing women, and all children are banned from all studies involving intentional exposure to pesticides. Our regulation “Protections for Subjects in Human Research” was amended in 2013. The regulation is consistent with the recommendations of a 2004 National Academy of Sciences (NAS) report and has been modeled primarily on EPA’s practice under the Federal Policy for the Protection of Human Subjects (the “Common Rule”). On February 8, 2013, EPA strengthened the standards for human research involving pesticides submitted by third parties for consideration in EPA decision-making. That is, the standards cover third parties, including pesticide companies and other research sponsors, who may want to submit to EPA human research involving pesticides. All information is on the EPA’s website (see Protections for Subjects in Human Research Involving Pesticides, also available in docket number EPA-HQ-OPP-2010-0785). The amendments made by EPA are: to broaden the scope and applicability of the rule; to further strengthen the standards for research to be considered in our decisions; to clarify the approach used in the science and ethics reviews of human research involving pesticides; and to formally disallow participation in testing by subjects who cannot consent for themselves. The amendments do not change the current Federal Policy for the Protection of Human Subjects (the “Common Rule”), which governs research with human subjects conducted or

178  Chapter 7 supported by the EPA and many other federal departments and agencies. These amendments implement the recommendations contained in a 2004 report from the NAS and satisfy our commitments under a 2010 settlement agreement with the Natural Resources Defense Council and other groups which challenged the 2006 rule.

7.6  Agriculture and Biological Control Owing to the increase in the world’s population, the agricultural production should increase to meet the need for food and energy and, at the same time, the use of pesticides and fertilizers of chemical origin should be decreased. In this context, there is interest in control of pests, diseases, and weeds (Alabouvette and Cordier, 2011) since the pests and diseases are responsible for 30%–40% loss in agricultural production in the tropics. Different approaches are used to prevent, mitigate, or control plant diseases in economically important crops, and the most conventional and common way of pest and disease control is through the use of pesticides; however, these pesticides and fungicides directly or indirectly enter the aquatic ecosystem and are biomagnified in the food chain, endangering the ecosystem and public health (Miriti et al., 2014). In contrast, biological control applies the use of microbial antagonists, like bacteria, fungi (Arbuscular Mycorrhizal Fungi), nematodes, insects, or mites (including viruses), for the control of weeds, to suppress a pest population and diseases of crop agricultural importance (Ehlers, 2011). Mechanisms of biological control using microbial agents include competition for an ecological niche or a substrate, production of inhibitory allelochemicals, and development of induced systemic resistance (ISR). There are four types of biological control strategies, namely: conservation (application of natural enemies), classical (introduction of exotic natural enemies to a new locale where they did not originate), augmentative (supplemental release of natural enemies at a critical time in season), and importation biological control (Kalia and Mudhar, 2011). In biological control, the organism that suppresses the pathogen is referred to as a BCA; the bacteria usually are classified as BCA and as plant growth promoters. Bacteria are used to control plant diseases; the members of major importance are the Enterobacteria, Streptomyces, Pseudomonas, Rhizobium, Azospirillum, Azotobacter, and Bacillus spp. In particular, the plant growth-promoting rhizobacteria (PGPR) are a promising strategy for plant protection (Saharan and Nehra, 2011). The benefits of PGPR are ample because they have a mode of action for disease control, plant health promotion, mechanisms of root colonization, and effects on plant physiology and growth; they may affect plant growth by producing auxins such as indole-3-acetic acid (IAA), cytokinins; or ethylene degrading precursors, ACC by CAC deaminase, while the biocontrol of plant pathogens may include the production of hydrogen cyanide, ISR, production of siderophore and antibiosis; So it can greatly decrease the use of conventional pesticides, leading to higher harvest yield (Liu et al., 2010; Ehlers, 2011; Kalia and Mudhar, 2011).

Biocontrol as an Efficient Tool for Food Control and Biosecurity  179

7.6.1 Opportunities Of all species of bacteria with biological control, the genus Bacilli are considered excellent for formulations applied to biological control. The bacteria of this genus are sporulating, found in different habitats, and have the peculiarity of being, in many cases, entomopathogenic. Currently, the metabolites produced by Bacillus thuringiensis, B. subtilis, B. megaterium, and B. firmus are used for biopesticides used to control nematodes of agricultural importance (Berlitz et al., 2014). Fungal biological control can be mediated by bacteria with inhibition of synthesis of fungal sterols and nucleic acids, as well as change of permeability of cell membrane and destruction of fungal cell wall (Kang et al., 2015). Some of the benefits attributed to biological control are the involvement of different mechanisms of disease suppression by a single microorganism, the complex interaction between the organisms, and the survival of BCA in the environment in which they are used, so they will be more durable than synthetic chemicals (Lee et al., 2013). Even if it is difficult to predict the result of interactions between plants and beneficial soil microorganisms and, even more, between species of microorganisms, thus far the microorganisms BCAs (MBCAs) have a history of safe use (Cano, 2011) because plant-associated bacteria cannot interact with other eukaryotic hosts like humans in a pathogenic way. Another attractive option is the use of specific bacteriophages (phages), viruses that specifically kill bacteria, providing a more targeted approach. They do not have a deleterious effect on the plant or target beneficial bacteria. Phages are viruses that specifically infect bacteria, yet have no direct negative effects on animals or plants. Meanwhile, the baculoviruses are pathogens that attack insects and other arthropods. They are usually extremely small and are composed of double-stranded DNA. The majority of baculoviruses used as BCAs are in the genus Nucleopolyhedrovirus. They do not show negative impacts on plants, mammals, birds, and fish, and more generally on nontarget insects. However, the high specificity of baculoviruses is also cited as a weakness for agricultural uses because of the narrow spectrum of its activity (Frampton et al., 2012; Regnault-Roger, 2012). In Table 7.2, some commercial products are presented, whose main ingredient is microorganisms, which have been of interest in the area for many years. Talking about microbial inoculant products, their presentation includes powders, suspensions, granules, liquids, and gels, while the final product is affected by factors such as the genetics and physiology of microorganisms, the composition of the medium used for multiplication, the growth phase, the material used as carrier (nature, particle size, and presence of contaminating microorganisms or viability of the bacteria), and the technology used for drying and preservation of bacteria (addition of nutrients and preservatives) in the substrates (Moreno-Gómez et al., 2012).

Trade Name AQUABAC (AQUABAC, n.d.)

Dipel® WG (Dipel WG, n.d.)

Active Ingredient

Concentration

Target Organisms

Mode of Action

Environmental Impact and Nontarget Toxicity

Is naturally Mosquito and Aedes aegypti, 8% solids, spores occurring and safe blackfly larvae Aedes darsalis, and insecticidal to the environment. are killed by Psorophora toxins. Equivalent to Does not persist in ingesting the columbiae, Culex 1200 International soil or water. Has protein crystal tarsalis, on Toxic Units (ITU/ (deltaendotoxin). no toxic effects on mosquito and mg) (4.84 Billion beneficial insects blackfly larvae. ITU/gallon or 1.2 such. Used for Against blackflies, Billion ITU/liter). 20 years with no fungus gnats, adverse effects on nuisance flies humans. (Psychoda spp. and Chironomus spp.) and nuisance aquatic midges (Chironomine). Control by Has organic status, Orgya antiqua, 6.4%. Bacillus ingested of larvae does not leave Cydia pomonella, It is containing thuringiensis, residues, prevents Cydia molesta, 32,000 subesp. the development Ectomyelois International Units kurstaki of resistance per milligram power ceratoniae, Proeulia to traditional auraria, Proeulia insecticides, it chrysopteris, is harmless to Proeulia triquetra, beneficial insects, is Otiorhychus safe for birds, fish, rugostriatus, bees, and other Copitarsia consueta, forms of wildlife in Diaphinia sp., the environment. Spodoptera sp., Orgya antiqua, Proeulia sp., Heliothis sp. Bacillus thuringiensis subspecies israelensis

Crops

Company

Alfalfa, almonds, asparagus, corn, cotton, dates, grapes, peaches and walnuts

AQUABAC

Nogal, Almond, melon, watermelon, Blueberry, Raspberry, Blackberry, Sarsaparilla, Apple, Pear, Membrillero, Kiwi, Vides, Cherry, Plum, Nectarine, Peach, Damascus.

BAYER

180  Chapter 7

Table 7.2: Biofertilizers in the current market

Bacillus thuringiensis aizawai GC-91

Fungifree AB (FMC, n.d.)

Bacillus subtilis

In humans or The toxin crystals Bonagota 1 billion viable are dissociating, laboratory animals. salubricola, spores/g (equivalent There is no known the protoxin Cryptoblabes to 38.0 g/kg to produce toxic molecules are endotoxin—25,000 gnidiella, Diaphania metabolites. “activated” by hyalinata, μL/mg potency) Little dangerous to the digestive Diaphania nitidalis, the environment enzymes of Ecdytolopha (CLASS IV) insects and toxins aurantiana, destroy the cell Grapholita molesta, membrane of Neoleucinodes the medium elegantalis, intestine. Plutella xylostella, Pseudoplusia includens, Spodoptera frugiperda and Tuta absoluta. (Equivalente Microbial It is not phytotoxic Colletotrichum a 10 g/kg) fungicide to crops indicated. gloeosporioides, Conteniendo no biological origin Colletotrichum acutatum, menos de 1 × 109 Leveillula taurica, UFC/g. 1.00 % Sphaerotheca macularis, Sphaerotheca humuli, and Erysiphe cichoracearum

Citrus, melon, cucumber, cabbage, tomato, potato, soybean, cotton, grape and apple

Biocontrole

Mango, avocado, papaya, lime, lemon, tangerine, orange, grapefruit, eggplant, peppers, tomatoes, strawberries, raspberries, zucchini, pumpkin, melon, cucumber and watermelon.

Agro&Biotecnia (A&B)

Continued

Biocontrol as an Efficient Tool for Food Control and Biosecurity  181

AGREE® (Bula, n.d.)

Target Organisms

Environmental Impact and Nontarget Toxicity

Trade Name

Active Ingredient

Concentration

RootShield Granules (BioWorks, n.d.)

Trichoderma harzianum Rifai strain T-22*

Contains at least 1.0 × 107 colony forming units per gram dry weight

Pythium, Rhizoctonia, Fusarium, Cylindrocladium and Thielaviopsis.

Grows onto plant roots as they develop and provides protection against plant root pathogens

For Terrestrial Use. Do not apply directly to water, or to areas where surface water is present, or to intertidal areas below the mean high water mark. DO NOT APPLY to sugarcane, pechay, rice, mushrooms, kiwi, tobacco, barley, oats, lemon, apple, and chickpea.

BAKTILLIS (BAKTILLIS, n.d.)

Bacillus subtilis

5.15 %. Content no less than 1 × 1012 CFU/ ml at 20°C

Fusarium spp., Mycosphaerella fijiensis, Botrytis cinérea, Leveillula taurica, Colletotrichum gloesporioides and Bremia lactucae

Biological fungicide based on metabolites, polypeptides and spores having the ability to inhibit the germination and growth of phytopathogenic fungi.

It is compatible with commonly used fungicides and insecticides, except with copper compounds and antibiotics.

Mode of Action

Crops

Company

Berries and small fruits, bulb vegetables, cereal grains, citrus fruits, cucurbit vegetables, fruiting vegetables, leafy and brassica (Cole) leafy vegetables, asparagus, legume vegetables, root and tuber vegetables, stone fruits and tree nuts: Tomato, chili, banana, strawberry, blueberry, raspberry, currant, blackberry, papaya, avocado and lettuce.

BioWorks, Inc.

BIOKRONE

182  Chapter 7

Table 7.2  Biofertilizers in the current market—cont’d

Biocontrol as an Efficient Tool for Food Control and Biosecurity  183

7.6.2 Drawbacks According to Patil and Solanki (2016), the biopesticides have the following drawbacks: • • • •

Usually are unable to control a broad range of pests present in the field. Owing to target specificity, the potential market for some products may be limited, which results in less market availability in the world. Require proper timing and procedures for having an effective application, as they may be sensitive to desiccation, heat, and UV exposure. Need some special formulation and storage procedures, which may complicate the production and distribution of certain products.

7.6.3 Regulation Trade regulation is another important point, because for the marketing of a biopesticide, it is required to evaluate and register it legally. In the registration process, the risk assessments associated with their properties and uses have to be evaluated. These risks are linked to the toxicity on the organisms and populations, as well as the exposure. Potential hazards for humans (operators, bystanders, consumers), wildlife, and the environment (fate in the air, soil, and water, nontarget organisms including the routes to which they are exposed) must be identified and evaluated depending on the uses of the end products. So despite the existence of new BCAs, there are still very few products on the market because of the regulatory status of BCAs, which must be registered according to guidelines originally developed for chemical pesticides. For example, in the European Union, the Directive 91/414 EEC applied to any type of plant protection products, including natural products such as plant extracts, semiochemicals and microorganisms (bacteria, viruses, and fungi) (EU, 1991). These requirements represent the main constraint of putting a BCA on the market. (Alabouvette and Cordier, 2011). Based among other things, the only difference between microbial and synthetic chemicals with respect to the record is that there is little difference between the active substance and the preparation formulated; it was amended the Directive 91/414 EEC (used in plant protection in the EU), now this directive was amended by the Commission Directive 2001/36/EC regarding the data requirements for the Annex I inclusion of microorganisms as active substances and national authorization of products (Annex IIB and IIIB). However, Directive 2001/36 follows the same scheme to prevent risks due to the application of chemical pesticides with negative effects to humans and the environment. Meanwhile, by contrast, the most of the BCAs are safe; there is a lack of evidence of proven deleterious effects. Meanwhile, the registration of Plant Protection Products in the EU is regulated by EU Regulation 1107/2009.

184  Chapter 7 In the USA, registration of the active microorganism procedure under code 40 CFR (Code of Federal Regulations) is regulated by the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) and implemented by US EPA (Kamilova et al., 2015). Meanwhile, in Mexico, the registration of biofertilizers is controlled by the Federal Commission for the Protection against Sanitary Risk (COFEPRIS), and has the program of the NOM-70-FITO-1995 which establishes the phytosanitary requirements and specifications for the importation, introduction, mobilization, and release of BCAs (DOF, 1996; NOM-182-SSA1-1998, National Advisory Committee on Standardization of Health Regulation and Development, 2000; Regnault-Roger, 2012). In order to promote the use of biofertilizers and to facilitate the integration of knowledge and technologies in this sense in Latin America, the countries of that region established in 2003 the Latin American network of organic farming and environment costumes. It is established that the inoculants should not be toxic; rather, it should be biodegradable and non-polluting, and present a minimal risk in the dispersion into the atmosphere or deep water, besides having qualities that allow having enough shelf life (1 or 2 years at room temperature). Generally, the African and South American countries and China do accept data present in the European or the US dossiers. However, all of them require efficacy trials performed locally. However, in Mexico, these products must be evaluated in different locations (Al Menos15–20) under different environmental conditions to validate the correct functioning of the product, some features that define a suitable use in the field shall be: strong promotion of plant growth, antagonism of multiple pathogens, pesticide resistance, competitive capacity, movement, and synergism capacity with other microorganisms. Based on the foregoing, in Mexico, consult NOM-182-SSA1-1998 (National Advisory Committee on Standardization of Health Regulation and Development, 2000) for the labeling of containers and have basic information such as: lot, production date, net content, expiration date, health registry, recommended crops, doses and instructions for use, amount of microorganism in CFU (colony forming units), genus and species of microorganisms (and if they have been genetically modified). With regard to the regulation of bacterial inoculants, the top quality parameters considered by India and regulated by the Ministry of Agriculture, stipulate some features to be met, which are shown in Table 7.3.

7.6.4 Trends Nowadays, we need strategies for the protection of agricultural crops against phytopathogens. The use of biopesticides is an ecological strategy that offers alternatives to solve the concerns posed by the excessive use of chemical pesticides, being widely accepted in sustainable agriculture as the best option today (Patil and Solanki, 2016). They control the harmful organisms that cause plant diseases without disturbing the ecological equilibrium. Therefore,

Table 7.3: Requirements for bacterial inoculants used in agriculture Bureau of Indian Standards

IS 14807 (2000): Phosphate Solubilizing Bacterial Inoculant (PSBI) 107 Viable phosphate solubilizing bacterial cells/g of the carrier material on dry mass basis

Have no contamination with other micro-organisms pH Shall be able to

105 Dilution

Packing

Between 6.5 and 7.5 Have phosphate solubilising capacity in the range of minimum 30% in terms of zone formation minimum 10 mm solubilization zone in a prescribed solid medium having at least 3 mm thickness Shall be packed in polyethylene packs, thickness of which shall not be less than 100 (μm)

IS 14806 (2000): Azospirillum inoculants

IS 8268 (2001): Rhizobium Inoculants

108 Viable Azotobacter cells/g of the carrier on dry-mass basis during the entire period of shelf-life from the date of manufacture 105 Dilution

107 Viable Azospirillum cells/g of the carrier material on dry mass basis 105 Dilution

107 Viable Rhizobium cells/g of the carrier on dry-mass basis till 6 months expiry period from the date of manufacture 105 Dilution

Between 6.5 and 7.5 Fix nitrogen in a minimal amount of 10 mg/g of sucrose utilized

Between 6.5 and 7.5 Show effective root development on all cultivar/ crops against which the inoculant is intended to be used.

Between 6.5 and 7.5 Show effective nodulation on all those species and/ or cultivars listed on the packet before the expiry date

Shall be packed in packaging material of low-density polyethylene/polypropylene bags thickness of which shall be 75–100 μm minimum

Shall be packed in polyethylene packs, thickness which shall not be less than 75–100 μm

Shall be packed in packaging material of low density polyethylene/ polypropylene bags thickness of which shall be 75–100 μm minimum

Continued

Biocontrol as an Efficient Tool for Food Control and Biosecurity  185

Minimum content

IS 9138 (2009): Azotobacter Chroococcum Inoculants

Bureau of Indian Standards Information for marking

IS 14807 (2000): Phosphate Solubilizing Bacterial Inoculant (PSBI) (a) Name of the product, especially as phosphate solubilizing bacterial inoculant; (b) Name and address of the manufacturer; (c) Crop(s) for which intended; (d) Type of the carrier used; (e) Batch number; (f) Date of manufacture; (g) Expiry date which shall not be less than 6 months from the date of manufacture; (h) Net mass in kg and area meant for; (i) Storage instructions worded as under “STORE IN COOL PLACE AWAY FROM DIRECT SUNLIGHT AND HEAT”; and (j) Any other information required under the Standards of Weights and Measures (Packaged Commodities) Rule, 1977

IS 9138 (2009): Azotobacter Chroococcum Inoculants

IS 14806 (2000): Azospirillum inoculants

IS 8268 (2001): Rhizobium Inoculants

(a) Name of the product, specifically as Azotobacter inoculant; (b) Non-leguminous crop for which intended; (c) Name and address of the manufacturer; (d) Type of the carrier; (e) Batch or Code number; (f) Date of manufacture; (g) Date of expiry (agreed between the manufacturer and the purchaser subject to minimum 6 months from the date of manufacture); (h) Net quantity and rate of application; (i) Storage instructions worded as “STORE IN A COOL PLACE AWAY FROM DIRECT SUN AND HEAT”; (j) Number of Azotobacter cells/g of carrier; and (k) Any other information

(a) Name of the product, especially as Azospirillum inoculant; (b) Name and address of the manufacturer; (c) Crop(s) for which intended; (d) Type of the carrier used; (e) Batch number; (f) Date of manufacture; (g) Expiry date which shall not be less than 6 months from the date of manufacture; (h) Net mass in kg and area meant for; (i) Storage instructions worded as under: “STORE IN COOL PLACE AWAY FROM DIRECf SUN LIGHT AND HEAT” (j) Any other information required under the Standards of Weights and Measures (Packaged Commodities)

(a) Name of the product, specifically as Rhizobium inoculant; (b) Leguminous crop for which intended; (c) Name and address of the manufacturer; (d) Type of the-carrier; (e) Batch or Code number; (f) Date of manufacture; (g) Date of expiry (agreed between the manufacturer and the purchaser subject to minimum 6 months from the date of manufacture); (h) Net quantity and the area meant for; (i) Storage instructions worded as under: “STORE IN COOL PLACE AWAY FROM DIRECT SUN AND HEAT”. (j) Any other information

186  Chapter 7

Table 7.3  Requirements for bacterial inoculants used in agriculture—cont’d

Biocontrol as an Efficient Tool for Food Control and Biosecurity  187 the initial effort of the countries should be to study new species with potential to carry biocontrol; so the progress in the area of biological control will benefit from adequate followups of the results of each project or work is done on this topic (Vitalis et al., 2015). Besides the use of suppressing microorganisms to improve the health of crops, biological control involves interactions between the plant, the rhizosphere microbial community, the pathogen, the biocontrol organism, and the physical environment. Numerous rhizosphere organisms are capable of producing compounds that are toxic to pathogenic microorganisms, such as strains of Azospirillum, Bacillus, Bradyrhizobium, Pseudomonas, Rhizobium, Streptomyces, Clonostachys, and Trichoderma, which can protect plants from pathogens and have the potential to directly promote crop yield. Various mechanisms of PGPR are used in biofertilization and biocontrol. Bacillus subtilis is one of the best examples of PGPR because it acts against a wide variety of pathogenic fungi (Tailor and Joshi, 2014). Unfortunately, there are several limitations to the use of PGPR as biological controllers in IPM programs. The principal limitation is the limited taxonomic, biological, and ecological knowledge of these species by field people in general (MuñozCárdenas et al., 2015). Nevertheless, there are many possibilities for combining several biocontrol agents with each other, or with physical or chemical control methods, or to offer an applicable strategy for the health of plants and economic growth (Andrés et al., 2016). The reduction of production costs and the obtaining of more competitive forms of biological control for pest control offers a promising future in the registration of organisms as biopesticides for their commercialization in more countries.

7.7 Perspectives Fresh fruits and vegetables are considered healthful and nutritious foods having no risk of foodborne illness associated with their consumption. However, foodborne illness outbreaks have been traced to these products, as well as to juices and milk (Bhattacharyya and Bandhopadhyay, 2010). Therefore, there is an urgent need to assess, control, and prevent the emerging microbiological and toxicological risks associated with food (Fusco et al., 2015) as well as the development of strategies to harmonize activities that guarantee food safety. Since 1996, HACCP System Regulation was signed by the US President codifying principles for the prevention and reduction of pathogens. However, existing food safety programs need to be revised to ensure that they are sustainable and effective. In this sense, a coordination of inspection, enforcement, and research may all contribute to food safety. On the other hand, the demand for food production has increased with a rapidly growing human population. This demand has been met by increasing the land area under cultivation, but it has become limited. For this reason, efforts have been made to increase productivity

188  Chapter 7 by fighting the losses inflicted by insects, weeds, and plant pathogens (Peshin, 2014). As an ecofriendly alternative to ensure environmental quality and human health, BCAs have emerged. However, it is necessary to highlight the importance of the isolation and selection of appropriate and new biocontrol agents from the products themselves. Besides, BCAs have different growth requirements, which makes its production, formulation and culture optimization a challenge. In case of predators, they could have an increase on artificial media but longer development times (Cohen and Smith, 1998). Thus, producers must develop specific methods for the production, formulation, and application of BCAs and determine which traits are most important for the most efficient biocontrol while keeping costs down. Finally, strategies combining BCAs with other preservative methods should be explored and optimized by focusing on the level and mode of inoculation to increase the safety and shelf life of foods

7.8 Conclusions Modern societies are characterized by rapid growth of their populations that demand higher levels of high quality food which promotes the production of biological control agents. However, the risks included in this chain are more; for this reason, the topics of biosafety and food control for production, distribution, and consumption are of great relevance in the design of strategies of each country, and each region is obligated to reach a guarantee for the people about the availability of the required foods. In any process where biological, physical, or chemical agents are used, there is always a latent risk, so it is extremely important to know the risk factors and find a way of preventing them. In recent times, biological threats are one of the challenges that has gained much attention, mainly due to the increased use of biological agents in a number of industrial sectors such as food production, agriculture, production of value-added compounds, etc. Finally, it is the duty of each government to develop strategies to address the management of standards and rules of biosafety, as well as the different strategies and specific characteristics of control in food production in traditional ways, including relevant information on emerging trends and alternatives of the conservation of these.

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Further Reading Campos-Herrera, R., El-Borai, F.E., Duncan, L.W., 2015. Modifying soil to enhance biological control of belowground dwelling insects in citrus groves under organic agriculture in Florida. Biol. Control 84, 53–63. Comité Consultivo Nacional de Normalización de Regulación y Fomento Sanitario. 2000. Norma Oficial Mexicana NOM-182-SSA1-1998, Etiquetado de Nutrientes Vegetales. Available from: www.salud.gob.mx/ unidades/cdi/nom/182ssa18.html. Diario Oficial de la Federación (DOF), 1996. PROYECTO de Norma Oficial Mexicana NOM-70-FITO-1995. Available from: http://dof.gob.mx/nota_detalle.php?-codigo=4881515&fecha=25/04/1996 (1 July 2016). Elmahdi, S., Kadir, J., Muda Mohamed, M.T., Vadamalai, G., Akter, S., 2014. Isolation, screening and characterization of effective microbes with potential for biological control of Fusarium wilt of Rock Melon. World J. Agricult. Res. 3 (1), 11–16. Environmental Protection Agency (EPA), 2016. Protections for Subjects in Human Research with Pesticides. https://www3.epa.gov/. Food and Drug Administration, 2015. (February 21). FSMA proposed rule for preventive controls for human food. Retrieved from, http://www.fda.gov/Food/GuidanceRegulation/FSMA/ucm334115.htm. Govindasamy, V., Franco, C.M.M., Gupta, V.V.S.R., 2014. Endophytic actinobacteria: diversity and ecology. In: Verma, V.C., Gange, A.C. (Eds.), Advances in Endophytic Research. Springer, New Delhi Heidelberg New York Dordrecht London, pp. 27–60. Heinzerling, L., McGarity, T.O., Shapiro, S., Steinzor, R., Patoka, M., 2013. Current good manufacturing practice and hazard analysis and risk-based preventive controls for human food (Docket ID No. FDA-2011-N-0920). Center for Progressive Reform, Washington, DC. Hunt, E.J., Loomans, A.J.M., Kuhlmann, U., 2011. An international comparison of invertebrate biological control agent regulation: what can Europe learn? In: Ehlers, R.-U. (Ed.), Regulation of Biological Control Agents. Springer, Dordrecht, pp. 79–112. Kamilova, F., Okon, Y., de Weert, S., Hora, K., 2014. Commercialization of microbes: manufacturing, inoculation, best practice for objective field testing, and registration. In: Lugtenberg, B. (Ed.), Principles of Plant-Microbe Interactions. Springer Cham, Heidelberg New York Dordrecht London, pp. 319–328. Liu, K., Garrett, C., Fadamiro, H., Kloepper, J.W., 2016. Antagonism of black rot in cabbage by mixtures of plant growth-promoting rhizobacteria (PGPR). BioControl. Springer. Loliam, B., Morinaga, T., Chaiyanan, S., 2012. Biocontrol of Phytophthora infestans, Fungal pathogen of seedling damping off disease in economic plant nursery. Hindawi Publishing Corporation, Psyche. 6 pages. Michel-Aceves, A.C., Otero-Sánchez, M.A., Martínez-Rojero, R.D., Rodríguez-Morán, N.L., Ariza-Flores, R., Barrios-Ayala, A., 2008. Producción Masiva De Trichoderma harzianum Rifai En Diferentes Sustratos Orgánicos. Rev. Chapingo Ser. Hortic. 14, 185–191. Moreira, R.R., Nesi, C.N., May De Mio, L.L., 2014. Bacillus spp. and Pseudomonas putida as inhibitors of the Colletotrichum acutatum group and potential to control Glomerella leaf spot. Biol. Contr. 72, 30–37.

CHAPTE R 8

Foodborne Diseases and Responsible Agents Md. Latiful Bari, Sabina Yeasmin University of Dhaka, Dhaka, Bangladesh

8.1 Introduction Foodborne diseases are a preventable public health challenge that causes illnesses and deaths each year worldwide. Foodborne diseases result from the consumption of food containing microbial agents such as bacteria, viruses, and parasites or the food contaminated by poisonous chemicals or biotoxins. More than 250 different types of viruses, bacteria, parasites, toxins, metals, and prions are associated with foodborne diseases in humans. Although viruses are responsible for more than 50% of all foodborne illnesses, generally, hospitalizations and deaths are due to bacterial agents. The diseases range from mild gastroenteritis to life-threatening neurologic, hepatic, and renal syndromes caused by either toxin from the “disease-causing” microbe, or by the human body’s reaction to the microbe itself (Nayenje and Ndip, 2013). An estimated 2.0 million deaths occurred due to gastrointestinal illness worldwide (Fleury et al., 2008). Although a majority of the foodborne illness cases are mild and self-limiting, severe cases can occur in high-risk groups resulting in high mortality and morbidity in this group. The high-risk groups for foodborne diseases include infants, young children, the elderly, and the immunocompromised persons. The battle against foodborne diseases is facing new challenges due to the globalization of the food market, climate change, and changing patterns of human consumption as fresh and minimally processed foods are currently preferred (Schelin et al., 2011). Foodborne illnesses have a negative impact on the public health as well as on the economy of a country. They also have a negative impact on the trade and industries of the affected countries. Identification of a contaminated food product can result in the recall of that specific food product leading to economic loss to the industry. Foodborne outbreaks may lead to closure of the food outlets or food industry resulting in job losses for workers, affecting the individuals as well as the communities. Localized foodborne disease outbreaks may become a global threat. The health of people in many countries can be affected by consuming contaminated food products, and

Food Safety and Preservation. https://doi.org/10.1016/B978-0-12-814956-0.00008-1 © 2018 Elsevier Inc. All rights reserved.

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196  Chapter 8 may negatively impact a country’s tourist industry. The foodborne disease outbreaks are reported frequently at the national as well as international level underscoring the importance of food safety (World Health Organization [WHO], 2011). Several factors are necessary for foodborne diseases to occur and among them are: (1) a pathogen; (2) a food vehicle; (3) conditions that allow the pathogen to survive, reproduce, or produce a toxin; and (4) a susceptible person who ingests enough of the pathogen or its toxin to cause illness. Emerging, or in some cases reemerging, food-borne problems are those that have appeared recently in a population; extended to new vehicles of transmission; started to increase rapidly in incidence or geographic range; or been widespread for many years but only recently been identified through new or increased knowledge or methods of identification and analysis of the disease agent (Fig. 8.1).

Foodborne diseases

Food poisoning

Chemical poisoning

Poisonous plant tissues

Food infections

Biological poisoning

Caused by various pathogenic microorganisms ingested with food

Poisonous animal tissues

Fig. 8.1 Schematic causes of foodborne diseases.

The most prominent emerging problems stem from bacteria, viruses, and protozoa. Other food safety problems include mycotoxins, pesticide residues, veterinary drugs, and unconventional agents such as prion (associated with transmissible spongiform encephalopathies) and environmental contaminants. The factors that play a role in the epidemiology of emerging foodborne problems include changes in the pathogens; development, urbanization, and new lifestyles; cuts in health systems; existing knowledge,

Foodborne Diseases and Responsible Agents  197 beliefs, and practices; demographic changes, travel, and migration; trade in food, animal feed, and animals; and poverty and pollution (Magkos et al., 2006). It is likely that emerging foodborne problems will become even more significant over the coming years. The following figure shows the schematic presentation of food-borne diseases.

8.2  Food Contamination and Infection Food-related infection occurs as a result of ingestion of pathogenic microorganisms with food. The ingested microorganisms multiply in the gut and can cause diseases like diarrhea, typhoid fever, and cholera; intestinal parasites can cause diseases such as amoebiasis and taeniasis (tapeworm disease); and zoonotic foodborne diseases (i.e., those that are transmitted to humans from other animals), for example, anthrax and bovine tuberculosis (Samad, 2011). There are many different kinds of foodborne diseases and they may require different treatments, depending on the symptoms they cause. Illnesses that cause acute watery diarrhea or persistent vomiting lead to dehydration if the person loses more body fluids and salts (electrolytes) than they are able to replace. It is therefore important to rehydrate the person, ideally with oral rehydration salts (ORS), or if this is not available, a simple mixture of clean water with some sugar and salt is advised (Ramakrishna et al., 2008). Electrolytes are salts in the body that conduct electricity; they are found in all cells, blood, and other body fluids and are essential for normal functioning.

8.2.1  Bacterial Infections Many common diarrheal diseases are caused by bacterial infections transmitted by ingestion of contaminated food and water. Prevention of these diseases should focus on good personal hygiene by all food handlers, including the consumer of the food. Some bacterial diseases such as anthrax, bovine tuberculosis, and brucellosis are particularly related to foods of animal origin; these are described in detail below: a) Escherichia coli O157. Referred to as enterohemorrhagic E. coli (EHEC), this pathogen produces toxins known as verotoxins. Cattle appear to be the main reservoir. Transmission to humans is principally through the consumption of contaminated foods, such as raw or undercooked meat products and raw milk. Fresh-pressed apple juice or cider, yoghurt, cheese, salad vegetables, and cooked maize have also been implicated. Fecal contamination of water and foods, as well as cross-contamination during food preparation, can lead to infection, as can person-to-person contact. It is a major cause of bloody and non-bloody diarrhea and often leads to long-term complications such as hemolytic uremic syndrome (Ferens and Hovde, 2011). b) Enteroaggregative Escherichia coli. Enteroaggregative E. coli (EAEC) has increasingly been recognized as an agent of a watery mucoid diarrhea—especially in children—in

198  Chapter 8

c)

d)

e)

f)

g)

developing and, recently, industrialized countries (Nataro et al., 1998). It is particularly associated with persistent diarrhea (lasting for more than 14 days), a major cause of illness and death. It is thought that EAEC adheres to the intestinal mucosa and elaborate enterotoxins and cytotoxins, which result in secretory diarrhea and mucosal damage. Recent studies support the association of EAEC with malnutrition and growth retardation in the absence of diarrhea (Haghi et al., 2014). Listeria monocytogenes. This ubiquitous microorganism has been isolated from various environments, including decaying vegetation, soil, animal feed, sewage, and water. It is resistant to diverse environmental conditions and can grow at temperatures as low as 3°C. It is found in a wide variety of raw and processed foods—such as milk and cheeses, meat (including poultry) and meat products, and seafood and fish products—where it can survive and multiply rapidly during storage (Ajayeoba et al., 2016). L. monocytogenes is responsible for opportunistic infections, preferentially affecting individuals, whose immune system is perturbed, including pregnant women, newborn babies, and the elderly. It primarily causes meningitis, encephalitis, or septicemia and, when pregnant women are infected, it can lead to abortion, stillbirth, or premature birth (Van de Venter, 2000). Salmonella enteritidis. This bacterium is the dominant cause of human salmonellosis in many parts of the world. Poultry, eggs, and egg products, in particular, are contaminated, but the microorganism has also been found in other foodstuffs such as ice cream. Crosscontamination, undercooking, and inadequate cooling procedures promote the spread and growth of salmonella during processing and handling. One important characteristic of S. enteritidis is its ability to contaminate the contents of intact egg shells (Kang et al., 2006). Manifestation of illness includes invasive disease and reactive arthritis. Multidrug-resistant Salmonella typhimurium DT 104. This microorganism has been isolated from cattle, poultry, sheep, pigs, and horses (Farzan et al., 2008). Antimicrobial therapy is used extensively to combat S. typhimurium infection in animals, and the evolution of a strain resistant to the commonly used antibiotics has made infections with S. typhimurium in food animals difficult to control. The primary route by which humans acquire infection is through the consumption of a large range of contaminated foods of animal origin (Van de Venter, 2000). Campylobacter jejuni. Most sporadic infections with this pathogen are associated with improper preparation or consumption of mishandled poultry products. Most C. jejuni outbreaks, which are far less common than sporadic illnesses, are associated with the consumption of raw milk or unchlorinated water. Campylobacteriosis may lead to Guillain-Barré syndrome, a cause of flaccid paralysis (McCarthy and Giesecke, 2001). The reservoirs of this organism include poultry, cattle, swine, sheep, rodents, and birds (Whiley et al., 2013). Vibrio vulnificus. The consumption of raw molluscan shellfish that are contaminated with this microorganism, which is a normal inhabitant of some marine environments, often

Foodborne Diseases and Responsible Agents  199 leads to primary septicemia and death (Nicholas A, 2011). Individuals most susceptible to infection with this agent include those with chronic liver disease or chronic alcoholism, or those who are immunosuppressed in some way. h) Streptococcus parasanguinis. Pure isolates of this bacterium were recovered from two sheep in Spain during a recent bacteriological survey for determining the prevalence of subclinical mastitis. Since this bacterium has been associated with the development of experimental endocarditis, its presence at relatively high concentrations in apparently healthy sheep’s milk may pose a health risk in persons with predisposing heart lesions (Fernández-Garayzábal et al., 1998).

8.2.2  Viral Infections Several different viruses may be transmitted by contaminated food via the fecal–oral route. Foodborne viral infections usually have an incubation period of between 1 and 3 days. They cause illnesses which are self-limited in people who are otherwise healthy (i.e., they recover naturally) but occasionally severe illness and even deaths may also occur. In the group of viral infections causing viral gastroenteritis (VGE), rotavirus is a common cause of vomiting and watery diarrhea. Dehydration is the likely consequence unless appropriate rehydration therapy is used. Caliciviruses such as norovirus (also known as Norwalk virus) also cause diarrhea. a) Hepatitis A & E. Viral hepatitis caused by Hepatitis A and E viruses is almost exclusively transmitted by the fecal–oral route. Hepatitis A is distinguished from other viral causes by its prolonged (2–6 weeks) incubation period and its ability to spread beyond the stomach and intestines into the liver. It often induces jaundice, or yellowing of the skin, and can occasionally lead to chronic liver dysfunction. The hepatitis E virus (HEV) usually enters the body through water or food, especially raw shellfish that has been contaminated by sewage. Anti-HEV activity has been determined in the serum of a number of domestic animals in areas with a high endemicity of human infection, indicating that this may be an emerging zoonosis (Van de Venter, 2000). b) Norovirus (Norwalk-like viruses). Noroviruses are a group of viruses that cause the “stomach flu,” or gastroenteritis, in people. Most foodborne outbreaks of norovirus illness are likely to arise though direct contamination of food by a food handler immediately before its consumption (Bresee et al., 2002). Noroviruses are found in the stool or vomit of infected people. People can become infected with the virus in several ways, including eating food or drinking liquids that are contaminated with norovirus; touching surfaces or objects contaminated with norovirus, and then placing their hand in their mouth; having direct contact with another person who is infected and showing symptoms. Noroviruses can survive on practically any surface including door handles, sinks, railings, and glassware (IPAC/Occupational Health, 2011). They occur throughout the year but are more common in winter and affect all age groups.

200  Chapter 8 c) Rotavirus is the major cause of severe diarrhea (gastroenteritis) in children throughout the world, more so in developing countries, with about 95% of children contracting the infection by 5 years of age (Festini et al., 2010). The death rate due to rotavirus infection is estimated at about 600,000 child deaths per year worldwide, with yearly death tolls highest in India, Nigeria, China, Pakistan, Congo, Ethiopia, and Bangladesh (Bernstein, 2009). Rotavirus infection is generally more severe and clinically significant in children aged 3–35 months, with the first infection being the most severe. Rotaviruses are highly communicable and they are mainly transmitted through the fecal–oral route. Since the virus is stable in the environment, transmission can occur via person-to-person contact, or through food, water, and contact with contaminated surfaces. Nosocomial rotavirus infection is also very common. Rotavirus infection was thought to be limited to the gastrointestinal tract. However, rotavirus shedding in nasopharyngeal secretions has been reported in children with or without gastrointestinal symptoms (Azevedo et al., 2005). d) Influenza A virus (H5N1): The highly pathogenic influenza A virus subtype H5N1 is an emerging avian influenza virus that has been causing global concern as a potential pandemic threat. It is often referred to simply as “bird flu” or “avian influenza,” even though it is only one subtype of the avian influenza-causing virus. H5N1 has killed millions of poultry in a growing number of countries throughout Asia, Europe, and Africa. Health experts are concerned that the coexistence of human flu viruses and avian flu viruses (especially H5N1) will provide an opportunity for genetic material to be exchanged between species-specific viruses, possibly creating a new virulent influenza strain that is easily transmissible and lethal to humans. The mortality rate for humans with H5N1 is 60%. The avian flu virus (H5N1) has been shown to survive in the environment for long periods of time. Infection may be spread simply by touching contaminated surfaces. Birds that are infected with this flu can continue to release the virus in their feces and saliva for as long as 10 days. Influenza A virus is usually transmitted via air droplets, and subsequently contaminates the mucosa of the respiratory tract. Health care workers and household contacts of patients with avian influenza may also be at an increased risk of the bird flu. e) Nipah virus (NiV) or Encephalitis: Nipah virus causes severe illness characterized by inflammation of the brain (encephalitis) or respiratory diseases. Nipah virus can be transmitted to humans from animals, and can also be transmitted directly from human-to-human; in Bangladesh, half of the reported cases between 2001 and 2008 were due to human-to-human transmission (Luby et al., 2009). Nipah virus can cause severe disease in domestic animals such as pigs. There is no treatment or vaccine available for either people or animals. Fruits or fruit products (e.g., raw date palm juice) contaminated with urine or saliva from infected fruit bats was the most likely source of infection. Nipah virus is presumably derived from a fruit bat virus that spread to swine and adapted over a few years in closely confined swine herds. Once established within the swine population, the virus had ample opportunity to adapt to human hosts. Effective treatment has not

Foodborne Diseases and Responsible Agents  201 been developed for Nipah infection; however, Ribavirin, an antiviral drug, may reduce mortality among patients with encephalitis caused by Nipah virus, although further study is needed (Broder et al., 2013). f) Polio virus can cause poliomyelitis disease, which mainly affects young children. The virus is transmitted by person-to-person spread mainly through the fecal–oral route or, less frequently, by a common vehicle (e.g., contaminated water or food) and multiplies in the intestine, from where it can invade the nervous system and can cause paralysis. Initial symptoms of polio include fever, fatigue, headache, vomiting, stiffness in the neck, and pain in the limbs. In a small proportion of cases, the disease causes paralysis, which is often permanent. There is no cure for polio; it can only be prevented by immunization.

8.2.3 Parasite A parasite is an organism that lives on or in a host organism and gets its food from or at the expense of its host. There are three main classes of parasites that can cause disease in humans: protozoa, helminths, and ectoparasites (Centers for Disease Control and Prevention, 2014). 8.2.3.1 Protozoa The name “proto-zoa” literally means “first animals” and early classification systems grouped the protozoa as basal members of the animal kingdom. More recently, the protozoa have been classified together with several algal and fungal groups in the kingdom Protista (protozoa representing the motile protists). Irrespective of contemporary classification systems, most parasitological texts continue to use the name protozoa for historical reasons. Protozoa are microscopic, one-celled organisms that can be free-living or parasitic in nature. They are able to multiply in humans, which contributes to their survival and also permits serious infections to develop from just a single organism. Transmission of protozoa that live in a human’s intestine to another human typically occurs through a fecal–oral route (e.g., contaminated food or water or person-to-person contact). Protozoa that live in the blood or tissue of humans are transmitted to other humans by an arthropod vector (e.g., through the bite of a mosquito or sand fly) (Centers for Disease Control and Prevention, 2014). The following protozoa are infectious to humans. a) Giardia is a microscopic parasite that causes the diarrheal illness known as giardiasis. Giardia (also known as Giardia intestinalis, Giardia lamblia, or Giardia duodenalis) is found on surfaces or in soil, food, or water that has been contaminated with feces (poop) from infected humans or animals. Giardia is protected by an outer shell that allows it to survive outside the body for long periods of time and makes it tolerant to chlorine disinfection. While the parasite can be spread in different ways, water (drinking water and recreational water) is the most common mode of transmission (Centers for Disease Control and Prevention, 2015).

202  Chapter 8 b) Balantidium coli is an intestinal protozoan parasite that can infect humans. These parasites can be transmitted through the fecal-oral route by contaminated food and water. B. coli infection is mostly asymptomatic, but people with other serious illnesses can experience persistent diarrhea, abdominal pain, and sometimes a perforated colon (Verweij and Stensvold, 2014). When traveling to endemic tropical countries, B. coli infection can be prevented by following good hygiene practices. Wash all fruits and vegetables with clean water when preparing or eating them, even if they have a removable skin. c) Cyclospora cayetanensis. This coccidian parasite occurs in tropical waters worldwide and causes watery, and sometimes explosive, diarrhea in humans. It was initially associated with waterborne transmission but has also been linked to the consumption of raspberries, lettuce, and fresh basil. The incubation period is 1 week after the ingestion of the contaminated food and the agent is shed in the feces for more than 3 weeks (Van de Venter, 2000). d) Toxoplasma gondii. The primary hosts of this protozoan are cats, and human infection takes place when contact is made with their feces. It can also occur through the ingestion of raw or undercooked meat from intermediate hosts, such as rodents, swine, cattle, goats, chicken, and birds. Toxoplasmosis in humans often produces mononucleosis-type symptoms, while buttran-splacental infection can result in fetal death if it occurs early in pregnancy. In immune-compromised individuals infection can cause pneumonitis, myocarditis, meningoencephalitis, hepatitis, chorioretinitis, or combinations of these. Cerebral toxoplasmosis is often seen in AIDS patients (Van de Venter, 2000). e) Cryptosporidium parvum. The mode of transmission of this coccidian protozoan is fecal to oral, including waterborne and foodborne means. The reservoirs include humans and domestic animals, including cattle. Oocysts can survive in the environment for long periods; they remain infective and are capable of resisting chemicals used to purify drinking water. They can, however, be removed from water supplies by filtration. Symptoms of cryptosporosis in humans include fever, diarrhea, abdominal pain, and anorexia. The disease usually subsides within 30 days, but may be prolonged and continue to death in immune-deficient individuals (Van de Venter, 2000; Centers for Disease Control and Prevention, 2014). 8.2.3.2 Helminths Helminths are large, multicellular organisms that live at the expense of the host and are generally visible to the naked eye in their adult stages. Like protozoa, helminths can be either free-living or parasitic in nature. In their adult form, helminths cannot multiply in humans. There are three main groups of helminths (derived from the Greek word for worms) that are human parasites: They may occur in foods in the form of eggs, larvae, or other immature forms (Gebrie and Engdaw, 2015). Tapeworms are one of the most common causes of

Foodborne Diseases and Responsible Agents  203 foodborne parasitic diseases in developing countries. The following helminthes are infectious to humans: a) Beef tapeworm: Taenia Saginata (the beef tapeworm) is the most common cause of tapeworm disease in Ethiopia (Gebrie and Engdaw, 2015). Immature forms of the tapeworm develop in the muscles of animals that have eaten tapeworm eggs while grazing on infected grass. People are infected when they eat raw or undercooked beef (Fig. 8.2). The adult tapeworms develop in the person’s small intestine and segments of the worms containing eggs are deposited in the environment when the person defecates. This is how the cycle is continued (Qekwana et al., 2016).

Fig. 8.2 Eating raw beef can be dangerous because it might be infected with beef tapeworm (The Open University, 2016).

b) Dog tapeworm: Hydatid disease, caused by dog tapeworm, is transmitted when a person ingests the eggs of Echinococcusgranulosus in food contaminated with dog feces. This disease may cause symptoms in women that resemble “false pregnancy,” because its effect is to enlarge the liver and cause the abdomen to swell so the woman may appear to be pregnant (Hassan et al., 2013). The infection may also lodge in the lung or the brain. The prevention of disease caused by dog tapeworm is through personal hygiene when handling food and thorough washing of raw foods, especially if they have come into contact with soil. c) Fish tapeworm: Fish tapeworm (Diphyllobothriumlatum) infects people through the consumption of raw fish and is more common in the lake areas of Ethiopia where the diet is highly dependent on fish. The symptoms of infection with the fish tapeworm are similar to those of other tapeworm infections, i.e., abdominal discomfort or pain, nausea, vomiting, or diarrhea and loss of appetite and weight loss (Craig, 2012). People should be advised only to eat fish that has been properly cooked.

204  Chapter 8 8.2.3.3 Ectoparasites Parasites that live on the outside of the host, either on the skin or the outgrowths of the skin, are called ectoparasites. Although the term ectoparasites can broadly include bloodsucking arthropods such as mosquitoes (because they are dependent on a blood meal from a human host for their survival), this term is generally used more narrowly to refer to organisms such as ticks, fleas, lice, and mites that attach or burrow into the skin and remain there for relatively long periods of time (e.g., weeks to months). Arthropods are important in causing diseases in their own right, but are even more important as vectors, or transmitters, of many different pathogens that in turn cause tremendous morbidity and mortality from the diseases they cause. a) Trichinosis has been an important reportable pathogen associated with undercooked pork (Mattiucci et al., 2013). Other parasites of concern include flatworms or nematodes (associated with fish), cestodes or tapeworms (usually associated with beef, pork, or fish), and trematodes or flukes (more of a concern in developing countries). b) Anisakiasis: Anisakiasis is an infection of the human intestinal tract caused by the ingestion of raw or undercooked fish containing larval stages of the nematodes Anisakis simplex or Pseudoterranovadecipiens. Infections caused by the latter roundworm are not a serious threat to human health, but those caused by A. simplex are more problematic because this agent penetrates the gastrointestinal tissue and causes disease that is difficult to diagnose. The primary hosts are warm-blooded marine mammals such as seals, walruses, and porpoises (Mattiucci et al., 2013). Their larvae pass via krill to fish such as cod, pollack, halibut, rockfish, salmon, and herring.

8.2.4  Unconventional Agents Prions: Transmissible spongiform encephalopathies in animals and humans are caused by an unconventional virus or prion. These conditions include scrapie in sheep, bovine spongiform encephalopathy (BSE or mad cow disease) in cattle, and Creutzfeldt-Jakob disease (CJD) in humans. It is commonly accepted that BSE was first caused in the United Kingdom when cattle were fed carcass meal from scrapie-infected sheep. It is also accepted that humans contracted the nonclassic form of CJD after consuming cattle meat, in particular nerve tissue (Table 8.1).

8.3  Food Poisoning Food poisoning can be from chemical or biological sources. If we eat food that contains harmful chemicals, or biological toxins (poisons) from plants, animals, or microorganisms, that food can make us sick. Some common sources of food poisoning are caused by contaminants already in the food when the raw materials are harvested, for example: •

Bacterial toxins produced by bacteria such as Clostridium botulinum and Clostridium perfringens, which are commonly found in the natural environment, for example, in soil.

Foodborne Diseases and Responsible Agents  205 Table 8.1: Foodborne infections, causative agents and commonly affected foodstuffs (The Open University, 2016) Disease Category

Disease

Causative Agent(s)

Bacterial

Typhoid fever Paratyphoid fever Shigellosis

Salmonella typhi Salmonella paratyphi Shigella species

Viral

Parasitic (unicellular)

Parasitic (multicellular)

Unconventional agents

Foods Commonly Involved

Raw vegetables and fruits, salads, pastries, unpasteurised milk and milk products, meat All foods handled by unhygienic workers, potato or egg salad, lettuce, raw vegetables Cholera Vibrio cholera Fruits and vegetables washed with contaminated water Non-typhoid Salmonella species, Eggs, poultry, undercooked meals, salmonellosis e.g., Salmonella unpasteurised dairy products, sea foods, typhimurium sausages Brucellosis Brucellaspecies, mostly Milk and dairy products from infected Brucellamelitensis animals Anthrax Bacillus anthracis Contaminated raw and undercooked meat from sick and dying oxen, cows, sheep, goats, camels, etc. Bovine Mycobacterium bovis Unpasteurised milk, dairy products or meat tuberculosis from tuberculosis-infected cows E.coli infection Escherichia coli Beef, dairy products, fresh products, raw produce (potatoes, lettuce, sprouts, fallen apples), salads Listeriosis Listeria monocytogenes Milk, cheese, ice cream, poultry, red meat Viral Rotavirus, caliciviruses Any food contaminated with the virus gastroenteritis including norovirus, (VGE) astrovirus Viral hepatitis Hepatitis A and E Raw shellfish from polluted water, viruses sandwiches, salad and desserts Poliomyelitis Polio virus Any food contaminated with the virus Rift valley fever Rift valley fever virus Any food contaminated with blood or aerosols from infected domestic animals or their aborted fetuses Amoebiasis Entamoebahistolytica Any food soiled with feces Trichinosis Trichinellaspiralis Insufficiently cooked pork and pork products Ascariasis Ascarislumbricoides Foods contaminated with soil, especially foods that are eaten raw, such as salads, vegetables Giardiasis Giardia lamblia Any contaminated food item Toxoplasmosis Toxoplasma gondii Raw or undercooked meat and any food contaminated with cat feces Cryptosporidiosis Cryptosporidium parvum Any contaminated food item Hydatid disease Echinococcusgranulosus Any food contaminated with dog feces Diphyllobothriasis Diphyllobothriumlatum Raw or uncooked fish Taeniasis Taenia species Raw beef, raw pork (tapeworm infection) Anisakiasis Pseudoterranovadecipiens Raw or undercooked fish containing larval roundworm stages BSE or mad cow Bovine spongiform Cattle meat, sheep, cow, etc. disease encephalopathy

206  Chapter 8 • • • •

Chemical toxins, for example, insecticides sprayed onto growing crops. Heavy metals, for example, lead and mercury, particularly in fish caught near chemical processing facilities. Certain toxic plant tissues, for example, poisonous mushrooms. Toxic animal tissues, for example, the poison glands of certain fish, crabs, etc.

Chemical food poisoning can also occur if foodstuffs have been in contact with toxic chemicals during food production, processing, storage, and handling. The symptoms of food poisoning can range from mild headache to severe flu-like symptoms. The most common signs and symptoms are nausea, stomach cramps, diarrhea, fever, chills, and vomiting. A person with food poisoning may have any combination of these symptoms depending on the cause or the agent involved. The illness may begin from 1 to 72 h after eating the food (The Open University, 2016).

8.3.1  Bacterial Food Poisoning Some pathogenic bacteria, including Bacillus cereus (Bottone, 2010), C. botulinum (Nigam and Nigam, 2010), and C. perfringens (Eriksen et al., 2010), form spores that can survive adverse environmental conditions. The spores germinate to form viable cells that increase to large numbers. Spore-forming pathogens are significant because when the spores occur in foods, they are more difficult to kill. For example, although B. cereus bacteria survive up to 122°F, much higher temperatures are required to kill the spores of B. cereus. The bacteria that produce foodborne intoxications include B. cereus, C. botulinum, and Staphylococcus aureus. a) Staphylococcal food poisoning: Staphylococcal food poisoning is caused by one of the many species of staphylococcal bacteria and is the most common and major type of food poisoning you are likely to encounter. This type of food poisoning can result from the preparation of food more than half a day in advance of needs, storage at ambient temperature, inadequate cooling, or inadequate reheating. It begins with symptoms such as nausea, vomiting, stomach cramping, and diarrhea. These can persist for days and lead to dehydration, loss of electrolytes, and even death if not treated promptly. Control measures are promoting and monitoring the personal hygiene of food handlers, safe and hygienic conditions in food preparation areas, and keeping cooked or processed foods covered and in cool conditions until consumed (Argudín et al., 2010). b) Botulism: Foodborne botulism is a form of food poisoning caused by C. botulinum. It occurs in poorly canned foods, including home-canned foods, and honey. It is advisable not to eat food from deformed or bulging cans and not to give honey to young children (Ağaçayak, 2011).

Foodborne Diseases and Responsible Agents  207

8.3.2  Fungal Food Poisonings Mycotoxins are the toxic products of certain microscopic fungi which, in some circumstances, develop on or in foodstuffs of plant or animal origin. Filamentous fungi and molds are able to produce an enormous number of secondary metabolites, including antibiotics and mycotoxins. The term mycotoxin refers to those secondary metabolites which, at a low concentration, are toxic to humans and animals (Sánchez-Hervás et al., 2008). The existence of mycotoxin-producing fungi in plants is not always favorable for contamination with mycotoxins. In order for fungi to produce these secondary metabolites, they have to be stressed by some factor, such as nutritional imbalance, drought, or water excess (Dutton, 2009). They are ubiquitous and widespread at all levels of the food chain. Hundreds of mycotoxins have been identified and are produced by some 200 varieties of fungi. In terms of their implications for human health and the economy, mycotoxins are by far the most important contaminants of the food chain. Of particular importance in current toxicological studies are the combined and possible synergistic effects that some of the mycotoxins may have on human and animal life. Mycotoxins have been implicated as causative agents of human foodborne intoxication, as well as human hepatic and extrahepatic carcinogenesis (Wild and Gong, 2010); clinical symptoms include diarrhea, liver, and kidney damage, pulmonary edema, vomiting, hemorrhaging, and tumors (Bryden, 2012). The most frequent toxigenic fungi are Aspergillus, Penicillium, and Fusarium species (Sánchez-Hervás et al., 2008). The foodborne mycotoxins of greatest significance in Africa and other tropical countries are the fumonisins (FB), aflatoxins (AFs), and trichothecenes (Wagacha and Muthomi, 2008). These toxins contaminate various foodstuffs, including maize, cereals, groundnuts, and tree nuts feed during production, harvest, storage, or processing (Sánchez-Hervás et al., 2008); mycotoxins can also occur in milk, meat, and their products as a result of animals consuming mycotoxin contaminated feeds (Wild and Gong, 2010). The toxins frequently occur in maize, a staple food in most parts of Africa, Asia, and Latin America; hence, their contamination translates to high-level chronic exposure in these countries (Wild and Gong, 2010). The most common foodborne disease symptoms and mode of contamination by fungi other than mushroom are shown in Table 8.2. a) Mushroom toxins: Mushroom poisoning is caused by consumption of raw or cooked fruiting bodies (mushrooms, toadstools) of a number of species of higher fungi. The term “toadstool” is commonly used for poisonous mushrooms. For individuals who are not trained experts in mushroom identification, there are generally no easily recognizable differences between poisonous and nonpoisonous species. Folklore notwithstanding, there is no reliable rule of thumb for distinguishing edible mushrooms from poisonous ones. The toxins involved in mushroom poisoning are produced naturally, by the fungi themselves. Most mushrooms that cause human poisoning cannot be made nontoxic by cooking, canning, freezing, or any other means of processing. Thus, the only way

208  Chapter 8 Table 8.2: Food poisoning, toxin type, causative agents, and commonly affected foodstuffs Disease Category Bacterial toxins

Fungal toxins

Diseases Staphylococcal food poisoning Perfringens food poisoning Botulism food poisoning Escherichia coli food poisoning Bacillus cereus food poisoning Ergotism

Aflatoxin food poisoning

Natural toxins in foods

Neurolathyrism Mushroom poisoning

Chemical toxins

Shellfish toxins, and tetrodotoxin (pufferfish) Scombrotoxin Ciguatera toxin Chemical poisoning

Toxin Type and Causative Agent Entero-toxins from Staphylococcus aureus Strain of Clostridium welchii/C. perfringens Toxin of Clostridium botulinum Enterohaemorrhagic Escherichia coli O157:H7 Enterotoxins of Bacillus cereus A toxin (ergot) produced by a group of fungi called Clavicepspurpurea Aflatoxin produced by some groups of fungus (e.g., Aspergillusflavus, Aspergillusparasiticus) Beta-oxalyl amino-alanine Phalloidine and alkaloids found in some poisonous mushrooms Ingested toxic dinoflagellates

Production of histamine Heavy metals (e.g., lead, mercury, cadmium) Pesticides and insecticides

Additives

Other toxins

Environmental contaminants Biotechnology

Dioxins Chlorinated biphenyls Furans Food allergens Genetically modified foods

Foods Commonly Involved Milk and milk products, sliced meat, poultry, legumes Inadequately heated or reheated meat, poultry, and legumes Home-canned foods, low-acid vegetables, corn, and peas Ground beef, dairy products and raw beef Cereals, milk and dairy products vegetables, meats, cooked rice Rye, wheat, sorghum, barley

Cereal grains, groundnuts, peanuts, cottonseed, sorghum

Lathyrussativus (guaya) Poisonous mushrooms such as species of Amanita phalloidesand Amanita muscaria Caused by the consumption of mussels, clams and scallops, etc. Due to bacteria spoilage of fish Tropical fish Fish, canned food Foods contaminated by utensils or coated with heavy metals Residues on crops, vegetables, fruits Accidental poisoning where some chemicals may be mistaken for food ingredients When contaminated containers are used to hold stored foods Various food items where unauthorized additives may be added as coloring agents, sweeteners, preservatives, flavoring agents, etc. Environment contaminants enter the food chain via plants or animals Gluten containing cereals include wheat, rye, barley, oats, and hybrid strains of these cereals Genetically modified foodstuffs

Foodborne Diseases and Responsible Agents  209 to avoid poisoning is to avoid consumption of toxic species. Mushroom poisonings are generally acute, although onset of symptoms may be greatly delayed in some cases, and are manifested by a variety of symptoms and prognoses, depending on the amount and species consumed. Each poisonous species contains one or more toxic compounds that are unique to few other species. Therefore, cases of mushroom poisonings generally do not resemble each other, unless they are caused by the same or very closely related mushroom species. The chemistry of many mushroom toxins (especially the less deadly ones) is still unknown, and identification of mushrooms is often difficult or impossible; however, mushroom poisonings are generally categorized by their physiological effects. Cultivated commercial mushrooms of various species have not been implicated in poisoning outbreaks, although they may result in other problems, such as bacterial food poisoning associated with improper canning. Mushroom poisonings are almost always caused by ingestion of wild mushrooms that have been collected by nonspecialists. Illnesses have occurred after ingestion of fresh, raw mushrooms; stir-fried mushrooms; home-canned mushrooms; mushrooms cooked in tomato sauce (which can render the sauce itself toxic, even when no mushrooms are consumed); and mushrooms that were blanched and frozen at home. Accurate figures on the relative frequency of mushroom poisonings are difficult to obtain, and the fact that some cases are not reported must be taken into account. During the 10-year period from 2001 to 2011, 83,140 mushroom ingestions were reported to US Poison Control Centers; of these, 64,534 (77.6%) were pediatric ingestions and 48,437 (58.3%) occurred in children younger than 6 years. The toxin group was identified in 12,147 (14.6%) ingestions (Hatten et al., 2012). Between 1959 and 2002, there were more than 28,000 reported mushroom poisonings around the world, resulting in 133 deaths (Diaz, 2005). In April 2008, an outbreak of mushroom poisonings in the Upper Assam part of India claimed more than 30 lives (Dutta et al., 2013). Known cases are sporadic and large outbreaks are rare. Poisonings tend to be grouped in the spring and fall, when most mushroom species are at the height of their fruiting stages. Unfortunately, a number of factors (not discussed here) often make identification of the causative mushroom impossible. In such cases, diagnosis must be based on symptoms alone. To rule out other types of food poisoning and to conclude that the mushrooms eaten were the cause of the poisoning, it must be established that everyone who ate the suspect mushrooms became ill and that no one who did not eat the mushrooms became ill. Wild mushrooms, whether they were eaten raw, cooked, or processed, should always be regarded as prime suspects. The mushroom toxins can, with difficulty, be recovered from poisonous fungi, cooking water, stomach contents, serum, and urine. Procedures for extraction and quantitation are generally elaborate and time-consuming, and, in most cases, the patient will have recovered by the time an analysis is made on the basis of toxin chemistry. The exact chemical natures of most of the toxins that produce milder symptoms are unknown.

210  Chapter 8 b) Aflatoxins: Aflatoxins (AFs) are mycotoxins produced by certain fungi and can cause serious illness in animals and humans. The four major aflatoxins are AFB1, AFB2, AFG1, and AFG2. In adverse weather or under poor storage conditions, these toxins are produced mainly by certain strains of Aspergillus flavus and A. parasiticus in a broad range of agricultural commodities, such as corn and nuts. The name “aflatoxin” reflects the fact that this compound was first recognized in damaged peanuts contaminated with Aspergillus flavus (Menza et al., 2015). The aflatoxins then were described according to other mechanisms (i.e., on the basis of their blue or green fluorescence under UV light and relative chromatographic mobility after thin-layer chromate–graphic separation). Another aflatoxin, aflatoxin M1 (AFM1), is produced by mammals after consumption of feed (or food) contaminated by AFB1. Cows are able to convert AFB1 into AFM1 and transmit it through their milk. Although AFM1 in milk is, by far, not as hazardous as the parent compound, a limit of 0.5 parts per billion is applied, largely because milk tends to constitute a large part of the diet of infants and children. In the United States, strict regulations in place since 1971, as well as FDA monitoring of the food supply and the population’s consumption of a diverse diet, have prevented human health problems. However, aflatoxin-induced chronic and acute disease is common in children and adults in some developing countries (Menza et al., 2015). c) Fumonisins. Fumonisins are a group of fusarium mycotoxins occurring worldwide in maize and maize-based products. Their causal role in several animal diseases has been established. Available epidemiological evidence suggests that there is a link between dietary fumonisin exposure and human esophageal cancer in some locations with high disease rates (Scott, 2012). Fumonisins are mostly stable during food processing. d) Zearalenone. This fungal metabolite is mainly produced by Fusarium graminearum and F. culmorum, which are known to colonize maize, barley, wheat, oats, and sorghum. These compounds can cause hyperoestrogenism and severe reproductive and infertility problems in animals, especially in swine, but their impact on human health is difficult to evaluate (Atanasova-Penichon et al., 2016). e) Trichothecenes. These mycotoxins are produced by many species of the genus Fusarium. They occur worldwide and infect many different plants, notable of which are the cereal grains, especially wheat, barley, and maize. There are over 40 different trichothecenes, but the most well known are deoxynivalenol and nivalenol. In animals, they cause vomiting and feed refusal, and also affect the immune system. In humans, they cause vomiting, headache, fever, and nausea (Yazar and Omurtag, 2008). f) Ochratoxins. These compounds are produced by Penicillium verrucosum and by several species of Aspergillus. The major dietary sources are cereals, but significant levels of contamination may be found in grape juice and red wine, coffee, cocoa, nuts, spices, and dried fruits. Contamination may also carry over into pork and pig blood products and into beer. Ochratoxin is potentially nephrotoxic and carcinogenic, the potency varying markedly among species and sexes. It is also teratogenic and immunotoxic (Cabanes et al., 2010).

Foodborne Diseases and Responsible Agents  211

8.4  Chemical Contaminants in Food Chemical contaminants can be present in foods mainly as a result of the use of agrochemicals, such as residues of pesticides and veterinary drugs, contamination from environmental sources (water, air, or soil pollution), cross-contamination or formation during food processing, migration from food packaging materials, presence or use of unapproved food additives and adulterants.

8.4.1  Pesticide Residues The use of pesticides, such as insecticides, fungicides, or herbicides, has become an integral part of modern agriculture to increase crop yields and quality by controlling various pests, diseases, and weeds (Gill and Garg, 2014; Maksymiv, 2015). Registration of new pesticides is a strictly regulated process that evaluates their toxicity and environmental fate, and sets maximum residue limits (tolerances) in raw and processed commodities (Damalas and Eleftherohorinos, 2011). There are over 1400 known pesticides. Some of them should no longer be used but may still be present in the environment. Older pesticides are being reevaluated based on currently available scientific data. Approved uses of pesticides following Good Agricultural Practices should result in pesticide residues below maximum residue limits established in a given country. However, global sourcing of raw commodities and global distribution of food products complicate the situation because pesticide registrations, uses, and limits can be and are different in different countries. Consequently, an approved use in one country may result in an illegal pesticide residue in a food imported into another country, such as the recent case of the fungicide carbendazim in orange juice imported into the United States from Brazil. Furthermore, pesticides can be misused or present in food due to contamination during application (spray drift), storage, or transportation or from environmental sources, such as contaminated water or soil. i) Coumatetralyl and warfarin in pork—maximum residue limits: introduce temporary maximum residue limits (MRLs) for residues of two rodenticides—coumatetralyl and warfarin—in the Food Standards Code. Enforcement agencies are currently investigating how these chemicals are being used on farm to determine why residues have been found in livers.

8.4.2  Veterinary Drug Residues Similar to pesticides, veterinary drugs are agrochemicals that undergo a thorough registration process, resulting in setting of their maximum residue limits/tolerances in animal-derived foods. The major classes of veterinary drugs include antibiotics, anthelmintics, coccidiostats, nonsteroidal anti-inflammatory drugs, sedatives, corticosteroids, beta-agonists, and anabolic

212  Chapter 8 hormones. These drugs, which are administered to live animals, can remain as residues in animal tissues (Mastovska, 2013). Liver and kidney are highly susceptible to residues given their biological function. Certain antibiotics, such as penicillin, can cause severe allergic reactions in sensitive individuals, which is an important reason for enforcing their residue limits in foods of animal origin. Another important justification for limiting antibiotic usage in food-producing animals is to reduce the risk of pathogenic microorganisms becoming resistant to antibiotics. Most veterinary drugs are not of acute toxicological concern, but some substances, such as nitrofurans, chloramphenicol, clenbuterol, and diethylstilbestrol, have been banned in most countries due to their carcinogenicity (Mohsina et al., 2015). Concern about endocrine-disrupting effects has become another reason for regulation of certain veterinary drugs, such as beta-agonists and hormones.

8.4.3  Environmental Contaminants Chemicals such as dioxins, chlorinated biphenyls, furans, and heavy metals may contaminate the environment as a result of industrial activities (Kataria et al., 2015). From the environment, these chemicals may enter the food chain via plants or animals and cause a variety of health problems. These are considered as emerging problems in countries that are in the early stages of industrialization. Environmental contaminants can be man-made or naturally occurring substances present in air, water, or soil. They can enter the food chain and even bioaccumulate. Some can pose an acute health risk if present at higher concentrations, but the major concern related to the presence of environmental contaminants in foods is their potential endocrine disruption, developmental, carcinogenic, and other chronic effects. Examples of environmental contaminants that enter the food chain include heavy metals, polychlorinated biphenyls (PCBs), “dioxins” (polychlorinated-dibenzo-dioxins and dibenzofurans), persistent chlorinated pesticides (e.g., DDT, aldrin, dieldrin, heptachlor, mirex, chlordane), brominated flame retardants (mainly poly-brominated-diphenyl-ethers), polyfluorinated compounds, polycyclic aromatic hydrocarbons (PAHs), perchlorate, pharmaceutical, and personal care products or haloacetic acids and other water disinfection by-products (Mastovska, 2013). The manufacture and use of PCBs and other persistent organic pollutants (POPs) have been banned for years, but they remain in the environment due to their high stability. PAHs can be found in the environment as a result of industrial pollution or can originate from oil spills; thus, they were of concern in seafood after the oil spill accident in the Gulf of Mexico in 2010. i) Dioxins: Dioxins, a collective term for a group of environmental contaminants that includes certain dioxin, furan, and dioxin-like PCB congeners, are found throughout the world. Dioxins and furans are released into the air from combustion processes (Lopes et al., 2015). The wide use of PCBs as dielectric and coolant fluids in the past has resulted in their presence in the environment. They persist in the environment for a long

Foodborne Diseases and Responsible Agents  213 time and can get into food but the FDA-2005 report showed a minimum health risk of dioxins to human. Dioxins also break down in our bodies and we excrete them. More than 96% of dioxins in the environment come from air emissions. Dioxins then fall to the ground and occur in trace amounts on soil, plant, and water surfaces (Science Communication Unit, University of the West of England, Bristol, 2013). In Australia, the major sources of dioxin emissions in the air are bushfires and burning agricultural stubble. Plants do not generally absorb dioxins (Tran et al., 2010). However, dioxins can enter the food chain when animals eat plants on which dioxins have fallen. In oceans, rivers, and lakes, filter-feeding animals can absorb dioxins when they filter sediments floating in the water.

8.4.4  Heavy Metals as Contaminants Metals such as arsenic, cadmium, lead, and mercury are natural occurring chemical compounds (Tchounwou et al., 2012). They can be present at various levels in the environment, for example, soil, water, and atmosphere. Metals can also occur as residues in food because of their presence in the environment, as a result of human activities such as farming, industry, or car exhausts or from contamination during food processing and storage (EFSA (European Food Safety Authority), 2013). People can be exposed to these metals from the environment or by ingesting contaminated food or water. Their accumulation in the body can lead to harmful effects over time. i) Mercury: Almost all of the mercury in food occurs in seafood. A dramatic instance of mercury poisoning occurred in the Minimata Bay area in Japan. Fish and shellfish that were heavily contaminated by industrial waste caused poisoning in many of the people who ate them, resulting in damage to the central nervous system and, in some instances, death (Gupta and Gajbhiye, 2008). Surveys of the levels of mercury and other heavy metals in food are regularly carried out and have shown that generally the levels are below the maximum amounts permitted by health authorities. Occasionally, higher levels are detected and the food is withdrawn from sale. ii) Lead: Lead occurs widely in the environment and it can enter our bodies through drinking water and the air we breathe, as well as through food. Children are the group at greatest risk, because even at levels below those that produce the usual signs of poisoning, lead can cause behavioral abnormalities (van der Kuijp et al., 2013). The levels of lead that cause these effects are uncertain, so it is difficult to estimate what amount is “safe.” In some areas, particularly where there is heavy lead pollution in the air from leaded petrol, lead levels may be hazardous for children. Legislation to limit the total environmental lead burden is being enacted in many countries. iii) Cadmium: Cadmium is present at very low levels in a wide variety of foods. Poisoning due to cadmium in food is rare. The upper “acceptable” limit for cadmium in food recommended by the World Health Organization is generally complied with. The

214  Chapter 8 kidneys of animals are generally higher in cadmium than are other foods. Contamination of rice, soya bean, and seafood with cadmium from local industrial and mining operations has caused cadmium poisoning (Gupta and Gajbhiye, 2008). iv) Arsenic: Arsenic is a chemical element found in water, air, food, and soil as a naturally occurring substance or due to contamination from human activity. It occurs in organic and inorganic forms. The organic forms are of relatively low toxicity while the inorganic forms present a greater hazard (Tchounwou et al., 2012). Since both appear naturally in soil and ground water, small amounts are unavoidably found in some food and drinks. Arsenic compounds were more widely used in the past, for example in pesticides and veterinary drugs, but there are currently no registered uses for food crops or for animal production in Australia and New Zealand. Inorganic arsenic is registered for use in timber preservatives and for controlling termites in timber, and arsenic derivatives are used in herbicides for turfs and lawns, and cotton. A limit of 1 mg/kg applies to seaweed and molluscs, while for fish and crustacea inorganic arsenic is not allowed above a level of 2 mg/kg. There are also arsenic limits in the Code for cereals such as rice. These limits, which are set at levels consistent with protecting public health and safety and which are reasonably achievable, cover the major foods that are likely to contribute to arsenic exposure (Table 8.3).

Table 8.3: Sources of environmental contamination in food

Origin

Food in Which a Contaminant Is Likely to Be Found Industrial:

Mercury Lead Cadmium Polychlorinated biphenyls

Fish All foods, water Fish, shellfish, kidney Fish, poultry, milk, eggs

Possibility of Health Hazard From Amounts in Food, Assuming a Normal Varied Diet Low Low Very low Very low

Agricultural: Pesticides Antibiotics Hormones

All foods Milk Some poultry

Low Very low Very low

Food processing: Cleaning agents Lubricants Packing materials Solvent residues Extraneous substances (rodent excreta, hair, insects, etc.)

Any processed food

Very low

Foodborne Diseases and Responsible Agents  215

8.4.5  Food Processing-Induced Contaminants Certain toxic or undesirable compounds can be formed in foods during their processing, such as during heating, baking, roasting, grilling, canning, hydrolysis, or fermentation (Gupta and Gajbhiye, 2008). Precursors of these contaminants can occur naturally in the food matrix, such as in the case of acrylamide being formed during the Maillard reaction between the amino acid asparagine and a reducing sugar (especially in potato- and cereal-based, heat-treated products). Alternatively, certain processing contaminants, such as nitrosamines, can be formed by interaction of natural food components with food additives (Nerín et al., 2016). Carcinogenic and genotoxic chlorpropanols, such as 3-monochloropropane-1,2diol (3-MCPD), are formed during the acid hydrolysis of wheat, soya, and other vegetable protein products. Examples of other processing contaminants include PAHs (in grilled and smoked products), ethyl carbamate (in yeast-fermented alcoholic beverages and other products), or furan (in a variety of heat-treated foods, especially coffee and canned/jarred food). Food processing may also be a source of cross-contamination, such as contamination of nonallergenic foods with known food allergens (Allen et al., 2014). i) Acrylamide: Acrylamide is a chemical that can form in some foods during high-temperature cooking processes, such as frying, roasting, and baking. It has been detected in a range of foods including fried or roasted potato products, coffee, and cereal-based products (including sweet biscuits and toasted bread) (Krishnakumar and Visvanathan, 2014). International food regulators are working with industry to reduce acrylamide levels. New farming and processing techniques are being investigated to produce lower levels of acrylamide, for example, lowering cooking temperatures, using enzymes that reduce acrylamide formation, and obtaining raw materials with lower reducing sugar levels. However, reducing acrylamide in some foods, such as coffee, is difficult without changing its taste. Generally, more acrylamide accumulates when cooking is done for longer periods or at higher temperatures. Cooking cut potato products, such as frozen French fries or potato slices, to a golden yellow color rather than a brown color helps reduce acrylamide formation. Brown areas tend to contain more acrylamide. ii) 3-MCPD (3-monochloropropane-1,2-diol): 3-MCPD may be formed as a result of a reaction between a source of chlorine (e.g., chlorinated water or salt) in the food or a food contact material and a lipid source (Zhang et al., 2013). This reaction is encouraged during the heat processing of foods, including the roasting of cereals and malts used for brewing. It is also known to occur in acid-hydrolyzed vegetable protein (HVP) when produced using hydrochloric acid. The contaminant has now been found to occur at low levels in a number of foods and ingredients and the actual mechanisms for its formation in some of these are not fully understood. Once formed, the stability of 3-MCPD has been shown to be dependent upon the pH and temperature to which it has been exposed

216  Chapter 8 (Lee and Khor, 2014). The higher the pH and the higher the temperature of the heat treatment, the greater will be the rate of degradation of 3-MCPD. iii) Perchlorate: Perchlorate is a naturally occurring and man-made chemical that can affect the functioning of the thyroid gland at sufficiently high doses. It is present in some public drinking water systems and in some foods (Huber et al., 2011). Perchlorate occurs naturally in arid regions, in nitrate fertilizer deposits in soils, and in potash ore content regions. It also forms naturally in the atmosphere. Perchlorate is manufactured and used as an industrial chemical and can be found in rocket propellants, explosives, fireworks, and road flares. It has been found in some public drinking water systems and in food. Perchlorate can interfere with the human body’s ability to absorb iodine into the thyroid gland which is a critical element in the production of thyroid hormone (Curely, 2009). The Environmental Protection Agency (EPA, 2005) recommended an RfD of 0.7 μg/kg bw/day for perchlorate for the human population without appreciable deleterious effects during a lifetime. iv) Ethyl carbamate (EC, urethane) is a naturally occurring component of all fermented foods and beverages. Since EC has shown a potential for carcinogenicity when administered in high doses in animal tests (Lachenmeier et al., 2010), the wine industry is interested in reducing EC levels in their products. Arginine, usually one of the most abundant yeast available amino acids in grape juice (Wang et al., 2008), is taken up by wine yeast as a nutrient and may be metabolized yielding urea if present in excess amounts. If the urea cannot be further metabolized and accumulates above a critical concentration, yeast strains release it from their cells into the wine during or at the end of fermentation. Urea can spontaneously react with the alcohol in wine to form EC. The chemical reaction between urea and ethanol is exponentially accelerated at elevated temperatures. To a lesser extent citrulline, an amino acid which is not incorporated into yeast protein, and is formed during arginine biosynthesis, can serve as an EC precursor. Lactic acid bacteria can also be a source of citrulline under wine-making conditions (Terrade and Mira de Orduña, 2006). However, the key reaction for EC formation in wine is between urea and ethanol. Also, urease activity is severely limited under normal wine conditions, specifically with respect to low pH and ethanol. Urease is especially inhibited by high concentrations of malic acid, and fluoride residues in excess of 1 mg/L. Any combination of these factors can make it practically impossible to reach the desired low urea levels in reasonable time, even at a very high enzyme dosage. A complete elimination of EC is not possible. v) Natural contaminants in honey (Paterson’s curse/salvation Jane honey toxins): Some types of honey contain high levels of naturally occurring plant toxins, known as pyrrolizidine alkaloids (PAs), which may cause adverse health effects (Mol et al., 2011). PAs are found in many foods and are naturally produced in more than 600 plants. The toxins may get into the honey when bees forage on the flowers that are rich in pyrrolizidine alkaloids such as Paterson’s Curse, also known as Salvation Jane.

Foodborne Diseases and Responsible Agents  217 vi) Furan is a chemical contaminant that forms in some foods during traditional heat treatment techniques, such as cooking, jarring, and canning. The presence of furan is a potential concern because, on the basis of high-dose animal tests, furan is considered possibly carcinogenic to humans (Bakhiya and Appel, 2010). vii) Others: Another source of contaminants may be the interactions of cleaning and sanitizing agents with other naturally occurring compounds. For example, the carcinogen bromate forms in water when naturally occurring bromide reacts with ozone used for disinfection; haloacetic acids form when chlorine disinfectants react with organic acids.

8.4.6  Migrants From Contact Materials Food packaging prevents contamination, allows food to be transported easily, and extends shelf life. Packaging also provides a surface for labeling and identification of products. Packaging materials also need to ensure that food is not contaminated from substances that could migrate from the packaging into food. Direct contact of foods with packaging materials can result in chemical contamination caused by migration of certain substances into foods. Examples of migrants of health concern may include bisphenol A or phthalates from plastic materials, 4-methylbenzophenone and 2-isopropylthioxanthone from inks (Bradley et al., 2013), mineral oil from recycled fibers (Biedermann and Grob, 2010), or semicarbazide from a foaming agent in the plastic gaskets (Li et al., 2015) that are used to seal metal lids to glass packaging. i) Benzene in flavored beverages: Benzene is a common industrial chemical used in manufacturing plastics and some types of rubbers, detergents, drugs, and pesticides. It is a carcinogen that can cause cancer in humans (Liu et al., 2010). Natural sources of benzene include volcanoes and forest fires. It is also found in crude oil, petrol, and cigarette smoke. Benzene may also be found in nonalcoholic beverages including soft drinks at very low levels. It can form at very low levels (ppb level) in some beverages that contain both benzoate salts and ascorbic acid (vitamin C) or erythorbic acid (a closely related substance (isomer) also known as d-ascorbic acid). Exposure to heat and light can stimulate the formation of benzene in some beverages that contain benzoate salts and ascorbic acid (vitamin C). Sodium or potassium benzoate may be added to beverages to inhibit the growth of bacteria, yeasts, and molds (Kregiel, 2015). Benzoate salts also are naturally present in some fruits and their juices, such as cranberries, for example. Vitamin C may be present naturally in beverages or added to prevent spoilage or to provide additional nutrients. ii) Bisphenol A (BPA): Bisphenol A (BPA) is a chemical used in the lining of some food and beverage packaging to protect food from contamination and extend shelf life. It is also used in nonfood products. Small amounts of BPA can migrate into food and beverages from containers. In January 2015, the European Food Safety Authority (EFSA) released a report on the agency’s comprehensive reevaluation of BPA exposure and toxicity.

218  Chapter 8 EFSA’s reevaluation concluded that BPA poses no health risk to consumers of any age group (including unborn children, infants, and adolescents) at current exposure levels. However, foods packaged in polycarbonate plastics, steel cans with epoxy lining, and some glass jars with metal lids may result in BPA production and contaminate the food products. iii) Fluoride in bottled water: The Food Standards Code allows between 0.6 and 1.0 mg of fluoride (including naturally occurring and added fluoride) per liter of bottled water (Almulla et al., 2016). This is the same level recommended for drinking water to provide benefits for dental health. An FSANZ risk assessment study found that there is a history of safe use of fluoride in tap water at this level. Fluoridated bottled water (at the approved levels) is nutritionally equivalent to fluoridated tap water. Not all bottled water contains fluoride. However, all bottled water with fluoride added must be clearly labeled. Bottled water with added fluoride is safe for everyone and can be used to make up infant formula. Remember to follow the preparation instructions on the formula label. iv) Melamine: Melamine is use in the manufacturing of some cooking utensils, plates, plastic products, paper, paperboard, and industrial coatings, among other things. Plastic tableware made in China was found to contain 20,000 parts per billion of melamine. This type of tableware is manufactured with a substance called melamine-formaldehyde resin. It forms molecular structures that are molded, with heat, to form the shape of the tableware. A small amount of the melamine used can be “left over” from this chemical reaction and remain in the plastic. This leftover melamine can migrate very slowly out of the plastic into food that comes into contact with the tableware. The melamine-formaldehyde tableware does not migrate into most foods; however, when highly acidic foods are heated to extreme temperatures (e.g., 160°F or higher), the melamine can migrate into food. Thus, foods and drinks should not be heated on melamine-based dinnerware in microwave ovens. Melamine contamination may put people at risk of conditions such as kidney stones and kidney failure, and of death. Signs of melamine poisoning may include irritability, blood in urine, little or no urine, signs of kidney infection, and/or high blood pressure (Buka et al., 2009).

8.4.7  Food Additives and Adulterants Food additives are substances added to food to preserve flavor or enhance its taste and appearance. Some additives have been used for centuries; for example, preserving food by pickling (with vinegar), salting, as with bacon, and preserving sweets or using sulfur dioxide as with wines. Any unapproved food additive used in a beverage or other conventional food causes the food to be adulterated under existing international food law. The food and chemical industries have said for decades that all food additives are well tested and safe. Most additives are safe. However, the history of food additives is riddled with additives that, after many years of use, were found to pose health risks. Those listed below have been banned.

Foodborne Diseases and Responsible Agents  219 The moral of the story is that when someone says that all food additives are well tested and safe, you should take their assurances with a grain of salt. Food adulteration: Food adulteration can happen accidentally when unapproved additives are introduced to the food, or the wrong additive is introduced through formulation error. This results in mislabeled food. Perhaps a larger health issue is when foods are adulterated intentionally for economic reasons to sell a low-value food or material for more or to mask food spoilage. Some adulteration may just mislead or cheat consumers, such as adding high fructose corn syrup to honey, but some may be harmful to them. The most notorious example from recent years is the addition of melamine to whey and other protein concentrates to increase their apparent protein content analyzed as total nitrogen (Buka et al., 2009). Other examples include the use of toxic Sudan dyes in adulterated chili powders (Nisa et al., 2015) or adulteration of virgin olive oil with hazelnut oil (Arlorio et al., 2010), which can cause unexpected allergic reactions in sensitive individuals (Table 8.4). Table 8.4: Banned food additives Additive

Function

Agene (nitrogen trichloride)

Flour bleaching and aging agent

Natural or Synthetic

Year Banned

Synthetic

1949

Problem Dogs that ate bread made from treated flour suffered epileptic-like fits; the toxic agent was methionine sulfoxime

Artificial colorings Butter yellow Green 1 Green 2 Orange 1 Orange 2 Orange B

Red 1 Red 2 Red 4

Artificial coloring Artificial coloring Artificial coloring Artificial coloring Artificial coloring Artificial coloring

Synthetic

1919

Toxic, later found to cause liver cancer

Synthetic

1965

Liver cancer

Synthetic

1965

Synthetic

1956

Insufficient economic importance to be tested Organ damage

Synthetic

1960

Organ damage

Synthetic

1978 (ban never finalized)

Artificial coloring Artificial coloring Artificial coloring

Synthetic

1961

Contained low levels of a cancer-causing contaminant. Orange B was used only in sausage casings to color sausages, but is no longer used in the United States Liver cancer

Synthetic

1976

Possible carcinogen

Synthetic

1976

High levels damaged adrenal cortex of dog; after 1965 it was used only in maraschino cherries and certain pills; it is still allowed in externally applied drugs and cosmetics Continued

220  Chapter 8 Table 8.4: Banned food additives—cont’d Natural or Synthetic

Year Banned

Artificial coloring

Synthetic

1956

Artificial coloring Artificial coloring

Synthetic

1919

Synthetic

1973

Artificial coloring Artificial coloring Artificial coloring

Synthetic

1959

Cancer (it had been used to stamp the Department of Agriculture’s inspection mark on beef carcasses) Intestinal lesions at high dosages

Synthetic

1959

Heart damage at high dosages

Synthetic

1959

Heart damage at high dosages

Additive

Function

Red 32

Sudan 1 Violet 1

Yellow 1 and 2 Yellow 3 Yellow 4

Problem Damages internal organs and may be a weak carcinogen; since 1956 it continues to be used under the name Citrus Red 2 only to color oranges (2 ppm) Toxic, later found to be carcinogenic

Other additives Cinnamylanthranilate Cobalt salts Coumarin Cyclamate

Artificial flavoring Stabilize beer foam Flavoring Artificial sweetener

Diethyl pyrocarbonate Preservative (DEPC) (beverages) Dulcin Artificial (p-ethoxy-phenylurea) sweetener Ethylene glycol Solvent Monochloroacetic Preservative acid Nordihydroguaiaretic Antioxidant acid (NDGA)

Oil of calamus

Flavoring

Polyoxyethylene-8stearate (Myrj 45) Safrole

Emulsifier

Thiourea

Flavoring (root beer) Preservative

Synthetic

1982

Liver cancer

Synthetic

1966

Toxic effects on heart

Tonka bean Synthetic

1970

Liver poison

1969

Synthetic

1972

Synthetic

1950

Bladder cancer, damage to testes; now not thought to cause cancer directly, but to increase the potency of other carcinogens Combines with ammonia to form urethane, a carcinogen Liver cancer

Synthetic Synthetic

1998 1941

Kidney damage Highly toxic

Desert plant

1968 (FDA), 1971 (USDA) 1968

Kidney damage

Root of calamus Synthetic

1952

Intestinal cancer

Sassafras

1960

High levels caused bladder stones and tumors Liver cancer

Synthetic

c.1950

Liver cancer

Foodborne Diseases and Responsible Agents  221

8.4.8  Intentional Contaminants This type of contamination is perhaps the most difficult to handle since adulteration is unexpected and difficult to anticipate. Adulterants may be used to sell lower-value products as the original product, mask products that are already past their prime, or add a cheaper compound to a food or ingredient and sell it for a higher value. Recent examples include the use of Sudan red in spices (Nisa et al., 2015), melamine in milk, and the use of inedible Japanese star anise (which contains sikimi-toxin) instead of the closely related edible and innocuous Chinese star anise. i) Sudan dyes: Sudan dyes are a group of industrial dyes consisting of a number of red colors (e.g., Sudan I, II, III, and IV, etc.). The International Agency for Research on Cancer (IARC) evaluated the safety of Sudan I, II, III, and IV in 1987 and considered that they were unclassifiable as to their carcinogenicity to humans (Cao et al., 2014). Although there is some evidence that Sudan dyes may cause cancer in experimental animals and may cause damage to the genes (Pan et al., 2015), there is currently inadequate evidence that they cause cancer in humans. The use of Sudan dyes as coloring matters in food is not permitted under the Coloring Matter in Food Regulations. In the case of chili and chili products, the contamination was traced back to adulterated chili powder that originated from India. It was suspected that Sudan I was fraudulently used to enhance and maintain the color of the product. The price of chili powder is largely linked to the intensity of the color and its maintenance. Indeed vegetable products lose their color over time and thus may become less appealing to consumers after some time. ii) Formaldehyde in Food: Formaldehyde is a chemical commonly used in industry for the manufacture of plastic resins that can be used in the wood, paper, and textile industries. Formalin, which is a solution of about 37% formaldehyde, serves as a disinfectant and preservative for household products. Formaldehyde is sometimes added inappropriately in food processing for its preservative (Akand et al., 2015) and bleaching effects. The common incriminated food items are soybean sticks, mung bean vermicelli, and hydrated food such as tripe, chicken paws, etc. However, this chemical also occurs naturally in the environment. As a metabolic intermediate, formaldehyde is present at low levels in most living organisms. Formaldehyde can be found naturally in food up to the levels of 300–400 mg/kg, including fruits and vegetables (e.g., pear, apple, green onion), meats, fish (e.g., Bombay duck, codfish), crustacean and dried mushroom, etc. Ingestion of a small amount of formaldehyde is unlikely to cause any acute effect. Acute toxicity after ingestion of large amount can cause severe abdominal pain, vomiting, coma, renal injury, and possible death. The main health concern of formaldehyde is its cancer causing potential. The IARC considered that there was sufficient evidence for carcinogenicity (International Agency for Research on Cancer (IARC), 2014) in humans upon occupational exposure via inhalation. On the other hand, the WHO in 2005 when setting its Drinking Water Guidelines considered that there was no definitive evidence for carcinogenicity upon ingestion.

222  Chapter 8

8.4.9  Food Biotechnology The production of genetically modified foodstuffs offers tremendous opportunities and benefits for future food production. However, the emergence of this new technology has also given rise to a number of problems, although such problems are often regarded as potential or perceived, rather than real. The concerns that have been expressed relate mainly to changes in the nutritional quality of food, an increase in toxicity or hazards with respect to food intolerances or food allergies, and the development of antimicrobial resistance (Qaim and Kouser, 2013). Taking everything into consideration, GM crops are alive; they can migrate and spread worldwide. In this regard, clear signals should be sent to biotech companies to proceed with caution and avoid causing unintended harm to human health and the environment. It is widely believed that it is the right of consumers to demand mandatory labeling of GM food products, independent testing for safety and environmental impacts, and liability for any damage associated with GM crops.

8.4.10  Food Allergies Some foods and food ingredients or their components can cause severe allergic reactions including anaphylaxis. A food allergy occurs when a person’s immune system reacts to allergens that are harmless to other people. Most food allergies are caused by peanuts, tree nuts, milk, eggs, sesame seeds, fish and shellfish, soy, and wheat (Waserman and Watson, 2011), and these must be declared on the food label, however small the amount present in the food. Food allergies can be life-threatening. For people who have a food allergy, the only way to manage the allergy is to avoid the food allergen. For this reason, there are laws in place, for example, mandatory labeling requirements to help people who have a food allergy avoid food allergens. •

•

Gluten-containing cereals need to be declared on the label, so people with Celiac Disease and cereal allergies can identify these products (Elli et al., 2015). Gluten containing cereals include wheat, rye, barley, oats, and hybrid strains of these cereals (e.g., triticale). Sulfite preservatives must also be declared on the label if added at 10 (or more) milligrams per kilogram of food. Food allergens such as sulfites and derivatives of egg, fish, milk, and tree nuts may be used as fining agents in the wine production process. While these substances are largely removed through filtration, very small residual amounts may be present in the final product (Vally and LA Misso, 2012).

8.4.11  Food Intolerances A true food allergy causes an immune system reaction that affects numerous organs in the body. It can cause a range of symptoms. In some cases, an allergic reaction to a food can be severe or life-threatening. In contrast, food intolerance symptoms are generally less serious

Foodborne Diseases and Responsible Agents  223 and often limited to digestive problems. Adverse reactions to foods which are not the same as allergic may include: rashes and swelling of the skin, asthma, and stuffy or runny nose, irritable bowel symptoms, colic, bloating, and diarrhea, migraines, headaches, lethargy, and irritability. The causes of food intolerance include: (1) absence of an enzyme needed to fully digest a food. Lactose intolerance is a common example; (2) irritable bowel syndrome. This chronic condition can cause cramping, constipation, and diarrhea; (3) food poisoning. Toxins such as bacteria in spoiled food can cause severe digestive symptoms; (4) sensitivity to food additives. For example, sulfites used to preserve dried fruit, canned goods, and wine can trigger asthma attacks in sensitive people; (5) recurring stress or psychological factors. Sometimes the mere thought of a food may make people sick. The reason is not fully understood; and (6) celiac disease has similar features of a true food allergy because it involves the immune system. However, symptoms are mostly gastrointestinal, and people with celiac disease are not at risk of anaphylaxis. This chronic digestive condition is triggered by eating gluten, a protein found in wheat and other grains.

8.5  The Future of Foodborne Diseases In the past few years, the world has witnessed an upsurge in incidences associated with food safety, which are all attributed to different intrinsic and extrinsic factors (Fig. 8.3).

Vancomycin-resistant S. aureus

Cryptosporidiosis

Multidrug-resistant tuberculosis Drug-resistant malaria SARS

Cyclosporiasis

Diphtheria

E. coli O157:H7

E. coli O157:H7

Hepatitis C vCJD Lyme disease

Human monkeypox

H5N1 influenza

Typhoid fever

West Nile virus

Vancomycinresistant S. aureus

Anthrax bioterrorism

Lassa fever

Rift Valley fever HIV

Nipah virus

Whitewater arroyo virus

Hendra virus

Hantavirus pulmonary syndrome Dengue

Enterovirus 71

Human monkeypox Yellow fever

Cholera

Marburg haemorrhagic fever

Ebola haemorrhagic fever

Plague

Fig. 8.3 Worldwide foodborne diseases incidents. Source: Adopted from Morens et al. (2004) with permission.

224  Chapter 8 Today, with the increase in knowledge and available databases on food safety issues, the world is witnessing tremendous efforts toward the development of new, economical, and environment-friendly techniques for maintaining the quality of perishable foods and agro-based commodities. The intensification of food safety concerns reflects a major global awareness of foods in world trade. Several recommendations have been put forward by various world governing bodies and committees to solve food safety issues, which are all mainly targeted at benefiting consumers. In addition, economic losses and instability to a particular nation or region caused by food safety issues can be huge. Various “non-dependent” risk factors can be involved with regard to food safety in a wide range of food commodities such as fresh fruits, vegetables, seafood, poultry, meat, and meat products. Additionally, food safety issues involve a wide array of issues including processed foods, packaging, postharvest preservation, microbial growth, and spoilage (Akand et al., 2015; International Agency for Research on Cancer (IARC), 2014), food poisoning, handling at the manufacturing units, food additives, presence of banned chemicals and drugs, and more. Rapid change in climatic conditions is also playing a pivotal role with regard to food safety issues, and increasing the anxiety about our ability to feed the world safely.

8.6 Conclusion Foodborne diseases can cause short-term symptoms, such as nausea, vomiting, and diarrhea (commonly referred to as food poisoning), but can also cause longer-term illnesses, such as cancer, kidney or liver failure, brain, and neural disorders. These diseases may be more serious in children, pregnant women, elderly people, and those who have a weakened immune system. Children who survive some of the more serious foodborne diseases may suffer from delayed physical and mental development, impacting their quality of life permanently. Nonetheless, increased foodborne incidences pose a global threat to regulatory authority and reinforce the need for governments, the food industry, and individuals to do more to make food safe and prevent foodborne diseases. Since food safety is a shared responsibility of all the stakeholders along the food chain, there remains a significant need for education and training on the prevention of foodborne diseases among food producers, suppliers, handlers, and the general public. In addition, all the stakeholders along the food chain must work closely with national governments to help set and implement food safety strategies and policies that will in turn supply safe food for the world populations.

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Foodborne Diseases and Responsible Agents  227 Kregiel, D., 2015. Health safety of soft drinks: contents, containers, and microorganisms. Biomed. Res. Int. 2015. 128697. 15 pages. https://doi.org/10.1155/2015/128697. Krishnakumar, T., Visvanathan, R., 2014. Acrylamide in food products: a review. J. Food Process Technol. 5, 7. https://doi.org/10.4172/2157-7110.1000344. Lachenmeier, D.W., Lima, M.C., Nóbrega, I.C., Pereira, J.A., Kerr-Corrêa, F., Kanteres, F., Rehm, J., 2010. Cancer risk assessment of ethyl carbamate in alcoholic beverages from Brazil with special consideration to the spirits cachaça and tiquira. BMC Cancer 10, 266. https://doi.org/10.1186/1471-2407-10-266. Lee, B.Q., Khor, S.M., 2014. 3-Chloropropane-1,2-diol (3-MCPD) in soy sauce: a review on the formation, reduction, and detection of this potential carcinogen. Compr. Rev. Food Sci. Food Saf. 14 (1), 48–66. Li, G., Tang, C., Wang, Y., Yang, J., Wu, H., Chen, G., Kong, X., Kong, W., Liu, S., You, J., 2015. A rapid and sensitive method for semicarbazide screening in foodstuffs by HPLC with fluorescence detection. Food Anal. Methods 8, 1804–1811. https://doi.org/10.1007/s12161-014-0063-9. GaoA, Xin Zuo, Qingjie Liu, Xue Lu, Wei Guo, Lin Tian. 2010. Methylation of PARP-1 promoter involved in the regulation of benzene-induced decrease of PARP-1 mRNA expression. Toxicol. Lett. 195 (2–3), 114–118. Lopes, E.J., Okamura, L.A., Yamamoto, C.I., 2015. Formation of dioxins and furans during municipal solid waste gasification. Braz. J. Chem. Eng. 32 (1). https://doi.org/10.1590/0104-6632.20150321s00003163. Luby, S.P., Gurley, E.S., Jahangir Hossain, M., 2009. Transmission of human infection with Nipah virus. Clin. Infect. Dis. 49 (11), 1743–1748. https://doi.org/10.1086/647951. Magkos, F., Arvaniti, F., Zampelas, A., 2006. Organic food: buying more safety or just peace of mind? A critical review of the literature. Crit. Rev. Food Sci. Nutr. 46 (1), 23–56. https://doi.org/10.1080/10408690490911846. Maksymiv, I., 2015. Pesticides: benefits and hazards. J. Vasyl Stefanyk Precarpathian Natl. Univ. 2 (1), 70–76. https://doi.org/10.15330/jpnu.2.1.70-76. Mastovska, K., 2013. Modern analysis of chemical contaminants in food. Food Safety Mag. http://www.foodsafetymagazine.com/magazine-archive1/februarymarch-2013/ modern-analysis-of-chemical-contaminants-in-food/. Mattiucci, S., Fazii, P., De Rosa, A., Paoletti, M., Salomone Megna, A., Glielmo, A., et al., 2013. Anisakiasis and gastroallergic reactions associated to Anisakis pegreffii infection, Italy. Emerg. Infect. Dis. 19, 496–499. McCarthy, N., Giesecke, J., 2001. Incidence of Guillain-Barré syndrome following infection with Campylobacter jejuni. Am. J. Epidemiol. 153 (6), 610–614. https://doi.org/10.1093/aje/153.6.610. Menza, N.C., Margaret, M.W., Lucy, K.M., 2015. Incidence, types and levels of aflatoxin in different peanuts varieties produced in Busia and Kisii Central Districts, Kenya. Open J. Med. Microbiol. 5, 209–221. https:// doi.org/10.4236/ojmm.2015.54026. Mohsina, Z., Yang, S., Tarique, T.M., Qiu, J., 2015. Use of banned veterinary drugs in feed: food safety challenges and strategies in china: a review. Eur. Acad. Res. 3 (3), 2871–2892. Mol, H.G.J., Van der Kamp, H., Van der Weg, G., Van der Lee, M., Punt, A., De Rijk, T.C., 2011. Screening of plant toxins in food, feed and botanicals using full-scan high-resolution (Orbitrap) mass spectrometry. J. AOAC Int. 94, 1722–1740. Morens, D.M., Folkers, G.K., Fauci, A.S., 2004. The challenge of emerging and re-emerging infectious diseases. Nature 430, 242–249. Nataro, J.P., Steiner, T., Guerrant, R.L., 1998. Enteroaggregative E. coli (EAEC) has increasingly been recognized as an agent of a watery mucoid diarrhoea—especially in children—in developing and, recently, industrialized countries. Emer. Infect. Dis. 4 (2). Available at: https://wwwnc.cdc.gov/eid/article/4/2/98-0212_article. Nayenje, M.E., Ndip, R.N., 2013. The challenges of foodborne pathogens and antimicrobial chemotherapy: a global perspective. Afr. J. Microbiol. Res. 7 (14), 1158–1172. Nerín, C., Aznar, M., Carrizo, D., 2016. Food contamination during food process. Trends Food Sci. Technol. 48, 63–68. Nicholas A, D., 2011. Vibrio vulnificus oysters: pearls and perils. Clin. Infect. Dis. 52 (6), 788–792. https://doi. org/10.1093/cid/ciq251. Nigam, P.K., Nigam, A., 2010. Botulinum toxin. Indian J. Dermatol. 55, 8–14. Nisa, A.U., Zahra, N., Akhlaq, F., 2015. Detection of Sudan dyes in different spices. Pakistan. J. Food Sci. 25 (3), 144–149.

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Further Reading Smith, K.F., Goldberg, M., Rosenthal, S., Carlson, L., Chen, J., Chen, C., Ramachandran, S., 2014. Global rise in human infectious disease outbreaks. J. R. Soc. Interface 11, 20140950. https://doi.org/10.1098/ rsif.2014.0950. Wilson, N.O., Hall, R.L., Montgomery, S.P., Jones, J.L., 2015. Trichinellosis surveillance—United States, 2008–2012. MMWR Surveill. Summ. 64, 1–8.

CHAPTE R 9

Challenges in Emerging Food-Borne Diseases K.D. Devi Nelluri, Navya Sree Thota KVSR Siddhartha College of Pharmaceutical Sciences, Vijayawada, Andhra Pradesh, India

9.1 Introduction Food security and nutrition are inseparably connected, especially in places where food supplies are not safe. At the point when food turns out to be scarce, hygiene, safety, and nutrition are frequently overlooked as individuals shift to less nutritious diet plans and expend more unhealthy food in which chemical, microbiological, zoonotic, and other risks represent a health hazard. The World Health Organization and Food and Agriculture Organization characterized food safety as “food that is free from all hazards, whether chronic or acute, that may make food injurious to the health of the consumer” (Elmi, 2004). Food production must conform to the conditions given by food legislation prioritizing the food safety (Steinhauserova and Borilova, 2015). Food sanitation and food safety have become the global issue at present (Nsoesie et al., 2014; Saccol et al., 2013). Also, their idea is in expanding pattern, as a result of the expanding outbreaks of food-borne diseases (FBDs) around the world. An FBD Outbreak is characterized as the occurrence of two or more instances of a comparative illness coming about because of common food (Korea Centers for Disease Control and Prevention, 2012). These are disorders including nausea, vomiting, abdominal cramps, and diarrhea brought about by the ingestion of food defiled by chemical substances or microorganisms and/or their poisons (Hennekinnea et al., 2015). Types of food-borne illness: Pathogens can bring about various types of FBDs. • • •

Food-borne infection: Once a food contaminated is consumed, sickness can be brought on by the pathogens themselves. Food-borne intoxication: The poisons produced by the pathogens in the food. Food-borne toxin mediated disease: This is caused by poisons produced in the body by pathogens.

Food Safety and Preservation. https://doi.org/10.1016/B978-0-12-814956-0.00009-3 © 2018 Elsevier Inc. All rights reserved.

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232  Chapter 9

9.2 Epidemiology FBDs can be seen worldwide with a high prevalence in developing countries (Kappagoda and Ioannidis, 2012, 2014). Although it is impossible to estimate the worldwide burden of FBDs (World Health Organization, 2008a), numerous FBD outbreaks have been reported to the national surveillance and reporting systems in diverse countries such as the United States, European countries, China, and Japan (World Health Organization, 2008b; Centers for Disease Control and Prevention, 2013; European Centers for Disease Control and Prevention, 2013; Chen et al., 2010; Shinagawa, 2010). Between the years 2002 and 2011, 67% of the food-borne outbreaks that could be ascribed to a particular food were caused by foods under Food and Drug Administration (FDA) regulation (CSPI, Centre for Science in the Public Interest, 2014). The World Health Organization in 2009 has reported that >200 diseases can be spread by polluted food or water with the level of food-borne infections being opened up by expanded worldwide exchange in food and population mobility (World Health Organization (WHO), 2016). It has, however, assessed that food-borne and waterborne diseases combined kill about 2.2 million people annually, of whom 1.9 million are children (World Health Organization (WHO), 2010). In 2013, an aggregate of 5196 weak and strong evidence outbreaks were accounted for by 24 EU MS6. The outbreaks included 43,183 human cases, 5946 hospitalizations, and 11 deaths (European Food Safety Authority (EFSA) and European Centre for Disease Prevention and Control (ECDC), 2015). Salmonella remained the most regularly identified causative organism (22.5%) followed by viruses (18.1%) and bacterial poisons (16.1%) as appeared in Fig. 9.1. Recent estimates inferred that foods consumed in the United States that were debased with 31 known agents of FBDs brought on 9.4 million illness, 55,961 hospitalizations, and 1351 deaths every year (Scallan et al., 2011). The Official number of food-borne outbreaks in 2013 was 5196 for Europe and 818 for the United States (Spirica et al., 2015). In Korea, a total of 2862 FBDOs were accounted for between 2007 and 2012. Of these, 1794 FBDOs influencing 48,897 patients happened locally and 1068 FBDOs happened outside Korea (Moon et al., 2014). The prevention, maintenance, and treatment of diseases from street food-borne illnesses were reported to result in the heavy drain on the purse of individuals and governments in the developing countries due to the huge spending involved (Ekanem, 1998). Information on the measure of sickness is especially hard to decide in developing countries because of inadequate observation and poor reporting frameworks. The Indian subcontinent is thought to be a center of food- and waterborne tropical diseases, extraordinarily brought on by the individuals from the Enterobacteriaceae family, attributable to the changes in temperature and humidity. The food- and waterborne diseases are less

Challenges in Emerging Food-Borne Diseases  233 Unknown causative agent 28.9%

Other causative agent 6.4%

Campylobacter 8% Bacterial exotoxins 16.1%

Staphylococcal toxins 7.4%

Bacillus toxins 5.4% Clostridium toxins 3.3%

Viruses 18.1% Salmonella 22.5%

Fig. 9.1 Distribution of food-borne outbreaks per causative agent in the EU during 2013. Data from EFSA, 2015. The European Union summary report on trends and sources of zoonoses, zoonotic agents, and food-borne outbreaks in 2013. EFSA J. 13, 1–162.

predominant in temperate climates, because of the event of a cold season, which controls the quick increase of possibly pathogenic microscopic organisms. As per the World Health Organization, >33% of the aggregate populations in developing nations are, consistently influenced by FBDs. Studies showed that food handlers play a noteworthy reason in contaminating food (Campos et al., 2009), and that the greater part of food poisoning cases happen in schools and colleges in and around the world (Sani and Siow, 2014). The burden of FBDs is hard to estimate but is in the billions of dollars for loss of life or reduced quality of life, particularly for diseases that are endemic (Todd, 2014).

9.3 Etiology Food-related pathogens are an exceptionally diverse population. They can be found in the environment where food and feed are created and taken care of. The World Health Organization (2007) reported that in the year 2005 alone, almost 1.8 million individuals passed on the result of diarrheal cases internationally, and the majority of them consumed contaminated food and water. Food-borne ailments can be brought on by microorganisms and their poisons, marine organisms and their poisons, fungi and their related poisons, and chemical contaminants. Despite the fact that food decay microorganisms are not specifically bringing about any food-related outbreaks, some of them can be opportunistic human pathogens (Al-Kharousi et al., 2016). In the past 20 years, many food-borne episodes have

234  Chapter 9 been caused by bacteria, viruses, and protozoa, and numerous more pathogens are being presented through food contamination consistently every year (Marusi, 2011). Before 1960, the significant reasons for gastrointestinal illness were recognized as Salmonella spp., Shigella spp., Clostridium botulinum, and Staphylococcus aureus. In the mid-1960s, Clostridium perfringens and Bacillus cereus were included and after that, in the 1970s, rotavirus and norovirus (NoV). In the 1980s and 1990s, there was a whirlwind of additions including Campylobacter, Yersinia, Listeria monocytogenes, and new strains of Escherichia coli, for example, O157:H7, Cryptosporidia, and Cyclospora. It seems exceptionally plausible that new food-borne pathogens (FBP) will be discovered in the 21st century (Newell et al., 2010). There are >200 known microbial, chemical, or physical agents that can bring about illness when ingested (Acheson, 1999) Figs. 9.2 and 9.3 show the circulation of food-borne outbreaks by a portion of the causative agents. Interestingly, in >66% of food-borne illness outbreaks, no pathogen is identified (Olsen et al., 2000). The weight of diseases created by FBP remains to a great extent obscure and our comprehension of the microbial organisms of intestinal illness stays constrained (Newell et al., 2010). 0

200

400

600

800

1000

1200

1400

1600

Yersinia Parasites E. coli, pathogenic(VTEC) Other bacterial agents Other causative agents

Weak-evidence outbreaks Strong-evidence outbreaks

Campylobacter Bacterial toxins Viruses Salmonella Unknown

Fig. 9.2 Distribution of food-borne outbreaks by causative agents (EFSA annual report, 2013).

Challenges in Emerging Food-Borne Diseases  235 Other Escherichia coli, Enteroaggregative Clostridium botulinum Staphylococcus spp. Vibrio cholerae Shigella Bacillus cereus

No.of outbreaks reporting outbreak factor

Listeria monocytogenes Staphylococcus aureus enterotoxin

Total no.of outbreaks

Vibrio parahaemolyticus Campylobacter Clostridium pefringens E. coli, Shiga toxin producing Salmonella Viral 0

50 100 150 200 250 300 350 400

Fig. 9.3 Distribution of food-borne outbreaks by causative agents (US annual report, 2013).

Essential hints to deciding the etiology of FBDs are the: • • • •

incubation period, duration of the resultant ailment, predominant clinical indications, and population required in the episode.

The basic pathogen sort that causes food-borne illness is bacteria, parasites, and infections:

9.3.1  Food-Borne Bacterial Agents All through the 1990s and until today, three noteworthy food-borne bacterial targets Salmonella spp., Campylobacter spp., and, what’s more, E. coli have continued, directing the most research and surveillance attention from government organizations and, to an expansive degree, the most awareness from the food industry. More recently, there has been a developing worry about L. monocytogenes. These bacterial pathogens together constitute the greatest burden of FBDs for which the etiology is known. They are extraordinarily distinctive regarding the study of disease epidemiology, physiology, ecology, host association, and virulence properties; however, together they empower some

236  Chapter 9 generic conclusions to be drawn on the general steadiness of food-borne bacterial infection in the course of the most recent 20 years (Table 9.1). Despite the fact that these are the major bacterial pathogens checked, numerous others are likewise transmitted through food. Whenever, such minor FB pathogens, similar to L. monocytogenes, can get to be significant issues. Examining the explanations behind such moves in examples of food-borne infection gives significant data of the future about risk management strategies (Hennekinne et al., 2012). Some bacteria, for example, Salmonella, E. coli, and Campylobacter, can be found in raw meat. Among these pathogens, coagulase positive staphylococci and their enterotoxins are responsible for various food poisoning outbreaks (Ponka et al., 1999). Table 9.1: CDC estimates of food-borne illness in the United States 2011 Pathogen Type Bacteria

Pathogen Bacillus cereus, food-borne Brucella spp. Campylobacter spp. Clostridium botulinum, food-borne Clostridium perfringens, food-borne E. coli (STEC) O157 E. coli (STEC) non-O157 Enterotoxigenic E. coli (ETEC) Diarrheagenic E. coli other than STEC and ETEC Listeria monocytogenes Mycobacterium bovis Salmonella spp., nontyphoidal S. enterica serotype typhi Shigella spp. Streptococcus spp. Group A, food-borne Streptococcus Vibro cholera, toxigenic V. vulnificus V. parahemolyticus Vibrio spp., other Yersinia enterocolitica

Estimated Annual Illnesses

Estimated Annual Hospitalizations

Estimated Annual Deaths

63,000 840 850,000 55 970,000 63,000 110,000 18,000 12,000

20 55 8500 42 440 2100 270 12 8

0 1 76 9 26 20 1 0 0

1600 60 1,000,000 1800 130,000 240,000

1500 31 19,000 200 1500 1100

250 3 380 0 10 6

11,000 84 96 35,000 18,000 98,000

1 2 93 100 83 530

0 0 36 4 8 29

Data from Scallan, E., Hoekstra, R.M., Angulo, F.J., Tauxe, R.V., Widdowson, M.-A., Roy, S.L., et al., 2011. Food-borne illness acquired in the United States—major pathogens. Emerg. Infect. Dis. J. 17, 7e15.

9.3.2  Food-Borne Parasitic Agents Parasitism is a symbiotic relationship between two organisms in which the parasite benefits for growth and reproduction to the harm of the host. Around 300 types of parasitic worms and

Challenges in Emerging Food-Borne Diseases  237 >70 types of protozoa have been depicted that can infect people. Enteric parasitic diseases can be transmitted by the fecal-oral course by eating characteristically contaminated food or by means of uptake of free-living parasitic stages from nature like eggs, pimples, and oocysts (Tables 9.2 and 9.3).

Table 9.2: CDC estimates of food-borne illness in the United States 2011: food-borne illnesses, hospitalizations, and deaths Pathogen Type

Pathogen

Parasites

Cryptosporidium spp. Cyclospora cayetanensis Giardia intestinalis Toxoplasma gondii Trichinella spp. Astrovirus Hepatitis A virus Norovirus Rotavirus Sapovirus

Viruses

Estimated Annual Estimated Annual Illnesses Hospitalizations 58,000 11,000 77,000 87,000 160 15,000 1600 5,500,000 15,000 15,000

210 11 230 4400 6 87 99 15,000 350 87

Estimated Annual Deaths 4 0 2 330 0 0 8 150 0 0

Data from Scallan, E., Hoekstram, R.M., Angulo, F.J., Tauxe, R.V., Widdowson, M.-A., Roy, S.L., et al., 2011. Food-borne illness acquired in the United States—major pathogens. Emerg. Infect. Dis. J. 17, 7e15.

Table 9.3: Parasites in different foods Foods

Protozoa

Beef

Toxoplasma gondii Cryptosporidium parvum Toxoplasma gondii Cryptosporidium parvum Toxoplasma Cryptosporidium (sheep goat) Toxoplasma Cryptosporidium

Pork, other meat

Milk Fish/squid

Crabs, shrimps, shellfish

Cryptosporidium spp. Giardia lamblia Toxoplasma gondii

Nematodes

Cestodes

Trematodes

Taenia saginata

Fasciola hepatica

Trichinella spp. (horse, wild boar bear, walrus, crocodile) Gnathostoma (frogs)

Taenia solium/asiatica Alaria alata (wild boar)

Paragonimus (wild boar)

Anisakis spp. Gnathostoma

Diphyllobothrium

Clonorchis Opisthorchis Paragonimus Echinostomes

Gnathostoma

Continued

238  Chapter 9 Table 9.3: Parasites in different foods—cont’d Foods

Protozoa

Nematodes

Cestodes

Trematodes

Snails/slugs Fruit/ vegetables (raw)

Cyclospora Cryptosporidium spp. Giardia lamblia Toxoplasma gondii Entamoeba histolytica Balantidium coli Trypanosma cruzi Cyclospora Cryptosporidium Giardia lamblia Toxoplasma gondii Balantidium coli

Angiostrongylus Ascaris Toxocara Baylisascaris spp. Trichuris trichiura

Echinococcus Taenia solium

Echinostomes Fasciola hepatica Fasciolopsis

Ascaris

Echinococcus

Fasciola Fasciolopsis

Water

Data from Newell, D.G., Koopmans, M., et al., 2010. Food-borne diseases—the challenges of 20 years ago still persist while new ones continue to emerge. Int. J. Food Microbiol. 139, S3–S15.

9.3.3  Food-Borne Viral Agents Food-borne viral contamination causes rather explosive outbreaks. Properties of food-borne infections: • • • •

Viruses do not develop in food. Most food-borne infections are (exceptionally) irresistible, spreading quickly, starting with one individual then onto the next. There is no methodical surveillance for the food-borne viral diseases. There is a scope of clinical syndromes connected with various food-borne infections.

Food might be contaminated by contact with human fecal matter at the source (Parashar et al., 2001) or by unhygienic control by a food handler excreting the virus (Velebit et al., 2015). Within food, viruses cannot replicate since they require living cells for this; thus, food is not sensorily changed (Yu et al., 2010). The virus can be excreted without symptoms, so improper control by an asymptomatic food handler can prompt an outbreak (Daniels et al., 2000; Atmar and Estes, 2006). As of now, NoV is viewed as the main source of food-borne illness and acute nonbacterial gastroenteritis around the world (Teunis et al., 2008). NoV is greatly infectious, with an expected irresistible dosage as low as 18 viral particles (Petrović and D'Agostino, 2016), and transmission happens through three general courses: individual-to-individual, food-borne, and waterborne (Table 9.4). NoV is followed by hepatitis A virus and hepatitis E virus is progressively being recognized as an emerging viral FBP that incorporates zoonotic transmission through pork items (Kirbisa and Krizmana, 2015).

Challenges in Emerging Food-Borne Diseases  239 Table 9.4: Common food-borne viruses and their characteristics Virus/Family

Risk Level

Genome

Type of Illness

Source

Transmission/Food Vehicle

Norovirus/ Caliciviridae Hepatovirus A/ Picornaviridae

High

ssRNA

Gastroenteritis

High

ssRNA

Hepatitis A

Human stool, vomit Human stool

Orthohepevirus A/Hepeviridae

Low to moderate

ssRNA

Hepatitis E

Pig liver

Fecal-oral/berry fruit, deli meat, shellfish Fecal-oral and person to person/deli meat, raw beef, water, shellfish, fruit, and vegetable Environmental/pork

Data from Velebit, B., Radin, D., Teodorovic, V., 2015. Transmission of common food-borne viruses by meat products. Procedia Food Sci. 5, 304–307.

9.3.4  Molds, Toxins, and Contaminants Bacteria, viruses, and parasites cause most food poisoning instead of toxic substances in the food. In any case, a few instances of food poisoning can be connected to either natural poisons (e.g., those in a few mushrooms and puffer fish) or chemical poisons, (e.g., pesticides or melamine). While a few molds are alluring in nourishments (e.g., blue cheddar), some can produce poisons that cause disease. Mycotoxins are thought to be an important food safety issue in food obtained from plants considering that very nearly a quarter of the worldwide annual maize crop can end up being infected with mycotoxins. The impacts of environmental change on mycotoxins might be hard to foresee as it is normal that these are perplexing, mold and area particular. An expansion in temperature may wipe out mycotoxin production by some mold species, while in colder parts of the world the rate of diseases may increase.

9.3.5 Allergens Food allergy or sensitivity is an anomalous reaction to a food activated by your body’s immune framework. Unfavorably susceptible responses to food can now and again cause genuine sickness and even death. The foods that most often trigger allergic reactions are: wheat, milk, soybeans, eggs, fish (e.g., bass, flounder, cod), crustacean shellfish (e.g., crab, lobster, shrimp), peanuts, tree nuts (e.g., almonds, walnuts, pecans).

9.4  Food-Borne Antimicrobial Resistance These days, the food-borne zoonoses of most prominent concern are campylobacteriosis, salmonellosis, and Verotoxigenic E. coli, and antimicrobial resistance in these pathogens is an emerging health issue (NARMS, 2009).

240  Chapter 9 Zoonotic diseases are infections that are normally transmitted among animals and humans. The most serious danger for zoonotic disease transmission happens at the human-animal interface through immediate or roundabout human introduction to animals, their items (e.g., meat, milk, eggs), and/or their surroundings. Overviews led by the National Antimicrobial Resistance Monitoring System (NARMS) demonstrate that retail meat and poultry items are often contaminated with types of multidrug resistance Campylobacter, Salmonella, Enterococcus, and E. coli (Sørum and L’Abée-Lund, 2002). Resistance in food-related bacteria reflects the resistance circumstance in microbes from all the different environments from which food for human utilization originates (Akinbode et al., 2011) as shown in Fig. 9.4. Unhealthy activities navigate the entire chain of street food business from agricultural raw materials to the last retail road food and have been fingered in the outbreak of illness and diseases (Rocourt, 2014).

Animals get antibiotics and develop resistant bacteria in their guts

Resistant bacteria spreads in households

Resistant bacteria remain on food

Resistant bacteria in sewage can infect animals through water used on crops

People get antibiotics and develop resistant bacteria in their guts

People contaminate food while working in food producing industry

Food can be source of infection for people and animals

Resistant bacteria in human feces spreads through sewage and contaminates water

Fig. 9.4 The occurrence of resistance in bacteria and transfer via humans, animals, and foodstuffs.

Challenges in Emerging Food-Borne Diseases  241 Regardless of the pathogen, various factors have been added to or are adding to changing patterns in food-borne illnesses. These include: • •

• • • • •

• • •

Rapid population development and a demographic movement toward an aging population. A progressively worldwide business sector in vegetables, organic products, meat, ethnic food, and even farm animals, some of which begin from nations without proper microbiological safety procedures. Improved transport logistics and conditions, which enable agents to survive on food items and reach the purchaser in a viable form. A progressively transient human population conveying its intestinal flora around the world. Changing dietary patterns, for example, the utilization of raw or lightly cooked food, and the interest in exotic foods, for example, bush meats. The shift from low- to high-protein foods as countries develop financially with a concomitant and global greater dependency on meat and fish products. Higher extents of immunologically compromised people either as a result of changing demographics creating an inexorably elderly population or the generation of highly susceptible groups with immunosuppressive diseases or treatments. Changing cultivating practices, for instance, escalation to create cheaper food or a shift to free-range or organic animal production to respond to consumer welfare concerns. The expanding interruption of man on native wildlife habitats. Climate change, for instance, bringing novel vectors into temperate regions, or temperature-related changes in contamination levels (Chauhan et al., 2015).

9.5  Clinical Presentations Susceptibility to FBD is the inability of the host to prevent or overcome the invasion by pathogenic microorganisms transmitted by food (CDC and MMWR, 2001). FBDs created by microorganisms are an undeniably perceived issue including a wide range of sicknesses, for example, the acute impacts on the gastrointestinal tract and additionally different manifestations, for example, fever, vomiting, nausea, diarrhea as well as bloody diarrhea, lack of hydration, and kidney failure in severe cases (Rocourt, 2014).

9.5.1  Acute Effects • • • • • •

high fever (temperature >101.5°F, measured orally) blood in the stools prolonged vomiting that avoids holding fluids down prolonged bloody diarrhea (three or more unformed stools every day) weight loss neurologic involvement such as paresthesias, motor weakness, and cranial nerve palsies

242  Chapter 9 • • •

severe abdominal pain. persistent loose stools (lasting >14 days) diarrhea prompting signs of dehydration, decreased urination, a dry mouth and throat, etc.

Noninflammatory diarrhea is connected with acute watery diarrhea without fever, a few patients may present with severe fever. Noninflammatory diarrhea is described by mucosal hypersecretion or diminished ingestion without mucosal devastation and by and large includes the small intestine. Some influenced patients might be dehydrated as a result of extreme watery loose bowels and may show up genuinely sick. This is more regular in the young and the elderly. Most patients experience insignificant dehydration and show up somewhat sick with insufficient physical findings. Sickness ordinarily happens with a sudden onset and brief length of illness. Fever and systemic side effects generally are absent (with the exception of indications related directly to intestinal fluid loss). Inflammatory diarrhea (obtrusive gastroenteritis; bloody stool and fever might be present) is portrayed by mucosal invasion with resulting inflammation and is brought on by intrusive or cytotoxigenic microbial pathogens. The diarrheal ailment typically includes the internal organ and might be connected with fever, abdominal pain and tenderness, headache, nausea, vomiting, malaise, and myalgia. Stools might be bloody and may contain fecal leukocytes.

9.5.2  Chronic Effects A large portion of the general population enduring food-borne infections will recover with no chronic impacts from their sickness. For a few, be that as it may, the impacts can be devastating and even dangerous (Kingsley and Chen, 2009). The serious impacts connected with a few regular sorts of food poisoning are: •

•

Kidney-related problems Hemolytic-uremic syndrome (HUS) is a serious complication that for the most part happens when a contamination in the digestive framework produces dangerous substances that destroy red blood cells, bringing about kidney harm. HUS may happen after disease with a few sorts of E. coli bacteria. It is most common in kids and is the most widely recognized reason for acute kidney failure in them. Arthritis and related problems Some persons with Shigella or Salmonella contamination develop pain in the joints, aggravation of the eyes, and painful urination. This condition is called reactive arthritis. It can keep going on for a considerable length of time or years, and can prompt constant joint pain, which is hard to treat. Persons with Campylobacter disease may likewise develop chronic arthritis.

Challenges in Emerging Food-Borne Diseases  243 •

Brain- and nerverelated problems A Listeria contamination can prompt meningitis. In the event that a newborn infant is infected with Listeria, long-term outcomes may incorporate mental retardation, seizures, paralysis, visual deficiency, or deafness. Guillain-Barré syndrome is a condition that influences the nerves of the body. This happens when a person’s immune system assaults the body’s own nerves. It can bring about paralysis that keeps going on for several weeks and requires serious consideration. More than 40% of Guillain-Barré disorder cases might be caused by a contamination with Campylobacter. • Death In the United States, around 3000 individuals pass on every year because of sicknesses connected with food poisoning. Five sorts of life forms represent 88% of the deaths for which the cause is known: Salmonella, Toxoplasma, Listeria, norovirus, and Campylobacter. Different sorts of food-borne illness may bring about death too. For instance, some Vibrio diseases may contaminate the body’s circulation system and cause an extreme, life-threatening illness. About half of these contaminations are fatal, and death can happen within 2 days.

9.6  Diagnosis of Food-Borne Diseases The presentation of a patient with an FBD is almost similar to that of one with viral infection. Likewise, viral infections are common to the point that it is sensible to accept that a percentage of those diagnosed to have a viral infection have really contracted an FBD. The viral infection must be excluded to suspect the FBD and make proper public health move. Each episode of FBD starts with a list of patients who may not be seriously sick. A doctor or health care professional, who come across this individual might be the only one with the chance to make an early finding. Since contagious diarrhea can be infectious and is effectively spread, fast and early detection of an etiologic agent may control an illness outbreak. Early identification of a case of foodborne illness can avoid further exposures. Irresistible and noninfectious agents must be considered in patients suspected of having an FBD. Signs and symptoms happen, alone or in combination, and laboratory testing may give essential diagnostic hints. A differential diagnosis of gastrointestinal tract illness ought to incorporate hidden medical conditions additional to food-borne causes. The differential diagnosis of patients giving neurological side effects due to an FBD is complex. The possible food-related causes to consider incorporate ingestion of contaminated fish, mushroom poisoning, and chemical

244  Chapter 9 poisoning. Since the ingestion of specific poisons (e.g., botulinum poison, tetrodotoxin) and chemicals (e.g., organophosphates) can be life threatening, a differential diagnosis must be made rapidly with concern toward aggressive treatment and life support measures (e.g., respiratory supportive care, antitoxin administration, atropine) and possible doctor’s facility affirmation. • •

• •

•

Stool cultures are shown if the patient is immune-compromised, febrile, has bleeding looseness of the bowels, severe abdominal pain, or if the sickness is clinically serious. Stool examination for parasites is demonstrated for patients with suggestive travel histories, who are immune-compromised, who suffer persistent diarrhea, or when the diarrheal illness is unresponsive to proper antimicrobial treatment. Blood cultures ought to be acquired when bacteremia or systemic infection is suspected. Viruses are tough to distinguish, as they are too small to see under a light microscope and are hard to culture. Infections are normally distinguished by testing feces for genetic markers that demonstrate whether a particular infection is available. Direct antigen detection tests and molecular biology techniques are accessible for fast distinguishing proof of certain bacterial, viral, and parasitic agents in clinical specimens. In a few circumstances, microbiologic and chemical laboratory testing of vomitus or involved food items likewise is justified.

9.7  General Management In individuals whose immune system is completely functional and normal, most foodborne illnesses are self-restricting and a complete recovery is typically made but immune compromised hosts will probably secure contamination after introduction to pathogens, have more serious sickness once the disease is set up, have scattered disease instead of confined contamination, and a higher death rate is observed. There is a wide range of sorts of food-borne sicknesses and they may require different therapies, contingent upon the dependable pathogen and side effects they cause. The decision of antimicrobial treatment ought to be based on: • • • •

clinical signs and symptoms organism recognized in clinical samples antimicrobial susceptibility tests; and appropriateness of treating with an antitoxin (some enteric bacterial diseases are best not treated)

The definitive diagnosis can be made just through stool culture or more propelled laboratory testing. Be that as it may, these outcomes ought not to postpone empirical treatment if an FBD is suspected. Empirical therapy ought to concentrate on symptom management, rehydration if the patient is clinically dehydrated, and antimicrobial treatment.

Challenges in Emerging Food-Borne Diseases  245 Indeed, antimicrobials have no impact on viruses and utilizing the antibiotics to treat a viral disease could bring about more harm than good. Many emerging zoonotic pathogens are becoming increasingly resistant to antimicrobial agents, largely because of the widespread use of antibiotics in the animal reservoir. Illnesses that are fundamentally diarrhea and vomiting can prompt lack of hydration if the individual loses more body liquids and salts (electrolytes) than they take in. • •

• •

Replacing the lost liquids and electrolytes and staying aware of liquid intake is important. If the diarrhea is severe, oral rehydration arrangements ought to be drunk to replace the fluid losses and avoid dehydration. Sports drinks do not replace the fluid loss effectively and should not be utilized for the treatment of diarrhea. Preparations of bismuth subsalicylate can reduce the term and seriousness of diarrhea. If looseness of the bowels and cramps happen, without bloody diarrhea or fever, taking an antidiarrheal medicine may give symptomatic relief; however, these medicines should be avoided if there is high fever or blood in the stools since they may aggravate the sickness (Samapundo et al., 2015).

Numerous diarrheal diseases are created by infections and will improve in 2 or 3 days without antitoxin treatment. The following are some of the specific treatments considered for bacterial, viral, parasitic, and noninfectious causes of food-borne illness:

9.8  Bacterial Agents •

Bacillus anthracis

Clinical syndrome: Nausea, vomiting, malaise, bloody diarrhea, acute abdominal pain. Treatment options: Penicillin is the first choice for naturally acquired gastrointestinal anthrax. Ciprofloxacin is the second option. •

Bacillus cereus (preformed enterotoxin)

Clinical syndrome: Sudden onset of severe nausea and vomiting. Diarrhea may be present. Treatment options: Supportive care. •

Bacillus cereus (diarrheal toxin)

Clinical syndrome: Abdominal cramps, watery diarrhea, nausea. Treatment options: Supportive care. •

Brucella abortus, Brucella melitensis, and Brucella suis

Clinical syndrome: Fever, chills, sweating, weakness, headache, muscle and joint pain, diarrhea, bloody stools during acute phase.

246  Chapter 9 Treatment options: Rifampin and doxycycline daily for >6 weeks. Infections with complications require combination therapy with rifampin, tetracycline, and an aminoglycoside. •

Campylobacter jejuni

Clinical syndrome: Diarrhea, cramps, fever, and vomiting; diarrhea may be bloody. Treatment options: Supportive care. For severe cases, antibiotics such as erythromycin and quinolones may be indicated early on in the diarrheal disease. Guillain-Barré syndrome can be a sequela. •

Clostridium botulinum—children and adults (preformed toxin)

Clinical syndrome: Vomiting, diarrhea, blurred vision, diplopia, dysphagia, and descending muscle weakness. Treatment options: Botulinum antitoxin is helpful if given early in the course of the illness. •

Clostridium botulinum—infants

Clinical syndrome: In infants 8 years). •

Vibrio parahaemolyticus

Clinical syndrome: Watery diarrhea, abdominal cramps, nausea, vomiting. Treatment options: Supportive care. Antibiotics are recommended in severe cases: tetracycline, doxycycline, gentamicin, and cefotaxime. •

Vibrio vulnificus

Clinical syndrome: Vomiting, diarrhea, abdominal pain, bacteremia, and wound infections. Treatment options: Supportive care and antibiotics; tetracycline, doxycycline, and ceftazidime are recommended.

248  Chapter 9 •

Yersinia enterocolytica and Yersinia pseudotuberculosis

Clinical syndrome: Appendicitis-like symptoms (diarrhea and vomiting, fever, and abdominal pain) occur primarily in older children and young adults. Patients may have a scarlatiniform rash with Y. pseudotuberculosis. Treatment options: Supportive care. If septicaemia or other invasive disease occurs, antibiotic therapy with gentamicin or cefotaxime (doxycycline and ciprofloxacin also effective) should be done.

9.9  Viral Agents •

Hepatitis A

HAV is also transmitted through contaminated food or water. Since HAV is present in the blood during acute infection, blood-borne transmission is also possible but rare. Clinical syndrome: Diarrhea, dark urine, jaundice, and flu-like symptoms, that is, fever, headache, nausea, and abdominal pain. Treatment options: Supportive care. Prevention is possible with immunization. Hepatitis A vaccination can be given to anyone 2 years of age and older, and has the advantage of providing long-term protection (at least 20 years). Hepatitis A vaccine is an inactivated HAV preparation. The first dose of vaccine provides protective anti-HAV levels within 30 days for >90% of vaccine recipients. •

Noroviruses (and other caliciviruses)

Clinical syndrome: Nausea, vomiting, abdominal cramping, diarrhea, fever, myalgia, and some headache. Diarrhea is more prevalent in adults and vomiting is more prevalent in children. Treatment options: There is no antiviral agent that can be used to treat NoV infections. Supportive care such as oral or intravenous fluids for rehydration should be provided. • Rotavirus Clinical syndrome: Vomiting, watery diarrhea, low-grade fever. Temporary lactose intolerance may occur. Infants and children, the elderly, and immune compromised are especially vulnerable. Treatment options: Supportive care. Severe diarrhea may require fluid and electrolyte replacement. •

Other viral agents (astroviruses, adenoviruses, parvoviruses)

Clinical syndrome: Nausea, vomiting, diarrhea, malaise, abdominal pain, headache, fever. Treatment options: Supportive care.

Challenges in Emerging Food-Borne Diseases  249

9.10  Parasitic Agents •

Angiostrongylus cantonensis

Clinical syndrome: Severe headaches, nausea, vomiting, neck stiffness, paresthesias, hyperesthesia, seizures, and other neurologic abnormalities. Treatment options: Supportive care. Repeat lumbar punctures and use of corticosteroid therapy may be used for more severely ill patients. •

Cryptosporidium

Clinical syndrome: Diarrhea (usually watery), stomach cramps, upset stomach, slight fever. Treatment options: Supportive care, self-limited. If severe, consider paromomycin for 7 days. For children aged 1–11 years, consider nitazoxanide for 3 days. •

Cyclospora cayetanensis

Clinical syndrome: Diarrhea (usually watery), loss of appetite, substantial loss of weight, stomach cramps, nausea, vomiting, fatigue. Treatment options: TMP-SMX for 7 days. •

Entamoeba histolytica

Clinical syndrome: Diarrhea (often bloody), frequent bowel movements, lower abdominal pain. Treatment options: Metronidazole and a luminal agent (iodoquinol or paromomycin). •

Giardia lamblia

Clinical syndrome: Diarrhea, stomach cramps, gas. Treatment options: Metronidazole. •

Toxoplasma gondii

Clinical syndrome: Generally asymptomatic, lymphadenopathy, and/or a flu-like illness. In immune-compromised patients, central nervous system (CNS) disease, myocarditis, or pneumonitis is often seen. Treatment options: Asymptomatic healthy, but infected, persons do not require treatment. Spiramycin or pyrimethamine plus sulfadiazine may be used for pregnant women. Pyrimethamine plus sulfadiazine may be used for immune-compromised persons in specific cases. Pyrimethamine plus sulfadiazine (with or without steroids) may be given for ocular disease when indicated. Folinic acid is given with pyrimethamine plus sulfadiazine to counteract bone marrow suppression. •

Toxoplasma gondii (congenital infection)

250  Chapter 9 Toxoplasma gondii may be transmitted transplacentally to the fetus if the mother acquired toxoplasmosis during pregnancy. There is almost no risk of transplacental transmission if the mother was infected prior to conception. Women with positive IgG antibody tests for toxoplasmosis at the onset of pregnancy are not at risk for developing acute toxoplasmosis. Treatment options: Treatment of the mother may reduce severity and/or incidence of congenital infection. Most infected infants have few symptoms at birth. Later, they will generally develop signs of congenital toxoplasmosis (mental retardation, severely impaired eyesight, cerebral palsy, seizures), unless the infection is treated. •

Trichinella spiralis

Clinical syndrome: nausea, diarrhea, vomiting, fatigue, fever, abdominal discomfort followed by muscle soreness, weakness, and occasional cardiac and neurologic complications. Treatment options: Supportive care plus mebendazole or albendazole.

9.11  Noninfectious Agents • Antimony Clinical syndrome: Vomiting, metallic taste. Treatment options: Supportive care. • Arsenic Clinical syndrome: Vomiting, colic, diarrhea. Treatment options: Gastric lavage, BAL (dimercaprol). • Cadmium Clinical syndrome: Nausea, vomiting, myalgia, increase in salivation, stomach pain. Treatment options: Supportive care. •

Ciguatera fish poisoning (ciguatera toxin)

Clinical syndrome: abdominal pain, nausea, vomiting, diarrhea, paresthesias, reversal of hot or cold, pain, weakness, bradycardia, hypotension, increase in T wave abnormalities. Treatment options: Supportive care, IV mannitol. Children are more vulnerable. • Copper Clinical syndrome: Nausea, vomiting, blue or green vomitus.

Challenges in Emerging Food-Borne Diseases  251 Treatment options: Supportive care. • Mercury Clinical syndrome: Numbness, weakness of legs, spastic paralysis, impaired vision, blindness, coma. Pregnant women and the developing fetus are especially vulnerable. Treatment options: Supportive care. •

Mushroom toxins, short-acting (museinol, muscarine, psilocybin, copriusartemetaris, ibotenic acid)

Clinical syndrome: Vomiting, diarrhea, confusion, visual disturbance, salivation, diaphoresis, hallucinations, disulfiram-like reaction, confusion, visual disturbance. Treatment options: Supportive care. •

Mushroom toxin, long-acting (amanitin)

Clinical syndrome: Diarrhea, abdominal cramps, leading to hepatic and renal failure. Treatment options: Supportive care, life-threatening, may need life support. •

Nitrite poisoning Pesticides (organophosphates or carbamates)

Clinical syndrome: Nausea, vomiting, cyanosis, headache, dizziness, weakness, loss of consciousness, chocolate-brown colored blood. Treatment options: Supportive care, methylene blue. •

Pesticides (organophosphates or carbamates)

Clinical syndrome: Nausea, vomiting, abdominal cramps, diarrhea, headache, nervousness, blurred vision, twitching, convulsions, salivation, and meiosis. Treatment options: Atropine; 2-PAM (Pralidoxime) is used when atropine is not able to control symptoms and is rarely necessary for carbamate poisoning. •

Puffer fish (tetrodotoxin)

Clinical syndrome: Paresthesias, vomiting, diarrhea, abdominal pain, ascending paralysis, respiratory failure. Treatment options: Life-threatening, may need respiratory support. •

Scombroid (histamine)

Clinical syndrome: Flushing, rash, burning sensation of skin, mouth, and throat, dizziness, urticaria, paresthesias.

252  Chapter 9 Treatment options: Life-threatening, may need respiratory support. Supportive care and antihistamines. •

Shellfish toxins (diarrheic, neurotoxic, amnesic)

Clinical syndrome: Nausea, vomiting, diarrhea, and abdominal pain accompanied by chills, headache, and fever. Tingling and numbness of lips, tongue, and throat, muscular aches, dizziness, and reversal of the sensations of hot and cold, neurologic problems, such as confusion, memory loss, disorientation, seizure, coma. Treatment options: Supportive care, generally self-limiting. The elderly are especially sensitive to ASP. •

Shellfish toxins (paralytic shellfish poisoning)

Clinical syndrome: Diarrhea, nausea, vomiting leading to paresthesias of mouth, lips, weakness, dysphasia, dysphonia, respiratory paralysis. Treatment options: Life-threatening, may need respiratory support. •

Sodium fluoride

Clinical syndrome: Salty or soapy taste, numbness of mouth, vomiting, diarrhea, dilated pupils, spasms, pallor, shock, collapse. Treatment options: Supportive care. • Thallium Clinical syndrome: Nausea, vomiting, diarrhea, painful paresthesias, motor polyneuropathy, hair loss. Treatment options: Supportive care. • Tin Clinical syndrome: Nausea, vomiting, diarrhea. Treatment options: Supportive care. • Vomitoxin Clinical syndrome: Nausea, headache, abdominal pain, vomiting. Treatment options: Supportive care. • Zinc Clinical syndrome: Stomach cramps, nausea, vomiting, diarrhea, myalgia. Treatment options: Supportive care.

Challenges in Emerging Food-Borne Diseases  253

9.12  Emerging Challenges and Technologies Issues that were once local are now progressively national or worldwide and they are progressively obvious. Harvest, transport, processing, storage and finally during food preparation and storage by consumers are all elements that add to the many challenges of keeping the food supply safe. One of the real development sections in the food retail industry is minimally processed food. This moderately new market pattern has strived to grow new advances or new utilizations of customary innovations to protect the qualities of excellence in the items, increase the time span of usability, and enhance their microbiological safety. The majority of these innovations for gentle preservation are based on the hurdle concept. For example: Common salt, for quite a long time used to hinder microbes, does not impel antiviral impact. Interestingly, it has been exhibited that it ensures NoV and HAV amid highweight disinfection of meat (García-Canas et al., 2012). The idea of food security gets to be basic when the food is prepared and served to hospitalized patients since they are more prone to food-borne ailments than the overall public as the vast majority of them have low-resistance. What is more, food-borne sicknesses may bring about gastroenteritis which may impede the retention of supplements in patients. This may prompt the malnutrition condition of the patient, and in some patients the condition may become worse. So these food-borne sicknesses turn out to be an exceptionally basic issue in doctor’s facilities. Food safety is an extraordinary worry and concern of the food business; however, health organizations give less consideration (Abubakar et al., 2007). In a recent research study, it was found that in 60% of the cases, flies and animals were obvious around the stalls and 65% did not have access to drinking water. Food was served with bare hands and a majority did not wash their hands after handling cash. Also, 70% of the sellers did not chill precooked food. The conditions in which street food vendors work are generally unsuitable from a food security perspective and an effort ought to be made to give them with the satisfactory base including consumable water, toilets and waste disposal and transfer facilities. The emerging challenges of FBDs are boundless. The distinguishing proof, aversion, control, and monitoring of FBP and microbial parasites remain one of the food industry’s most pressing issues. Although numerous biochemical and microbiological advances are now accessible for the normal appraisal of food items, these are not sufficiently precise to decide new microbiological hazards in foods (Suo et al., 2010).

254  Chapter 9 •

Conventional techniques usually utilized as a part of the field depend on the development of target pathogens or indicator microorganisms on particular media. Be that as it may, these techniques require several days for completion and are tedious. Also, culture-based techniques sometimes lack specificity in selecting or recognizing unknown pathogens in food (Horakova et al., 2008; Liu et al., 2008).

New molecular-based techniques for detecting pathogens in food and water have various advantages over conventional culture-based techniques, including the capacity to recognize and list pathogens quickly and continuously, the capacity to negatively screen tests without the need for culture, the ability to connect recognition with a particular strain ID and affirmation, and the chance to identify just viable cells. Several DNA-based techniques have been created to detect pathogenic microscopic organisms. The polymerase chain response (PCR) is a very helpful and imperative method for the discovery of DNA of a particular microorganism (Llop et al., 1999; Mao et al., 2007; Sawada et al., 1992; Takaishi et al., 2003; Versalovic et al., 1995; Fricker et al., 2007; Fukushima et al., 2007). Real-time PCR frameworks for quantitative investigations of pathogenic microscopic organisms have been developed (Lopez and Pardo, 2010; Call et al., 2003; Cremonesi et al., 2009). However, it is one of the speediest genetic-based analytical methods utilized against microorganisms, especially in the industries, for example, RTE meat and poultry, refrigerated food and insignificantly prepared produce. PCR, which offers high specificity and amazingly fast investigative turnaround times, may turn out to be more prominent as economical test strategies and instruments are presented for commercial use. The proceeded with changes in PCR and other DNA fingerprinting advances, including tests, ribotyping, and beat field gel electrophoresis, will help the industry in distinguishing noteworthy food-borne risks. However, these methodologies cannot simultaneously distinguish different pathogenic microorganisms in parallel utilizing a single experimental cycle. Recently, microarray technology has empowered high-throughput discovery of various pathogens in a large number and has been generally connected to DNA recognition and genotyping because of its miniature arrangement, high execution, and simplicity of computerization (Drost et al., 2009; Hacia et al., 2000; Huang et al., 2006; Jin et al., 2006; Kim et al., 2003; Park et al., 2004; Pasquini et al., 2008; Peplies et al., 2003; Tiberini et al., 2010; Resch-Genger et al., 2008; Chan et al., 2002). The labeled particles utilized as a part of DNA microarrays to identify binding events are generally fluorescent dyes. In spite of the fact that they give a sensitive, safe, and minimal cost identification framework, they experience several limitations. First, organic dyes are sensitive to photo bleaching and are regularly not sufficiently brilliant for the evaluation of a particular signal over background. Second, the fluorescence ranges of organic dyes are not

Challenges in Emerging Food-Borne Diseases  255 symmetric and each fluorophore is described by its particular ideal wavelength of excitation, which restrains its multiplexing capacity (Tan et al., 2002). Use of nanotechnology for checking and division of nourishment borne pathogens is a dynamic territory of examination. •

Advances in nanotechnology have given a novel and promising class of semiconductor nanocrystal quantum spots (QDs). QDs have various points of interest over traditional dyes, for example, a high quantum yield, long photostability, and high extinction coefficient (Pathak et al., 2001; Gerion et al., 2003). Many investigations demonstrated that QDs have better sensitivity and photostability compared to traditional dyes (Xiao and Barker, 2004; Lee et al., 2004; Hahn et al., 2005; Gazouli et al., 2010; Zhao et al., 2009; Wang et al., 2015; Augustine et al., 2016).

Fluoroimmunoassay strategy utilizing multicolor quantum dots (qds) as fluorescent probes for simultaneous, sensitive, and rapid identification of three types of major pathogenic bacteria. This multiplex fluoroimmunoassay technique has been connected to various types of food samples to demonstrate its anti-interference capacity and extensive variety of application (Badilita et al., 2014). Magnetic nanoparticles are brought into ordinary pathogen detection techniques to make them simple, fast, exceedingly selective, and sensitive. When incorporated with PCR, immunoassay, spectrometry, and biosensors, these magnetic nanoparticles make a quick or online detection of pathogens. Superparamagnetic nanoparticles have enhanced the detection sensitivity of pathogens utilizing the PCR method by 10–100 times (Inoue et al., 2015). Metabolomics has attained tremendous fame in the most recent 10 years for the investigation of food ingredients, food processing, and food pathogens (Vikram et al., 2004; Özogul et al., 2016; Singh, 2013). Nutraceuticals, botanicals, vitamins, and dietary supplements are the greatest development zones in the food business, and rising food security confirmation issues connected with these items will require new advances in testing and innovation. As good as ever, extraction systems to upgrade evacuation of contamination from organic solvents while as yet keeping up a measure of active ingredients will be a need for industries in this field. Other expected enhancements in innovation incorporate high performance liquid chromatography and liquid chromatography/mass spectrometry frameworks for botanicals and aflatoxin examinations. ELISA strategies grew particularly for difficult analysis, for example, in procedure control for B vitamins and bioassay methods for synthetic constituents in herbal and nutraceutical investigation to guarantee that the item is effectively described, free of contaminants and nontoxic.

256  Chapter 9 Water quality and security issues are developing as critical issues on the world stage. Water quality testing capacities as test strips, portable or compact meters, and automated analyzers have enhanced in the previous couple of years. Such instruments distinguish potential contaminants and measure process control adequacy. Tests are accessible for measuring aluminum, arsenic, chloride, lead, pH, nitrate, phosphate, and different chemicals. All FBP are capable of decarboxylating more than one amino acid and the inhibition impact of zeolite on AMN and pas production by pathogens relies upon the zeolite doses and the bacterial strains. This could be utilized as a part of the food sections to avert undesirable compound production (AMN and amines) by pathogenic microscopic organisms which constitute a danger to the purchaser’s well-being and can bring about several FBDs (Vikas et al., 2012). Advancements to enhance the shelf life are developed to guarantee food security amid its supply are time-temperature integrator (TTI) devices, which go about as “smart” labels with respect to the temperature presentation history of the item as it goes from spot of production, through different supply channels to the buyer. By recording and outwardly indicating changes in mechanical, compound, enzymatic, or microbiological frameworks, TTIs can be utilized to screen the temperature presentation of individual food packages, containers, or pallet loads, and the information accumulated from every phase of product supply could be utilized to recognize food security issues and actualize remedial activities at the proper stages. Notwithstanding the TTIs available at present, an assortment of other enhanced compact temperature recording devices became important apparatuses in food security applications. Plant can serve as a good therapeutic agent against various infections (Pandey et al., 2012; Akthar et al., 2014; Costa et al., 2010). The substances obtained from therapeutic plants have been utilized for quite a long time as a part of conventional medication to treat various infections and a majority are used for their antimicrobial properties. The study proposes that test plants could be a potential possibility for producing new antimicrobial medications against the extensive variety of pathogenic microscopic organisms and parasitic strains (Lund and O’Brien, 2011). In developing nations, food poisoning and food-borne contamination brought on Salmonella spp., E. coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus and Candida spp. have been perceived as a significant issue for the weakening of the nature of food products (Schelz et al., 2010; Yang et al., 2009). Utilization of medicines as antimicrobials is cost-effective, environmentally safe, and an option to control microbial contaminations in resistance cases (Zhao et al., 2015). Thus, the medicinal plants can serve as a potential supply for the improvement of novel chemotherapeutic agents (Hudson et al., 2009).

Challenges in Emerging Food-Borne Diseases  257 Sugarcane bagasse contains natural substances that can altogether restrain FBP growth and development. The after effects of scanning electron microscopy and transmission electron microscopy demonstrated that the sugarcane bagasse extract may change cell morphology and internal structure (Feagins et al., 2008). While no single, fast technology or innovation is accessible right now to substantiate whether a harvest, food, or food ingredient has been genetically altered, present controls require quantitative detection frameworks, for example, quantitative competitive PCR or real-time PCR for DNA identification and discovery. ELISA frameworks utilizing immunochemical methodologies are as of now used to detect target proteins. Since detection techniques enhance to meet food security affirmation and export testing necessities and new labeling prerequisites all over the world, the requirement for check of those frameworks for use by the food industry will increase.

9.12.1  Resistance of Food-Borne Viruses and Systems for Counteractive Action Food-borne virus has a more complex structure and is less resistant to ecological factors. NoV, HAV, and HEV, being deprived of a lipid envelope and having a protein coat, are just unaffected by traditional measures taken to control food-borne microorganisms, for example, chilling and freezing. Processing of meat in altered climate likewise does not impact a decrease of viral contamination. Virus infectivity is lost when dried on surfaces but continues for 30 days or more when dried on paper, material, plastic, aluminum, and earthenware production. Length of survival is connected to the kind of surface, air dampness, and the presence or absence of organic material, and in addition to the structure of the viral capsid proteins. Apart from ultrahigh temperature treatment, no strategies would totally inactivate >3 log of foodborne infection, and if food gets to be contaminated during the preparation, the infection will stay sufficiently dynamic to bring about contamination. UV light is a viable and clean apparatus for food-borne infection control but has drawbacks because of the absence of meat matrix penetration. Ozone is very powerful against these three RNA infections because of activation of protein peroxidation of capsid, and some of the time ozonized water is utilized to flush raw meat; be that as it may, it adjusts the taste and shade of food (Hu et al., 2016). Appropriate cooking of porcine liver destroys the infectivity of HEV. Boiling in water for 5 min and stir-frying for 5 min at 191°C to an internal temperature of 71°C inactivates this viral contamination. Be that as it may, incubating of contaminated liver for 1 h at 56°C was not a suitable heat treatment (Waters et al., 2011). Since pork is typically cooked until well done, it is not

258  Chapter 9 viewed as likely that hepatitis E is a typical FBD. It is clear that some sporadic diseases result from consuming undercooked meat and from cross-contamination amid food processing. Testing of antiviral adequacy is cumbersome with regard to human NoV. This infection is not cultivable, so some surrogate cultivable strains (murine or canine NoV) are utilized to access interventions. These every now and again react contrastingly to stress created by antiviral treatment when contrasted with human adjusted strains. The main instrument that remains for discovery of NoV is real-time reverse transcription PCR which is sufficiently sensitive to distinguish as low as 10 viral duplicates for each gram. In any case, totally inactivated viral particles that represent no danger to general well-being contain RNA, henceforth bringing about a positive virus assay. The RNA will be degraded eventually, but it is unknown to what extent this will take in various environments. To address this variety of rising food security challenges, the greater part of the players in the food processing, manufacturing, and supply chain will need to stay cautious in setting up effective approaches, conventions, and techniques to recognize and decrease the occurrence of FBDs bringing on dangers. The food business has long comprehended the advantages to the matter of a far-reaching, science-based food safety program. As food security administration practices and frameworks turn out to be more refined and custom-made to every particular connection in the food processing and supply chain, the interest for science-based advances will increment in a variety of regions.

9.13  Preventive Measures Once a food-borne sickness is identified, various critical inquiries should be offered an explanation to build up a sound way to deal with counteractive action. The definitive objective for public health and food security authorities is not simply halting FBD outbreaks once they happen, but preventing them from happening in any case. Long-term counteractive action of food-borne episodes takes the activities of many partners in the food production chain, extending from farm to table. Food safety methodologies are basic to reducing pathogens brought about by FBD. However, there is no real way to totally dispense with the danger of expending contaminated food products. At the point when prevention efforts come up short, fast identification of the contaminated product (CP) is key (Hennekinne et al., 2012). The systemic methodology for food safety is shown in Table 9.5. Scientific improvements in the field of agriculture prompted the expanded use of modernday chemicals: insecticides, pesticides, herbicides, veterinary drugs, and many exogenous

Table 9.5: A system approach to food safety Sources

Pathways

Drivers

Pathogens

Farms

Globalization

Reduced geographical barriers to spread (of new variants)

Inadequate sanitation: higher pathogen loads Intensified contact structures

Minimal processing

Adaptation

Laboratory methods

Discovery of new pathogens or variants omics approaches

Population contact structures

Food choice

Animal friendly and organic production

Irrigation water quality Transfer of virulence factors Antimicrobial resistance Species jumps (spillover from epizootics or exploitation of new agricultural areas) Psychrotrophs Re-emerging pathogens

Reduced AMR

Waste recycling New reservoirs

Outcomes Preparation/ Consumption

Long and complex supply chains Varying hygiene levels

Increased risk

Less kill steps

Increased risk if not well controlled Increased observed risk

Water, energy savings cleaning, process Ingredient water quality Increased survival

Increased risk Increased infectivity

Contact zoonosis (MRSA,Q-fever)

Exotic/ethinic foods Regional products

Re-emergence (Trichinella, Toxoplasma) Higher (Campylobacter) or lower prevalence (Salmonella)

Public Health

Increased risk

Increased risk

No or mild processing less heat treatment Increased preprocessing and packaging

Convenience foods year round availability Healthy foods Less fat salt sugar Eating outside home

Increased risk

Risk no clear

Data from Havellar, A.H., Burl, S., Jong, A., Jonge, R., Zwietering, M.H., Kuile, B.H., 2010. Future challenges to microbial food safety. Int. J. Food Microbiol. 139, S79–S94.

Challenges in Emerging Food-Borne Diseases  259

Water, waste, and energy Evolution

Processing/ Distribution

260  Chapter 9 chemicals with undesirable compounds, for example, plant toxicants and mycotoxins, which can penetrate the food supply chain. Their residual effects on foods have magnified the associated risk, and they need to be scientifically monitored, with precise limits for labeling foods as safe for human consumption. Preventive measures and safety ventures at every level: •

•

Producing and collecting food • quality confirmation programs at egg ranches • safe agricultural farm practices • efforts to keep shellfish harvest beds free of sewage defilement Processing nourishments • inspection frameworks at meat handling plants

US retail meat and poultry are much of the time contaminated with multidrug-resistant S. Aureus (Barry-Ryan, 2015). A few microscopic organisms, for example, Salmonella, E. coli, and Campylobacter can be found in raw meat; consequently, there is a need to cook these foodstuffs altogether to keep away from food poisoning outbreaks (Romani et al., 2006). So there is a need for the use of pasteurization, canning, cooking, illumination, and different strides to slaughter pathogens in food production and processing.

9.13.1  Preventive Measures in Distribution and Preparation of Food Buyer particulars for safety of food in food acquiring contracts •

Training about food safety and sanitation measures • Separate the food to maintain a strategic distance from cross-contamination. Crosspollution is the word for how microorganisms, infections, and parasites can be spread starting with one food item then onto the next. This is particularly genuine when taking care of raw meat, poultry, fish, and eggs, so keep these food and their juices far from prepared and ready-to-eat foods. • Proper hand-washing methodology and facilities. Hand-washing appears to significantly lessen levels of contamination in homes and work places, and minimize the transmission of FBDs. • Cook to a legitimate temperature. Foods are appropriately cooked when they are heated for a sufficiently long time and at a sufficiently high temperature to kill the unsafe pathogens that cause a food-borne ailment. • Prompt Refrigeration. Chilly temperatures keep unsafe pathogens from growing and increasing. Along these lines, refrigerating not higher than 40°F and solidifying at 0°F is appropriate. • Encouraging food specialists not to work when they are sick • Food safety instruction for purchasers

Challenges in Emerging Food-Borne Diseases  261 The WHO is currently extending the five keys to safer food idea to cover extra gatherings over the farm to table continuum to advance safe handling practices of food. The five keys practices are: 1. 2. 3. 4. 5.

personal cleanliness avoiding animal fecal contamination in fields using treated fecal waste assess the risks from irrigation water keep harvest and storage systems clean and dry.

To control the food safety and spoilage, various preservation strategies have been developed. The use of natural antimicrobials is one such recent strategy, with increasing research and commercial applications (Jha, 2016). Chemical additives have dependably been utilized as antimicrobials to hinder the development of FBP, which may lead to chemical harm in people. Some studies have reported that common phytochemicals, for example, phenolics, demonstrated huge antibacterial, antiviral, and disinfectant activities (FAO, 2003). Sustaining food safety standards will rely on constant vigilance maintained by monitoring and surveillance at the same time, with the rising significance of other sustenance-related issues, for example, food security, weight and environmental change, and rivalry for resources later on to empower this might be furious. Food guidelines are administrative instruments, frequently called the food laws, which are authorized to secure customers against dangerous items, corruption, and misrepresentation; to guarantee quality consistence; and in addition to ensure legit food makers, processors, merchants, and dealers. They additionally encourage the development of merchandise within and between nations by giving a typical lexicon to food quality and well-being. Guidelines are intended for some reasons. The principal concern is quality and security; however, any standard or control cannot be set to the apex level of value; along these lines, above-normal quality is for the most part mulled over in requirement on a more extensive scale. Reasonable points of confinement are frequently characterized in the greater part of food standards to recognize maximum and minimum levels and consequently give a guide to keeping away from food having a hazard or any health risk. Establishing these points of confinement or levels relies upon the planned target of examination by law implementation organizations or agencies and bodies required in authorizing the laws or models. Scientific advancements in the field of agriculture had prompted expanded utilization of cutting-edge chemicals like pesticides, herbicides, veterinary medications, and numerous exogenous chemicals with undesirable mixes, for instance, plant toxicants and mycotoxins, which can enter the food production network. Their remaining consequences for food have amplified the related danger, and they should be scientifically checked, with premise limits for naming foods as safe for human utilization.

262  Chapter 9 Standards help to ensure that food is wholesome and contains whatever the label claims, and they minimize the chances of deceit in terms of quality and economic value and associated risk. Food-borne episodes are examined for two primary reasons. The first is to distinguish and control a progressing source by emergency activity: product review, eatery conclusion, or other but however complete solutions. The second reason is to figure out how to keep future comparable outbreaks from happening. Over the long term, this second reason will have a considerably great effect on general health than basically identifying and ending the episodes. Interventions amid outbreaks regularly rely upon having adequate epidemiologic information to act with certainty, without waiting for a complete research center test, especially if deadly sicknesses are included. Outbreak Investigations may discover new pathogens, new food vehicles, and unsuspected holes in the food safety framework. They can enhance experimental comprehension of how the contamination happened at particular focuses in the food supply chain, of the odds that it might happen once more, and how it might be decreased or prevented. Frequently, outbreak examinations bring up issues that lead to new research to better see how contamination occurs and how it can be avoided or reduced. The present situation has many ramifications for the act of general health, beginning at the local level. One is that when diffuse episodes are identified, a local health department may need to explore a couple of cases that are a part of a bigger outbreak in spite of their evidently small local impact. Second, a local outbreak may proclaim the initially identified indication of a national or even international event. Counteractive action can be “implicit” to the business by distinguishing and controlling the key focuses from field, farm, or fishing ground to the dining table at which pollution can either happen or be avoided out. The general procedure known as Hazard Analysis and Critical Control Points (HACCP) replaces the system of final product review. Some basic control methodologies are clearly self-evident, once the reality of microbial contamination is recognized.

9.14 Conclusion Eating healthy is a journey shaped by many factors, including our stage of our life, situations, preferences, access to food, culture, traditions, and the personal decisions we make over time. As we discussed above, the ultimate goal for public health and food safety officials is not just stopping FBD outbreaks once they occur, but preventing them from happening in the first place. Food security exists when all individuals, at all times, have admittance to adequate safe and nutritious food to meet their dietary needs and food preferences for a dynamic and

Challenges in Emerging Food-Borne Diseases  263 sound life. Many of the most significant drivers of health and health care costs are outside of the scope of health care alone. For this, there is a need to create settings where healthy choices are available and affordable to the community. Professionals, policymakers, partners, industries, families, and individuals can help others in their journey to make healthy eating a part of their lives and avoid the constantly emerging challenges of FBDs and their outbreaks in the future.

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Further Reading Djenane, D., Yangüela, J., Roncalés, P., Aider, M., 2013. Use of essential oils as natural food preservatives: effect on the growth of Salmonella enteritidisin liquid whole eggs stored under abuse refrigerated conditions. J. Food Res. 2 (3), 65–78. Mrityunjoy, A., Kaniz, F., Fahmida, J., Shanzida, J.S., Uddin, M.A., Noor, R., 2013. Prevalence of Vibrio cholera in different food samples in the city of Dhaka, Bangladesh. Int. Food Res. J. 20 (2), 1017–1022. Rahman, F., Noor, R., 2012. Prevalence of pathogenic bacteria in common salad vegetables of Dhaka Metropolis. Bangladesh J. Bot. 41 (2), 159–162. Sarker, N., Islam, S., Hasan, M., Kabir, F., Uddin, M.A., Noor, R., 2013. Use of multiplex PCR assay for detection of diarrhoeagenic Escherichia coli in street vended food items. Am. J. Life Sci. 1 (6), 267–272.

CHAPTE R 10

Opportunistic Food-Borne Pathogens Vincenzina Fusco⁎, Hikmate Abriouel†, Nabil Benomar†, Jan Kabisch‡, Daniele Chieffi⁎, Gyu-Sung Cho‡, Charles M.A.P. Franz‡ ⁎

Institute of Sciences of Food Production, National Research Council of Italy (CNR-ISPA), Bari, Italy University Jaen, Jaén, Spain ‡Max Rubner-Institute, Kiel, Germany

†

10.1 Introduction Opportunistic pathogens are microorganisms that are usually harmless in healthy, immunocompetent persons but may become virulent in compromised hosts such as the immunocompromised, or people with underlying disease. These microorganisms may cause severe infections or diseases, such as hospital acquired infections, including bloodstream infections, pneumonia, surgical site infections, diarrhea, and urinary tract infections. Many human groups have been identified as being more susceptible to opportunistic infections, often as a consequence of modifications in their immune system. These include the elderly (mainly due to a progressive immunosenescence), infants, and young children (mainly due to the immaturity of their immune system), pregnant women (due to the immunotolerance to the fetus), and people who are immunocompromised as a result of disease (cancer and HIV patients, people affected by hunger and hidden hunger, etc.) or as a result of medical interventions (chemotherapy and radiotherapy for cancer, immunosuppressive agents for organ transplant recipients, long-term antibiotic treatments, etc.). These vulnerable groups of persons may be nearly 20% of the population in countries such as the United Kingdom and United States (Lund, 2015). Moreover, their number is expected to significantly increase worldwide, due to the demographic shift toward an elderly population and the expected growing number of immunocompromised people who are more susceptible to opportunistic infections. In addition, many factors such as the exposure to antibiotics or the consumption of high-fat and high-sugar diets may provoke a reduction of the human microbiota biodiversity and heighten the susceptibility to opportunistic pathogens (Josephs-Spaulding et al., 2016). Such pathogens may then grow and overtake residential microorganisms, which results in dysbiosis and eventually leads to disease (Josephs-Spaulding et al., 2016). The settings where opportunistic infections occur most are residential homes (domestically acquired infections) (Harrison et al., 2013), hospitals (health care associated or nosocomial infections) (Sydnor and Perl, 2011), nurseries (neonatal health care-associated infections) (Birt et al., 2016), Food Safety and Preservation. https://doi.org/10.1016/B978-0-12-814956-0.00010-X © 2018 Elsevier Inc. All rights reserved.

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270  Chapter 10 and community health care settings (Mackay et al., 2014) such as nursing homes (Montoya and Mody, 2011). As recently highlighted by Berg et al. (2014), although opportunistic pathogens belong to a high number of different species of bacteria, virus and fungi, they have in common some of the following characteristics: (i) elicit antagonistic activity against other microorganisms, (ii) are versatile in their nutritional needs, (iii) cultivable, (iv) copiotrophs, (v) highly competitive, (vi) able to form biofilms, (vii) hypermutators, and (viii) have antibiotic and toxin resistance (Berg et al., 2014). Considering that most of these strainspecific features are acquired through horizontal gene transfer (Juhas, 2015), it is conceivable that further species of novel “superbugs” will emerge in the near future as opportunistic pathogens. Opportunistic pathogens mainly occur in natural environments including soil and vegetation (Berg et al., 2014) and may be transferred via foods (Lund and O'Brien, 2011; Lund, 2015). Apart from well-known and studied opportunistic pathogens, such as Pseudomonas aeruginosa (Huber et al., 2016) and methicillin-resistant Staphylococcus aureus (Doulgeraki et al., 2017), numerous species of different taxa, even generally recognized as safe (GRAS, United States, http://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS) species or with qualified presumption of safety (QPS, E.U. https://www.efsa.europa.eu/en/topics/topic/ qps) status have been found capable of acting as opportunistic pathogens. Although Gram-positive lactic acid bacteria (LAB) have been used since ancient times to produce fermented foods (Stiles and Holzapfel, 1997), and strains of this group even have probiotic activity (Khani et al., 2012), some species belonging to the Enterococcus, Lactobacillus, and Weissella genera, occurring in several environments including foods, have been found to act as opportunistic pathogens (Abriouel et al., 2015; Franz et al., 2003; Fusco et al., 2015; Goldstein et al., 2015). On the other hand, the Gram-negative Enterobacteriaceae such as Klebsiella, Enterobacter, Citrobacter, and Serratia are members of the normal intestinal microbiota of humans and animals and usually are harmless commensals. They are also found in several environments including foods, where they act as spoiling bacteria. Nevertheless, species of this group, such as Serratia marcescens, Klebsiella pneumonia, Enterobacter cloacae, and Citrobacter freundii, are among the most frequently occurring opportunistic pathogens. The vegetable microbiome has recently been recognized as a probable reservoir of several of these opportunistic species (Berg et al., 2014). Indeed, human infections and outbreak due to the consumption of vegetables and fruits contaminated with these opportunistic pathogens are increasing together with their increased consumption (Olaimat and Holley, 2012), prompting the need of adequate strategies to ensure the safety of these products. In this chapter, an overview of the main opportunistic pathogens which occur in foods is provided, and the question of whether there is a food-borne route for possible transmission and infection will be examined for each of the opportunistic pathogen species mentioned below.

Opportunistic Food-Borne Pathogens  271

10.2  Gram-Positive Opportunistic Pathogens 10.2.1  Enterococcus faecalis and Enterococcus faecium The enterococci are important in environmental, food, and clinical microbiology, as they have a very robust nature and can exist in diverse environments. For example, enterococci can occur in surface waters and seawater, as well as in municipal and hospital wastewaters. However, there are many possible sources for enterococci apart from sewage, including animal waste, invertebrates, plants, and soils (Harwood et al., 2004). Owing to their intestinal habitat in food animals, they can contaminate animal derived foods such as milk, or meat at the time of slaughter, and can lead to the contamination of and persistence in animal products and in food producing environments. Enterococci are typical opportunistic human pathogens. They can cause infections in patients with severe underlying disease, who have received surgery or who are immunocompromised (Morrison et al., 1997). They are commonly associated with nosocomial infections and cause bacteremia, endocarditis, urinary tract, and other infections (Morrison et al., 1997; Murray, 1990; Top et al., 2008). Enterococci are among the most prevalent organisms associated with nosocomial infections, accounting for ~12% of these in the United States (Linden and Miller, 1999). Enterococcus (Ent.). faecium and Ent. faecalis cause the vast majority of enterococcal nosocomial infections (Prieto et al., 2016). Ent. faecalis was earlier noticed to predominate (>60%) among human infections, while Ent. faecium was associated with the remainder (Jett et al., 1994; Top et al., 2008; Sievert et al., 2013). However, the ratio of Ent. faecalis to Ent. faecium infections changed toward Ent. faecium in the United States in the late 1990s (Mundy et al., 2000; Treitman et al., 2005), while in Europe the first reports of increased infection due to Ent. faecium were published in the mid-1990s (Top et al., 2008). Other enterococcal species are rarely associated with human disease, but strains of Ent. avium, Ent. casseliflavus, Ent. durans, Ent. gallinarum, Ent. hirae, Ent. mundtii, and Ent. raffinosus have been reported in association with infections (Top et al., 2008). A multitude of enterococcal virulence factors have been described over the past years and include factors promoting colonization (surface adhesins, e.g., pili (ebp-pili), aggregations substance, AS; enterococcal surface protein, Esp; Enterococcus endocarditis antigen, EfaA; adhesin to collagen, Ace; fibronectin binding protein, Fnm) and invasion (AS) of host tissue, translocation through epithelial cells (AS), and evasion of the hosťs immune response (Gelatinase-Gel, Esp, Ace, cytolysin-Cyl, capsule) (Table 10.1). Furthermore, certain strains produce pathological changes either directly, by cell toxin production (e.g., Cyl), or indirectly, by inflammation (sex pheromones) (Jett et al., 1994; Franz et al., 2003; Montealegre et al., 2015). Enterococci are also known for their ability to quickly acquire and disseminate genes encoding resistance toward antibiotics (Werner et al., 2013). Intrinsic antibiotic resistances in enterococci include resistance to cephalosporins, β-lactams, sulfonamides, and low

272  Chapter 10 Table 10.1: Virulence factors which may be present in some Enterocccus strains and association with stage of virulence Virulence Determinant

(Suggested) Association With Stage of Virulence

Aggregation substance (AS)

Adhesion to eukaryotic cells (adhesin)/promotes colonization Invasion of eukaryotic cells (invasin) Adhesion to extracellular matrix proteins (may promote translocation) Increases survival in immune cells (evasion of host immune response) Eukaryotic cell toxin Lyses immune cells (evasion of host immune response) Can hydrolyse various biological peptides, for example, collagens and fibrin (role in translocation?) Can hydrolyse antibacterial peptides (evasion of host innate immune response) Adhesin, promotes colonization Exhibits characteristics of MSCRAMM’s—role in evasion of immune response? Adhesion to extracellular matrix proteins (may promote translocation) Exhibits MSCRAMM characteristics: role in evasion of immune response?

Cytolysin (Cyl) Gelatinase (Gel)

Enterococcal surface protein (Espfs and Espfm) Adhesin to collagen of Ent. faecalis (Ace) or Ent. faecium (Acm) Endocarditis antigen from Ent. faecalis or Ent. faecium (EfaAfs and EfaAfm) Hyaluronidase Pheromones Ent. faecium secreted antigen (Sag) Superoxide and hydrogen peroxide Capsule

Adhesin: role in endocarditis

Degrades hyaluronic acid, a major extracellular matrix constituent: role in translocation? Cause inflammation, induce superoxide production Adhesion to extracellular matrix proteins May cause cell/DNA damage, improves colonization Evasion of host immune response

levels of clindamycin and aminoglycosides, while acquired resistance includes resistance to chloramphenicol, erythromycin, high levels of clindamycin and aminoglycosides, tetracycline, high levels of β-lactams, fluoroquinolones, and glycopeptides such as vancomycin (Murray, 1990; Leclercq, 1997). Only a few β-lactamase producing Ent. faecalis and Ent. faecium isolates have been isolated (Murray et al., 1986; Murray, 1992; Sarti et al., 2012) and the high-level ampicillin resistance of Ent. faecium is explained rather by mutations in PBP5 which results in a lower affinity for ampicillin (Al-Obeid et al., 1990; Ligozzi et al., 1996), or overproduction of PBP5 (Klare et al., 1992; Fontana et al., 1994; Top et al., 2008). Quinolone resistance is also an acquired resistance and ciprofloxacin resistance results from a chromosomal mutation in the DNA gyrase (gyrA) or topoisomerase IV (parC) genes (Top et al., 2008). Resistance to macrolides such as erythromycin can occur by ribosomal modification and genes encoding methylases, such as ermB, the most widely

Opportunistic Food-Borne Pathogens  273 distributed erm gene in enterococci located on a transposon Tn917 (Shaw and Clewell, 1985), and genes encoding ermA or ermC are also associated with resistance to erythromycin. In addition, efflux pumps such as msrC, mefA, mefE, or phosphotransferase mphA may also be involved in erythromycin breakdown. The erm(B) gene also confers cross-resistance to group B streptogramins and lincosamides (Werner et al., 2002). ermA, ermB, and msrC are the most common erythromycin-resistance determinants detected among enterococci isolated from foods (Werner et al., 2013). Resistance to streptogramin B in the majority of Ent. faecium and related spp. is encoded by acetyltransferase genes vat(D) and vat(E) (formerly known as satA and satG, respectively), which are probably plasmid-located, as resistances can be transferred by filter-mating (Hammerum et al., 1998; Werner et al., 2000; Simjee et al., 2002). High-level aminoglycoside resistances in enterococci are characterized by MICs for gentamicin of ≥1000 μg/mL and/or streptomycin of ≥2000 μg/mL. The corresponding transferable genes of these high-level resistances encode aminoglycoside modifying enzymes (phosphotransferases, acetyltransferases, and nucleotidyltransferases), of which the bifunctional enzyme Aac(6′)/aph(2″) is especially important. It mediates high-level resistance to all aminoglycosides and can also be found often in food enterococci strains, especially those of meat origin (Werner et al., 2013). Tetracycline resistance is encoded by different tet genes responsible for ribosomal protection [(tet(M), tet(O), tet(S)] or efflux mechanisms [tet(K) and tet(L)]. The tet(M) gene often occurs on the Tn916–1545 family of conjugative transposons, which have a broad host range (Wilcks et al., 2005; Hummel et al., 2007), while the other determinants may be located on the chromosome [tet(L)] or on conjugative plasmids [tet(L), tet(O), tet(S)] (Klare et al., 2003). The tet(L), tet(M), and tet(O) genes are predominant tetracycline resistance determinants in enterococci isolates from foods (Werner et al., 2013), while tet(K) occurs more often in staphylococci (Klare et al., 2003). Resistance to chloramphenicol is mediated by O-acetyltransferases (plasmid encoded cat genes) of streptococcal or staphylococcal origin, or by an efflux mechanism. The presence of cat genes has been shown in food enterococcal isolates in a number of studies (Aarestrup et al., 2000; Hummel et al., 2007; Jamet et al., 2012). Vancomycin resistance is of special concern, because this antibiotic was considered a last line therapy for treatment of multiple-resistant enterococcal infections (Van Tyne and Gilmore, 2014). In addition, this antibiotic was given as an alternative to ampicillin or penicillin/ aminoglycoside treatment to persons with allergy against penicillin or ampicillin (Morrison et al., 1997). Six types of glycopeptide resistance (vanA, vanB, vanC, vanD, vanE, and vanG) have been described in enterococci and can be distinguished on the basis of the sequence of the structural gene encoding the resistance ligase (Depardieu et al., 2004). The emergence of vancomycin-resistant enterococci (VRE) in hospitals has led to infections that cannot be treated with conventional antibiotic therapy, and thus such strains pose a serious medical concern. Moreover, the vanA and vanB resistance determinants are located on transposons (Tn1546 or a member of the Tn3 family for vanA, Tn1547 or Tn5382 for vanB) integrated

274  Chapter 10 either into the chromosome or in plasmids, and these are transferable. There have been numerous reports of association of enterococci with vancomycin resistance in foods and the transfer of resistant strains from food to humans was demonstrated (Bertrand et al., 2000; Descheemaeker et al., 1999; Freitas et al., 2011; Van den Bogaard et al., 1997). Particularly worrisome is the fact that a multitude of mechanisms are available among enterococci to transfer antibiotic resistance genes. Mobile elements are responsible for horizontal transfer of antibiotic-resistance genes between microorganisms. Plasmids and transposons are highly organized mobile elements, whose presence indicates potential intra- and interspecies transfer of resistance genes (Alekshun and Levy, 2007; Devirgiliis et al., 2011). Insertion sequences (IS) are simpler mobile elements that can harbor antibiotic resistance genes and are capable of autonomous transposition (Devirgiliis et al., 2011). IS elements have been found on the chromosome and on plasmids, or on both, but their horizontal transfer usually occurs only when associated with conjugative elements. Transposons play an important role in transfer of resistances toward macrolide, tetracycline, and glycopeptide antibiotics of enterococci, while plasmids were described to be responsible for transfer of chloramphenicol, tetracycline, and streptogramin resistances in these bacteria (Werner et al., 2013). As mentioned above, enterococci occur in a great variety of foods based on animal sources, these being either dairy or meat foods. As a result of enterococci occurring in water, soils, and plants, the enterococci can also occur on plant foods. This means that enterococci can prevail, multiply, and transfer between different ecosystems, each associated with different and specific environmental conditions. Such bacteria which are able to exist in the vertebrate gut and which are also free-living, and which are thus not adapted to a single ecosystem, have been called cosmopolitan bacteria (Ley et al., 2008). This means that inevitably foods containing high numbers of enterococci (e.g., up to 107 CFU/g cheese or in fermented meats) are probably consumed possibly at least once weekly by the average consumer. The questions arising from this fact are whether these strains harbor virulence determinants or antibiotic resistances, and whether there is a food route of transmission. Can enterococci from foods survive the gastrointestinal passage and are they able to colonize the gastrointestinal tract? Can food strains which harbor virulence factors or antibiotic resistance determinants cause human infection or can such determinants be transferred to commensal bacteria in the gut? Does the presence of enterococci in foods constitute a risk? There is evidence that enterococci from the environment or from foods can survive in, and at least transiently establish themselves, in the human gastrointestinal tract. A study of Gelsomino et al. (2003) showed that identical strains could be isolated from farm milk, cheese and human fecal samples, suggesting a food route of transmission. In various studies, genetically indistinguishable enterococci were found in both animals and humans, also suggesting that animal-derived enterococci may colonize the human gut (Bates et al., 1994; Descheemaeker et al., 1999; Jensen et al., 1998; Van den Bogaard et al., 1997). However, there are controversial results in different geographical regions with respect to

Opportunistic Food-Borne Pathogens  275 vancomycin-resistant enterococci (VRE) strains. Tzavaras et al. (2012) showed that broiler and clinical VRE isolates in Greece belonged to clearly unrelated populations, as supported by PFGE patterns, as well as antimicrobial resistance. Similar results were also shown by other investigators (Kwon et al., 2012; Ribeiro et al., 2007; Willems et al., 2000). Freitas et al. (2011), on the other hand, were able to demonstrate clonal relationships between hospital- and swine-associated VRE (strains belonging to CC17 and CC5 Ent. faecium and CC2 Ent. faecalis clusters), indicating that this route of transmission does exist. López et al. (2009) showed that three vancomycin-resistant Ent. faecium strains isolated from food animals (chicken, veal, rabbit), with coresistance to erythromycin or tetracycline or both, also belonged to the so far clinically associated CC17 cluster. Finally, Bertrand et al. (2000) showed that Ent. faecalis strains from different French cheeses showed clonal relationship to and shared high-level kanamycin resistance to French clinical outbreak isolates, again indicating that transfer from food, even nonmeat type of food, does occur. Prieto et al. (2016) examined the emergence of antibiotic-resistant enterococci and their population structures, and indicated that Ent. faecium and Ent. faecalis clonal populations evolved and disseminated to become global pathogens. For Ent. faecium, specific clonal subpopulations were found to be associated with hospitalized patients and these are rarely encountered in the community (Prieto et al., 2016). Thus, from this, it may be deduced that the data so far do not indicate foods to be a major route for transmission of such clinical associated Ent. faecium subpopulations. In addition, wgMLST (whole genome multilocus sequence typing) data also revealed the distinct clustering of human commensal, animal, and human clinical strains (Prieto et al., 2016). WGS-based studies proposed that the Ent. faecium population was divided into two species-level subdivisions. The subpopulations were termed clade A or hospital-associated clade which primarily contains isolates from hospitalized patients, and clade B or community-associated clade which mostly contains isolates from healthy, nonhospitalized individuals (Galloway-Peña et al., 2012; Palmer et al., 2012). Notably, with this analysis, animal isolates do not group in a single clade A2 but form multiple distinct clusters located between the human commensal and clinical isolates. This indicated that clade A2 may not be monophyletic, as was previously postulated (Lebreton et al., 2013; Prieto et al., 2016). Interestingly, clade A1 Ent. faecium strains were found to have lower fitness in natural environments, where they are outcompeted by other Ent. faecium clones (Leclercq et al., 2013). Similarly, clade B strains outcompeted clade A strains in an animal model of gut colonization in the absence of selection by antibiotics (Montealegre et al., 2015). These findings point to a niche specialization to the hospital environment of clade A1 strains, which thus may come as a fitness cost in nonhospital environments. Together, these data further support that a food role for transmission of nosocomial Ent. faecium strains is unlikely. Ent. faecalis clones associated with hospital infections, on the other hand, appear not to be exclusively found in hospitals, but are present also in healthy humans and animals (Prieto et al., 2016). Furthermore, virulence factor genes of Ent. faecalis strains are also not unique

276  Chapter 10 to clinical strains, but are also present in strains from commensal niches (Prieto et al., 2016). Taken together, these data may indicate that for Ent. faecalis strains a food-borne route of spread of opportunistically pathogenic Ent. faecalis strains is possible.

10.2.2  Weissella Species The genus Weissella (W.) includes Gram-positive, catalase-negative, and non-endospore forming coccoid or rod-shaped LAB (Collins et al., 1993; Björkroth et al., 2014). Twenty validated species of Weissella are known to date (Fusco et al., 2015; Lee et al., 2015a, b) (Table 10.2). Certain Weissella strains are being found to have probiotic (Fusco et al., 2015; Andrabi et al., 2016; Hong et al., 2016) activity. Despite these positive roles, strains of W. viridescens, W. cibaria, and W. confusa have been demonstrated to be capable of acting as opportunistic pathogens in humans (Table 10.3), while strains of W. paramesenteroides and W. cibaria have been isolated from the milk of cows with clinical mastitis (Wald et al., 2016) and that of W. ceti has been recognized as the etiological agent of a disease in farmed rainbow trout (Oncorhynchus mykiss), known as weissellosis (Fusco et al., 2015), which causes septicemia with a high mortality rate (Welch et al., 2014). Moreover, W. confusa caused a systemic infection in a mona monkey (Cercopithecus mona) (Vela et al., 2003) and neonatal sepsis in a foal (Lawhon et al., 2014). W. viridescens has been identified in the feces of children with celiac disease (Sanz et al., 2007) and in the blood of patients with bacteremia Table 10.2: Weissella species known to date Species

Reference

W. beninensis W. ceti W. cibaria W. confusa W. diestrammenae W. fabaria W. ghanensis W. halotolerans W. hellenica W. jogaejeotgali W. kandleri W. koreensis W. minor W. oryzae W. paramesenteroides W. soli W. thailandensis W. uvarum W. viridescens

Padonou et al. (2010) Vela et al. (2011) Björkroth et al. (2002) Collins et al. (1993) Oh et al. (2013) De Bruyne et al. (2010) De Bruyne et al. (2008) Collins et al. (1993) Collins et al. (1993) Lee et al. (2015a, b) Collins et al. (1993) Lee et al. (2002) Collins et al. (1993) Tohno et al. (2013) Collins et al. (1993) Magnusson et al. (2002) Tanasupawat et al. (2000) Nisiotou et al. (2014) Collins et al. (1993)

Opportunistic Food-Borne Pathogens  277 Table 10.3: Diseases caused by Weissella spp. Etiological Agent

Disease

Reference

W. viridescens W. cibaria W. confusa

Bacteremia Bacteremia Bacteremia Fatal case of endocarditis Infective endocarditis of native valves Bacteremia in patient with peritoneal neuroblastoma subjected to chemotherapy and ileus surgery Postoperative osteomyelitis with chronic discharge in a young female Large case series involving 10 patients with bacteremia Sepsis in a 48-year-old male operated for adenocarcinoma of the gastro-esophageal junction maintained on total parenteral nutrition Bacteremia in patients with acute lymphocytic leukemia undergoing autologous stem cell transplantation Bacteremia in patients with hepatocellular carcinoma occurring after liver transplant Bacteremia in a patient with a prosthetic joint infection Bacteremia in a Crohn’s disease patient

Kulwichit et al. (2007) Kulwichit et al. (2007) Kulwichit et al. (2007) Flaherty et al. (2003) Shin et al. (2007) Svec et al. (2007) Kulwichit et al. (2008) Lee et al. (2011) Kumar et al. (2011)

Salimnia et al. (2011) Harlan et al. (2011) Medford et al. (2014) Vasqueza et al. (2015)

(Kulwichit et al., 2007), whereas W. cibaria has been isolated from the blood, urine, and lung of patients with bacteremia (Kulwichit et al., 2007) and has been associated with dog ear otitis (Björkroth et al., 2002). W. confusa is being reported since 1990 either in cases of polymicrobial infections (Bantar et al., 1991; Björkroth et al., 2002; Green et al., 1990; Olano et al., 2001; Riebel and Washington, 1990) or as the sole etiological agent (Table 10.2) in a broad range of infections. However, considering that weissellas may be misidentified with other vancomycin-resistant, Gram-positive bacteria such as enterococci and leuconostocs (Fairfax et al., 2014; Fusco et al., 2015; Kulwichit et al., 2007; Lee et al., 2011). If inadequate identification methods are used, the actual incidence of the infections due to weissellas is most likely underestimated. Immunocompromised patients and patients with underlying disease have been involved in the majority of W. confusa infections reported to date. The main risk factors for the development of infections due to weissellas include malignancy and recent chemotherapy, orthopedic procedures, central line catheter insertion, total parental nutrition, long-term use of steroids, organ transplant, chronic alcoholism, and renal insufficiency, burn, and diabetes (Kamboj et al., 2015). Considering that the gastrointestinal tract of humans (Albesharat et al., 2011; Gomathi et al., 2014; Lee et al., 2012; Nistal et al., 2012; Rubio et al., 2014; Walter et al., 2001; Wang et al., 2008; Zhang et al., 2014) and human vagina (Jin et al., 2007; Nam et al., 2007; Silvester and Dicks, 2003) are among the natural habitats of weissellas, a damage of the gastrointestinal and vaginal mucosal barriers, even as a result of surgery, may favor the entrance of weissellas into underlying tissue and then result in infections. Exposure to antibiotics, especially vancomycin, to which weissellas are

278  Chapter 10 resistant, is another important predisposing factor to Weissella infections. Given their role as opportunistic pathogens, studies should be undertaken to unveil the pathogenicity of these LAB. In this perspective, further insight into the pathogenesis of W. ceti has been provided by genomic studies. Ladner et al. (2013) found in the genome of the W. ceti NC36 five collagen adhesin genes, a mucus-binding protein encoded gene, and a platelet-associated adhesin gene which are probably virulence-related. Figueiredo et al. (2015) performed a comparative genome analysis of 4 W. ceti strains isolated from different rainbow trout farms in Brazil (3) and the United States (1), and found genes putatively encoding proteins involved in processes of bacterial physiology and pathogenesis such as antibiotic resistance (sulfonamide-resistance protein and several multidrug efflux pumps), survival in the water environment under stressful temperatures; cell lysis and bacterial spread inside the host (hemolysins and their regulators); contact with the host cells (adhesins) and resistance to immune-cell-mediated stresses. Abriouel et al. (2015) analyzed 13 genome sequences available on the NCBI database of W. ceti (four strains), W. koreensis (two strains), W. cibaria, W. confusa, W. halotolerans, W. hellenica, W. koreensis, W. horyzae, W. paramesenteroides, and W. thainlandensis (one strain per each species) and screened them for the presence of virulence determinants. They found collagen adhesin genes in three W. ceti strains and in one W. confusa strain. The most parsimonious phylogenetic tree obtained by aligning the relevant DNA sequences with those of other LAB (Lactococcus, Leuconostoc, Enterococcus, and Lactobacillus) revealed a close relatedness of the collagen adhesion genes of Weissella, Enterococcus, and Leuconostoc, leading to the hypothesis of a same evolutionary origin of these proteins in these groups of microorganisms. However, as discussed by Abriouel et al. (2015), further studies are needed to demonstrate if these genes are expressed and are located on mobile genetic elements. The W. ceti NC36 and W. confusa LBAE C39-2 harbor genes encoding a mucus-binding protein, which is involved in the adhesion process of the bacterium to the host. The presence of both the adhesin and the mucus-binding protein may be a favorable or detrimental feature, depending on whether these occur in probiotic or pathogenic strains, respectively. Genes encoding for aggregation substances were found in the W. oryzae genome (Abriouel et al., 2015). Genes encoding a protein similar to the staphylococcal surface protein A were found in three of the W. ceti genomes analyzed, while genes coding for hemolysin A and hemolysin-like proteins were found in all the Weissella genomes (Abriouel et al., 2015). Moreover, genes coding for fosfomycin- and methicillin-resistance proteins were also found in almost all analyzed Weissella genomes, while one of the four W. ceti genomes, as well as the W. confusa, W. halotolerans, W. hellenica, W. koreensis, W. oryzae, and W. paramesenteroides genomes, harbored multidrug transporters involved in fosfomycin and deoxycholate resistance (Abriouel et al., 2015). The W. confusa, W. cibaria, and W. paramesenteroides genomes, on the other hand, were also shown to harbor the vanZ-resistance gene (Abriouel et al., 2015). Recently, Zornetta et al. (2016) demonstrated that a botulinum neurotoxin (BoNT) like gene found in the genome of W. oryzae SG25T by bioinformatic methods encodes for a

Opportunistic Food-Borne Pathogens  279 metalloprotease similar to the Clostridial BoNTs. However, further studies are needed to investigate the possible role of W. oryzae as an emerging food-borne pathogen. Although these genome-based analyses have provided further insight into the pathogenic potential of some weissellas, a higher number of genomes from different strains per each Weissella species should be analyzed and the in silico analyses should be followed by in vivo studies aimed at verifying the expression of the virulence genes. Moreover, data on the actual incidence of Weissella spp. in clinical infections should be provided. However, to reach this goal, appropriate methods of detection and identification of weissellas, such as DNA based approaches (such as the 16S rRNA gene sequencing (Fairfax et al., 2014; Kulwichit et al., 2008; Lee et al., 2011; Medford et al., 2014; Vasqueza et al., 2015), the W. confusa species-specific PCR (Fusco et al., 2011), and the real-time PCR specific for W. viridescens (Gómez-Rojo et al., 2015) and for W. ceti (Snyder et al., 2015)) should be applied. In addition, other techniques, such as fluorescent amplified fragment length polymorphism (Fusco et al., 2011), repetitive element-PCR fingerprinting using (GTG)5-PCR (Bounaix et al., 2010) or Matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) approaches (Fairfax et al., 2014; Lee et al., 2015a, b) for the identification and typing of weissellas, should be applied by clinical laboratories in order to avoid misidentifications of these microorganisms that inevitably occur when morphological and biochemical tests are applied, or when biochemical-based identification kits are used (Fairfax et al., 2014; Fusco et al., 2011, 2015; Lee et al., 2011; Medford et al., 2014; Shin et al., 2007).

10.2.3  Lactobacillus Species Lactobacilli are Gram-positive rods or coccobacilli (Hugenholtz, 1998), facultatively anaerobic, nonspore-forming, and catalase-negative bacteria that produce lactic acid as the major fermentation product (>50% of the metabolites produced), which are characteristic of the LAB group (Klaenhammer and de Vos, 2011). Lactobacillus is the largest genus among LAB with over 221 species described to date in the List of Prokaryotic names with Standing in Nomenclature “LPSN” (September 2016, http://www.bacterio.net) and they are phylogenetically diverse. Their large history of safe use already for millennia as starter cultures in several fermented foods (reviewed in Tamang and Kailasapathy, 2010) allowed them the GRAS status in the United States and the QPS status of many species in Europe. Owing to their safe use and their variable catabolic activities, lactobacilli have broad use in food fermentations. Furthermore, in the past decades, Lactobacillus spp. also had important industrial applications as probiotic adjuncts (Casas and Dobrogosz, 1997, 2000). Examples are L. paracasei Shirota (Nanno et al., 2011) and L. rhamnosus GG (Szajewska and Chmielewska, 2013), the well-known probiotics which confer health benefits to the host upon administration (Hill et al., 2014). In comparison to the amount of species known

280  Chapter 10 and investigated, only few Lactobacillus species were screened for their probiotic potential. Lactobacillus spp. have several health benefits including prevention and treatment of digestive disorders (infantile and adult diarrhea, antibiotic-associated diarrhea, inflammatory bowel syndrome), urinary tract infections (candidiasis, vaginitis), and also other novel applications such as for the increase of longevity (Erkosar and Leulier, 2014), reduction of radiation injury (Ciorba et al., 2012), treatment of celiac and Crohn’s diseases (Duar et al., 2015; Schultz et al., 2004), cancer, and stress (Da Silva et al., 2014; Khazaie et al., 2012). On the other hand, owing to the incidence of a number of cases of human infections caused by Lactobacillus spp., the European Food Safety (EFSA, 2012a, b) recommended that the QPS status of lactobacilli should be based on the “body of knowledge” of the species and its safety, including its history of infection or its possession of transferable antibiotic resistance or virulence genes (EFSA, 2012a). In this sense, both functionality and safety of Lactobacillus spp. are strain dependent. Lactobacilli that occur naturally in the human and animal mouth or gastrointestinal or urogenital tracts could be translocated to other organs, causing infection. Moreover, they are also ingested in high number (>108 CFU/mL) in fermented foods, which can also increase quantitatively and qualitatively the diversity of the gastrointestinal environment (Ali and Mustafa, 2009). Lactobacillus is a rare pathogen and the infections caused are almost always detected in patients with serious underlying illnesses or the immunocompromised. These lactobacilli mostly caused infections after dental manipulation, recent surgery, transplantation, diabetes, colonoscopy, or cancer. They can be occasional opportunistic pathogens causing localized infection, bacteremia associated or not with endocarditis, although they remain to be clinically important causes of infection. Several cases of endocarditis were reported since 1950 (Baron et al., 1986; Cannon et al., 2005; reviewed in Salvana and Frank, 2006) and different species were involved, although L. casei and L. rhamnosus were the most relevant species (Salvana and Frank, 2006; See et al., 2006) (Table 10.4). Lactobacillus endocarditis has been linked to heart diseases and prosthetic valves, although the most important predisposing condition in almost half of the patients was dental infection (Cannon et al., 2005). Lactobacillus causing bacteremia is more frequent than endocarditis (Cannon et al., 2005; Salvana and Frank, 2006) and the predisposing factors were immunosuppression, prior prolonged hospitalization, and prior surgical interventions. No relation was established between bacteremia caused by Lactobacillus and probiotic consumption in some cases (Aroutcheva et al., 2016; Robin et al., 2010; Salminen et al., 2002), although the consumption of probiotics should be stopped before colonoscopy or any digestive intervention (Franko et al., 2013). However, in other clinical scenarios, the consumption of lactobacilli as probiotics such as by immunosuppressed patients or those with inflammatory digestive diseases (ulcerative colitis) may increase the risk of Lactobacillus bacteremia (Vahabnezhad et al., 2013). This has necessitated the use of the discriminatory molecular genotyping methods (pulsed field

Opportunistic Food-Borne Pathogens  281 Table 10.4: Diseases caused by Lactobacillus spp. Lactobacillus spp.

Disease

Reference

Lactobacillus spp. L. rhamnosus L. casei L. fermentum L. acidophilus L. delbrueckii L. plantarum L. casei Lactobacillus sp. L. acidophilus L. casei L. curvatus L. fermentum L. jensenii L. paracasei paracasei L. rhamnosus L. jensenii L. acidophilus L. paracasei L. acidophilus L. rhamnosus L. paracasei L. plantarum L. casei L. rhamnosus L. rhamnosus L. rhamnosus L. rhamnosus L. rhamnosus L. acidophilus L. delbrueckii

Bacteremia

Antony et al. (1996) Salminen et al. (2006) and Falci et al. (2015) Salminen et al. (2006) Salminen et al. (2006) Rasul et al. (2012) DuPrey et al. (2012) Shinar et al. (1984) Chong et al. (1991) and See et al. (2006) Reviewed in Salvana and Frank (2006)

Endocarditis

Peritonitis

Pneumonia Arteritis Deep abdominal abscesses Pancreatitis Meningitis Pyelonephritis

Fradiani et al. (2010) and Patnaik et al. (2015) Nishijima et al. (2012) and Encarnacion et al. (2016) Franko et al. (2013) Schleifer et al. (1989) Sanyal and Bhandari (1992) Neef et al. (2003) Tena Gómez (2012) Martins et al. (2016) Querol et al. (1989) Holliman and Bone (1988) Cannon et al. (2005) Brahimi et al. (2008) Robin et al. (2010) Rasul et al. (2012) Duprey et al. (2012)

gel electrophoresis, 16S rRNA gene sequencing, and DNA fingerprint analysis “rep-PCR”) to clearly determine whether there is a link between the patient’s isolate and the probiotic strains (Aroutcheva et al., 2016). However, an increased endocarditis risk was reported in patients chewing the contents of probiotic capsules before dental treatment (Mackay et al., 1999). In general, although probiotics have an excellent overall safety record, they should be administered with care in certain patient groups (particularly neonates born prematurely or with immune deficiency) (Boyle et al., 2006). In both the cases of bacteremia and endocarditis, empiric antibiotic therapy is not recommended (Salvana and Frank, 2006), and the choice of antibiotics is highly dependent on the in vitro susceptibility testing of the lactobacilli (Salminen et al., 2004). A combination of gentamicin and penicillin was reported

282  Chapter 10 as the most effective treatment of endocarditis and bacteremia caused by Lactobacillus spp. in the majority of cases. Furthermore, other less frequent diseases such as meningitis, arteritis, pneumonia, peritonitis, deep abdominal abscesses, pyelonephritis, and pancreatitis (Table 10.4) were related to Lactobacillus spp.

10.3  Gram-Negative Opportunistic Pathogens 10.3.1  Klebsiella pneumoniae Klebsiella (K.) pneumonia is a member of the Enterobacteriaceae family and is an important opportunistic pathogen that can cause community and clinical infections (Brown and Seidler, 1973; Gupta et al., 2003; Gundogan and Yakar, 2007; Jung and Matthews, 2016; Keynan and Rubinstein, 2007; Podschun and Ullmann, 1989). Klebsiella pneumoniae is often isolated from the environment, for example, water, sewage, and soil (Broberg et al., 2014), but also occurs in humans in the gastrointestinal tract, skin, and nasopharynx (Pitout et al., 2015). It is the cause of serious community onset infections such as necrotizing pneumoniae, pyogenic liver abscesses, and endogenous ophthalmitis, and during the 1970s it also became a serious cause of nosocomial infections, especially urinary tract, respiratory tract, and bloodstreamassociated infections (Hennequin and Robin, 2016; Keynan and Rubinstein, 2007; Pitout et al., 2015). In the past three decades, a distinctive syndrome of community acquired invasive infections, primarily in the form of pyogenic liver abscess, has emerged (Chang and Chou, 1995). These infections are caused by hypervirulent (hvKP) isolates mainly of serotypes K1 and K2. Serotype K1 strains typically belong to particular clones such as clonal complex 23 (CC23), which comprises the sequence types (STs) ST23 and ST57. Serotype K2 strains also belong to several STs, some of which are linked to hypervirulence, that is, ST86, ST375, and ST380 (Hennequin and Robin, 2016). The main virulence factors of K. pneumoniae are capsule, fimbriae, lipopolysaccharide, siderophores (enterobactin, aerobactin, salmochelin, yersiniabactin), and efflux (Podschun and Ullmann, 1989; Hennequin and Robin, 2016). However, since the 1980s, K. pneumoniae infections have also become more and more difficult to control due to the emergence of antibiotic resistances. The antibiotics of choice to treat Klebsiella infections are cephalosporins, fluoroquinolones, and trimethoprim-sulfamethoxazole, and resistance to these agents has already created major therapeutic problems (Pitout et al., 2015). Of special importance has been the development of resistance toward carbapenem antibiotics (imipenem, meropenem, biapenem, ertapenem, and doripenem), since these were used as the last line of effective therapy for treating multidrugresistant K. pneumoniae (Lee et al., 2016; Mathlouthi et al., 2016; Pitout et al., 2015). Resistance to carbapenems is mediated mostly by two mechanisms: (1) production of carbapenem-hydrolyzing β-lactamases (de-repressed cephalosporinases or ESBL) with nonsignificant carbapenemase activity, combined with decreased permeability due to porin

Opportunistic Food-Borne Pathogens  283 loss or alteration or (2) production of carbapenem-hydrolyzing β-lactamases (Mathlouthi et al., 2016; Nordmann et al., 2011). The carbapenemases can be subdivided on the basis of their dependency on divalent cations for enzyme activation into the metallocarbapenemases (zinc-dependent class B carbapenemases) and nonmetallo-carbapenemases (zinc-independent class A, C, and D carbapenemases (Lee et al., 2016). The class A K. pneumoniae carbapenemases (KPCs) have been identified worldwide in K. pneumoniae and were almost exclusively reported to occur in this bacterium (Pitout et al., 2016; Tängdén and Giske, 2015). More than 20 class A KPC type β-lactamases are currently recognized, although KPC-2 and KPC-3 remain the most commonly isolated variants (Pitout et al., 2016). These β-lactamases (especially KPC-2 and KPC-3) have been described in several enterobacterial species, especially in Klebsiella spp. and to a lesser extent in Enterobacter spp. (Queenan and Bush, 2007). In Klebsiella, these are mainly plasmid-encoded (Lee et al., 2016) and the blaKPC were reported to occur in numerous plasmid types such as IncF, IncX, IncA/C, IncR, and ColE1 (Pitout et al., 2015), but the predominant plasmid type is IncF with FIIK replicons (Lee et al., 2016). Transposon Tn4401 has been shown to be the main genetic structure enhancing the spread of blaKPC-type genes onto different plasmid scaffolds as mentioned above. Tn4401 is 10 kb in length and is delimited by two 39-pb imperfect inverted repeats. It contains a Tn3 resolvase gene and two IS ISKpn6 and ISKpn7 (Pitout et al., 2016). The class B β-lactamases or metallo-β-lactamases (MBLs) identified in K. pneumoniae have also been identified in various other enterobacterial spp. (Nordmann et al., 2011; Pitout et al., 2016) and are mainly NDM-VIM- and IMP-type enzymes. The first group is the New Delhi metallo-β-lactamases (NDM) which are the most commonly identified worldwide (Pitout et al., 2016). Since the first description of NDM-1, >10 variants have been described. Bacteria with MBLs are often resistant to penicillins, carbapenems, cephalosporins, and cephamycins but remain susceptible to monobactams and their activities are inhibited by chelators such as EDTA and dipicolinic acid (Pitout et al., 2016). The MBL genes (blaIMP, blaVIM, and blaNDM) are found on different broad host range plasmid types (e.g., IncA/C, IncN) with different genetic features (Pitout et al., 2016). The only class D carbapenem-hydrolysing β-lactamase found in K. pneumoniae isolates is OXA-48 (and derivatives, i.e., OXA-181, OXA-204, and OXA-232). OXA-48 efficiently hydrolyses narrow-spectrum β-lactams such as penicillins, weakly hydrolyses carbapenems, and spares broad-spectrum cephalosporins (Poirel et al., 2004). It was found in all the members of the Enterobacteriaceae, but mostly in nosocomial K. pneumoniae and community isolates of E. coli (Pitout et al., 2016). The blaOXA-48 gene is located on the Tn1999 composite transposon and its current dissemination is mainly due to the epidemic IncL/M plasmid (pOXA-48a), which was shown to be highly transferable (Poirel et al., 2012; Potron et al., 2014).

284  Chapter 10 Carbapenemases possess variable hydrolytic activities, with the MBLs and KPC enzymes hydrolyzing carbapenems more efficiently than the OXA-48 enzymes. However, high-level carbapenem resistance additionally requires permeability deficiencies regardless of which type is produced (Nordmann and Poirel, 2014; Pitout et al., 2016). High-risk K. pneumoniae clones are clones with increased pathogenicity and survival, coupled with acquisition of antibiotic resistance determinants and an enhanced ability to spread. The blaKPC genes so far have been found in >100 STs, but the worldwide pandemic spread has primarily been driven by isolates which are members of the clonal complex 258 (CC258) (the founding member of which is ST292) (Munoz-Price et al., 2013; Pitout et al., 2016). CC258 consists of one predominant ST, that is, ST258 and to a lesser extent of ST11, ST340, and ST512, which are single-locus variants of ST258. Whole genome sequencing and SNP analysis of the core genomes of CC258 isolates showed that K. pneumoniae ST258 belongs to two well-defined lineages named clades I and II. Clade I was associated with KPC-2 and clade II with KPC-3 and the genetic divergence was shown to occur in a 215-kb region that includes the genetic material encoding capsule polysaccharide biosynthesis (Deleo et al., 2014; Pitout et al., 2016). Additional investigation showed that ST258 clade II is actually a hybrid clone which was created by a recombination event between ST11 and ST442 and that ST258 clade I strains evolved from a clade II strain as a result of replacement of the capsule cps region from ST42 (Chen et al., 2014; Pitout et al., 2016). As mentioned above, K. pneumoniae often occurs in the community and in the environment, as well as in sewage and in water. Thus, there seems to be ample opportunity for entering the food chain at various points. The question thus is, whether there is food-borne transmission of K. pneumoniae. Indeed, recent reports seem to point out a possible link between foods as a source of infections with K. pneumoniae. As mentioned above, there appear to be two distinct groups of K. pneumoniae, typified by either the multidrug-resistant lineages (e.g., CC258), which are predominantly associated with the nosocomial setting, or the group containing the hvKP lineages (e.g., CC23) which often are community acquired infections. STs associated with hvKP lineages have also been isolated from raw retail turkey (ST25) or bovine infections (ST65) (Davis et al., 2015; Holt et al., 2015), demonstrating their occurrence in livestock and the potential for dissemination via animal-based foods. However, plasmids encoding antibiotic resistances, such as ESBL and carbapenemase resistances, have been detected in hvKP strains and their prevalence is increasing (Davis and Price, 2016). This means that now there are also strains with enhanced capacity to cause disease which do not respond to antibiotics and that therefore it is also important to estimate the prevalence and to characterize the virulence and antibiotic resistance characteristics of Klebsiella strains occurring among livestock or the environment (Davis and Price, 2016). Antibiotic-resistant K. pneumoniae strains have indeed been isolated from various food sources including retail meats, seafood, and vegetables (Davis et al., 2015; Davis and Price,

Opportunistic Food-Borne Pathogens  285 2016; Zurfluh et al., 2015). For example, ESBL-resistant strains have been isolated from mastitic cows, retail poultry samples, cultured shrimp, and fresh sprouts, and these strains often also harbored other antibiotic resistances or were even multidrug resistant (Davis and Price, 2016). The question remains whether K. pneumonia from food can be transmitted by this route to cause infection. Overdevest et al. (2014) identified K. pneumoniae isolates of the same ST in a human blood specimen and a sample of retail chicken meat. In this case, however, the ESBL genes that the strains carried on a plasmid differed, which could imply that the strains were not connected, or that the variability in plasmid content among isolates of the same ST does not preclude their having originated from the same source population (Davis and Price, 2016). Another study involved whole genome analyses to show close phylogenetic relationships between K. pneumoniae from retail meats and human clinical strains. Davis et al. (2015) showed that four strains contained both meat source and clinical isolates which displayed a similar, although relatively low-level, virulence in a mouse model. Taken together, the authors suggested that the close phylogenetic relationships and similar in vivo virulence of meat source and clinical isolates suggest that there is a low barrier to transmission between these two environments (Davis et al., 2015). However, this barrier seems to be low not only for meat-based foods. Raw foods such as fresh produce also often harbor high populations of antibiotic-resistant bacteria and most people eat the foods without adding a processing barrier such as cooking to inactivate microorganisms. This may facilitate the microorganisms to reach the human gastrointestinal tract. Various studies have shown that Klebsiella pneumoniae is a predominant commensal microorganism on fresh produce (Boehme et al., 2004; Falomir et al., 2013; Kim et al., 2015; Österblad et al., 1999; Zurfluh et al., 2015) and antibiotic-resistant strains are frequently characterized among these (Boehme et al., 2004; Kim et al., 2015; Zurfluh et al., 2015). Jung and Matthews (2016) showed experimentally that the blashv18 gene could be transferred between Klebsiella pneumonia in raw foods such as alfalfa sprouts and chopped lettuce. Thus, transfer of antibiotic-resistance genes from Klebsiella pneumoniae in foods and in the gut is possible, and may contribute to virulence of the strains. Food-borne transmission of virulent and antibiotic-resistant K. pneumonia strains needs to be accounted for more in risk assessments of foods in which K. pneumoniae strains occur and which are possibly eaten raw.

10.3.2  Enterobacter Species Similar to Klebsiella spp., Enterobacter spp. belongs to the family of Enterobacteriaceae and the bacterial cells are rod-shaped or coccibacilli, Gram-negative, and facultatively anaerobic (Mokracka et al., 2011). The species Enterobacter (E.) cloacae and E. aerogenes have in past years taken on a clinical significance as opportunistic pathogens and have emerged also as nosocomial pathogens (Mezzatesta et al., 2012). Some reports indicate that E. cloacae and E. hormaechei are the most frequently isolated Enterobacter strains isolated from clinical specimens (Davin-Regli and Pagès, 2015). In fact, the importance

286  Chapter 10 of members of the E. cloacae complex strains as nosocomial pathogens is well known and strains belonging to this complex have been associated with severe opportunistic infections in debilitated persons, often in intensive care units (Mokracka et al., 2011). These bacteria cause pneumonia and urinary tract, wound, skin, and soft tissue, as well as ophthalmic and bloodstream infections (Fraser et al., 2008). The virulence mechanisms and factors which contribute to the pathogenicity of E. cloacae have not been fully elucidated (Davin-Regli and Pagès, 2015). Its ability to form biofilms and to secrete a variety of toxins (enterotoxins, hemolysins, pore-forming toxins), however, has been recognized to be important in virulence (Mezzatesta et al., 2012; Davin-Regli and Pagès, 2015). Also, the development of antibiotic resistances is considered to have contributed to E. cloacae strains becoming nosocomial pathogens. E. cloacae is naturally resistant to ampicillin, amoxicillin, first-generation cephalosporins, and cefoxitin as a result of the constitutive expression of a low-level AmpC β-lactamase type cephalosporinase. Owing to the transfer of extended spectrum β-lactamases (ESBL) and carbapenemases into the species, E. cloacae has become the third broad spectrum Enterobacteriaceae species involved in nosocomial infections after Escherichia coli and K. pneumonia (Potron et al., 2013; Davin-Regli and Pagès, 2015). Some strains of E. cloacae, which harbored a chromosomally encoded AmpC gene (blaCMY-10), integrated the gene on a large plasmid (130 kb), thus facilitating a possible mobile genetic exchange (Lee et al., 2003). Furthermore, since the 1990s, clonal E. aerogenes strains emerged with resistance to common β-lactam antibiotics due to ESBL, for example, TEM-24 or other TEM types, as well as CTX-M types (e.g., CTX-M-2) (Bertrand et al., 2003; Bosi et al., 1999; Davin-Regli and Pagès, 2015; Pitout et al., 1998). The TEM-24 mediated resistance remains preferentially associated with a conjugative plasmid in E. aerogenes (Biendo et al., 2008; Kanamori et al., 2012). Today, E. cloacae is the most frequently observed clinical isolate and can be associated with epidemic plasmids which bear the most prevalent resistance genes and express new βlactamases or carbapenemases (Davin-Regli and Pagès, 2015). They are capable of overproducing AmpC-β-lactamase by blocking the repression of the chromosomally located gene, or by the acquisition of a transferable AmpC gene on plasmids, conferring resistance to third-generation cephalosporins (Davin-Regli and Pagès, 2015; Zaher and Cimolai, 1997). Various plasmid-borne ESBL conferring resistance to third-generation cephalosporins, but not cefamycins, are responsible for the global resistance to the carbapenems and beta lactamases, except the carapenems (Pitout et al., 1997). Various ESBL of TEM, SHV, and CTX-M types have been characterized in E. cloacae. Among ESBL producers, some subclones were identified as CTX-M-3 and CTX-M-15 producers, while other TEM or SHV (e.g., SHV-12) types were associated with epidemic isolates (Davin-Regli and Pagès, 2015). E. cloacae occur in different environments such as water, sewage soil, or foods. In foods, E. aerogenes and E. cloacae strains, including antibiotic-resistant, ESBL, or carbapenemase—producing strains, have been isolated from products such as fresh produce, seafood, raw and bulk tank milk as well as meats (Geser et al., 2012; Li et al., 2016). The species also occurs in the

Opportunistic Food-Borne Pathogens  287 intestinal tracts of animals and humans and is also a pathogen in plants and in insects. This diversity of habitats is mirrored by the genetic variety of E. cloacae, but recently MLST and PFGE epidemiological methods showed that there was a worldwide spread of several epidemic clonal complexes (Falomir et al., 2013; Izdebski et al., 2015; Janecko et al., 2016; Mezzatesta et al., 2012; Nüesch-Inderbinen et al., 2015; Odenthal et al., 2016; Zurfluh et al., 2015). Whether food is a source of opportunistic infections with Enterobacter spp. is, however, not yet clear and requires further study.

10.3.3  Serratia marcescens The genus Serratia (S.) is also a member of the Enterobacteriaceae family and S. marcescens is generally easy to characterize and differentiate from other Enterobacteriaceae as most strains are red pigmented on nutrient agar. Serratia stains are motile, nonendospore forming Gram-negative rods and have been isolated from various sources such as water, soil, plants, and animals. In addition, they have been reported as common members of intestinal microflora from bats (Klite, 1965) and isolated from grapes and leafy vegetables (Barata et al., 2012). The study of Cobo Molinos et al. (2009) showed that the Serratia spp. were the predominant bacteria at early stages in soybean sprouts production when investigated using culture-independent DGGE methods. Serratia (S.) marcescens is the type species of the genus Serratia and 17 species are currently recognized within this genus (Kämpfer and Glaeser, 2016). Many strains of S. marcescens are pigmented and possess red pigment, so the organism has been used as a tracer organism in medical laboratory experiments as nonpathogenic bacteria (Mahlen, 2011). Until the late 1950s, the species of the genus Serratia were rarely isolated from human patients and the type species, S. marcescens, was rarely associated with primary invasive infection. During the past four decades, however, Serratia spp. have been isolated from animal infections such as mastitis in cows, conjunctivitis in horses, and septicemia in goats (Kämpfer and Glaeser, 2016). S. marcescens is normally a common enteric bacterium occurring in the gastrointestinal tract of clinically healthy individuals. On the other hand, bacteria of this species are also opportunistic pathogens when they colonize or infect other organs in the body, such as the respiratory and urinary tracts of hospitalized patients (Ochieng et al., 2014). S. marcescens has been implicated in a wide range of serious infections, including pneumonia and endocarditis in hospital outbreaks (Jones, 2010). The study of Sader et al. (2014) showed that Serratia spp. could cause infections at an average incidence of 6.5% of all Gram-negative infections in intensive care units and an average incidence of 3.5% in nonintensive care units in the United States and Europe. Thus, this strain has emerged as an important nosocomial pathogen. The formation of fimbriae, the production of potent siderophores, the presence of cell wall antigens, the ability to resist the bactericidal action of serum, and the production of proteases are possible pathogenicity factors found in S. marcescens (Grimont and Grimont, 2006). However, the pathogenicity of this species is not yet fully understood. Other important

288  Chapter 10 features associated with the virulence of S. marcescens are multiple antimicrobial resistances toward different groups of antibiotics (Merkier et al., 2013). A chromosomal AmpC beta-lactamase and acquired plasmid-mediated ESBL in S. marcescens have been reported from a Bulgarian hospital (Ivanova et al., 2008) and a representative metallo beta-lactamase producing S. marcescens also has been isolated in Japan (Osano et al., 1994). In addition, Serratia spp. showing resistance to the third-generation cephalosporin antibiotics are isolated from different fresh retail vegetables and most strains of isolates harbored a blaFONA variant (van Hoek et al., 2015). The emergence of multiple antibiotics-resistant S. marcescens from normal raw food material and infection in hospital suggests that the use of antibiotics may have led to the development of antibiotic-resistant strains from the food chain to the hospital. However, there is no concrete proof or study which shows that this actually is the case and thus should be carefully monitored in the future.

10.3.4  Citrobacter freundii The genus Citrobacter belongs to the Enterobacteriaceae family and, as the name suggests, is able to utilize citrate as a sole carbon source (Borenshtein and Schauer, 2006). The Citrobacter strains are motile by peritrichous flagella and are most closely related to Salmonella and Escherichia. Citrobacter occurs in the intestine of humans and animals, and, as a consequence of animals/human fecal shedding, it can also be found in varied environments such as water, soil, and sewage. The genus Citrobacter comprises 12 species and the latest described species is Citrobacter (C.) pasteurii CIP55.13 T isolated from human diarrhea in the United States (Clermont et al., 2015). C. freundii is a member of the genus Citrobacter as type species, and the species is subdivided into seven biotypes (Brenner et al., 1999). Similar to other species of the Enterobacteriaceae family such as Salmonella and Escherichia, a large number of somatic (O) and flagella (H) antigens of this species were characterized, and atypical C. freundii strains, which showed similar biochemical and antigenic features of the genus Salmonella and E. coli, have been reported (Gilchrist, 1995). Since the 1980s, heat-stable toxins and Shiga-like toxin-producing C. freundii were isolated from humans and these heat-stable peptides, which were constituted from 18 amino acids, were identical to the heat-stable enterotoxin from E. coli (Guarino et al., 1989). Apart from being toxinogenic, C. freundii is a prominent opportunistic pathogen in the Enterobacteriaceae family that is also responsible for infections in the urinary and respiratory tracts, central nervous system, as well as for intra-abdominal and bloodstream infections (Kanamori et al., 2011). Relatively little is known about the virulence of C. freundii. Recently, aggregative adherence cytotoxicity was demonstrated for a specific C. freundii isolate and the strain was found to contain a complete type six secretion system (T6SS) located on a genomic island. This T6SS was shown to regulate the flagellar system, enhanced motility, was involved in adherence to host cells, and induced cytotoxicity to host cells (Bai et al., 2012; Liu et al., 2015).

Opportunistic Food-Borne Pathogens  289 On the basis of in vitro antimicrobial susceptibility tests, aminoglycosides, fluoroquinolones, carbapenems, and the fourth-generation cephems have been used to treat C. freundii infections. The resistance to carbapenems was one of the emerging limitations in antimicrobial therapy. In the 1970s, isolates of C. freundii were usually susceptible to ampicillin and carbenicillin, but resistant to cephalothin (Holmes et al., 1974). Choi et al. (2008) described the presence of an inducible chromosomally located AmpC β-lactamase gene in C. freundii and increasing ability of resistance toward broad spectrum cephalosporins. Similar to the β-lactamase producing enterobacterial spp., class A, B, and D β-lactamase have been identified in C. freundii containing a multidrug-resistant plasmid. The complete nucleotides sequence of plasmid isolated from C. freundii containing a class A type β-lactamase blaKPC-2 gene was recently reported and the blaKPC-2 insertion event of this plasmid was described to occur at the end of a Tn4401 element with the ISKpn6a and Kpc-2 genes and an adjacent Tn3-like segment (Yao et al., 2014). The occurrence of carbapenemresistant Enterobacteriaceae with metallo-β-lactamases (class B) has been mainly reported from Asia, but the IMP-type of MBL producing C. freundii was identified between March 2012 and March 2013 in Germany (Peter et al., 2014). Two of three C. freundii isolates were highly resistant to meropenem (MIC >32 mg/L), whereas one isolate was intermediate susceptible to meropenem, with an MIC of 8 mg/L. These isolates harbored the plasmid contained blaIMP-8 gene with a noncoding point mutation at nucleotide position 18; thus, this plasmid might facilitate the blaIMP-8 gene transfer within enterobacterial spp. and other Citrobacter spp. In addition, a class D β-lactamase producing C. freundii was isolated from a nosocomial wastewater plant and this isolate was found to be blaOXA-372 positive (Antonelli et al., 2015). Moreover, this study showed that the hospital wastewater plant could be a reservoir of β-lactamases within the highly complex microbial community and the mobile antibiotic-resistant elements could be transferred in this wastewater plant by different Enterobacteriaceae. Antibiotic-resistant C. freundii have been isolated from various vegetables such as lettuce, salad, and ready-to-eat-salad (Boehme et al., 2004; Campos et al., 2013; van Hoek et al., 2015). Third-generation cephalosporin antibiotics such as cefotaxime and ceftazidime-resistant C. freundii were isolated from chicory in 2004 (Boehme et al., 2004), and an ampC producing, third generation of cephalosporin-resistant C. freundii were isolated, for example, from lettuce during 20,122,013 in the Netherlands (van Hoek et al., 2015). In addition, a multidrug-resistant C. freundii was isolated from mixed salad and possessed a plasmid-mediated quinolone resistance with the qnrB9 gene conferring ciprofloxacin resistance (Campos et al., 2013). Therefore, raw foods that people eat without any microbiological inactivation steps such as cooking need multilevel strategies to decrease the contamination of opportunistic microorganisms, which often are multidrug-resistant. So far, it is not known whether there has been a food-borne route of C. freundii transmission in a human opportunistic infection, but as for the other closely related Enterobacteriaceae genera such as Klebsiella, Enterobacter, and Serratia, this may be possible and should be further investigated.

290  Chapter 10

10.3.5  Acinetobacter baumannii Species of the genus Acinetobacter (A.) belong to the γ-Proteobacteria, and group in the order Pseudomonadales and family Moraxellaceae (Bouvet and Joly-Guillou, 2000). The description of the different species of the genus Acinetobacter has for many years been problematic (Krizova et al. 2015). There are currently 42 validly published species names, which include at least one pair of synonyms (Touchon et al. 2014; Krizova et al. 2015). The species A. baumannii, A. haemolyticus, and A. calcoaceticus have been isolated from a number of human infections (Doughari et al. 2011) and are thus of clinical importance. In routine diagnostics, however, it is not possible or economically feasible to differentiate accurately between A. calcoaceticus, A. baumannii, A. pittii, and A. nosocomialis strains, as these are highly similar with respect to their phenotypic and biochemical properties. Therefore, these have commonly been grouped together in the so-called A. calcoaceticus-A. baumannii complex (Sheng et al. 2011). A. baumannii is one of the six most important multidrug-resistant microorganisms in hospitals worldwide (Talbot et al., 2006; Antunes et al., 2014) and in 2005 was estimated to cause 2%–10% of all Gram-negative bacterial hospital infections (Antunes et al., 2014). Multidrug-resistant A. baumannii are responsible for severe hospital acquired infections (bloodstream, skin, soft tissue, wound, urinary tract, pulmonary, and device-related infections) and are frequently isolated from immunocompromised patients hospitalized in intensive care units (Antunes et al., 2014; Potron et al., 2015). The emergence of A. baumannii as a major nosocomial pathogen was attributed not only to bacterial characteristics, such as multidrug resistance and its survival for long periods on surfaces and medical equipment, but also to human factors, such as host health status and its association with person-to-person transmission (Medell et al., 2012; Antunes et al., 2014; Tuon et al., 2015). The increase in antibiotic resistance of A. baumannii has consequently reduced therapeutic options for inhibition of this pathogen (Dijkshoorn et al., 2007). The bacterium is known to survive for an extended period in the nosocomial setting and can even cause recurrent outbreaks of, for example, pneumonia, especially in emergency and intensive care units (Peleg et al., 2008; Ahmed et al., 2015). Person-to-person transmission thus appears to be the predominant or probably currently the only route for infection currently known. Antibiotics are, however, also being used for therapeutical purposes in livestock production on a relatively large scale, and this has been linked to the emergence and spread of resistant bacteria from animals and foods stemming from animals to people (Hamouda et al., 2011). For Acinetobacter, however, a previous study (Hamouda et al., 2011) showed that A. baumannii strains from a Scottish abattoir did not possess epidemiological characteristics that are similar to strains isolated from clinics, concluding that A. baumannii isolates from animals investigated in that study were not precursors of the strains causing hospital acquired infections. A. baumannii is also known to occur in animal products such as bulk

Opportunistic Food-Borne Pathogens  291 tank milk (Straley et al., 2006; Gurung et al., 2013; Tamang et al., 2014), as well as in infant milk formula (Wang et al., 2009; Araujo et al., 2014). Nevertheless, a connection between antibiotic-resistant Acinetobacter spp., foods, and community or hospital infections has so far not been shown.

10.4 Conclusions Numerous Gram-positive and Gram-negative bacteria which also occur in foods may act as opportunistic pathogens. Horizontal transmission of virulence factor genes may increase both the number and the type of opportunistic pathogens. A contributing complicating factor is the growing number of immunocompromised people. This number is expected to rise even further due to an increase in the global aged population, which is projected to grow between 2015 and 2030 by 56% (from 901 million to 1.4 billion), reaching nearly 2.1 billion (United Nations, 2015) by 2050. Such an increase in vulnerable people leads to the hypothesis that the number of opportunistic pathogen infections will also continue to increase in the near future, and this will be further exacerbated by the development of multiplying antibiotic-resistant strains. Considering that many opportunistic pathogens occur on produce (vegetables) usually consumed raw or minimally processed, adequate studies should be undertaken to ascertain if there is a food source for such dangerous strains. Once ingested with foods, these strains may survive the gastrointestinal tract and reach the gut, where they can disseminate or cause infections. Genotypic studies based on genotyping techniques such as REA-PFGE (restriction endonuclease analysis-pulsed field gel electrophoresis) or MLST (multilocus sequence typing) and wgMLST should be used to support such investigations on food-borne transmission, as these can accurately relate specific clonal complexes with origin, source, and virulence. Moreover, further investigations are needed to unveil how the food sourse itself may influence the role of the bacteria in virulence, since it was recently demonstrated that growth in foods may actually elicit changes toward an increased virulence in some bacteria (Ketola et al., 2016). Finally, adequate measures should be undertaken to avoid opportunistic food-borne pathogen infections such as (i) the introduction for vulnerable people of low microbial diets (avoiding foods that may contain opportunistic pathogens) or safer weaning foods, either changing the food production processes or the source of raw materials and (ii) the introduction of educational programs on food safety to help immunocompromised people to prevent food-borne opportunistic infections.

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304  Chapter 10 Tuon, F.F., Rocha, J.L., Merlini, A.B., 2015. Combined therapy for multi-drug-resistant Acinetobacter baumannii infection—is there evidence outside the laboratory? J. Med. Microbiol. 64, 951–959. Tzavaras, I., Siarkou, V.I., Zdragas, A., Kotzamanidis, C., Vafeas, G., Bourtzi-Hatzopoulou, E., Pournaras, S., Sofianou, D., 2012. Diversity of vanA-type vancomycin-resistant Enterococcus faecium isolated from broilers, poultry slaughterers and hospitalized humans in Greece. J. Antimicrob. Chemother. 67, 1811–1818. United Nations, 2015. World Population Aging 2015. United Nations, New York. Vahabnezhad, E., Mochon, A.B., Wozniak, L.J., Ziring, D.A., 2013. Lactobacillus bacteremia associated with probiotic use in a pediatric patient with ulcerative colitis. J. Clin. Gastroenterol. 47, 437–439. Van den Bogaard, A.E., Mertens, P., London, N.H., Stobberingh, E.E., 1997. High prevalence of colonization with vancomycin- and pristinamycin-resistant enterococci in healthy humans and pigs in the Netherlands: is the addition of antoibiotics to animal feeds to blame? J. Antimicrob. Chemother. 40, 454–456. van Hoek, A.H., Veenman, C., van Overbeek, W.M., Lynch, G., de Roda Husman, A.M., Blaak, H., 2015. Prevalence and characterization of ESBL- and AmpC-producing Enterobacteriaceae on retail vegetables. Int. J. Food Microbiol. 204, 1–8. Van Tyne, D., Gilmore, M.S., 2014. Virulence plasmids of nonsporulating gram-positive pathogens. Microbiol. Spectr. 2, 1–16. Vasqueza, A., Pancholib, P., Balada-Llasatb, J.M., 2015. Photo quiz: confusing bacteremia in a Crohn’s disease patient. J. Clin. Microbiol. 53, 759. Vela, A.I., Porrero, C., Goyache, J., Nieto, A., Sánchez, B., Briones, V., Moreno, M.A., Domínguez, L., FernándezGarayzábal, J.F., 2003. Weissella confusa infection in primate (Cercopithecus mona). Emerg. Infect. Dis. 9, 1307–1309. Vela, A.I., Fernández, A., de Quirós, Y.B., Herráez, P., Domínguez, L., Fernández-Garayzábal, J.F., 2011. Weissella ceti sp. nov., isolated from beaked whales (Mesoplodon bidens). Int. J. Syst. Evol. Microbiol. 61, 2758–2762. Wald, R., Baumgartner, M., Urbantke, V., Wittek, T., Stessl, B., 2016. Detection of Weissella spp. in milk samples of two dairy cows with clinical mastitis. A case report. Ein Fallbericht. Tierarztl. Prax. Ausg. G Grosstiere Nutztiere 44, 307–312. Walter, J., Hertel, C., Tannock, G.W., Lis, C.M., Munro, K., Hammes, W.P., 2001. Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 67, 2578–2585. Wang, C., Zhang, C.W., Chen, H.C., Yu, Q., Pei, X.F., Liu, H.C., 2008. Phylogeny analysis and identification of two bacterial strains sourcing from human intestine and having resistance to acid and bile. Sichuan Da Xue Xue Bao Yi Xue Ban 39, 263–266. Wang, M., Cao, B., Gao, Q., Sun, Y., Liu, P., Feng, L., Wang, L., 2009. Detection of Enterobacter sakazakii and other pathogens associated with infant formula powder by use of a DNA microarray. J. Clin. Microbiol. 47, 3178–3184. Welch, T.J., Marancik, D.P., Good, C.M., 2014. Weissellosis. In: FHS Blue Book: Suggested Procedures for the Detection and Identification of Certain Finfish and Shellfish Pathogens, 2014 edition 1.3.4. AFS-FHS (American Fisheries Society-Fish Health Section), pp. 1–10. Werner, G., Klare, I., Heier, H., Hinz, K.H., Böhme, G., Wendt, M., Witte, W., 2000. Quinupristin/dalfopristinresistant enterococci of the satA (vatD) and satG (vatE) genotypes from different ecological origins in Germany. Microb. Drug Resist. 6, 37–47. Werner, G., Klare, I., Witte, W., 2002. Molecular analysis of streptogramin resistance in enterococci. Int. J. Med. Microbiol. 292, 81–94. Werner, G., Coque, T.M., Franz, C.M., Grohmann, E., Hegstad, K., Jensen, L., van Schaik, W., Weaver, K., 2013. Antibiotic resistant enterococci-tales of a drug resistance gene trafficker. Int. J. Med. Microbiol. 303, 360–379. Wilcks, A., Andersen, S.R., Licht, T.R., 2005. Characterization of transferable tetracycline resistance genes in Enterococcus faecalis isolated from raw food. FEMS Microbiol. Lett. 243, 15–19. Willems, R.J., Top, J., van Den Braak, N., van Belkum, A., Endtz, H., Mevius, D., Stobberingh, E., van den Bogaard, A., van Embden, J.D., 2000. Host specificity of vancomycin-resistant Enterococcus faecium. J Infect Dis 182, 816–823.

Opportunistic Food-Borne Pathogens  305 Yao, Y., Imirzalioglu, C., Hain, T., Kaase, M., Gatermann, S., Exner, M., Mielke, M., Hauri, A., Dragneva, Y., Bill, R., Wendt, C., Wirtz, A., Domann, E., Chakraborty, T., 2014. Complete nucleotide sequence of a Citrobacter freundii plasmid carrying KPC-2 in a unique genetic environment. Genome Announc. 2, e01157-14. Zaher, A., Cimolai, N., 1997. ERIC-PCR typing profiles of Enterobacter cloacae are stable after development of advanced cephalosporin resistance. Int. J. Antimicrob. Agents 9, 165–167. Zhang, Z., Peng, X., Li, S., Zhang, N., Wang, Y., Wie, H., 2014. Isolation and identification of quercetin degrading bacteria from human fecal microbes. PLoS One 9 (3), e90531. Zornetta, I., Azarnia Tehran, D., Arrigoni, G., Anniballi, F., Bano, L., Leka, O., Zanotti, G., Binz, T., Montecucco, C., 2016. The first non Clostridial botulinum-like toxin cleaves VAMP within the juxtamembrane domain. Sci. Rep. 6, 30257. https://doi.org/10.1038/srep30257. Zurfluh, K., Poirel, L., Nordmann, P., Klumpp, J., Stephan, R., 2015. First detection of Klebsiella variicola producing OXA-181 carabepenase in fresh vegetable imported from Asiat o Switzerland. Antimicrob. Resist. Infect. Control. 4, 38.

Further Reading Bouvet, P.J.M., Grimont, P.A.D., 1986. Taxonomy of the genus Acinetobacter with the recognition of Acinetobacter baumannii sp. nov., Acinetobacter haemolyticus sp. nov., Acinetobacter johnsonii sp. nov., and emended descriptions of Acinetobacter calcoaceticus and Acinetobacter lwoffii. Int. J. Syst. Bacteriol. 36, 228–240. Bouvet, P.J.M., Jeanjean, S., 1989. Delineation of new proteolytic genomic species in the genus Acinetobacter. Res. Microbiol. 140, 291–299. CLSI, 2015. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fifth Informational Supplement. CLSI document M100-S25. Clinical and Laboratory Standards Institute, Wayne, PA. EUCAST, 2016. The European Committee on Antimicrobial Susceptibility Testing. Breakpoint Tables for Interpretation of MICs and Zone Diameters. Version 6.0. http://www.eucast.org. Fishbain, J., Peleg, A.Y., 2010. Treatment of Acinetobacter infections. Clin. Infect. Dis. 51, 79–84. Food and Agriculture Organization (FAO)/World Health Organization (WHO), 2006. In: Enterobacter Sakazakii and Salmonella in Powdered Infant Formula. Second Risk Assessment Workshop. Meeting Report, MRA Series 10, World Health Organization, Geneva, Switzerland. http://www.who.int/foodsafety/publications/ micro/mra10/en/index.html. Heritier, C., Poirel, L., Fournier, P.E., Claverie, J.M., Raoult, D., Nordmann, P., 2005. Characterization of the naturally occurring oxacillinase of Acinetobacter baumannii. Antimicrob. Agents Chemother. 49, 4174–4179. Higgins, P.G., Dammhayn, C., Hackel, M., Seifert, H., 2010. Global spread of carbapenem-resistant Acinetobacter baumannii. J. Antimicrob. Chemother. 65, 233–238. Holzapfel, W.H., 2002. Appropriate starter culture technologies for small-scale fermentation in developing countries. Int. J. Food Microbiol. 75, 197–212. Huys, G., Cnockaert, M., Nemec, A., Dijkshoorn, L., Brisse, S., Vaneechoutte, M., Swings, J., 2005a. RepetitiveDNA-element PCR fingerprinting and antibiotic resistance of pan-European multi-resistant Acinetobacter baumannii clone III strains. J. Med. Microbiol. 54, 851–856. Huys, G., Cnockaert, M., Vaneechoutte, M., Woodford, N., Nemec, A., Dijkshoorn, L., Swings, J., 2005b. Distribution of tetracycline resistance genes in genotypically related and unrelated multiresistant Acinetobacter baumannii strains from different European hospitals. Res. Microbiol. 156, 348–355. Kajala, I., Shi, Q., Nyyssölä, A., Maina, N.H., Hou, Y., Katina, K., Tenkanen, M., Juvonen, R., 2015. Cloning and characterization of a Weissella confusa dextransucrase and its application in high fibre baking. PLoS One 10 (1), e0116418. Kajala, I., Mäkelä, J., Coda, R., Shukla, S., Shi, Q., Maina, N.H., Juvonen, R., Ekholm, P., Goyal, A., Tenkanen, M., Katina, K., 2016. Rye bran as fermentation matrix boosts in situ dextran production by Weissella confusa compared to wheat bran. Appl. Microbiol. Biotechnol. 100, 3499–3510.

306  Chapter 10 Kent, R.M., Fitzgerald, G.F., Hill, C., Stanton, C., Ross, R.P., 2015. Novel approaches to improve the intrinsic microbiological safety of powdered infant milk formula. Nutrients 7, 1217–1244. Kostinek, M., Specht, I., Edward, V.A., Schillinger, U., Hertel, C., Holzapfel, W.H., Franz, C.M., 2005. Diversity and technological properties of predominant lactic acid bacteria from fermented cassava used for the preparation of Gari, a traditional African food. Syst. Appl. Microbiol. 28, 527–540. Lin, M.F., Lan, C.Y., 2014. Antimicrobial resistance in Acinetobacter baumannii: from bench to bedside. World J. Clin. Cases 2, 787–814. Ma, Z., Zhou, L., Wang, H., Luo, L., 2015. Investigation of the genomic diversity of OXA from isolated Acinetobacter baumannii. Int. J. Clin. Exp. Med. 8, 4429–4432. Minervini, F., De Angelis, M., Di Cagno, R., Gobbetti, M., 2014. Ecological parameters influencing microbial diversity and stability of traditional sourdough. Int. J. Food Microbiol. 171, 136–146. Parte, A.C., 2014. LPSN—list of prokaryotic names with standing in nomenclature. Nucleic Acids Res. 42, D613–616. https://doi.org/10.1093/nar/gkt1111. Pereira, A.L., Silva, T.N., Gomes, A.C., Araujo, A.C., Giugliano, L.G., 2010. Diarrhea-associated biofilm formed by enteroaggregative Escherichia coli and aggregative Citrobacter freundii: a consortium mediated by putative F pili. BMC Microbiol. 10, 57. Roca, I., Marti, S., Espinal, P., Martinez, P., Gibert, I., Vila, J., 2009. CraA, a major facilitator superfamily efflux pump associated with chloramphenicol resistance in Acinetobacter baumannii. Antimicrob. Agents Chemother. 53, 4013–4014. Shi, Q., Hou, Y., Juvonen, M., Tuomainen, P., Kajala, I., Shukla, S., Goyal, A., Maaheimo, H., Katina, K., Tenkanen, M., 2016. Optimization of isomaltooligosaccharide size distribution by acceptor reaction of Weissella confusa dextransucrase and characterization of novel α-(1→2)-branched isomaltooligosaccharides. J. Agric. Food Chem. 64, 3276–3286. Sianglum, W., Kittiniyom, K., Srimanote, P., Wonglumsom, W., 2009. Development of multiplex pcr assays for detection of antimicrobial resistance genes in Escherichia coli and enterococci. J. Rapid Meth. Aut. Mic. 17, 117–134. Siegmund-Schultze, N., 2015. Nosokomialinfektionen mit multiresistenten Bakterien: acinetobacter auf dem Vormarsch. Dtsch. Aerztebl. 112. A-184/B-162/C-157. Valacoba, E., Almuzara, M., Gulone, L., Traglia, G.M., Figueroa, S.A., Sly, G., Fernández, A., Centrón, D., Ramírez, M.S., 2013. Emergence and spread of plasmid-borne tet(B)::ISCR2 in minocycline-resistant Acinetobacter baumannii isolates. Antimicrob. Agents Chemother. 57, 651–654. Wolter, A., Hager, A.S., Zannini, E., Czerny, M., Arendt, E.K., 2014. Influence of dextran-producing Weissella cibaria on baking properties and sensory profile of gluten-free and wheat breads. Int. J. Food Microbiol. 172, 83–91.

CHAPTE R 11

Food Poisoning and Intoxication: A Global Leading Concern for Human Health Mohammad AL-Mamun, Tuhina Chowdhury, Baishakhi Biswas, Nurul Absar University of Science and Technology Chittagong (USTC), Chittagong, Bangladesh

11.1 Overview Food is a substance consisting of essential nutrients such as carbohydrates, protein, lipid, minerals, vitamins, and condiment to sustain growth, repair, and vital processes and to furnish energy to live. Some countries define food as a substance that is intended to be, or reasonably expected to be processed, partially processed, or unprocessed for consumption by humans. Major foods or foodstuffs are obtained through agriculture where only 4% of 250,000–300,000 are considered edible plant species, and only 150–200 are used by humans for cultivation (FAO, 1999) to intake, while milk, egg, meat, and fish are obtained from animals. Some cultures consume blood in the form of blood sausage or stews as jugged hare (Davidson, 2014). Usually fruits are eaten raw whereas most of the vegetables and animal foods and foodstuffs are prepared through cooking which involve washing, cutting, trimming, and adding other foods or ingredients to make the foods tasty. Some foods are also manufactured as packaged foods such as prepared meat, fried fish, fruits pickling and juices, beverages, chocolate products, potato chips, etc., which involve salting, curing, curdling, drying, pickling, fermenting, and smoking to prepare these foods which are available in many restaurants and food shops, and usually consumed in most of the cultures. However, sometimes these foods can be a cause of illness, even death due to possible contamination or adulteration. The WHO estimates that almost 1 in 10 people fall ill from eating contaminated foods and causes 420,000 deaths every year globally, where about 30% are children under the age of 5 (WHO, 2015a, 2015b). According to the Centers for Disease Control and Prevention (CDC), >250 known foodborne diseases caused by 31 microbial pathogens (bacteria, fungus, viruses, or parasites) and prions and 48 million people gets sick, 128,000 are hospitalized, and 3000 die each year in the United States (CDC, 2016; Scallan et al., 2011a, 2011b). The rampant use of chemicals in cultivation, farming, manufacturing, cooking, packaging, distribution, and sale are also another major concern for food poisoning that leads to serious health risks and life threatening long-term diseases such as cancer (WHO, 2015a—Fact sheet). Now food safety has become a global issue. Food Safety and Preservation. https://doi.org/10.1016/B978-0-12-814956-0.00011-1 © 2018 Elsevier Inc. All rights reserved.

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11.2  Foodborne Diseases Foodborne diseases can be defined as the illness due to the ingestion of spoiled or poisonous food, contaminated by microorganisms or toxicants, which may occur at any stage during food processing from production to consumption. Contamination may also occur from the environment like using chemicals or polluted water and soil for cultivation or from air. Clinical symptoms represent a wide spectrum of illness, from acute to long-term sickness, which include gastrointestinal infections, immunological or neurological disorders, multiorgan failure, and even cancer.

11.3  Types of Foodborne Diseases The preponderance of foodborne illness is usually caused by harmful bacteria, virus, or parasite including chemicals (CDC, 2010). Based on the causes it can be classified into two categories: 1. Foodborne infections 2. Foodborne intoxications

11.4  Foodborne Infections Illness resulting from contaminated foods by pathogenic microbes can be referred to as foodborne infections. Disease causing microorganisms are ingested through food and infect the gastrointestinal tract by releasing toxins, damaging the intestinal epithelium, and causing gastroenteritis (Frazier and Westhoff, 1988). – – – –

Microorganisms: Bacteria, viruses, and parasites Incubation period: Hours to days Symptoms: Diarrhea, nausea, vomiting, abdominal pain, cramping, fever Transmission: Can spread from person to person through feco-oral route, cross contamination of food – Factors related to contamination: Inadequate cooking, poor personal hygiene, bare hand contact, mixing of raw and cooked food

11.4.1  Bacterial Foodborne Infections Bacteria, single-celled microscopic organisms, are self-sufficient and able to grow not only in the suitable environment but also need available nutrients which are abundant in food. Bacteria can contaminate food at any time during harvesting, processing, storage, and shipping as well as during the preparation of food in restaurants or kitchens if food preparers do not wash their hands, kitchen utensils, cutting boards, or other kitchen surfaces properly. Raw foods such as meat, poultry, fish and shellfish, eggs, unpasteurized milk, and dairy products are the most favorable foodstuff for pathogenic bacterial growth and the cause of illness. Some of the most common foodborne diseases caused by bacteria and their possible sources and symptoms are illustrated in Table 11.1.

Food Poisoning and Intoxication: A Global Leading Concern for Human Health  309 Table 11.1: Bacterial infections Bacteria

Sources and Symptoms

Bacillus cereus

Gram positive, facultative anaerobe, and endospore forming bacteria Food sources: Meats, Stews, Gravies, Vanilla Sauces Symptoms: In diarrheal type: abdominal cramps, watery diarrhea, nausea In emetic type: nausea, vomiting Duration: 24–48 h Gram negative and nonspore forming coccobacilli Food sources: Unpasteurized milk, soft cheese made from the milk of infected animals Symptoms: Fever, chills, sweating, weakness, malaise, muscle pain, endocarditis and myocarditis, spondilytis Duration: Few weeks to months or years Gram negative and nonspore forming Food sources: Raw and undercooked poultry, unpasteurized milk, contaminated water Symptoms: Diarrhea (sometimes bloody), cramps, fever, vomiting Duration: 2–10 days Gram positive, anaerobic, and spore forming bacteria Food sources: Mostly found in improperly canned foods, home canned vegetables, fermented fish, baked potatoes in aluminum foil Symptoms: Vomiting, diarrhea, blurred vision, double vision, difficulty is swallowing, muscle weakness, respiratory failure leads to death Duration: Variable Gram positive, anaerobic, and spore forming bacteria Food sources: Meats, poultry, gravy, dried or precooked foods, time, and temperature abused foods Symptoms: Intense abdominal cramps, watery diarrhea Duration: Usually 24 h Gram negative, shiga toxin producing bacteria Food sources: Naturally occur in undercooked beef (Hamburger), unpasteurized milk and juice, raw fruits, and vegetables (e.g., Srouts), contaminated water Symptoms: Severe diarrhea (often bloody), abdominal pain and vomiting, and can lead to kidney failure. More common in children 4 years to younger Duration: 5–10 days

Brucella Spp.

Campylobacter jejuni

Clostridium botulinum

Clostridium perfringens

Escherichia coli O157:H7

Continued

310  Chapter 11 Table 11.1: Bacterial infections—cont’d Bacteria

Sources and Symptoms

Listeria monocytogenes

Gram positive, facultative bacterium Food sources: Commonly occur in unpasteurized milk and soft cheeses made with that milk, ready to eat deli meats Symptoms: Fever, muscle aches, nausea, diarrhea, immune-compromised patients may develop bacteremia or meningitis; Pregnant women may have mild flu like illness or infection, and can lead to premature delivery or stillbirth Duration: 9–48 h for gastrointestinal diseases, 2–6 weeks for invasive diseases Gram negative, nonspore-forming bacterium Food sources: Commonly occur in eggs, poultry, meat, unpasteurized milk, or juice, cheese, contaminated raw fruits, and vegetables Symptoms: Abdominal cramps, diarrhea, fever, vomiting Duration: 4–7 days Food sources: Meats, poultry, eggs, milk and dairy products, cross contamination Symptoms: Abdominal pains, diarrhea, constipation, headache, achiness, loss of appetite Duration: 2–4 weeks Gram negative, nonmotile and nonspore-forming bacteria Food sources: Contaminated drinking water, uncooked foods, and cooked foods that are not reheated after contact with an infected food handler Symptoms: Abdominal cramps, fever, diarrhea, stool may contain blood, and mucus Duration: 24–48 h Gram positive, nonmotile and catalase positive bacteria Food sources: Unrefrigerated or improperly refrigerated meats, potato, and egg salads, cream pastries Symptoms: Sudden onset of severe nausea and vomiting. Abdominal cramps. Diarrhea and fever may be present Duration: 24–48 h Gram negative bacteria Food sources: Shell fish, salad, cheese, meats, water, beef, raw milk, vegetables, and fruits Symptoms: Watery or bloody diarrhea Duration: 24–48 h Food sources: Undercooked or raw seafood, such as shellfish Symptoms: Watery (occasionally bloody) diarrhea, abdominal cramps, nausea, vomiting, fever Duration: 2–5 days

Salmonella

Salmonella typhi Salmonella Paratyphi A

Shigella

Staphylococcus aureus

Vibrio cholera

Vibrio parahaemolyticus

Food Poisoning and Intoxication: A Global Leading Concern for Human Health  311 Table 11.1: Bacterial infections—cont’d Bacteria

Sources and Symptoms

Vibrio vulnificus

Food sources: Undercooked or raw seafood, such as shellfish (especially oysters) Symptoms: Vomiting, diarrhea, abdominal pain, blood borne infection. Fever, bleeding within the skin, ulcers requiring surgical removal. Can be fatal to persons with liver disease, or weakened immune systems Duration: 2–8 days Food sources: Meats, oysters, fish, crabs, raw milk Symptoms: Frequently occurs in children, vomiting, fever, abdominal pain, bloody stools, headache Duration: Few days to 3 weeks

Yersinia enterocolitica

Source: Scallan, E., Griffin, P.M., Angulo, F.J., Tauxe, R.V., Hoekstra, R.M., 2011a. Foodborne illness acquired in the United States— unspecified agents. Emerg. Infect. Dis. 17(1), 16–22 and Scallan, E., Hoekstra, R. M., Angulo, F. J., Tauxe, R. V., Widdowson, M., Roy, S. L, Jones J.L., Griffin, P.M., 2011b. Foodborne illness acquired in the United States—major pathogens. Emerg. Infect. Dis. 17(1), 7–15. Website: 10.3201/eid1701.P11101; Centers for Disease Control and Prevention (CDC), 2010. Surveillance for foodborne disease outbreaks—United States, 2007. Morb. Mortal. Wkly Rep. 59(31), 973–979; Food and Drug Administration (FDA), 2012. Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural Toxins, second ed., Website: https://www.fda.gov; Food Safety and Inspection Service (FSIS), 2015. Foodborne Illness and Disease, United States Department of Agriculture (USDA), Website: https://www.fsis.usda.gov; National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), 2012. Foodborne Illness, National Institutes of Health (NIH), U.S. Department of Health and Human Services, NIH Publication: 12–4730. Website: https://www.niddk.nih.gov; Farrar, J., Hotez, P., Junghanss, T., Kang, G., Lalloo, D., White, N.J., 2013. Manson's Tropical Diseases E-Book. Elsevier Health Sciences.

11.4.2  Viruses in Foodborne Infections Viruses are much smaller than bacteria and contain genetic material. Usually, viruses do not grow in food because they need living cells for replication. Almost all the viral foodborne diseases are strictly human pathogens (Table 11.2) that are transmitted to humans via food through fecal contamination, but cannot replicate. Most of the foodborne viruses are infectious and spreads so fast from one individual to another and are capable of causing significant illness and mortality in humans (Newell et al., 2010).

11.4.3  Parasitic Foodborne Infections Enteric parasitic infections are transmitted by the fecal-oral route by taking intrinsically contaminated food products. Parasites are different from bacterial pathogens as they do not replicate outside the host. Infection caused by parasite is a symbiotic relationship between two organisms. Most of the foodborne parasites (Table 11.3) has a global distribution, even in developed countries estimating the risk due to the lack of knowledge and awareness among health professionals and necessary researches. Relatively mild symptoms, long incubation periods, and inadequate laboratory detection methods are typical for parasitic diseases and therefore, infection and illness is scarce and often recognized as unknown outbreaks.

312  Chapter 11 Table 11.2: Viral infections Viruses

Sources and Symptoms

Hepatitis A (HAV)

Food sources: Contaminated drinking water, fruit and fruit juices, milk and milk products, uncooked foods, cooked foods, that are not reheated, after contact with an infected food handler, Shellfish from contaminated water Symptoms: Fever, headache, nausea, abdominal pain, diarrhea, dark urine, jaundice, and flu like symptoms Duration: 2 weeks–3 months Food sources: Waterborne and foodborne. e.g., Zoonotic spread involving consumption of undercooked wild boar meat, Pork livers, contaminated tomatoes and strawberries due to using of human and swine feces for irrigation, and agriculture Symptoms: Symptoms are most often seen in patients between the ages of 15–40, Jaundice, malaise, anorexia, abdominal pain, hepatomegaly, vomiting, and fever Duration: 2 weeks Food sources: Consumption of contaminated water, municipal water, well water, stream water, commercial ice, lake water, swimming pool, recreational surface water exposure as well as floodwater, Salad, fruit, and oysters Symptoms: Vomiting (often explosive), watery diarrhea, abdominal cramps, nausea, chills, muscle aches Duration: Generally persist for 12 to 60 h, with a mean period of 24 to 48 h Food sources: Foods such as salads, fruits handled with infected people. Person to person fecal oral spread is the most important means of transmission Symptoms: Vomiting, watery diarrhea, fever, dehydration, hypovolemic shock and in severe cases, death Duration: 3 to 7 days

Hepatitis E Virus (HEV)

Norovirus (NoV)

Rotavirus

Source: Scallan, E., Griffin, P.M., Angulo, F.J., Tauxe, R.V., Hoekstra, R.M., 2011a. Foodborne illness acquired in the United States— unspecified agents. Emerg. Infect. Dis. 17(1), 16–22 and Scallan, E., Hoekstra, R. M., Angulo, F. J., Tauxe, R. V., Widdowson, M., Roy, S. L, Jones J.L., Griffin, P.M., 2011b. Foodborne illness acquired in the United States—major pathogens. Emerg. Infect. Dis. 17(1), 7–15. Website: 10.3201/eid1701.P11101; Centers for Disease Control and Prevention (CDC), 2010. Surveillance for foodborne disease outbreaks—United States, 2007. Morb. Mortal. Wkly Rep. 59(31), 973–979; Food and Drug Administration (FDA), 2012. Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural Toxins, second ed., Website: https://www.fda.gov; Food Safety and Inspection Service (FSIS), 2015. Foodborne Illness and Disease, United States Department of Agriculture (USDA), Website: https://www.fsis.usda.gov; National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), 2012. Foodborne Illness, National Institutes of Health (NIH), U.S. Department of Health and Human Services, NIH Publication: 12–4730. Website: https://www.niddk.nih.gov; Farrar, J., Hotez, P., Junghanss, T., Kang, G., Lalloo, D., White, N.J., 2013. Manson's Tropical Diseases E-Book. Elsevier Health Sciences.

Food Poisoning and Intoxication: A Global Leading Concern for Human Health  313 Table 11.3: Parasitic infections Parasites

Sources and Symptoms

Taxoplasma gondii

Primary sources: Feces of domestic and wild cats, birds, rodents, pigs, sheep, and cattle; feeding on oocyst—contaminated feed, water and soil Food sources: Ingestion of undercooked or raw meats, fruits, and vegetables contaminated with cat feces or by handling of cat’s feces Symptoms: Acute toxoplasmosis: Sore lymph nodes and muscle pains Ocular toxoplasmosis: Blurred or reduced vision, tearing of, or redness in the eye, pain, and sensitivity to light Duration: Last for several weeks Food sources: Ingestion of water or food contaminated with feces of infected humans or animals Symptoms: Sometimes asymptomic. Malodorus diarrhea, malaise cramps, flatulence, and weight loss Duration: Generally 2–6 weeks Food sources: E. histolytica cysts contaminated drinking water and foods, sometimes raw foods may act as source of infection Symptoms: Mild diarrhea to severe, dysentery with mucus and blood, weight loss, liver tenderness Duration: May reside in intestine for years without causing symptoms. Can be last to few days to several weeks Food sources: Contamination mainly occurs from oocysts (animal manure) to lives and food sources. In addition to various foods, such as fresh produces juices and milk may be contaminated. Contaminated water Symptoms: Profuse, watery diarrhea, nausea, vomiting and cramping, fever Duration: 2–14 days Food sources: Imported fresh produce raspberries, basil and lettuce Symptoms: Watery diarrhea, explosive bowl movement, appetite, weight loss, abdominal cramping, bloating Duration: Days to months Primary sources: Infected beef and pork Cysticercosis: Adult T. solium tape worm in human intestine can produce hundreds to thousands of eggs, per day, that can survive for months under harsh environmental conditions Symptoms: Usually asymptomatic, but may cause abdominal pain, nausea, diarrhea, change in appetite, general malaise. Seizures, increased intracranial pressure, headache, and altered mental status Duration: Live for months

Giardia lamblia

Entamoeba histolytica

Cryptosporidium parvum

Cyclospora cayetanensis

Taenia spp. Tania solium Tania saginata

Continued

314  Chapter 11 Table 11.3: Parasitic infections—cont’d Parasites

Sources and Symptoms

Nanophyetus salmincola

Food sources: Live in raw or undercooked fish Symptoms: Abdominal pain, diarrhea, vomiting Duration: Without treatment, symptoms may last several months

Source: Scallan, E., Griffin, P.M., Angulo, F.J., Tauxe, R.V., Hoekstra, R.M., 2011a. Foodborne illness acquired in the United States— unspecified agents. Emerg. Infect. Dis. 17(1), 16–22 and Scallan, E., Hoekstra, R. M., Angulo, F. J., Tauxe, R. V., Widdowson, M., Roy, S. L, Jones J.L., Griffin, P.M., 2011b. Foodborne illness acquired in the United States—major pathogens. Emerg. Infect. Dis. 17(1), 7–15. Website: 10.3201/eid1701.P11101; Centers for Disease Control and Prevention (CDC), 2010. Surveillance for foodborne disease outbreaks—United States, 2007. Morb. Mortal. Wkly Rep. 59(31), 973–979; Food and Drug Administration (FDA), 2012. Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural Toxins, second ed., Website: https://www.fda.gov; Food Safety and Inspection Service (FSIS), 2015. Foodborne Illness and Disease, United States Department of Agriculture (USDA), Website: https://www.fsis.usda.gov; National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), 2012. Foodborne Illness, National Institutes of Health (NIH), U.S. Department of Health and Human Services, NIH Publication: 12–4730. Website: https://www.niddk.nih.gov; Farrar, J., Hotez, P., Junghanss, T., Kang, G., Lalloo, D., White, N.J., 2013. Manson's Tropical Diseases E-Book. Elsevier Health Sciences.

11.4.4  Prions—Transmissible Spongiform Encephalopathies (TSEs) The transmissible spongiform encephalopathies (TSEs), also known as “prion disease” caused by prion protein (PrP), are neurodegenerative diseases. Prions are misfolded protein materials that create numerous holes in the affected brain tissue and give the brain a sponge-like appearance (Prusiner, 1991). Both animals and human can be affected by TSEs. The TSEs in animals include Scrapie in sheep and Bovine spongiform encephalopathy (BSE)—widely known as “mad cow” in cattle. In humans, only the variant Creutzfeldt-Jakob diseases (vCJD) is transmitted through food (meat products or from affected person) rather than Kuru, fatal familial insomnia, Gerstmann-Straussler-Scheinker syndrome, and Creutzfeldt-Jakob diseases (CJD) (CDC, 2015; WHO, 2012—Fact Sheet No.180). Symptoms: vCJD usually present with psychiatric problems such as depression, unpleasant sensations in the limbs or face, problems in walking, and muscle coordination. Victims become forgetful and experience severe problems with processing information and speaking. Patients are hospitalized as they are increasingly unable to care for themselves until death occurs (Prusiner, 1991; Saba and Booth, 2013). Duration: Months to years.

11.5  Leading Factors of Microbial Growth in Food The growth of microbes depends on some influential factors in the food, where some factors act as stimulator while some are inhibitors (Hamad, 2012). As, for example, tomatoes are suitable for the growth of some Lactobacillus species, as the conditional factors for its growth

Food Poisoning and Intoxication: A Global Leading Concern for Human Health  315 are available in tomato. Again, lysozyme in egg, agglutinin in milk, eugenol in cloves, etc., act as natural inhibitors of microbial growth. Factors that influence microbial growth in food are as follows (ICMSF, 1980, 1996): – – – –

Intrinsic factors: Physical and chemical properties of the food. Extrinsic factors: Storage conditions. Implicit factors: Physiological properties of microorganisms. Process factors: Washing, cutting, heating, etc.

11.5.1  Intrinsic Factors Characteristics of food that maintain the microbial growth are called intrinsic factors which include the hydrogen ion concentration (pH), water activity (aw), redox potential (Eh), nutrient contents, antimicrobial constituents, and biological structures (Hamad, 2012)

11.5.2  Hydrogen Ion Concentration (pH) Increasing the acidity of food, either through fermentation or by the addition of weak acids, has been used as a preservation method since ancient times. In natural state, most of the foods such as meat, fish, and vegetables are slightly acidic whereas fruits are moderately acidic. As pathogens do not grow or grow very slowly at pH levels below 4.6, so hydrogen ion concentration (pH) is paramount while using acidity as a preservation method for acidic foods (Hamad, 2012; ICMSF, 1980, 1996; Jay, 2000). pH usually interacts with aw, salt, temperature, Eh, and preservatives to inhibit the growth of pathogens. Foods with inherently low pH (12 h – The symptoms are relieved by taking antihistamine drugs

Ciguatoxin

Scombrotoxin

Source: Mudambi, S.R., Rao, S.M. and Rajagopal, M.V., 2006. Food borne diseases. Food Science, Chapter 19, Delhi, India: New Age International; Food and Drug Administration (FDA), 2012. Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural Toxins, second ed., Website: https://www.fda.gov.

11.6.3  Bacterial Foodborne Intoxications Pathogenic bacteria are also responsible for producing toxicities (Table 11.12) in the food.

Food Poisoning and Intoxication: A Global Leading Concern for Human Health  329 Table 11.12: Bacterial intoxications Bacteria

Important Features and Diseases

S. aureus

– Gram positive, facultative anaerobe, and nonspore-forming bacteria – Some strains produce five types of highly heat stable serological enterotoxins in food like staphylococcal enterotoxins A, B, C, D, and E – Contamination mainly occurs in food from the food handler’s nose, nasal mucosa, skin, saliva, and feces Symptoms: – Rapidly produce symptoms like nausea, abdominal cramping, vomiting, and diarrhea – In more severe cases dehydration, headache, muscle cramping and transient changes in blood pressure, and pulse rate may occur – Duration of illness is 24–72 h. Sometimes required hospitalizations – Gram positive, facultative anaerobe, and endospore forming – Some strains produce two types of enterotoxin: diarrheal and emetic – Meats, milk, vegetables, and fish have been associated with the diarrheal type of intoxications – Especially rice products and other starchy products like potato, pasta, and cheese are related with emetic intoxications Symptoms: – For diarrheal type: Watery diarrhea, abdominal cramp, and pain – For emetic type: Nausea, and vomiting – Gram positive, anaerobe, and spore-forming bacteria – Poisoning occurs due to enterotoxigenic strains type A and some type of C and D strains that have a variety of origins including human and animal feces, water, sewage marine sediments, etc. – Enterotoxin is synthesized during sporulation – Food poisoning strains are heat resistant and serving at 100°C for 1 h Cause of intoxications: – By the ingestion of food containing large numbers of vegetative cells of enterotoxigenic strains – These cells multiply in the intestine and sporulate to release enterotoxin – Spores can resist boiling for 4 or more hours – If the spores are present as contaminants on raw meat they resist boiling or steaming, and on slow cooling the spores will germinate into rapidly multiplying bacterial cells, which produce large amounts of toxin Vehicle foods: – Red meats, chicken, fish, fruits, vegetables, and spices – Food that has been prepared one day and served on the next day Mode of transmission: – Directly from slaughter animals – Contamination from slaughter meat from containers, handlers, dust, and water – Cross contamination in the kitchen environment Symptoms: – Two types of symptoms may be seen – Gastroenteritis form: Watery diarrhea, mild abdominal cramps –  Enteritis necroticans: Abdominal pain and distention, diarrhea (sometimes bloody), vomiting, and patchy necrosis of the small intestine – Illness takes duration of 1–2 days

Bacillus cereus

Clostridium perfringens

Continued

330  Chapter 11 Table 11.12: Bacterial intoxications—cont’d Bacteria

Important Features and Diseases

Clostridium botulinum

– Gram positive bacilli and spore forming anaerobe – Poisoning caused by enterotoxin producing strains – Poisoning strains are two types: Proteolytic and non-proteolytic – Proteolytic strains produce active botulinal toxin while non-proteolytic strains produce inactive pro-toxin that require activation by trypsin – Botulinus toxin is one of the most lethal poisons where its lethal dose has been calculated for an adult person is 10 μg – It is highly neurotoxic, heat stable and resistant to pepsin, and acidic environment – It can resist the action of gastric and intestinal juices Vehicle foods: – Spoiled canned meat, hams, bacon stacked without air access are particularly liable to be infective – Home-made fermented foods with smoked, pickled, and canned foods that are allowed to stand and then eaten without adequate cooking Mode of transmission: – Contamination of food due to improper handling – Insufficient heating of food to destroy spores – Spores present in animal tissues, e.g., meat and fish Symptoms: –  Infant botulism: Occurs in infant 225 mg of zinc – Excess amount of zinc may cause vomiting, diarrhea, abdominal pain, neurologic degeneration, osteoporosis – Additional metals may cause poisoning include antimony, aluminum, platinum and tin. Common symptoms of poisoning from these metals may include gastrointestinal, renal and neurological like headache, irritability, psychosis, stupor, coma, and convulsions

Others

Source: Mudambi, S.R., Rao, S.M. and Rajagopal, M.V., 2006. Food borne diseases. Food Science, Chapter 19, Delhi, India: New Age International; Hoffman, R.S., Howland, M.A., Lewin, N.A., Nelson, L.S., Goldfrank, L.R., 2014. Goldfrank's Toxicologic Emergencies, tenth ed., McGraw-Hill Education, NY; Tchounwou, P.B., Yedjou, C.G., Patlolla, A.K., Sutton, D.J., 2012. Heavy metal toxicity and the environment. EXS 101, 133–164; Schwartz, B.S., Hu, H. 2007. Adult lead exposure: time for change. Environ. Health Perspect. 115(3), 451–454.

11.6.5.3  Antibiotics and Hormone Antibiotic residues in food are becoming the highly concerned emerging contaminants due to increase in the antibiotic resistance incidences in the 21st century. The rampant use of antibiotics in clinical settings coupled with a large pool of immune-compromised patients as well as wide use of the antibiotics in livestock-production operations favors the rapid emergence of antibiotic-resistant microbial strains. In the United States in 2005, Methicillin-resistant S. aureus (MRSA) was associated with 18,650 deaths (Klevens et al., 2007) and MRSA-associated infections have also been widely reported globally (Diekema et al., 2001). Further compounding the problem is that the pathogens causing these diseases are gaining resistance to first line drugs for the treatment of diseases and in some instances found resistance to second and third line drug agents. Despite these, antibiotics are routinely used in livestock production for therapeutic treatment of disease (prophylaxis) and for growth promotion. The classes of antibiotics so used include tetracyclines, macrolides, lincosamides, polypeptides, streptogramins, cephalosporins, penicillins, sulfonamides, aminoglycosides, and fluroquinolones (Schmidt, 2002; Chee-Sanford et al., 2009), all of which include drug members that were originally intended for disease treatment in human but are also being used for livestock-production purposes which lead to increase in the emergence and spread of antibiotic-resistant bacteria. It is a cause for concern and deeper understanding in order to control and prevent the dissemination of resistance is required. Another type of residue is Sterol (hormone), used for increasing growth in animals raised for meat which has been found to be carcinogenic (Mudambi et al., 2006). 11.6.5.4  Radio Nuclides People are exposed daily to a wide range of naturally occurring radioisotopes of chemical elements that is unavoidable and considered to be one of the inherent risks in life.

338  Chapter 11 Radionuclides are dangerous due to their emission of sufficient energy and free radicals. They are highly reactive and responsible for adverse health effects associated with ionizing radiation. Radiological hazards include strontium-90, iodine-131, cesium-137, and various isotopes of plutonium. Consumers’ concern for radionuclides derived from nuclear testing has created a climate of fear of an all-out nuclear war. Although operational risks have been reduced over the past 20 years, natural calamities remain potential threats to nuclear facilities. As a result of unprecedented earthquake and tsunami in March 2011 that hit Fukushima, Japan, several nuclear reactors at the site released massive amounts of radioactive material into the environment and were found in a range of foods, including spinach, tea leaves, milk, cattle meat, and sea food. From Fig. 11.2, it can be assessed easily how radiation can be exposed to human and how it can contaminate food and water. The long-term adverse health effects associated with radionuclides include genetic mutations, teratogenic effects, and cancer (Belitz et al., 2009; Moy and Todd, 2014). 11.6.5.5  Additives and Preservatives Preservatives are the substances or chemicals widely used in food products as well as in pharmaceutical drugs, cosmetics, and biological samples to prevent microbial decompositions and adverse chemical changes. Still, public awareness of food preservatives is uneven. According to the Center for Disease Control (CDC) each year 76 million illnesses, 325,000 hospitalizations, and 5000 deaths linked to foodborne illness have been recorded (Theron and Lues, 2007). The increasing demand for ready-to-eat fresh food has been a challenge for both food manufacturers and distributors about the safety and quality of the foods. Artificial preservatives meet some of these challenges but causes negative side effects rather than preserving freshness. Harmful effects of some food additives and preservatives have been listed in Table 11.15. 11.6.5.6  Adulteration Through Hazardous Chemicals Food adulteration is a very old and common problem, which is often seen in both the low- and middle-income countries and sometimes even in some developed countries. The problem level is greater in low-economic zone like Bangladesh, Indonesia, India, Vietnam, and African countries. Food adulteration is a mostly practiced phenomenon in Bangladesh (Hossain et al., 2008). Consumers are helpless in front of unlawful acts of some unscrupulous importers, producers, wholesaler, or retailers, simply to increase profits with less capital and equipment (Hossain et al., 2008; Rashid, 2007a). Hazardous chemicals such as calcium carbide, sodium cyclamate, cyanide, and formalin are widely used for ripening green tropical fruits, to keep them fresh, and for preserving until sale (Amin et al., 2004; The Daily Prothom Alo, 2005). Low-cost textile dyes are used in coloring vegetables, fruits, popular sweetmeats, soft drinks, beverages, confectioneries to draw customers’ attention (Billah, 2007). Fishmongers are preserving fish with formalin to keep the body solid to cover up internal decomposition (Amin et al., 2004; Rashid, 2007b; Ullah, 2005). Intake of such types of chemically treated food may cause complex diseases and has direct consequences such as liver and kidney failure, autism, metabolic dysfunctions, and cancer (Hossain et al., 2008; Billah, 2007).

Food Poisoning and Intoxication: A Global Leading Concern for Human Health  339

Deposition increases due to heavy rainfall

Radioactive smokes are mixing with the winds and causing dense cloud and heavy rainfall

Direct exposer

Cultivation through contaminated water

Nuclear reactor

Radioactive wastes are mixing with fresh water and contaminating drinking water and fishes

Contaminated fishes

Cattles are eating contaminated grass

Fig. 11.2 Radiation exposures in food contamination.

340  Chapter 11 Table 11.15: Detrimental effects of some additives and preservatives Name

Uses

Diseases

Artificial sweeteners (Aspartame, Saccharine, Sucralose, Fructose Corn Syrup etc.) Refined sugar

Widely used in Soft drinks and beverages

Alter brain neurochemistry, obesity

Use for sweetening

Weight gain, obesity, bloating, fatigue, migraines, gallstones, gum diseases and cavities, arthritis, cardiovascular diseases, lowered immune functions Inhibits natural growth hormone, Monosodium glutamate (MSG) Flavor enhancer. Commonly dramatically promotes irreversible used in Chinese food, canned obesity, cause difficulties in vegetables, soups, and processed breathing, change pulse and heart meats rate, burning sensation, chest pain, Chinese restaurant syndrome, and neurotoxicity Brominated vegetative oil Used in soft drinks in suspension Reduce immune defenses and depletes histamine, myocarditis, fatty changes in liver, thyroid microfollicular hyperplasia, and renal proximal degeneration Reduce the level of polyunsaturated Partially hydrogenerated Used in migraines, crackers, oils (good fat) and trans fat create. vegetative oil candies, baked goods, snack Increase plasma lipid level, associated foods, fried foods, salad with coronary heart disease (CPD), dressings, and many processed breast cancer, colon cancer, and foods atherosclerosis Sodium benzoate (Benzoic acid) Used in soda and others beverage Carcinogenic. Levels of benzene increased when plastic bottles of soda expose to heat or combined with vitamin C. Cause serious damages to thyroidal gland, heart, spleen, liver, kidney, brain, and adrenal glands Butylated hydroxyanisole (BHA) Used in food packaging and Cancer causing preservative. Major cosmetics to prevent spoilage, endocrine disruptor and seriously and poisoning mess with hormones Sodium nitrates and sodium Used in processed meats like Colon cancer, metabolic syndrome nitrites bacon, lunch meat, ham, hot that leads to diabetes dogs, and sausage Artificial colors (Blue: 1 & 2; Used in food and beverages Thyroidal damages; adrenal, bladder, Green: 3; Red: 3; Yellow: 5 & 60 kidney, and brain cancers Antioxidant preservatives Used in fats and oils, processed Urticarial and eczema, breathing (Propyl gallate and foods and meat products to difficulty, gastrointestinal upsets tert-butylhydroquinone) prevent spoilage cholesterol in blood, increase tumor incidences and cancers

Food Poisoning and Intoxication: A Global Leading Concern for Human Health  341 Table 11.15: Detrimental effects of some additives and preservatives—cont’d Name

Uses

Diseases

Formalin (aqueous solution of formaldehyde)

Fruits, vegetables, and fishes are the common food items that are the main target of some deceitful peoples, food vendors, and merchants

Serious inhalation or ingestion can cause sore throat, larynx constriction, bronchitis and pneumonia, dermatitis or allergic reaction, ulceration, and necrosis of the mucous membrane, histopathological alteration in the stomach, gastrointestinal lesions like papillomata’s hyperplasia, and hyperkeratosis. Significantly related with cancer particularly nasopharyngeal and gastrointestinal cancer when administered with drinking water or food

Source: Schaumburg, H.H., Byck, R., Gerstl, R., Mashman, J.H., 1969. Monosodium L-glutamate: its pharmacology and role in the Chinese restaurant syndrome. Science 163(3869), 826–828. doi: 10.1126/science.163.3869.826; Husarova, V., Ostatnikova, D., 2013. Monosodium glutamate toxic effects and their implications for human intake: a review. JMED Res. 2013, 1–12; Munro, I.C., Hand, B., Middleton, E.J., Heggtveit, H.A., Grice, H.C., 1972. Toxic effects of brominated vegetable oils in rats. Toxicol. Appl. Pharmacol. 22(3), 423–429; Saleh, M.K., Hassan, M.A.A., Hamza, B.S., Al-Sereah, B.A., 2015. Clinical observation of toxicological pathology of vegetable oil in white male rats. Int. J. Emerg. Trends Sci. Technol. (IJETST) 2(6), 2552–2556; Kummerow, F.A., 2009. The negative effects of hydrogenated trans fats and what to do about them. Atherosclerosis, 205(2), 458–465, doi: 10.1016/j.atherosclerosis.2009.03.009; World Health Organization (WHO), 2000. Concise International Chemical Assessment Document 26. WHO, Geneva; Chung, J.G., 1999. Effects of butylated Hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on the acetylation of 2-aminofluorene and DNA-2-aminofluorene adducts in the rat. Toxicol. Sci. 51, 202–210; New Hampshire Department of Environmental Services (NHDES), 2006. Nitrate and Nitrite: Health Information Summary. Environmental Fact Sheet. Website: www.des.nh.gov; Amchova, P., Kotolova, H., Ruda-Kucerova, J., 2015. Health safety issues of synthetic food colorants. Regul. Toxicol. Pharmacol. 73(3), 914–922; Silva, M.M. and Lidon, F.C., 2016. An overview on applications and side effects of antioxidant food additives. Emirates J. Food Agric. 28(12), 823–832; Mamun, M.A.A., Rahman, M.A., Zaman, M.K., Ferdousi, Z., Reza, A.A., 2014. Toxicological effect of formalin as food preservatives on kidney and liver tissues in mice model. IOSR J. Environ. Sci., 8(9) Ver. II, 47–51; Griesemer, R.A., Ulsamer, A.G., Acros, J.C., 1982. Report of the federal panel on formaldehyde. Environ. Health Perspect. 43, 139–168; Rumchev, K.B., Spickett, J.T., Bulsara, M.K., Phillips, M.R., Stick, S.M., 2002. Domestic exposure to formaldehyde significantly increases the risk of asthma in young children. Eur. Respir. J. 20, 403–408.

11.7  Additional Sources of Contamination 11.7.1  Unhealthy Cooking Process Intake of nutrient-rich food does not mean always healthy diet. It loses its terms due to improper way of processing or unhealthy cooking which may be the cause of serious illness. Processing and cooking are now a challenge to keep food healthy. Some common actions in food preparation can be mentioned as unhealthy are as follows: – Cooking food in unhygienic conditions and environment. – Improper way of washing and cutting vegetables, fruits, fish, and meat (Uçar et al., 2016).

342  Chapter 11 – Deep frying in hot oil increases the saturated fat content of the food which increases cholesterol level and body weight and can cause stomach infections (Moreno and Fagan, 2015; de Souza et al., 2015). – Pan fry is also an unhealthy cooking process (Boskou and Elmadfa, 2010). – Cooking or heating food in plastic containers in microwave oven is also unhealthy. Plastic container contains cancer-causing substances which can get mixed with food at the time of heating or cooking inside the microwave oven (Moreira et al., 2014). – Charcoal barbecue, a popular way of cooking fish or meat is unhealthy. Charcoal smoke is carcinogenic and can cause respiratory diseases (McDonald, 2015; Prathomtong et al., 2016).

11.7.2  Intake of Reheating Food Intake of reheating leftover cooked food is a common phenomenon. People usually refrigerated the unused portion and reheated it before next meal, sometimes it can happen three to four times. With every reheating the food loses its nutritional quality and turn into toxin which may cause serious health effects. Research shows that heating over and over turns the nitrates of celery, spinach, and beets into toxic and release carcinogenic properties. Mushroom should be consumrd immediately after preparation. Eggs, the powerhouse of protein, can cause digestive problems after reheating. Botulism growth occurs in potatoes if left to cool down after frying without refrigeration, which cannot be killed by reheating or heating in microwave oven. Spores usually grow in uncooked rice, even in cooked rice. If the cooked rice is left at room temperature, spores multiply their growth and produce food poisoning. Reheating won’t get rid of this poison. Reheating cold refrigerated chicken meat can cause digestive trouble if it is not properly cooked. Overheating changes the protein compositions of meat (Roberts, 1982; Mann and Truswell, 2012).

11.7.3  Retail, Restaurant, and Travel Foods Meat, poultry products, and shell fish are significantly considered as the vehicles for the transmission of foodborne diseases (Duffy et al., 2006; Myint et al., 2006), which often occur from retail shops, restaurants, and travel foods made from contaminated raw foods, cross contamination during processing and storage, insufficient cooking, inadequate cooling, a lapse of >12 h between cooking and eating, improper handling by consumers or by infected food service workers, and container leakages increases the incidents of outbreaks (Sammarco et al., 1997). Studies revealed that different types of pathogens such as Salmonellas, E. coli, Campylobacter jejuni, Clostridium perfringens, Shigella, Staphylococcus, Listeria monocytogenes are often found in fresh meat and poultry products, which are most common in almost all retail shops and restaurants. Salmonella, Plesiomonas shigelloides, and Staphylococcus are also found in various types of shellfish like raw shrimp or crab meat while

Food Poisoning and Intoxication: A Global Leading Concern for Human Health  343 Vibrio gastroenteritis is the most commonly found pathogen in raw oysters (Altekruse et al., 2000; Wallace et al., 1999). Diseases due to these pathogenic microbes can cause serious, and sometimes fatal complications that make the immune-compromised patients, pregnant women and children vulnerable. Symptoms include bloody diarrhea, severe abdominal pain, hemolytic uremic syndrome (HUS), neurological disorders, long-term illness, and hospitalization (Duffy et al., 2006; Altekruse et al., 2000).

11.8 Outbreaks Lack of proper knowledge about food compositions and possible contaminations, poor hygiene, improper way of handling, unhealthy cooking, and storage are mainly responsible for most of the outbreaks and occurring frequently in every moment anywhere in the world including less developed, developing, or developed countries. Thousands of incidents are being born but only a few are recorded. The burden, especially the extent and cost of unsafe food is not well understood till now. Epidemiological data and laboratory capacity to detect the cause of foodborne disease outbreak are not available widely, particularly in the developing countries. As a result, many foodborne incidents often go unrecognized, unreported, or uninvestigated. Investigation and control of foodborne diseases are multidisciplinary tasks which demand experts from food chemistry and microbiology, food safety, laboratory medicine, clinical medicine, epidemiology, risk communication, and management (WHO, 2008). In that case, the initiatives by World Health organization (WHO) for evidence-based outbreaks data generation as well as the data from US FDA and US CDC enable us to know about the incidents and at the same time enable the policy makers to prioritize and allocate resources for food safety.

11.8.1  Notable Incidents From 2011 to 2017 References: Disease outbreak news. 2011—Vinegar from China contaminated with ethylene glycol led to 120 cases of illness including 11 deaths (The China Times, 2011). 2011—German E. coli O104:H4 outbreak, caused by contaminated fenugreek seeds. >3950 people were affected including 53 deaths (EFSA, 2012). 2011—United States Listeriosis outbreak. It was the second deadliest bacterial foodborne outbreak in US history which affected nearabout 28 states, 147 were infected including 33 deaths due to consumption of Listeria contaminated cantaloupe produced by Jensen Farms (CDC, 2012). 2013—European aflatoxin contamination. From February to March 2013, most of the European countries including Romania, Serbia, and Croatia reported nationwide contamination of milk for human consumption with aflatoxins (Mycotoxins, 2013).

344  Chapter 11 2013—Bihar school midday meal poisoning. On July 16, 2013, nearabout 23 students died and >25 fell ill due to the intake of pesticide contaminated midday meal (BBC, 2013). 2015—On July 13, 2015, nearabout 2000 in Caraga region of Philippines were affected after consuming durian mangosteen and mango flavored candies, most of them were school children (Rappler, 2015). 2015—United States E. coli O157:H7 outbreak. The spread of E. coli O157:H7 through contaminated celery which was consumed with chicken salad at various large retailers. Nearabout 155,000 products were recalled from >12 states, 19 people were affected from seven states and five were hospitalized (CNN, 2015). 2015—On April 15, 2015, The National Health and Family Planning Commission (NHFPC) of China notified WHO of 20 additional laboratory confirmed cases of human infection with avian influenza A (H7N9) virus from February 14 to March 21, 2015, including four deaths. The WHO advised to avoid poultry farms, wash hands with soap and water before taking foods, and to follow good food safety and good food hygiene practice (WHO, 2016). 2015—On October 21, 2015—The Ministry of Health and Social Welfare (MOHSW) of the United Republic of Tanzania has notified WHO of new foci of cholera outbreaks in the country. On October 13, 13 regions were affected and the cumulative number of cases was 4835 including 68 deaths. Water sanitation measures were implemented by MOHSW, WHO, US Centers for Disease Control (CDC), and the Field Epidemiology Programme (WHO, 2015). 2016—Punjab sweet poisoning. From April 20 to May 8, 2016 about 33 people died including 5 children due to the intake of “Laddu”—a baked confection which was poisoned by highly toxic insecticide—Chlorfenapyr (Mail Online, 2016). 2016—Campylobacteriosis incident in New Zealand. In August 2016 about 5500 were infected including three deaths due to contamination of unchlorinated drinking water with Campylobacter from sheep feces (DIA, 2017). 2016—Multistate outbreak of Hepatitis A linked to frozen strawberries. Overall, 143 cases were reported including 56 people were hospitalized from nine states of the United States due to the consumption of Hepatitis A contaminated strawberries (CDC, 2016). 2017—Outbreak of Listeriosis Linked to Soft Raw Milk Cheese. Total eight cases were counted including two deaths from four states of the United States (CDC, 2017a). 2017—Salmonella Kiambu outbreak. Illness started from May 17 to June 28, 2017 and reported on July 21, 2017 as 47 people were infected including 1 death and 12 people were hospitalized from 12 states due to intake of Salmonella kiambu strain contaminated Maradol Papayas (CDC, 2017b).

Food Poisoning and Intoxication: A Global Leading Concern for Human Health  345

11.9 Conclusion Food itself is a medicine due to its wide variety of nutrients and phytochemicals, but it should be safer for better health. Safer food also saves lives. With every bite one is potentially exposed to illness from either microbiological or chemical contamination. Intake of contaminated food and therefore, diseases are an important cause of morbidity and mortality, and a significant impediment to socioeconomic development worldwide. Globally, billions of people are at high risk of foodborne diseases and millions fall ill every year. Many also die as a result of consuming unsafe food (WHO, 2016). Concerns about food safety have skyrocketed in more affluent societies. However, the real tragedy of foodborne diseases is vital in poor and middle economic zone. Uses of impure water for cleaning and processing of food, unhygienic food-production processes and handling, inappropriate use of additives, preservatives, or chemicals, inadequate food storage infrastructure, and defective regulatory standards are the major contributors of foodborne diseases ranging from mild to debilitating and life threatening such as liver and kidney failure, neural disorders, paralysis, and cancers, including long period of absenteeism and premature death. The types, severity, and impacts of these illnesses are different through ages and diverse across regions, countries, and communities. Considerable differences were observed in the burden of foodborne diseases among the regions based on the child and adult mortality by WHO (FERG) and as reported in 2015, Africa (AFR, AFR D, and AFR E sub regions) have the highest burden of the foodborne diseases where the South-East Asian Regions are in second position (WHO, 2016). According to the WHO (2016 reports) and FERG reports of 2015, for the global estimates, 31 foodborne hazards causing 32 diseases are included, 11 being diarrheal, 7 invasive infectious, 10 helminthes, and 3 chemical disease agents were responsible for 600 million illness and 420,000 deaths in 2010. Again 150 million illness and 175,000 deaths and 12 million disability adjusted life years (DALYs) were reported by WHO South-East Asian Regions in 2010. Out of 10 children, three children under 5 years of age were found to be suffering from diarrhea due to Enterotoxigenic E. coli (11 million cases), Enteropathogenic E. coli (7.3 million cases), and Campylobacter Spp. (7 million cases) in 2010 in the South-East Asian Regions (Fig. 11.3). So, foodborne disease is one of the major concerns in 21 century. It does not recognize borders. A local incident can quickly become an international emergency due to the speed and range of product distribution, impacting health, international relations and trade. For many living at or below the poverty line, foodborne illness perpetuates the cycle of poverty. Beyond the individual level, it affects the economic development, challenging the agricultural and food (export) industries including tourists. Food contamination endangers billions of people at risk. Now global warming are forwarding this issue in a more complicated manner. Climatic factors influence the growth and survival of pathogens, as well as their transmission pathways (Jaykus et al., 2008). Higher ambient temperatures increase replication cycles of foodborne pathogens, and prolonged seasons may

346  Chapter 11 augment the opportunity for foodborne infections (Tirado and Schmidt, 2001). Pathogens are also altering their genome structures and changing disease patterns including multidrug resistance (Shuman, 2010; Githeko et al., 2000). As a result, medications and treatment are being complicated day by day, which signifies the importance of food safety that is still a challenging and questionable issue in low- and middle-economic zone. Enterotoxigenic E. coli Enteropathogenic E. coli Campylobacter spp. Shigella spp. Norovirus Non-typhoidal S. enterica Ascaris spp. Giardia spp. Hepatitis A virus Entamoeba histolytica 0

2 million

4 million

6 million

8 million

10 million

12 million

Fig. 11.3 Top 10 causes of foodborne illness in children under 5 years of age in South-East Asian Regions in 2010. Source: FERG Report, World Health Organization (WHO), 2015. WHO estimates of the global burden of foodborne diseases, Foodborne Disease Burden Epidemiology Reference Group (FERG), 2007–2015.

Food safety is a public health priority and a shared responsibility. Foodborne diseases are preventable. There are no single or global solutions to prevent the foodborne diseases. Nationwide policy development (based on geography, climate, potential contaminants or pathogens and their disease patterns) and establishment of regulatory frameworks and its implementation are the essential and foremost steps in food safety system. Proper education and training are needed among food producers, suppliers, handlers, and the general public, including women and school children. All food operators and consumers have to understand the roles that they must play to protect their health and for the wider community. Laboratory capacity must be strengthened to be able to detect the contaminants or pathogens, surveillance and collection of local data to validate regional estimates and translation of estimates of foodborne diseases into food safety policy. Strong coordination and cross border action across the entire food supply chain is highly required. A strengthened food safety system of a country can positively impact the food safety system of other countries (WHO, 2016). The International Health Regulations (IHR, 2005) are a legally binding instrument to ensure global health security (WHO, 2016). It calls upon WHO member states to build core capacities for the implementation of IHR (2005), including food safety events. WHO, FAO, and International Food Safety Authorities Networks (INFOSAN) are working with

Food Poisoning and Intoxication: A Global Leading Concern for Human Health  347 governments and partners to reduce the level of food contamination throughout different stages of the food chain and to ensure food safety from production to consumption in line with Codex Alimentarius (CA)—a collection of international food standards, guidelines, and codes of practice; and also ensuring effective and rapid communication during food safety emergencies. WHO’s basic principles for food safety that is widely known as “Five Keys to Safer Food” and that everyone should know all over the world to prevent the foodborne diseases are as follows (WHO, 2016): 1. Keep clean – Thoroughly wash raw fruits and vegetables with tap water. – Keep clean hands, kitchen, and chopping board all times. 2. Separate raw and cooked food – Do not mix raw meat and ready-to-eat food. – Do not mix raw meat, fish, and raw vegetables. 3. Cook thoroughly – Thoroughly cook all meat, poultry, and seafood, especially shell fish. – Reheat all leftovers until they are steaming hot. 4. Keep food at safe temperatures – Refrigerate cooked food within 2 h of preparation. – Never defrost food at room temperature; defrost frozen food in the refrigerator, cold water, or in the microwave. 5. Use safe water and raw materials – Use safe drinking water for preparing food. – Check use-by dates and labels while buying packed food.

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350  Chapter 11 Prusiner, S.B., 1991. Molecular biology of prion diseases. Science 252, 1515–1522. https://doi.org/10.1126/ science.1675487. PMID 1675487. Rao, M.B., Tanksale, A.M., Ghatge, M.S., Deshpande, V.V., 1998. Molecular and biotechnological aspects of microbial proteases. Microbiol. Mol. Biol. Rev. 62 (3), 597–635. Rashid, S., 2007a. Vejale Chheye Gechhe Desh (the country has been flooded with adulterated foods). The daily Janakantha, pp. 1 and 11, Bangladesh. Rashid, S., 2007b. Sorbotro Sorbonasa Formaline (Everywhere, the dangerous formalin). The Daily Janakantha, pp. 1–2, Bangladesh. Ray, B., 2004. (e-library 2005). Fundamental Food Microbiology, third ed. CRC Press Taylor & Francis Group, LLC, Boca Raton, FL. Ray, B., Bhunia, A., 2013. Factors influencing microbial growth in food. In: Fundamental Food Microbiology, fifth ed. CRC Press Taylor & Francis Group, LLC, Boca Raton, FL. Roberts, D., 1982. Factors contributing to outbreaks of food poisoning in England and Wales 1970–1979. J. Hyg. 89 (3), 491–498. https://doi.org/10.1017/S0022172400071059. Saba, R., Booth, S.A., 2013. The genetics of susceptibility to variant Creutzfeldt–Jakob disease. Public Health Genom. 16 (1–2), 17–24. https://doi.org/10.1159/000345203. PMID 23548713. Sammarco, M.L., Ripabelli, G., Grasso, G.M., 1997. Consumer attitude and awareness towards food-related hygienic hazards. J. Food Saf. 17 (4), 215–221. https://doi.org/10.1111/j.1745-4565.1997.tb00188.x. Scallan, E., Griffin, P.M., Angulo, F.J., Tauxe, R.V., Hoekstra, R.M., 2011a. Foodborne illness acquired in the United States—unspecified agents. Emerg. Infect. Dis. 17 (1), 16–22. Scallan, E., Hoekstra, R.M., Angulo, F.J., Tauxe, R.V., Widdowson, M., Roy, S.L., Jones, J.L., Griffin, P.M., 2011b. Foodborne illness acquired in the United States—major pathogens. Emerg. Infect. Dis. 17 (1), 7–15. Website: https://doi.org/10.3201/eid1701.P11101. Schmidt, C.W., 2002. Antibiotic-resistance in livestock: more at stake than steak. Environ. Health Perspect. 110 (7), A396–A402. Shalaby, A.R., 1996. Significance of biogenic amines to food safety and human health. Food Res. Int. 29 (7), 675–690. https://doi.org/10.1016/S0963-9969(96)00066-X. Shuman, E.K., 2010. Global climate change and infectious diseases. N. Engl. J. Med. 362, 1061–1063. https://doi. org/10.1056/NEJMp0912931. Skandamis, P.N., Nychas, G.J.E., 2012. Quorum sensing in the context of food microbiology. Appl. Environ. Microbiol. 78, 5473–5482. Soriano, J.M., Dragacci, S., 2004. Occurrence of fumonisins in foods. Food Res. Int. 37 (10), 985–1000. The Commonwealth Scientific and Industrial Research Organisation (CSIRO), 2015. Refrigerated Storage of Perishable Foods. Food Standards Australia New Zealand, Canberra, Australia. Website: https://www.csiro.au. The Daily Prothom Alo, 2005. Chemical die Aam Pakanor Ovijoge Chhoijoner Saja (six were punished for ripening mango using chemicals), Bangladesh, pp. 19–20. The International Health Regulations (IHR), 2005. Areas of Work for Implementation. World Health Organization. (June, 2007). Theron, M.M., Lues, J.F., 2007. Organic acids and meat preservation: a review. Food Rev. Int. 23, 141–158. Tirado, C., Schmidt, K., 2001. WHO surveillance programme for control of foodborne infections and intoxications: preliminary results and trends across greater Europe. J. Inf. Secur. 43 (1), 80–84. Uçar, A., Yilmaz, M.V., Çakıroğlu, F.P., 2016. Food safety—problems and solutions. In: Significance, Prevention and Control of Food Related Diseases. InTech, EU, Croatia. Chapter 1, https://doi.org/10.5772/63176. Ullah, H., 2005. Adulterated Food: Thought From an Expatriate. The Daily Star, Bangladesh. United States Environmental Protection Agency (USEPA), 2009. Reregistration Eligibility Decision (RED) for Malathion. Prevention, Pesticides and Toxic Substances (7508P), EPA: 738–R–06–030. Vos, J.G., Dybing, E., Greim, H.A., Ladefoged, O., Lambré, C., Tarazona, J.V., Brandt, I., Vethaak, A.D., 2000. Health effects of endocrine-disrupting chemicals on wildlife, with special reference to the European situation. Crit. Rev. Toxicol. 30 (1), 71–133. https://doi.org/10.1080/10408440091159176. PMID 10680769. Wallace, B.J., Guzewich, J.J., Cambridge, M., Altekruse, S., Morse, D.L., 1999. Seafood-associated disease outbreaks in New York, 1980-1994. Am. J. Prev. Med. 17 (1), 48–54.

Food Poisoning and Intoxication: A Global Leading Concern for Human Health  351 World Health Organization (WHO), 2008. Foodborne Disease Outbreaks: Guidelines for Investigation and Control. World Health Organization (WHO), 2012. Variant Creutzfeldt-Jakob Disease, Fact Sheet N0180. Website: http:// www.who.int. World Health Organization (WHO), 2015a. Food Safety, Fact Sheet N0399, (December 2015). World Health Organization (WHO), 2015b. WHO estimates of the global burden of foodborne diseases, Foodborne Disease Burden Epidemiology Reference Group (FERG), 2007–2015.

Further Reading Amchova, P., Kotolova, H., Ruda-Kucerova, J., 2015. Health safety issues of synthetic food colorants. Regul. Toxicol. Pharmacol. 73 (3), 914–922. Centers for Disease Control and Prevention (CDC), 2016. Burden of Foodborne illness in the United States. Centers for Disease Control and Prevention (CDC). last updated: September 1, 2016, Website: https://www. cdc.gov. Chung, J.G., 1999. Effects of butylated Hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on the acetylation of 2-aminofluorene and DNA-2-aminofluorene adducts in the rat. Toxicol. Sci. 51, 202–210. Clay, C.E., Board, R.G., 1991. Growth of salmonella enteritidis in artificially contaminated hens’ shell eggs. Epidemiol. Infect. 106, 271–281. European Food Safety Authority (EFSA), 2012. Scientific opinion on the risks for public and animal health related to the presence of citrinin in food and feed. EFSA J. 10 (3), 2605. Farrar, J., Hotez, P., Junghanss, T., Kang, G., Lalloo, D., White, N.J., 2013. Manson’s Tropical Diseases E-Book. Elsevier Health Sciences, China. Food and Drug Administration (FDA), 2012. Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural Toxins, second ed. Website: https://www.fda.gov. Food Safety and Inspection Service (FSIS), 2015. Foodborne Illness and Disease. United States Department of Agriculture (USDA). Website: https://www.fsis.usda.gov. Graeme, K.A., 2012. Toxic Plant Ingestions, Wilderness Medicine. Chapter 64, sixth ed. Elsevier Mosby, Philadelphia, PA. Graeme, K.A., 2014. Mycetism: a review of the recent literature. J. Med. Toxicol., 1–17. Griesemer, R.A., Ulsamer, A.G., Acros, J.C., 1982. Report of the federal panel on formaldehyde. Environ. Health Perspect. 43, 139–168. Hoffman, R.S., Howland, M.A., Lewin, N.A., Nelson, L.S., Goldfrank, L.R., 2014. Goldfrank’s Toxicologic Emergencies, tenth ed. McGraw-Hill Education, NY. Hunting, E.R., Kampfraath, A.A., 2013. Contribution of bacteria to redox potential (Eh) measurements in sediments. Int. J. Environ. Sci. Technol. 10, 55–62. https://doi.org/10.1007/s13762-012-0080-4. Husarova, V., Ostatnikova, D., 2013. Monosodium glutamate toxic effects and their implications for human intake: a review. JMED Res. 2013, 1–12. Kummerow, F.A., 2009. The negative effects of hydrogenated trans fats and what to do about them. Atherosclerosis 205 (2), 458–465. https://doi.org/10.1016/j.atherosclerosis.2009.03.009. Mamun, M.A.A., Rahman, M.A., Zaman, M.K., Ferdousi, Z., Reza, A.A., 2014. Toxicological effect of formalin as food preservatives on kidney and liver tissues in mice model. IOSR J. Environ. Sci. 8 (9), 47–51. Ver. II. Morris, J.G., Potter, M., 2013. Foodborne Infections and Intoxications. Elsevier Science, USA. Munro, I.C., Hand, B., Middleton, E.J., Heggtveit, H.A., Grice, H.C., 1972. Toxic effects of brominated vegetable oils in rats. Toxicol. Appl. Pharmacol. 22 (3), 423–429. National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), 2012. Foodborne Illness. National Institutes of Health (NIH), U.S. Department of Health and Human Services, NIH Publication: 12–4730. Website: https://www.niddk.nih.gov. New Hampshire Department of Environmental Services (NHDES), 2006. Nitrate and Nitrite: Health Information Summary. Environmental Fact Sheet, Website: www.des.nh.gov.

352  Chapter 11 Olson Jr., J.C., Nottingham, P.M., 1980. Temperatures in Microbial Ecology of Foods. vol. 1. Acedemic Press, NY. Rumchev, K.B., Spickett, J.T., Bulsara, M.K., Phillips, M.R., Stick, S.M., 2002. Domestic exposure to formaldehyde significantly increases the risk of asthma in young children. Eur. Respir. J. 20, 403–408. Saleh, M.K., Hassan, M.A.A., Hamza, B.S., Al-Sereah, B.A., 2015. Clinical observation of toxicological pathology of vegetable oil in white male rats. Int. J. Emerg. Trends Sci. Technol. (IJETST) 2 (6), 2552–2556. Schaumburg, H.H., Byck, R., Gerstl, R., Mashman, J.H., 1969. Monosodium L-glutamate: its pharmacology and role in the Chinese restaurant syndrome. Science 163 (3869), 826–828. https://doi.org/10.1126/ science.163.3869.826. Schwartz, B.S., Hu, H., 2007. Adult lead exposure: time for change. Environ. Health Perspect. 115 (3), 451–454. Silva, M.M., Lidon, F.C., 2016. An overview on applications and side effects of antioxidant food additives. Emirates J. Food Agric. 28 (12), 823–832. Tchounwou, P.B., Yedjou, C.G., Patlolla, A.K., Sutton, D.J., 2012. Heavy metal toxicity and the environment. EXS 101, 133–164. World Health Organization (WHO), 2000. Concise International Chemical Assessment Document 26. WHO, Geneva. World Health Organization (WHO), 2016. Burden of Foodborne Diseases in the South-East Asia Region. WHO Regional Office for South-East Asia, India.

Disease Outbreak News References BBC News, 2013. Violent Protests in India Over School Meal Deaths. Website: http://www.bbc.com. CDC, 2016. Multistate Outbreak of Hepatitis a Linked to Frozen Strawberries. Website: https://www.cdc.gov. CDC, 2017a. Multistate Outbreak of Listeriosis Linked to Soft Raw Milk Cheese Made by Vulto Creamery. Website: https://www.cdc.gov. CDC, 2017b. Multistate Outbreak of Salmonella Kiambu Infections Linked to Imported Maradol Papayas. https:// www.cdc.gov. Centers for Disease Contrl and Prevention (CDC), 2012. Multistate Outbreak of Listeriosis Linked to Whole Cantaloupes from Jensen Farms, Colorado. Website: https://www.cdc.gov. CNN, 2015. E. coli Recall Affects Major Retailers Across the U.S. Website: http://money.cnn.com. European Food Safety Authority (EFSA), 2012. E.coli: Rapid Response in a Crisis. Website: https://www.efsa. europa.eu. Mail Online, 2016. Pakistan Poisoned Sweets Death Toll Climbs to 33. Website: http://www.dailymail.co.uk. Mycotoxins, 2013. EU: Feed Contaminated With Aflatoxin. Website: www.allaboutfeed.net. Rappler, 2015. Number of People Sick From Davao Candies Jumps to 2,000. Website: https://www.rappler.com. The China Times, 2011. Eleven People Died From Poison in Vinegar. Website: http://www.thechinatimes.com. The Department of Internal Affairs (DIA), 2017. Government Inquiry into Havelock North Drinking Water, New Zealand. Website: https://www.dia.govt.nz. WHO, 2015. Cholera—United Republic of Tanzania, Disease outbreak news. Website: http://www.who.int. WHO, 2016. Human Infection With Avian Influenza a (H7N9) Virus—China. Disease outbreak news. Website: www.who.int.

CHAPTE R 12

Staphylococcal Food Poisoning Vincenzina Fusco⁎, Giuseppe Blaiotta†, Karsten Becker‡ *

Institute of Sciences of Food Production, National Research Council of Italy (CNR-ISPA), Bari, Italy University of Naples Federico II, Portici, Italy ‡University Hospital Münster, Institute of Medical Microbiology, Münster, Germany †

12.1  Staphylococcal Food Poisoning: Definition and Clinical Symptoms Staphylococcal food poisoning (SFP), also referred to as staphylococcal foodborne disease (FBD), is by definition a microorganism-related intoxication disease caused by the consumption of foods that contain sufficient amounts of preformed staphylococcal enterotoxins (SEs) that are sufficient to trigger the disease (Bergdoll, 1989). While putative first involvement of staphylococci in food poisoning has been described in 1884 after consumption of cheese and in 1914 after consumption of bovine raw milk (Barber, 1914; Barg and Harris, 1997), subsequent SFP reports started to come out in the 1930s starting with a report by Dack et al. (1930). Down to the present day, foodborne illness is a major public health concern around the world. Inadequately refrigerated, undercooked, or incompletely reheated food, which is contaminated with an enterotoxin-producing Staphylococcus aureus isolate, can result in food poisoning (Becker and Köck, 2014). Principally, SFP follows ingestion of SEs that have been released into contaminated foodstuff (Que and Moreillon, 2015). The most conspicuous characteristic is the abrupt onset of the principal symptoms comprising nausea, violent vomiting, and general malaise approximately 2–6 hours after food ingestion (Becker et al., 2015; Que and Moreillon, 2015). The incubation period depends on the amount of toxin ingested (Murray, 2005). This is accompanied by abdominal cramping, with or without diarrhea (Argudín et al., 2010) but lacks fever. The SFP is habitually self-limiting and, characteristically, symptoms wanes within the next 8–12 hours, but not exceeding 1–2 days (Que and Moreillon, 2015; Tranter, 1990). The SFP is generally not fatal; however, very rarely deadly outcome has been reported mostly due to severe dehydration and hypotension in elderly patients or younger children (Murray, 2005; Que and Moreillon, 2015). In terms of morbidity and mortality, older individuals are more susceptible than younger persons (Tranter, 1990). The differential diagnosis includes an acute gastroenteritis caused by emetic Bacillus cereus, which could give an identical clinical picture (Ehling-Schulz et al., 2015). Food Safety and Preservation. https://doi.org/10.1016/B978-0-12-814956-0.00012-3 © 2018 Elsevier Inc. All rights reserved.

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354  Chapter 12

12.2  Staphylococcal Enterotoxins The first discovered “classical” SEs, designated SEA through SEE, have already been described in the 1960–70s by the groups of Bergdoll and Casman (Bergdoll et al., 1959, 1965, 1971; Casman, 1960; Casman et al., 1967). A further one, initially designated as SEF (Bergdoll et al., 1981), has been later identified as TSST-1. Continuing the enterotoxin alphabet with SEG, many more SEs or SE-like toxins have been discovered within this superfamily to date (Jarrau et al., 2001). They also include molecules that exhibit high sequence similar to classical SEs but lack emetic properties or have not been tested for this feature. According to the standard nomenclature for the superantigens expressed by Staphylococcus proposed by the International Nomenclature Committee for Staphylococcal (INCSS) they should be designated as “staphylococcal enterotoxin-like” (SEl) superantigens (SAgs) (Lina et al., 2004). Today, SEs comprise a large family with more than 20 described toxins reaching the letter “Y.” Moreover, some of these SEs are polymorphic and comprise several serologically and/or molecularly defined variants such as SEC (SEC1, SEC2, SEC3, SECbovine, SECcanine, SECovine, and SECepi), SEG (SEGv, SEGL29P), SEI (SEIv), and SElU (SElUv) (Abe et al., 2000; Blaiotta et al., 2004a,b, 2006; Podkowik et al., 2016; Thomas et al., 2007a,b). Additional sequence variations—including the promoter region—exist and the degree of sequence conservation seems to be gene dependent (Johler et al., 2016). Based on the similarity of their amino acid sequences, SEs/SEls form three groups, i.e., the SEA group (SEA, SED, SEE, SEH, SElJ, SElN, SElO, SElP, and SES), the SEB group (SEB, SEC1–3, SEG, SER, and SElU), and the SEI group (SEI, SElK, SElL, SElM, SElQ, SElV), while SET and SElY is phylogenetically distinct from these groups (Ono et al., 2008, 2015). This subgroup, comprising SET and SElY, is related to the subgroup consisting of SElX, TSST-1, and the so-called staphylococcal superantigen-like proteins (SSLs) (Ono et al., 2015). The SEs and SEls are the members of the large SAg protein family, also referred to as pyrogenic toxin superantigen (PTSAg) family. These true exotoxins are serologically distinct; however, they share not only function but sequence homology, phylogenetic relationships, and structure (Balaban and Rasooly, 2000; Dinges et al., 2000; Hu and Nakane, 2014). The encoding genes of the SEs and SEls, respectively, are placed in the staphylococcal chromosome (SEB, SEG, SEI, SElM, SElN, SElO, SElU, SElV, SElY, and SElX) and/or found integrated in mobile genetic elements including plasmids (SEB, SED, SElJ, SER, SES, and SET), prophages (SEA, SEE, and SElP), and transposons (SEH); often together with further virulence factors (Balaban and Rasooly, 2000; Becker, 2005; Dinges et al., 2000; Pinchuk et al., 2010). Several SEs [SEB, SEC1–SEC3, SElK, SElL, SElQ, enterotoxin gene cluster (egc)-encoded SEs/SEls] are encoded on genomic islands (SaPI3, SaPIbov, and SePI). On SaPI3, an operon-designated (egc) has been detected that occurs in different subtypes (Jarrau et al., 2001; Letertre et al., 2003a). It forms a putative nursery of superantigens and contains five SE/SEl genes seg, sei, sem, sen, and seo, transcribed into a single polycistronic

Staphylococcal Food Poisoning  355 mRNA, as well as further SEls and pseudogenes (Jarrau et al., 2001; Kuroda et al., 2001; Williams et al., 2000). Themolecular weights of the SEs/SEls range from approximately 19.3 (SElX) to 28.6 (SElJ) kDa (Becker, 2005; Thomas et al., 2007a,b). Of note, SElW is often annotated as SEA in S. aureus genomes, although, its amino acid sequence is only 36% identical to that of SEA (Okumura et al., 2012). The most well-known SAgs are several streptococcal (e.g., streptococcal pyrogenic exotoxins), clostridial, and staphylococcal superantigens, however, Gram-negative bacteria such as Yersinia spp. and Mycoplasma arthritidis as well as eukaryotic microorganisms (Plasmodium falciparum, Toxoplasma gondii, and Candida albicans) are also able to express SAgs. Concerning S. aureus, this family includes, besides the SEs, also the toxic shock syndrome (TSS) toxin 1 (TSST-1) responsible for menstrual and nonmenstrual TSS. However, SEs, predominantly SEB, and SEC, have also been described as TSS-causing agents (DeVries et al., 2011; Schlievert, 1986). The SAgs are extremely potent stimulators of the immune system. As bivalent molecules, all PTSAgs share the ability to cross bridge both major histocompatibility complex (MHC) class II molecules on antigen-presenting cells and T cell receptors in an uncontrolled, nonantigen-specific manner causing a stimulation of up to 30%–50% of T cells (Fleischer and Schrezenmeier, 1988; Herman et al., 1991; Li et al., 1999); hence their designation as “superantigens” (Herman et al., 1991; Marrack and Kappler, 1990; White et al., 1989). Nano- to picogram concentrations of SAgs are sufficient to result in a massive release of proinflammatory cytokines after excessive polyclonal T-lymphocyte activation (Dinges et al., 2000; Llewelyn and Cohen, 2002). Unlike TSST-1, SEs are additionally defined by their emetic activity when ingested by humans or administered in the respective animal model (monkey feeding test) (Bergdoll, 1988; Lina et al., 2004). The SEs are tasteless, soluble in water, resistant to acids, desiccation, and proteolysis by digestive enzymes, and, of particular importance for food hygiene, highly heat stable (Worms, 1960; Tranter, 1990). The thermostable enterotoxin molecules can be inactivated only by prolonged boiling (heating to 95°C for 30–60 minutes is without loss of biological activity) (McCormick et al., 2001; Spaulding et al., 2013). In addition to the classical SEs, emetic activity in humans and/or other primates has been proven for SEG, SEH, SEI, SER, SES, and SET (Munson et al., 1998; Omoe et al., 2003; Ono et al., 2008; Su and Wong, 1995). Also, SElK, SElL, SElM, SElN, SElO, SElP, SElQ, and SElY are able to induce emetic reactions in monkeys or other animals, but, to a lesser extent compared with classical SEs (Omoe et al., 2013; Ono et al., 2015). However, other studies found no emetic activities for SElL and SElQ (Orwin et al., 2002, 2003). According to the recommendation of the INCSS, renaming as SEs instead of SEls could be considered for respective toxins (Omoe et al., 2013). Also from SFP outbreak investigations, there is some evidence that at least the egc-encoded newer SEls may cause typical symptoms including nausea and vomiting (Johler et al., 2015; Yan et al., 2012). For other SEls, respective data are not yet available.

356  Chapter 12

12.3  Epidemiology of SFP According to WHO definition, FBDs are “transmitted through ingested food” and “comprise a broad group of illnesses caused by enteric pathogens, parasites, chemical contaminants, and biotoxins” (WHO, 2008). Enterotoxigenic S. aureus strains contribute significantly to foodborne illness. In accordance with the zoonoses monitoring activities carried out in 32 European countries in 2013, a total of 5196 foodborne outbreaks were reported in the European Union (EU) comprising 43,183 human cases with 5946 hospitalizations and 11 deaths (EFSA and ECDC, 2015). For foodborne outbreaks caused by staphylococcal toxins, 13 European countries submitted data on 386 outbreaks with 1304 cases with an overall reporting rate of 0.13 per 100,000 population; of these, 70 were general outbreaks and 21 were household or domestic kitchen outbreaks (EFSA and ECDC, 2015). Thus, SEs were involved in 7.4% of all notified outbreaks in 2013. In the United States, passive surveillance on outbreak-associated illnesses estimated 323 laboratory confirmed cases with a hospitalization rate of 6.4% for the year 2006 (Scallan et al., 2011). While SFP outbreaks received considerable attention, most sporadic cases of SFP are undiagnosed or underreported. In particular, lack of routine examination of fecal specimens for S. aureus or its enterotoxins, problems with sensitivity, specificity, and practicability of enterotoxin-detecting assays as well as technical shortcomings in terms of sample collection and laboratory diagnostics may contribute to this situation (Kadariya et al., 2014). Thus, the true extent of SFP and related burden might not be adequately reflected in epidemiological studies on FBDs. Moreover, due to the drawbacks of the commercially available enterotoxin assays and their limitation for the detection of the classical SEs, many current studies are based on the detection of the SE-encoding genes, which inadequately reflect the real extent of the disease. The SFP epidemiology is closely linked to the prevalence of S. aureus in human hosts and animal food products while other CPS species and coagulase-negative staphylococci (CNS), if any epidemiologically significant role at all, only play a marginal role. The natural habitat of S. aureus is the human nasal cavity (Kaspar et al., 2016) and about 10%–35% of the general human population carry this pathogen permanently while the others are intermittently colonized (van Belkum et al., 2009). From the nose, S. aureus is transferred to the skin, mucous membranes, and other body areas and could cause mild to severe infections when overcoming the skin barrier (von Eiff et al., 2001). The transmission takes place to food products via contaminated hands. In contrast to pyogenic infections necessitating vital bacterial cells, the sole presence of the preformed SEs are sufficient to cause the SFP symptoms. However, for SFP genesis, the presence of SE-producing S. aureus cells is prerequisite at least once during the process of food preparation. Once produced in a sufficient amount, further processing such as heating has little or no influence on causing the disease.

Staphylococcal Food Poisoning  357 Data regarding the frequency of S. aureus enterotoxigenic strains vary considerably in terms of the specimen investigated, the approach used for direct or indirect detection (toxin detection vs. detection of the encoding genes; prevalence of serum antibodies), the number of SEs targeted by the detection approach used as well as the regional epidemiology of circulating S. aureus strains. Until the detection of the “postclassical” SEs, the percentage of SE-producing strains has been estimated to be approximately 25%, however, large deviations in both directions have been reported (Bergdoll, 1989). Applying nucleic acid-based detection approaches, the amount of those strains putatively able to produce SEA–SEE possessing encoding genes was found to be roughly 10% higher (Becker et al., 2003). Depending on the number of additionally included nonclassical SEs, the number of SE/SEl gene-possessing S. aureus strains increases drastically to more than or equal to 80% (Alibayov et al., 2014; Becker et al., 2003, 2004a,b). Thus, the possession of SE/SEl genes seems to be a habitual feature of S. aureus. Among all SEs, SEA is the most common toxin implicated in SFP (Schelin et al., 2011; Suzuki et al., 2014) followed by other classical SEs. Concerning the nonclassical SEs, outbreaks due to SEH-positive strains or due to strains possessing the egc cluster have been reported (Jørgensen et al., 2005; Johler et al., 2015). Non-S. aureus coagulase-positive staphylococcal species are of importance in veterinary medicine and are occasionally involved in human infections, e.g., those caused by animal bites (Becker et al., 2015). The SFP outbreaks due to these species have been anecdotally described (Khambaty et al., 1994), although a substantial percentage of strains of the S. intermedius complex were tested in phenotypically SEC-positive strains to analyze the occurrences of the classical SEs (Becker et al., 2001a,b). In this study, about 11% of the isolates including those from dogs, horses, and humans carried the canine variant of the sec gene, but no other SE genes. The same overall order of magnitude for the possession of SEs (SEC, also SEA or SED) by the members of S. intermedius complex has also been found in subsequent studies (Tanabe et al., 2013). Since the discovery of the classical SEs, there have been occasional reports on SE-producing CNS or, later on, reports on the detection of SE-encoding genes in CNS isolates from human and animal specimens as well as from foodstuffs (de Lourdes et al., 2006; Oliveira et al., 2010; Orden et al., 1992). Conversely, several studies could not confirm these observations (Becker et al., 2001a,b; Blaiotta et al., 2004a,b; Harvey and Gilmour, 1985; Nemati et al., 2008; Rosec et al., 1997). Until today, reports on well-characterized SE-producing CNS are missing, except the recent reports on two enterotoxigenic S. epidermidis isolates (Madhusoodanan et al., 2011; Podkowik et al., 2016). Similar to SaPIbov1 (Fitzgerald et al., 2001), SEC- and SElL-encoding genes were found on a pathogenicity island, designated SePI-1 (Madhusoodanan et al., 2011; Podkowik et al., 2016). The significance of the detection of SE/SEl-producing and/or SE/SEl genes-carrying isolates of non-S. aureus staphylococcal species for human health and food safety remains unclear, but—by the evidence of reported SFP cases and outbreaks currently available—are of rather subordinate importance.

358  Chapter 12

12.4  Detection of Staphylococcal Enterotoxins in CPS Despite the improved awareness and the progress made in the diagnosis of SEs, foodborne outbreaks caused by SEs are being reported worldwide. The EU legislation (EC regulations no. 2073/2005, 1441/2007) imposes the enumeration of CPS at specific step of the production process (process hygiene criteria) when the number of staphylococci is expected to be the highest and the screening of samples for the presence of SEs are well above the specified M values (food safety criteria). The enumeration of CPS in food microbiology is usually based on the use of selective agar medium such as Baird Parker with either egg yolk tellurite emulsion or rabbit plasma fibrinogen supplement, used in the relevant ISO protocols (ISO, 1999a,b), that due to a halo of the fibrin precipitation allow discrimination between coagulase-positive and coagulasenegative colonies during plate counting. The final taxonomic identification of CPS can be achieved by the analysis of DNA sequence or via species-specific PCR. Apart from the 16S DNA gene (Becker et al., 2004b), several genes such as heat shock protein 60 (hsp60) gene (Goh et al., 1997), the femA gene (Vannuffel et al., 1999), 16S–23S rRNA intergenic spacer region (Maes et al., 1997), the sodA gene (Poyart et al., 2001), the tuf gene (Martineau et al., 2001), the rpoB gene (Drancourt and Raoult, 2002; Mellmann et al., 2006), the gap gene (Ghebremedhin et al., 2008), and the kat gene (Blaiotta et al., 2010) have been targeted to achieve the sequence-based identification of Staphylococcus species and subspecies, respectively. Among these, the sodA, the hsp60, and the kat genes are the most suited ones for sequence-based identification of the CPS species (Blaiotta et al., 2010; Sasaki et al., 2007). An excerpt of simplex conventional and real-time PCRs developed so far and applied to identify and characterize CPS, together with the protocols to assess the presence of all the se and sel genes known to date, is illustrated in Tables 12.1 and 12.2. Although conventional PCR is still used especially in underdeveloped countries, it has been replaced in most laboratories by real time PCR, this being a more rapid, high throughput, and reliable method, allowing for real-time simultaneous quantitative detection of several target genes. The current trend is toward multiplex real-time PCR assays as these allow the identification and characterization of the target species (Table 12.3). Since matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become available for routine diagnostics of microorganisms, it has revolutionized and, in particular, accelerated species identification of microbes (Hillenkamp and Karas, 1990; Idelevich et al., 2014; van Belkum et al., 2015). As for other bacterial species, MALDI-TOF MS shows excellent sensitivity and specificity for the identification of clinically relevant staphylococcal species (Harris et al., 2010; Zhu et al., 2015). Also for

Table 12.1: Conventional PCR and real-time protocols for the detection of genes useable for the identification and characterization of coagulase positive/variable staphylococci (selection) Species a

S. aureus

Target Gene or Fragment femA

mecA

References Encoded Product

Conventional PCR

Real-Time PCR

Factor essential for the expression of methicillin resistance in MRSA PBP2A (Penicillin binding protein 2A) in MRSA

Riyaz-Ul-Hassan et al. (2008), Riva et al. (2015), Vannuffel et al. (1998)

Francois et al. (2003)

Murakami et al. (1991), Ndhai et al. (2013), Obaidat et al. (2015), Riva et al. (2015), Vannuffel et al. (1998)

Costa et al. (2005), Elsayed et al. (2003), Grisold et al. (2002), Kim et al. (2013b), Klaschik et al. (2015), McDonald et al. (2005), and Thomas et al. (2007a,b)

PBP2a mecC-variant in MRSA

kat 16S

Catalase 16S rRNA

tuf gap

sod

Elongation factor Tu Glyceraldehyde-3phosphate dehydrogenase β-Subunit of bacterial RNA polymerase 60-kDa heat shock protein (GroEL) Superoxide dismutase

nuc

Thermostable nuclease

aroA

5-Enolpyruvylshikimate-3phosphate synthase

rpoB hsp60

García-Álvarez et al. (2011), Kriegeskorte et al. (2012), Shore et al. (2011) Blaiotta et al. (2010) Becker et al. (2004b), Ghebremedhin et al. (2008), Martineau et al. (1998), Woo et al. (2001) Martineau et al. (2001) Blaiotta et al. (2010), Ghebremedhin et al. (2008) Blaiotta et al. (2010), Ghebremedhin et al. (2008) Ghebremedhin et al. (2008), Goh et al. (1996), Mellmann et al. (2006) Ghebremedhin et al. (2008), Poyart et al. (2001), Xing et al. (2014) Brakstad et al. (1992), Ercolini et al. (2004, 2005), Fusco et al. (2011), Schaumburg et al. (2014), Smyth et al. (2001), Wilson et al. (1991)

Hagi et al. (2010)

Alarcón et al. (2006), Costa et al. (2005), Elsayed et al. (2003), Fang and Hedin (2003), Hein et al. (2001), Kadiroğlu et al. (2014), McDonald et al. (2005), Poli et al. (2007), Studer et al. (2008), Thomas et al. (2007a,b)

Marcos et al. (1999) Continued

Staphylococcal Food Poisoning  359

mecC

Species

Target Gene or Fragment coa mupA blaZ spa

htrA lukS/lukF-PV

S. hyicus S. intermedius a

Sa442 sodA nuc

References Encoded Product

Conventional PCR

Coagulase enzyme Determinant of mupirocin resistance Beta-lactamase Staphylococcus aureus protein A

Ahmadi et al. (2010) Zhang et al. (2004)

Aires-de-Sousa et al. (2007), Blaiotta et al. (2006), Frénay et al. (1996), Hwang et al. (2010), Johler et al. (2011), Shopsin et al. (1999), Stephan et al. (2001)

Panton-Valentine leukocidin

Lina et al. (1999), von Eiff et al. (2004)

Superoxide dismutase Thermostable nuclease

Voytenko et al. (2006) Baron et al. (2004), Becker et al. (2005)

Real-Time PCR

Pereira et al. (2014) Okolie et al. (2015)

Chiang et al. (2007) McDonald et al. (2005), Fosheim et al. (2011) Grisold et al. (2002)

Recently, a Staphylococcus aureus complex has been defined bydelimitation of S. argenteus and S. schweitzeri from S. aureus according to Tong et al. (2015) (note that specificity of the other assays listed in the table has not been proven so far for the different members of this species complex). Compared to S. aureus, the 16S rRNA gene sequence is identical in S. argenteus and differs at one position in S. schweitzeri; differentiation is possible by MALDI-TOF mass spectrometry.

360  Chapter 12

Table 12.1: Conventional PCR and real-time protocols for the detection of genes useable for the identification and characterization of coagulase positive/variable staphylococci (selection) —cont’d

Staphylococcal Food Poisoning  361 Table 12.2: Conventional and real time simplex PCR protocols for the detection of staphylococcal enterotoxin and toxic-shock syndrome toxin genes References Target Gene or Fragment

Encoded Producta

sea

SEA

seb

SEB

sec

SEC

sec1

SEC1

sec2 sec3 sed

SEC2 SEC3 SED

see

SEE

seg

SEG

seh

SEH

Conventional PCR

Real-Time PCR

Adwan et al. (2005), Arcuri et al. (2010), Argudín et al. (2010), Ikeda et al. Blaiotta et al. (2004b), Bartolomeoli et al. (2009), (2005) Bendahou et al. (2009), Song et al. (2015), Johnson et al. (1991), Intrakamhaeng et al. (2012), Rall et al. (2008), Riva et al. (2015), Rosec and Gigaud (2002), Tsen and Chen (1992), Xing et al. (2014) Adwan et al. (2005), Arcuri et al. (2010), Argudín et al. (2010), Bartolomeoli et al. (2009), Bendahou et al. (2009), Blaiotta et al. (2004b), Intrakamhaeng et al. (2012), Johnson et al. (1991), Rall et al. (2008), Riva et al. (2015), Rosec and Gigaud (2002), Song et al. (2015), Xing et al. (2014), Wilson et al. (1991) Adwan et al. (2005), Arcuri et al. (2010), Argudín et al. (2010), Bartolomeoli et al. (2009), Bendahou et al. (2009), Blaiotta et al. (2004b), Chiang et al. (2006), Ercolini et al. (2004), Intrakamhaeng et al. (2012), Johnson et al. (1991), Rall et al. (2008), Riva et al. (2015), Rosec and Gigaud (2002), Song et al. (2015), Xing et al. (2014) Chen et al. (2001), Mäntynen et al. (1997), Pinto et al. (2005), Wilson et al. (1991) Chen et al. (2001) Chen et al. (2001) Adwan et al. (2005), Arcuri et al. (2010), Argudín et al. (2010), Bartolomeoli et al. (2009), Bendahou et al. (2009), Blaiotta et al. (2004b), Intrakamhaeng et al. (2012), Johnson et al. (1991), Rall et al. (2008), Riva et al. (2015), Rosec and Gigaud (2002), Song et al. (2015), Tsen and Chen (1992), Xing et al. (2014) Adwan et al. (2005), Arcuri et al. (2010), Argudín et al. (2010), Bartolomeoli et al. (2009), Bendahou et al. (2009), Blaiotta et al. (2004b), Johnson et al. (1991), Rall et al. (2008), Riva et al. (2015), Rosec and Gigaud (2002), Song et al. (2015), Tsen and Chen (1992), Xing et al. (2014) Arcuri et al. (2010), Argudín et al. (2010), Bania et al. (2006), Bendahou et al. (2009), Blaiotta et al. (2004b), Blaiotta et al. (2006), Ercolini et al. (2004), Fusco et al. (2011), Johnson et al. (1991), Little et al. (2008), McLauchlin et al. (2000), Rall et al. (2008), Rosec and Gigaud (2002), Song et al. (2015), Xing et al. (2014) Arcuri et al. (2010), Argudín et al. (2010), Bania et al. (2006), Bendahou et al. (2009), Ercolini et al. (2004), Rall et al. (2008), Rosec and Gigaud (2002), Song et al. (2015), Xing et al. (2014) Continued

362  Chapter 12 Table 12.2  Conventional and real time simplex PCR protocols for the detection of staphylococcal enterotoxin and toxic-shock syndrome toxin genes—cont’d References Target Gene or Fragment

Encoded Producta

sei

SEI

selj

SElJ

selk

SElK

sell

SElL

selm

SElM

seln

SElN

selo

SElO

selp selq ser ses set selu

SElP SElQ SER SES SET SElU

selu2 selv selw selx sely φent1

SElU2 SElV SElW SElX SElY Pseudogene φent1 Pseudogene φent2 TSST-1

φent2 tst egc

a

Enterotoxin gene cluster

Conventional PCR Arcuri et al. (2010), Argudín et al. (2010), Bania et al. (2006), Bendahou et al. (2009), Blaiotta et al. (2006), Ercolini et al. (2004), Fusco et al. (2011), Little et al. (2008), McLauchlin et al. (2000), Rall et al. (2008), Rosec and Gigaud (2002), Song et al. (2015), Xing et al. (2014) Arcuri et al. (2010), Bania et al. (2006), Bendahou et al. (2009), Blaiotta et al. (2004b), Rall et al. (2008), Rosec and Gigaud (2002), Song et al. (2015), Xing et al. (2014) Bania et al. (2006), Bendahou et al. (2009), Chiang et al. (2006), Song et al. (2015) Arcuri et al. (2010), Bania et al. (2006), Bendahou et al. (2009), Song et al. (2015) Akineden et al. (2008), Bania et al. (2006), Bendahou et al. (2009), Blaiotta et al. (2004b, 2006), Fusco et al. (2011) Akineden et al. (2008), Bania et al. (2006), Bendahou et al. (2009), Blaiotta et al. (2004b, 2006), Fusco et al. (2011), Song et al. (2015) Akineden et al. (2008), Bania et al. (2006), Bendahou et al. (2009), Blaiotta et al. (2004b, 2006), Fusco et al. (2011), Song et al. (2015) Bania et al. (2006), Chiang et al. (2008), Song et al. (2015) Bania et al. (2006), Chiang et al. (2008), Song et al. (2015) Argudín et al. (2010), Chiang et al. (2008), Song et al. (2015) Argudín et al. (2010), Ono et al. (2008), Song et al. (2015) Argudín et al. (2010), Ono et al. (2008), Song et al. (2015) Fusco et al. (2011), Chiang et al. (2008), Letertre et al. (2003a,b), Song et al. (2015)

Real-Time PCR

Letertre et al. (2003b)

Thomas et al. (2006) Thomas et al. (2006) Roetzer et al. (2016) Wilson et al. (2011) Ono et al. (2015) Letertre et al. (2003a,b) Blaiotta et al. (2006), Fusco et al. (2011), Letertre et al. (2003a,b) Argudín et al. (2010), Blaiotta et al. (2004b), Johnson et al. (1991) Argudín et al. (2010), Blaiotta et al. (2004b, 2006), Collery et al. (2009), Fusco et al. (2011), Little et al. (2008), McLauchlin et al. (2000)

SE, Staphylococcal enterotoxin; SEl, Staphylococcal enterotoxin-like; TSST-1, Toxic-shock syndrome toxin 1.

Fusco et al. (2011)

Staphylococcal Food Poisoning  363 Table 12.3: Conventional and real time multiplex PCR protocols for the identification, detection and characterization of CPS Species or Group S. aureus, S. epidermidis, S. haemolyticus, S. hominis, S. lugdunensis, S. saprophyticus Gram-positive cocci S. aureus, S. capitis, S. caprae, S. epidermidis, S. haemolyticus, S. hominis, S. lugdunensis, S. saprophyticus, S. warneri Staphylococcus genus and S. aureus CPS species Staphylococcus aureus

References

Target Gene or Fragment

Conventional PCR

fbl, ileS2, sap, nuc, mecA, mvaA, sep, hom

Campos-Peña et al. (2014)

mecA, S. aureus femA, S. epidermidis femA, 16S nuc

Na et al. (2015) Hirotaki et al. (2011)

Mason et al. (2001) Sasaki et al. (2010) Becker et al. (1998)

mecA, clfA, 16S nuc sea, seb, sec, sed, see, tst, eta, etb selm, seln, selo

Becker et al. (2004a)

16S rRNA, nuc, mecA

Montazeri et al. (2015)

SCCmec; sea and seb

Havaei et al. (2015)

SCCmec and sea-see

Riva et al. (2015)

SCCmec

McClure-Warnierm et al. (2013) Nagaraj et al. (2014)

sea, coa, sec, seb, sei, seg, tst, IAC, mecA SCCmec, mecA

Zhang et al. (2012)

sea, seb, sec

Zeinhom et al. (2015)

sea-see, seg, seh, sei, sej, selk, sll, selq, tst mecA, femA

Alibayov et al. (2014,b)

seb, sec-1, tst

Schmitz et al. (1998)

16S, mecA, femA

Xu et al. (2012)

mecA, aacA-aphD, tetK, tetM, erm(A), erm(C), vat(A), vat(B), vat(C), 16S sea, seb, sec, sed, see

Strommenger et al. (2003)

entA, entB, entC, entD, entE, femA

Real-Time PCR

Vannuffel et al. (1995)

Fooladi and Naderi (2010), Sharma et al. (2000) Kav et al. (2011), Mehrotra et al. (2000) Continued

364  Chapter 12 Table 12.3: Conventional and real time multiplex PCR protocols for the identification, detection and characterization of CPS—cont’d Species or Group

References

Target Gene or Fragment

Conventional PCR

sea, seb, sec, sed, see, seg, seh., sei, sej, tst eta, etb, tst, mecA, femA

Peles et al. (2007), Rosengren et al. (2010) Mehrotra et al. (2000)

Real-Time PCR

sea-see, tst

Chiefari et al. (2015)

nuc, mecA, 16S

Wang et al. (2014)

nuc, mecA, pvl

Velasco et al. (2014)

mecA, SCCmec-orfX, 16S

Kim et al. (2013a)

mecA, fem

Kwon et al. (2012)

16S, mecA, pvl,

Okolie et al. (2015)

SCCmec, orfX

Huletsky et al. (2004) Chen et al. (2012)

agr, gyrB sea, seb, sec1, sed, mecA, femB sea, seb, sec, sed, see, seg, seh, sei, sej nuc, mecA, tst,

Klotz et al. (2003) Letertre et al. (2003b) Fosheim et al. (2011)

the species identification of foodborne pathogens including staphylococci, this approach has proven useful, accurate, and rapid (Böhme et al., 2012; Regecová et al., 2014). In addition, several biosensors have been developed for the sensitive, real time, and on-site detection of S. aureus (Fusco and Quero, 2014). The latest platforms include an electrochemical immunosensor (Abbaspour et al., 2015), an electrochemical impedance sensor biochip (Primiceri et al., 2016), an electrochemical DNA-base biosensor (Sun et al., 2015), a nanoscale-film optical fiber immunosensor (Bandara et al., 2015), a loop-mediated isothermal amplification (LAMP)-surface plasmon resonance (SPR) biosensor (Nawattanapaiboon et al., 2015), a DNA-based fluorescence resonance energy transfer (FRET) biosensor based on grapheme quantum dots and gold nanoparticles biosensor (Shi et al., 2015), and a magnetically assisted surface-enhanced Raman scattering biosensor (Wang et al., 2015). However, none of these have been applied to detect S. aureus in food. Considering that SEs are thermostable and thus may persist in a food matrix after the vegetative cells have been inactivated, their detection may be crucial. Active SEs are usually

Staphylococcal Food Poisoning  365 detected by in vivo monkey or kitten bioassay (Bennett, 2005; Bergdoll, 1988). However, Rasooly and Hernlem (2012) demonstrated that tumor necrosis factor (TNF) protein could be used as biomarker for the quantitative detection of active SEA. Apart from the PCR-based detection of enterotoxin genes, serological methods based on the use of anti-enterotoxin polyclonal or monoclonal antibodies are most commonly used to detect SEs in food and mainly include radioimmunoassay, reversed passive latex agglutination, enzyme-linked immunosorbent (ELISA), and enzyme-linked fluorescent assays, with detection limits ranging from 0.5 to 2 ng enterotoxin per gram of food (Bergdoll and Reiser, 1980; Bennett, 2005; Di Pinto et al., 2004; Mathieu et al., 1992; Miller et al., 1978; Park et al., 1994; Vernozy-Rozand et al., 2004; Wieneke, 1991). Zhang et al. (2013) developed a chemiluminescence enzyme immunoassay to detect SEA in environmental, food, and clinical samples, while Jin et al. (2013) used chicken IgY antibodies specific for each SEA-SEE in ELISA assays, lateral flow device, and IgY-based immunopillar chips to detect these SEs in artificially contaminated dairy products. The IgY was also used to capture antibody by Reddy et al. (2014) in an immunocapture PCR assay to detect SEA in culture supernatants and in milk samples, while Mudili et al. (2015) combined the use of anti-SEB IgY antibodies with a biotinylated aptamer probe in a hybrid sandwich linked immune absorbent assay. However, commercial kits based on these detection methods are available only for the classical SEs (SEA-SEE). Moreover, in complex food matrices unrelated antigens or endogenous peroxides may react with the antibody leading to false positive results. This is the case, for example, of the immunoglobulin G (IgG)-binding staphylococcal protein A, which is cosecreted with SEs in food affecting the effectiveness of the assay (Dupuis et al., 2008), whose interference could be avoided using IgY instead of IgG as capture antibodies (Mizutani et al., 2012; Reddy et al., 2013). Moreover, false negative results may be obtained if the enterotoxin epitope is damaged so the enterotoxins is serologically but not biologically inactivated, as reported for SEA and SED in thermally processed foods (Bennet, 1992). False negative results in the immunological assays may occur also due to the aggregation of heattreated enterotoxins resulting in a reduced reactivity with antibodies (Anderson et al., 1996). Immuno-PCR (iPCR) applications first described by Sano et al. have been developed to increase the sensitivity of antigen detection assays by using PCR as signal amplification technique (Niemeyer et al., 2005; Sano et al., 1992). A quantitative real-time iPCR approach has been reported that was able to detect specifically smallest amounts of SEA and SEB as low as approximately 0.6–6 pg (4–40 amol/μL) by covalent binding of biotinylated reporter DNA to secondary detection antibodies, while specificity was reached by specific capture antibodies (Fischer et al., 2007). In addition to species identification, mass spectrometry techniques may allow the detection of all SEs. The protein standard absolute quantification (PSAQ) method, developed by Brun et al. (2007), using isotope-labeled proteins as internal standards for mass spectroscopy,

366  Chapter 12 allows the absolute quantification of target proteins. The PSAQ has been applied for the detection of SEA in semihard cheese made from cow’s milk (Dupuis et al., 2008), in a coconut-based dessert (Hennekinne et al., 2009), and in serum (Adrait et al., 2012). The MALDI-TOF MS has been used successfully to identify SEA in pasteurized milk specimens (Sospedra et al., 2011). Liquid chromatography coupled with mass spectrometry has been used to quantitatively detect SEA and SEB (Bao et al., 2012; Muratovic et al., 2015; Sospedra et al., 2012) in various food matrices. Biosensors for the detection of SEA (Pimenta-Martins et al., 2012) and biodefence toxin SEB have been developed (Deng et al., 2014; Tallent et al., 2013; Temur et al., 2012; Vinayaka and Thakur, 2012; Wu et al., 2013a,b). The current trend is to develop portable platforms allowing the simultaneous detection of several virulence factors of S. aureus or of different biodefence toxins. Jenko et al. (2014) developed an ELISA microarray assay to detect 10 toxins, namely shiga toxins 1 and 2, ricin, botulinum neurotoxins A, B, C, D, E, F, and SEB, while Stieber et al. (2015) developed an antibody-based microarray to detect staphylococcal marker and exotoxins including SEA and SEB.

12.5  Enterotoxin Detection in Coagulase Negative Staphylococci Enterotoxin production by non-S. aureus CPS, as S. intermedius and S. hyicus, has been reported since 1980s (Adesiyun et al., 1984; Hirooka et al., 1988). The CNS were rarely implicated in SFP outbreaks (Breckinridge and Bergdoll, 1971; Omori and Kato, 1959), but the enterotoxin-producing strains of S. capitis, S. caprae, S. chromogenes, S. cohnii, S. epidermidis, S. haemolyticus, S. hyicus (CNS), S. lentus, S. saprophyticus, S. sciuri, S. warneri, and S. xylosus have been detected since 1970s by immunological tests (Bautista et al., 1988; Breckinridge and Bergdoll, 1971; Crass and Bergdoll, 1986; Hoover et al., 1983; Marin et al., 1992; Olsvik et al., 1982; Orden et al., 1992; Valle et al., 1990). However, some researchers hypothesized that enterotoxigenic CNS may be mutant S. aureus, which do not express the coagulase (Gramoli and Wilkinson, 1978; Lotter and Genigeorgis, 1975; Victor et al., 1969). Moreover, the doubtless identification of staphylococcal isolates in the premolecular era in general and the specificity of the immunological assays used were being questioned. Therefore, to clearly demonstrate the toxin expression by CNS strains, Kreiswirth et al. (1987) have suggested the following three criteria that should be met: (i) the strains should be carefully identified as CNS; (ii) the production of toxin should be rigorously demonstrated; and (iii) the toxin gene should be detected by hybridization. Few studies have been performed to evaluate the occurrences of SEs genes, by PCR and/or DNA microarrays, in CNS isolated from food. No enterotoxins genes were detected in large

Staphylococcal Food Poisoning  367 collections of molecular identified CNS strains of S. lentus, S. haemolyticus, S. epidermidis, S. vitulus, S. pasteuri, S. succinus, S. warneri, S. equorum, S. xylosus, S. carnosus, and S. saprophyticus (Blaiotta et al., 2004a,b; Even et al., 2010). Rosec et al. (1997), applying PCR and immunoassay, showed that 51 CNS strains isolated from various foods did not produce classical enterotoxins (SEA-E). Moreover, although some CNS strains of S. carnosus, S. equorum, S. piscifermentans, and S. xylosus showed enterotoxin production (Immunoblot) (Zell et al., 2008), the corresponding genes could not be verified in the same strains by DNA microarray technology (Seitter et al., 2011). By contrast, enterotoxin C and/or D were detected in few strains of S. xylosus, S. cohnii, and S. epidermidis isolated from dry cured Spanish ham by immunoassay (Rodriguez et al., 1996). Moreover, strains of S. simulans, S. xylosus, S. equorum, S. lentus, and S. capitis, identified at the species level by phenotypic methods, from sheep milk or goat milk and from cheese, were shown to produce classical enterotoxin E and hybridized with see-related probe (Vernozy-Rozand et al., 1996). Marty et al. (2012) analyzing the occurrence of SE-encoding genes (sea-d, seg-j) in selected S. xylosus (n = 5) and S. equorum (n = 18) strains, isolated from Swiss sausages and Swiss meat products identified by 16S rDNA or rpoB gene sequencing, showed that only a strain of S. equorum harbors SEG-H-I-J toxins. By contrast, a high occurrence of SE-encoding genes was recorded in CNS isolated from raw and spontaneously fermented Camel milk in Kenya (seb, sed, seg, and sej) (Njage et al., 2013). Classical enterotoxin genes (sea-sed) detected by PCR and immunological tests in CNS strains isolated from food (de Lourdes et al., 2006,b) and dairy products implicated SFP outbreaks (Fereira Veras et al., 2008) in Brazil and no direct correlation was found when the results of the two methods were compared. Park et al. (2011), applying a multiplex PCR approach, showed that classical and new (including toxin-like) SE genes were widespread in molecular-identified CNS isolated from bovine intramammary infections (Idaho and Washington, USA). Classical SEs genes (sea-sed) occur in morpho-physiologically identified (RapID Staph system) CNS isolated from caprine subclinical mastitis in Brazil (Sampaio Salaberry et al., 2015). Mello et al. (2016) showed a high occurrence of SEs genes (sea-e, seg-j) in CNS isolated from bovine subclinical mastitis in different Brazilian states. Brazilian nosocomial S. epidermidis and S. haemolyticus isolates exhibited a high toxigenic potential, mainly containing the nonclassical enterotoxin genes seg and sei (Pinheiro et al., 2015). By contrast, in the United States, no enterotoxin (sea-e; seg-i) or enterotoxin-like (selj-u and selx) genes were detected in nasal S. epidermidis isolates from healthy human nares and diseased individuals and clinical CNS isolates (S. epidermidis and S. lugdunensis) (Stach et al., 2015). Classical (sea-sed) and new (see, seg-i) SE-encoding genes (PCR) and respective transcripts (RT-PCR) from CNS of clinical origin (Brazil), identified by ITS-PCR pattern comparison with type strains, were detected by de Oliveira et al. (2011) and Vasconcelos et al. (2011). A high occurrence (30%–50%) of SE genes was shown, but only in few cases the corresponding

368  Chapter 12 transcript was detected by RT-PCR (de Oliveira et al., 2011; Vasconcelos et al., 2011). Moreover, de Oliveira et al. (2011) sequenced the sea and/or sec PCR products of S. epidermidis, S. warneri, S. lugdunensis, and S. hominis and found 100% similarity with corresponding gene sequences of S. aureus. In all, 12 distinct combinations of SEs genes (including sea-e, seg-j, sell, sek-r, and seu) were found in molecularly identified (16S rDNA sequencing) CNS strains isolated from salami and from Minas Frescal cheese in Brazil (Casaes Nunes et al., 2015, 2016). However, no direct correlation between gene presence, mRNA transcription level (RT-PCR assay), and enterotoxin production (immunoassay) was found for classical enterotoxins. In fact, often the in vitro immunoassay results do not match those shown by RT-PCR assays (Casaes Nunes et al., 2015). Moreover, comparison of the nucleotide sequences of PCR products of sec and see, seg, seh, selm, and seln from CNS strains isolated from salami with those of S. aureus or S. pasteuri (see) showed a homology ranging from 65% to 98%: similarities of sec, seg, seh, selm, and seln between CNS and S. aureus was 98%, 60%, 98%, 65%, and 70%, respectively; similarity of see between CNS and S. pasteuri was 98% (Casaes Nunes et al., 2015). To date, the only well-characterized enterotoxigenic CNS are two S. epidermidis strains (FRI909 and 4S) (Madhusoodanan et al., 2011; Podkowik et al., 2016). The taxonomic affiliation of S. epidermidis FRI909, first isolated in the 1960s from a human clinical sample (Merlin Bergdoll, Food ResearchInstitute, Madison, WI), was recently confirmed by a number of biochemical tests and multilocus sequence typing by Madhusoodanan et al. (2011). These researches analyzing the draft genome sequence showed that the strain harbors sec and sell genes on an element similar to S. aureus pathogenicity island (designated SePI) and confirmed the expression of SEC and SElL by qRT-PCR and immunoblotting (Madhusoodanan et al., 2011). The taxonomic affiliation of S. epidermidis 4S, isolated from ready-to-eat meat products in Poland, was confirmed by simultaneous partial sequencing of tuf and 16S rDNA genes (Podkowik et al., 2016). As revealed by genome sequencing, the strain was found to possess stable enterotoxin sec and sell genes located in the region of 21426 bp similar to SePI of S. epidermidis FRI909 (Podkowik et al., 2016). S. epidermidis 4S sec gene was identical to sec gene from S. epidermidis FRI909, and its coding region was most closely related to S. aureus SEC3 (11 amino-acid substitutions) and most distantly related to SECovine (26 aminoacid substitutions) (Podkowik et al., 2016). The authors also demonstrated that SECepi can be produced in synthetic media, meat juices, and in milk at levels (as determined by Sandwich ELISA) sufficient to act as food safety hazard (Podkowik et al., 2016). In conclusion, according to Talon and Leroy (2011), enterotoxigenic capacity of CNS has always been a subject of controversy. Up to date, as described in this paragraph, multiple reports on CNS possessing genes homologous to S. aureus enterotoxins have appeared in the last decades, nonetheless the knowledge about their genetic context is still in its infancy (Podkowik et al., 2013).

Staphylococcal Food Poisoning  369 At first, different methods were used for the identification of CNS. It should be considered that the traditional identification methods, based on morpho-physiological and biochemical tests, are unreliable for staphylococcal identification (Blaiotta et al., 2003a,b, 2004a, 2010). As recommend by the ad hoc committee for the reevaluation of the definition of bacterial species (Stackebrandt et al., 2002), a multigenic approach should be applied for bacterial identification. We suggest that, in addition to 16S rDNA sequencing, at least one other housekeeping gene such as gap, kat, rpoB, sodA, hsp60, and tuf should be considered in sequencing scheme for staphylococcal identification (Blaiotta et al., 2010; Ghebremedhin et al., 2008). Methods used for screening might account for discrepancies between results relying on the detection of SE production by immunological methods and results based on the presence of the corresponding genes. Immunoassays to detect enterotoxins have been reported to lead to false diagnosis due to interferences, lack of specificity, and/or sensitivity (Dupuis et al., 2008). The proteins of CNS enterotoxin being presumably more related to those from S. aureus can be detected with cross-reacting antibodies, however, increasing the risk of false positive signals (Podkowik et al., 2013). In fact, SE detection based on immunological approaches is not always confirmed by DNA-based tests and vice versa. To overcome these limitations, a proteomic analysis of exo-proteins expressed by potential enterotoxigenic (PCR and or RT-PCR positive) CNS strains, especially for newly described enterotoxins/enterotoxins-like proteins, may be applied (Pocsfalvi et al., 2008). Different primer sets were used for PCR detection of the same SEs by different researches. For example, at least three different sets were described for seA PCR detection. The ability of polynucleotide DNA microarrays to detect sequence with similarities down to 70% in contrast to oligonucleotide-based microarrays seems to be more useful in the detection of CNS enterotoxins (Seitter et al., 2011). Finally, considering data reported by Oliveira et al. (2010), Madhusoodanan et al. (2011), Casaes Nunes et al. (2015), and Podkowik et al. (2016), one cannot exclude the existence of enterotoxin gene and/or enterotoxin gene variants (diverging from S. aureus sequences) in CNS species. In fact, these studies provided strong evidence for the presence and localization of enterotoxin-coding elements in CNS genomes and production of enterotoxins. Lateral transfer of superantigen genes by transduction of SaPIs from S. aureus to S. epidermidis and S. xylosus are documented events (Chen and Novick, 2009; Madhusoodanan et al., 2011). Therefore, the application the EC Regulation 2073/2005 on microbiological criteria for foodstuffs, modified by the Regulation 1441/2007, which consider five process hygiene criteria on CPS and one food safety criterion on classical SEs (to be tested when CPS enumeration is higher than 105 cfu/g) may be insufficient to guarantee food safety. However, as described by Park et al. (2011) and Podkowik et al. (2016), some enterotoxin genes seem to occur in unstable form in CNS. Therefore, the significance of these genes for food safety and public health remains unknown (Podkowik et al., 2013). In fact, no case of

370  Chapter 12 food poisoning involving exclusively strains of CNS of food origin has been reported in the last decades (Podkowik et al., 2013). What strategy should be used to clarify this controversial issue? The discrepancies regarding the enterotoxigenic potential of CNS accentuate the need for more detailed studies using the most innovative techniques available in many areas of research.

12.6  Expression of Enterotoxin Genes Expression patterns and amount of SEs produced vary depending on the strain, the type of enterotoxin, the mechanism of regulation, the food composition and structure as well as the food processing and storage conditions. Two variants of the sea gene (sea1 and sea2), which is located on the genome of a temperate bacteriophage of the Syphoviridae, are known. Strains producing high amounts of SEA harbor the sea1 gene, while the sea2 variant is harbored by strains producing low amount of SEA (Wallin-Carlquist et al., 2010b). It has been demonstrated that the sea gene expression and the amount of SEA produced by S. aureus strains are influenced by the life cycle of sea-carrying phages and that the prophage induction, using mitomycin C or acetic acid, increases the amount of phage replicative forms, the levels of sea1 gene copies and thus the relevant transcripts as well as the amount of SEA produced by the inducible high SEA-producer strains, mainly during the exponential phase of growth (Cao et al., 2012; Sumby and Waldor, 2003; Zeaki et al., 2015). The expression of the sec, seb, and sed genes is controlled by the accessory gene regulator (agr) system (Bayles and Iandolo, 1989; Gaskill and Khan, 1988; Regassa et al., 1991). The agr (reviewed by Le and Otto, 2015; Painter et al., 2014) activates the transcription of these genes during the transition from the exponential to the stationary phase of growth, while that of the sed gene is also mediated by the repressor of toxin, rot (Tseng et al., 2004). Also, the sarA, staphylococcal accessory regulator positively controls seb and sec expression. The expression of the sec gene is also positively regulated by the saeR/S (S. aureus exoprotein expression) gene regulatory system (Voyich et al., 2009), whereas the sigma factor sigB, by repressing the agr system, reduces seb expression (Schmidt et al., 2004). In the SFP clone CC81 subtype-1, Sato’o et al. (2015) found that the repressor of toxin rot functioned as stimulator of SHE production which did not affect the SEA production. Various parameters typical of certain food products and food processing conditions that affect the SE-encoding genes’ transcription and SE production have been studied, mainly for the classical SEs (Bang et al., 2008; Baird-Parker, 1971; Belay and Rasooly, 2002; Carpenter and Silverman, 1974, 1976; Domenech et al., 1992; Even et al., 2009; Hughes and Hurst, 1980; Onoue and Mori, 1997; Qi and Miller, 2000; Regassa and Betley, 1992, 1993; Rosengren et al., 2013; Sakai et al., 2008; Sihto et al., 2016). However, these studies have used nutritive broth media rather than real food matrices. Improvements in the protocols for the extraction

Staphylococcal Food Poisoning  371 and purification of RNA from food matrices purposes (Ablain et al., 2009; Duquenne et al., 2010; Monnet et al., 2008; Ulve et al., 2008) as well as improvement in the normalization strategy (mainly based on the selection of appropriate reference genes, usually evaluated by geNorm VBA applet for Microsoft Excel) (Valihrach and Demnerova, 2012; Vandesompele et al., 2002, 2009) have allowed to perform studies on the impact of the food composition, production, and storage on the expression of the SE genes. Cretenet et al. (2011) followed the growth and the virulence expression of S. aureus MW2 (harboring the sea, sec4, seg, seh, sel2, and sek genes) in a chemically defined medium (CDM) and in a ultra-filtrate (UF) retentate matrix obtained by microfiltrated skimmed raw milk (Ulve et al., 2008) in condition mimicking the temperature shift occurring for the production of soft cheeses (incubation at 30°C for 10 h and then at 12°C). They found that the dynamics of sec4, seh, and sel2 genes were significantly different in the UF retentate and in CDM, in which their expression levels were higher, whereas in the presence of L. lactis higher levels of sea gene expression were found in the UF retentate than in CDM (Cretenet et al., 2011). This is likely due to the acidified environment causing the sea-encoding prophage induction, which in turn increases the amount of phage replicative forms, the levels of sea gene copies, and thus, the relevant transcripts as well as the amount of SEA produced. Moreover, it was found that L. lactis downregulated the expression of the enterotoxins controlled by the agr system in the UF retentate matrix, mainly due to its ability to reduce the pH (Cretenet et al., 2011) and the redox potential (Nouaille et al., 2014). Valihrach et al. (2014) monitored the growth and the expression of eight regulation genes and six enterotoxin genes of S. aureus MW2 in TSB and in UHT 1.5% fat milk during 48 h of storage at 25°C under static conditions, and obtained the results similar to those reported by Cretenet et al. (2011). Wallin-Carlquist et al. (2010a) investigated the sea expression and SEA production in pure broth culture and in boiled ham, hot-smoked ham, Serrano dry-cured ham, and black pepper salami during their storage at 23°C for 1 week. While in the broth culture, the sea expression peaked in the late exponential phase to rapidly decline thereafter, a prolonged sea expression and SEA formation was found in smoked and boiled ham (with lower amounts of extracellular SEA found in the smoked ham than in the boiled ham), while in Serrano ham, the sea expression and SEA formation increased between the fifth and seventh day of incubation, and no sea expression or SEA were detected in black pepper salami, in which the inoculated S. aureus strain did not survive (Wallin-Carlquist et al., 2010a). An expression pattern similar to that of the prophage-carrying sea in boiled and smoked ham was found for the agr-regulated sed expression (Márta et al., 2011). The SEA formation and sea expression were assessed on frankfurter-type pork sausages added and not with 1% and 2% lactic acid inoculated either with high SEA producer or with low SEA producer strains during the storage at 15°C for 14 days (Zeaki et al., 2014). A prolonged SEA

372  Chapter 12 production, was found starting from the second day of inoculation in samples not added with lactic, while in samples treated with lactic acid the enterotoxin production started between the fourth and the sixth day from the inoculation when the cells entered the log-phase (Zeaki et al., 2014). The SEA amounts produced by the two high-producing strains were higher than those produced by the low-producing strain tested, indicating that, given the growth environment, the modulation of the expression of the various sea-harboring prophages affect the SEA formation (Zeaki et al., 2014). However, the amounts of enterotoxin produced by the strains were significantly less in the samples treated with lactic acid, thus indicating its suitability as antimicrobial agent for mitigating the risk of SEA food poisoning (Zeaki et al., 2014). Although many studies have been performed to understand mechanisms of production for the classical enterotoxins (SEA-SEE), little is known about the other SEs. However, with the exception of seh, ser, and sel, whose transcription levels were found increased in the postexponential phase, as suggested by Derzelle et al. (2009), the other SEs might not be controlled by the agr system and, since most of the newly described SE-encoding genes are located on phages, it is possible that their expression, as for sea, is controlled by the life cycle of the carrying phages. In particular, by the kinetic study of Derzelle et al. (2009), the transcript levels of the phage-encoded sej, sek, seq, and sep genes did not change while those of the egc-harboured seg, sei, sem, sen, seo, and seu slightly decreased after the log phase.

12.7 Conclusion The SEs particularly produced by S. aureus are the etiological agents of the SFP, one of the most widespread foodborne intoxications. Even today, the diagnostic of FBD caused by SEs is challenging. While the molecular era enabled us to detect the encoding genes of the classical and also of the recently described SEs/SEls, if cultivated isolates are available, the verification of the occurrence of preformed SEs in food specimens is still based on immunological assays, which are characterized by often insufficient sensitivities and specificities, even in the case of the classical SEs. For the newly described SEs and SEls, the capacity to cause SFP is still a matter of debate and the existing approaches for their detection are not suitable for routine testing. Thus, the development of specific and sensitive methods for the detection of enterotoxin suitable for routine use are required for the elucidation of foodborne outbreaks, individual cases of SFP, and to get more insights into the true epidemiology of this disease.

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Staphylococcal Food Poisoning  389 Wu, L., Gao, B., Zhang, F., Sun, X., Zhang, Y., Li, Z., 2013b. A novel electrochemical immunosensor based on magnetosomes for detection of staphylococcal enterotoxin B in milk. Talanta 106, 360–366. Xing, X., Li, G., Zhang, W., Wang, X., Xia, X., Yang, B., Meng, J., 2014. Prevalence, antimicrobial susceptibility, and enterotoxin gene detection of Staphylococcus aureus isolates in ready-to-eat foods in Shaanxi, People’s Republic of China. J. Food Prot. 77, 331–334. Xu, B., Liu, L., Liu, L., Li, X., Li, X., Wang, X., 2012. A multiplex PCR assay for the rapid and sensitive detection of methicillin-resistant Staphylococcus aureus and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci. J. Food Sci. 77, 638–642. Yan, X., Wang, B., Tao, X., Hu, Q., Cui, Z., Zhang, J., Lin, Y., You, Y., Shi, X., Grundmann, H., 2012. Characterization of Staphylococcus aureus strains associated with food poisoning in Shenzhen, China. Appl. Environ. Microbiol. 78, 6637–6642. Zeaki, N., Budi Susilo, Y., Pregiel, A., Rådström, P., Schelin, J., 2015. Prophage-encoded staphylococcal enterotoxin a: regulation of production in Staphylococcus aureus strains representing different sea regions. Toxins (Basel) 7, 5359–5376. Zeaki, N., Cao, R., Skandamis, P.N., Rådström, P., Schelin, J., 2014. Assessment of high and low enterotoxin A producing Staphylococcus aureus strains on pork sausage. Int. J. Food Microbiol. 182-183, 44–50. Zeinhom, M.M., Abdel-Latef, G.K., Jordan, K., 2015. The use of multiplex PCR to determine the prevalence of enterotoxigenic Staphylococcus aureus isolated from raw milk, feta cheese, and hand swabs. J. Food Sci. 80, 2932–2936. Zell, C., Resch, M., Rosenstein, R., Albrecht, T., Hertel, C., Gotz, F., 2008. Characterization of toxin production of coagulase-negative staphylococci isolated from food and starter cultures. Int. J. Food Microbiol. 127, 246–251. Zhang, C., Liu, Z., Li, Y., Li, Q., Song, C., Xu, Z., Zhang, Y., Zhang, Y., Ma, Y., Sun, Y., Chen, L., Fang, L., Yang, A., Yang, K., Jin, B., 2013. High sensitivity chemiluminescence enzyme immunoassay for detecting staphylococcal enterotoxin A in multi-matrices. Anal. Chim. Acta 796, 14–19. Zhang, K., McClure, J.A., Conly, J.M., 2012. Enhanced multiplex PCR assay for typing of staphylococcal cassette chromosome mec types I to V in methicillin-resistant Staphylococcus aureus. Mol. Cell. Probes 26, 218–221. Zhang, K., Sparling, J., Chow, B.L., Elsayed, S., Hussain, Z., Church, D.L., Gregson, C.B., Louie, T., Conly, J.M., 2004. New quadriplex PCR assay for detection of methicillin and mupirocin resistance and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci. J. Clin. Microbiol. 42, 4947–4955. Zhu, W., Sieradzki, K., Albrecht, V., McAllister, S., Lin, W., Stuchlik, O., Limbago, B., Pohl, J., Kamile Rasheed, J., 2015. Evaluation of the Biotyper MALDI-TOF MS system for identification of Staphylococcus species. J. Microbiol. Methods 117, 14–17.

Further Reading Banada, P.P., Chakravorty, S., Shah, D., Burday, M., Mazzella, F.M., Alland, D., 2012. Highly sensitive detection of Staphylococcus aureus directly from patient blood. PLoS One 7, e31126. Becker, K., Larsen, A.R., Skov, R.L., Paterson, G.K., Holmes, M.A., Sabat, A.J., Friedrich, A.W., Köck, R., Peters, G., Kriegeskorte, A., 2013. Evaluation of a modular multiplex-PCR methicillin-resistant Staphylococcus aureus detection assay adapted for mecC detection. J. Clin. Microbiol. 51, 1917–1919. Boerema, J.A., Clemens, R., Brightwell, G., 2006. Evaluation of molecular methods to determine enterotoxigenic status and molecular genotype of bovine, ovine, human and food isolates of Staphylococcus aureus. Int. J. Food Microbiol. 107, 192–201. Johler, S., Layer, F., Stephan, R., 2001. Comparison of virulence and antibiotic resistance genes of food poisoning outbreak isolates of Staphylococcus aureus with isolates obtained from bovine mastitis milk and pig carcasses. J. Food Prot. 74, 1852–1859. Kim, C.H., Khan, M., Morin, D.E., Hurley, W.L., Tripathy, D.N., Kehrli Jr., M., Oluoch, A.O., Kakoma, I., 2001. Optimization of the PCR for detection of Staphylococcus aureusnuc gene in bovine milk. J. Dairy Sci. 84, 74–83.

390  Chapter 12 Nema, V., Agrawal, R., Kamboj, D.V., Goel, A.K., Singh, L., 2007. Isolation and characterization of heat resistant enterotoxigenic Staphylococcus aureus from a food poisoning outbreak in Indian subcontinent. Int. J. Food Microbiol. 117, 29–35. Reischl, U., Linde, H.J., Metz, M., Leppmeier, B., Lehn, N., 2000. Rapid identification of methicillin-resistant Staphylococcus aureus and simultaneous species confirmation using real-time fluorescence PCR. J. Clin. Microbiol. 38, 2429–2433. Zhang, H., Ma, X., Liu, Y., Duan, N., Wu, S., Wang, Z., Xu, B., 2015. Gold nanoparticles enhanced SERS aptasensor for the simultaneous detection of Salmonella typhimurium and Staphylococcus aureus. Biosens. Bioelectron. 74, 872–877. Zhao, X., Meng, R., Shi, C., Liu, Z., Huang, Y., Zhao, Z., 2016. Analysis of the gene expression profile of Staphylococcus aureus treated with nisin. Food Control 59, 499–506.

CHAPTE R 13

Campylobacter: An Important Food Safety Issue Willian C. Silva⁎, Brenda N. Targino†, Amanda G. Gonçalves†, Marcio R. Silva‡, Humberto M. Hungaro† ⁎

University of Campinas, Campinas, São Paulo, Brazil †Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil ‡Brazilian Agricultural Research Corporation (Embrapa Dairy Cattle), Juiz de Fora, Minas Gerais, Brazil

13.1 Introduction Foodborne diseases are an important worldwide public health issue that can results in significant economic impact due to the resources spent on hospitalizations (Hudson et al., 2014; Scallan et al., 2011). The consumption of contaminated food with different agents such as viruses, parasites, and bacteria are the main causes of this type of illness (WHO, 2006). Among bacteria associated with foodborne diseases, Campylobacter has been the most frequent pathogen isolated in outbreaks in both developed and developing countries over the last 10 years (Kaakoush et al., 2015). Since its first description, the genus Campylobacter has grown to include several important human and animal pathogens. Also, after the publication of the first Campylobacter jejuni genome sequence, significant advances have been made in order to understand the pathogenicity, genetics, and physiology of these microorganisms. The genus Campylobacter comprises a diverse group of microaerophilic Gram-negative bacteria presenting a spirally curved shape, which colonizes the mucosal surfaces of the intestinal tracts, oral cavities, or urogenital tracts of most warm-blooded animals (Humphrey et al., 2007). They are prevalent in food-producing animals and domestic pets, including poultry, cattle, pigs, sheep, cats, and dogs (Epps et al., 2013). These bacteria are considered highly fastidious organisms since they show relatively high minimum growth temperatures, complex nutritional requirements, and limited defenses against environmental stress conditions (Rowe and Madden, 2014). However, they have various survival mechanisms and virulence factors that make it possible to overcome the defense barriers of the host organism to cause diseases as well as survive the environmental stress. Different species of this genus has an association with at least one type of illness in humans and/or domestic animals.

Food Safety and Preservation. https://doi.org/10.1016/B978-0-12-814956-0.00013-5 © 2018 Elsevier Inc. All rights reserved.

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392  Chapter 13 Campylobacter may cause a foodborne disease named of campylobacteriosis, usually selflimiting, but that can progress to severe complications such as Guillain-Barré syndrome (GBS) and reactive arthritis (Moore et al., 2005). The mechanisms leading to this disease are complex and not fully known but include several virulence factors (Bolton, 2015). In the past few years, the increasing antimicrobial resistance in Campylobacter especially strains isolated from food has raised some concerns by health authorities, since the infections caused by these resistant strains have an association with a longer duration of illness, an increased risk of invasive disease, and death (Kaakoush et al., 2015). The primary pathway of Campylobacter transmission in humans is the consumption of contaminated poultry and poultry products. This microorganism has frequently been isolated from samples of these kinds of foods worldwide (Sahin et al., 2015; Suzuki and Yamamoto, 2009). However, significant variations in the prevalence and count of Campylobacter observed in these samples, which can be attributed to different factors, such as the ways of bird breeding, failures in hygiene practices throughout the production chain, sampling techniques and differences between the analytical methods (Daskalov and Maramski, 2012; Stern and Line, 1992; Tang et al., 2011). Monitoring of contamination through appropriate methodologies is an important tool to measure the problem, allowing to determine the level of contamination, the main control points, and to establish the risks in the poultry production chain (Nauta et al., 2005; Uyttendaele et al., 2006). Besides, different strategies can contribute to decreasing the risk of campylobacteriosis in humans such as the control of the dissemination of Campylobacter species in the poultry industry by reducing the environmental exposure and colonization of the chicks (Kaakoush et al., 2015).

13.2  The Genus Campylobacter The genus Campylobacter belongs to the family Campylobacteriaceae, the order Campylobacterales, the class Epsilonproteobacteria, and the phylum Proteobacteria, consisting of a vast and diverse group of bacteria currently comprising 27 species, 9 subspecies, and 3 biovars (Table 13.1). However, the taxonomic structure of the genus is constantly changing since its inception due to the discovery of novel species or reclassification of some species into other genera. Two other novel species isolated from prairie dogs and cetaceans were proposed, but they have not definitively been included in this genus (Beisele et al., 2011; Goldman et al., 2011). Campylobacter was first reported by Theodor Escherich in 1866, who observed and described nonculturable spiral-shaped bacteria in the colon of children with an enteric disease called “cholera infantum.” Thenceforth, these microorganisms were identified from the uterine mucus of a pregnant sheep in 1906, aborted bovine fetuses in 1913, feces of cattle with diarrhea in 1927, and feces of pigs with diarrhea in 1944. They are classified as a group of vibrio-like bacteria and named as “Vibrio fetus,” “Vibrio bubulus,” “Vibrio sputorum,” “Vibrio jejuni,” or “Vibrio coli” by researchers according to the isolation source and characteristics observed. It also includes cell morphology, growth requirements,

Campylobacter: An Important Food Safety Issue   393 Table 13.1: Species and subspecies within the genus Campylobacter and their known sources and disease associations in humans and animals Disease Associationsa Species

Known Source(s)

Human

Veterinary

Campylobacter avium Campylobacter canadensis Campylobacter coli

Poultry (chicken and turkey) Whooping cranes

None as yet None as yet

None as yet None as yet

Pig, poultry (chicken, duck, turkey, and seagull) ostriches, cattle, sheep, goat, monkey, and dog Human and domestic pets (cat and dog)

Gastroenteritis, acute cholecystitis, meningitis, bacteremia, and sepsis

Gastroenteritis, infectious hepatitis None as yet

Campylobacter corcagiensis Campylobacter cuniculorum Campylobacter curvus

Lion-tailed macaques

Gastroenteritis, IBD, Barrett’s esophagitis, periodontitis, brain abscesses, and reactive arthritis None as yet

Rabbits

None as yet

None as yet

Human and dog

None as yet

Campylobacter fetus subsp. fetus

Cattle, horse, sheep, reptiles (pet turtle), and kangaroo

Campylobacter fetus subsp. testudinum Campylobacter fetus subsp. venerealis Campylobacter gracilis

Reptiles

Gastroenteritis, ulcerative colitis, Barrett’s esophagitis, periodontitis, and bronchial and liver abscesses Gastroenteritis, septicemia, abortion, meningitis, epidural and brain abscesses, endocarditis, and peritonitis Gastroenteritis, cellulitis

Cattle, sheep

Septicemia

Human

Campylobacter helveticus

Dog and cat

Periodontitis, empyema, abscesses, and IBD Gastroenteritis and periodontitis

Campylobacter hominis

Human

Campylobacter hyointestinalis subsp. hyointestinalis Campylobacter hyointestinalis subsp. lawsonii Campylobacter iguaniorum Campylobacter insulaenigrae Campylobacter jejuni subsp. doylei

Cattle, deer, pig, hamster

Gastroenteritis in immunocompromised Gastroenteritis

Pig

None as yet

None as yet

Reptiles

None as yet

None as yet

Seals, porpoises, and other marine mammals Human

Gastroenteritis

None as yet

Gastroenteritis and septicemia

None as yet

Campylobacter concisus

None as yet

Bovine and ovine spontaneous abortion None as yet Bovine infectious infertility None as yet Feline and canine gastroenteritis None as yet Porcine and bovine enteritis

Continued

394  Chapter 13 Table 13.1  Species and subspecies within the genus Campylobacter and their known sources and disease associations in humans and animals—cont’d Disease Associationsa Species

Known Source(s)

Human

Veterinary

Campylobacter jejuni subsp. jejuni

Poultry, cattle, pig, sheep, dog, cat, ostrich, wild birds, mink, rabbit, insects, and water

Spontaneous abortion, avian hepatites, and gastroenteritis

Campylobacter lanienae

Cattle, pig, sheep, and human Shellfish

Gastroenteritis, IBD, celiac disease, reactive arthritis, GuillainBarré syndrome, septicaemia, meningitis, urinary tract infection, abortion, proctitis, and septicemia Gastroenteritis Gastroenteritis

None as yet Avian gastroenteritis Porcine necrotic enteritis and ileitis None as yet None as yet

Campylobacter lari subsp. concheus Campylobacter lari subsp. lari Campylobacter mucosalis

Poultry, wild birds, dog, shellfish, and horse Pig and dog

Gastroenteritis and septicemia

Campylobacter peloridis Campylobacter rectus

Shellfish Human and dog

Campylobacter showae

Human and dog

Campylobacter sputorum bv. sputorum Campylobacter sputorum bv. faecalis Campylobacter sputorum bv. paraureolyticus Campylobacter subantarcticus Campylobacter troglodytis Campylobacter upsaliensis

Human, cattle, pig, and sheep Sheep and bull

Gastroenteritis Gastroenteritis, IBD, periodontitis, empyema thoracis, and vertebral abscess Periodontitis, Crohn’s disease, ulcerative colitis, intraorbital abscess, and IBD Gastroenteritis, abscesses

Gastroenteritis

None as yet

None as yet

None as yet

Spontaneous abortionb None as yet

Cattle

Gastroenteritis

None as yet

Birds in the subantarctic (albatrosses and penguins) Chimpanzee

None as yet

None as yet

None as yet

None as yet

Dog and cat

Gastroenteritis, septicaemia, abscesses, and spontaneous abortion Gastroenteritis, IBD, Crohn's disease, ulcerative colitis, septicemia, and oral, perianal and soft tissue abscesses None as yet

Canine and feline gastroenteritis

Campylobacter ureolyticus

Human and horse

Campylobacter volucris

Black-headed gulls

None as yet

None as yet

The table was adapted from Humphrey, T., O’Brien, S., Madsen, M., 2007. Campylobacters as zoonotic pathogens: a food production perspective. Int. J. Food Microbiol. 117(3), 237–257. doi:10.1016/j.ijfoodmicro.2007.01.006 and updated from the works of Man, S.M., 2011. The clinical importance of emerging Campylobacter species. Nat. Rev. Gastroenterol. Hepatol. 8(12), 669–685. doi:10.1038/nrgastro.2011.191; On, S.L.W., 2013. Isolation, identification and subtyping of Campylobacter: where to from here? J. Microbiol. Methods 95(1), 3–7. doi.org/10.1016/j.mimet.2013.06.011; Fitzgerald, C., 2015. Campylobacter. Clin. Lab. Med. 35(2), 289–298. doi:10.1016/j.cll.2015.03.001. a Association of species with a disease is not necessarily a causal agent confirmation. b Disease association assigned to the species.

Campylobacter: An Important Food Safety Issue   395 and basic biochemical tests (Butzler, 2004; Skirrow, 2006). The first well-documented foodborne disease outbreak associated with Campylobacter occurred in Illinois in 1938 due to consumption of milk, affecting 355 inmates of two adjacent state institutions. Because of their particular characteristics, such as the G + C content of DNA, microaerophilic growth, and nonfermentative metabolism, these microorganisms were included in an exclusive genus called Campylobacter, proposed by Seabald and Vernon in 1963 to distinguish them from the Vibrio spp. The name of the genus was derived from the Greek word “kampyo’s,” which means curved (Keener et al., 2004). The members of the Campylobacter genus are small Gram-negative rods of a spirally curved shape, size between 0.2 and 0.9 μm wide and 0.5–5 μm long, nonspore forming, usually motile by a single flagellum at one or both poles of the cells (Fig. 13.1).

Fig. 13.1 Morphology of the cells of Campylobacter jejuni in the process of dividing. Note the spirally curved shape and the flagella.Source: Humphrey, T., O’Brien, S., Madsen, M., 2007. Campylobacters as zoonotic pathogens: a food production perspective. Int. J. Food Microbiol. 117(3), 237–257. https://doi.org/10.1016/j.ijfoodmicro.2007.01.006.

The flagella may be 2–3 times the length of the cells and confer a characteristic corkscrew motion. Among all known species, only Campylobacter gracilis is nonmotile and Campylobacter showae shows multiple flagella. The bacteria of this genus are primarily microaerophilic with a respiratory-type metabolism, growing best in an atmosphere containing approximately 3%–6% O2. Thus, oxygen is required for energy production, but the microorganism cell can only tolerate this gas at levels below normal atmospheric pressure (Rowe and Madden, 2014). Campylobacter is often capnophilic, that is, in the presence of 2%–10% CO2 their growth enhances (Fitzgerald, 2015). Some species also depends on the presence of hydrogen (H2) for growth, which is supplemented at a final concentration ≤10% (Macé et al., 2015). They are fastidious, require complex nutritional environments, and may form a coccoid form in old cultures and under conditions of stress, getting into the viable but nonculturable

396  Chapter 13 state (VBNC). Regarding the growth temperature, most species are considered mesophilic growing at temperatures from 25°C to 45.5°C. The main species pathogenic for man and transmitted commonly through food, including Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis, are classed as thermotolerant Campylobacter due to the optimal growth temperature of around 42°C (Levin, 2007). Campylobacter grows optimally in environments with water activity (aw) of 0.997 and pH 6.5–7.5, but do not survive at a pH below 4.9 and above 9.0 (Silva et al., 2011). These microorganisms are highly sensitive to desiccation, osmotic stress, and aeration, and do not tolerate sodium chloride concentrations greater than 2% (w/v), neither can grow at a water activity below 0.987 (Murphy et al., 2006; Rowe and Madden, 2014; Silva et al., 2011). They are also sensitive to thermal treatment compared with other foodborne pathogens and cannot survive pasteurization or most culinary treatments (Jacobs-Reitsma et al., 2008; Moore and Madden, 2000). For example, the D-values for thermotolerant Campylobacter at 55°C and 60°C in sterile chicken extract and milk were ≤1 min (Baserisalehi et al., 2006). The z-values range from 2.8°C to 5.8°C (Li et al., 2002; Sörqvist, 2003). Campylobacter is unusually sensitive to different environmental stress conditions because they lack many of the adaptive responses presented by most of the other foodborne pathogens (Park, 2002). All these characteristics previously shown restrict the ability of these microorganisms to multiply outside the host, so they cannot grow in foods during processing or storage (Ganan et al., 2012). Campylobacter is chemoorganotrophs, nonproteolytic, nonlipolytic, nonsaccharolytic, and obtain energy mainly from the oxidation of amino acids or tricarboxylic acids intermediates. They do not ferment or oxidize carbohydrates because they lack the glycolytic enzyme 6-phosphofructokinase (PFK), a critical glycolytic enzyme (Jeon et al., 2010). These microorganisms can also conserve energy via respiration, oxidizing hydrogen, and formate for the reduction of the electron acceptors such as fumarate, nitrate, sulfites and, if at low concentrations, oxygen, to generate proton motive force for electron transport phosphorylation (Epps et al., 2013). Members of the Campylobacter genus may live as commensal organisms in the gastrointestinal (GI) tract of a wide range of hosts, such as pets, farm animals, and wild animals, and are frequently found in contaminated food, which indicates that these bacteria are at risk of zoonotic transmission to humans (Bolton, 2015). Many of these species have been associated with human disease, such as gastroenteritis, inflammatory bowel disease (IBD), and periodontitis (Fitzgerald, 2015; Man, 2011). Campylobacter jejuni and Campylobacter coli are the most well-known species related to foodborne disease outbreaks worldwide. Interestingly, these two species colonize intestinal tracts of domestic livestock and wild animals, almost always without any harmful effects (Humphrey et al., 2007; Meade et al., 2009). A growing number of Campylobacter species other than Campylobacter jejuni and Campylobacter coli has also been recognized as emerging human and animal pathogens, particularly due to advances in molecular biology, development of innovative isolation, and detection methodologies (Man, 2011).

Campylobacter: An Important Food Safety Issue   397 According to Fitzgerald (2015), Campylobacter species may be classified into four groups: (1) thermotolerant strains, which are the main species of clinical and public health importance; (2) species that infrequently cause disease in humans and are associated with livestock animals; (3) species either implicated in periodontal disease or isolated from humans; and (4) species that have not been isolated from food or water and are not associated with human illness. Table 13.1 presents a full list of the species within the genus Campylobacter and their reservoirs and human and animal diseases. Among all Campylobacter species, Campylobacter avium, Campylobacter canadenses, Campylobacter cuniculorum, Campylobacter subantarcticus, Campylobacter volucris, Campylobacter troglodytis, Campylobacter corcagiensis, and Campylobacter iguaniorum are the only species that are not yet associated with diseases in humans or animals (Fitzgerald, 2015; Man, 2011; On, 2013).

13.3  Virulence and Survival Factors The mechanisms of survival and infection used by Campylobacter to overcome the barriers of the host organism and mediate diseases in humans are complex and not fully known, whatever we know about this process is due to studies performed mostly with Campylobacter jejuni. The colonization and infection processes require several virulence factors. These include motility, chemotaxis, adherence, and invasion of the host cell, toxin production, structures of the cell envelope, iron uptake system, multidrug and bile resistance, and mechanisms of responses to stress (Bolton, 2015; Dasti et al., 2010; Epps et al., 2013). Campylobacter must bypass the mechanical and immunological barriers of the GI tract of the host in order to establish a successful infection. The first contact of the bacteria with the host tissues occurs in the intestine, which comprises mucus layer and intestinal epithelial cells serving as the first line of defense. In humans, the infection occurs predominantly in the small intestine, whereas, in poultry, the primary colonization site is the cecum (Meade et al., 2009). The motility and chemotaxis systems are essential for this pathogen to penetrate this main barrier toward specific sites for a cellular invasion to take place. The presence of one or two polar flagella and the spiral cell shape provide propulsive torque and corkscrew cell movement, facilitating the microorganism get across the mucus layer (Ferrero and Lee, 1988; Guerry, 2007). Chemotactic response drives the cell movement, which guides the microorganism to migrate toward favorable environments and also keep away from unfavorable circumstances (Rowe and Madden, 2014). Some of the chemoattractants that mediate Campylobacter chemotaxis are mucins, glycoproteins of the mucus, amino acids, and other metabolic substrates, donors, and acceptors of electrons (Bolton, 2015; Hermans et al., 2012). The chemotaxis system uses a single two-component histidine protein kinasedependent signal transduction pathway and methyl-accepting chemotaxis proteins (MCPs) (Hamer et al., 2010; Lertsethtakarn et al., 2011). The MCPs are responsible for sensing

398  Chapter 13 chemoattractants in the environment, and its combination with these molecules triggers a phosphorylation signaling pathway causing activation of histidine protein kinase and a response regulator that controls the direction of the flagellar rotation (Bolton, 2015). Several different proteins and regulatory systems are associated with structure and function of the flagellum, as well as, its anchoring to the bacterial cell, movement, and guidance (Bolton, 2015; Dasti et al., 2010). The flagellum is a complex structure composed of a hook-basal body that includes anchoring proteins in the cytoplasm, the inner and outer membrane of the cell, motor proteins, and the extracellular filament structural components. The hook-basal body complex is composed of several different proteins including those that make up the rings of anchoring the flagellum (FliM, FliN, FliY, FliG, FliF, FlgH, and FlgI), secretion system (FlhA, FlhB, FliO, FliP, FliQ, and FliR), motor components (MotA and MotB) and motor switch components (FliM and FliY), and structural components of hook (FlgE and FliK). FlaA and FlaB are the major structural proteins of the extracellular filament of the flagellum and encoded by genes flaA and flaB, which is also used for typing Campylobacter isolates (Bolton, 2015). After microorganisms have crossed the mucous layer, they encounter the intestinal epithelial cells and can bind to them. Cellular adhesion is a complex process, in which adhesions of the bacterial cell surface interact with the host cell receptors leading to an irreversible and specific binding (Ganan et al., 2012). This adherence to epithelial cells of the GI tract is fundamental for Campylobacter to resist intestinal peristalsis and expulsion (Jeon et al., 2010). There are several factors that mediate the adhesion of Campylobacter to host GI epithelial cells. CadF is an outer membrane protein of Campylobacter that mediates cell adhesion by binding to fibronectin, a glycoprotein of the extracellular matrix of the GI tract. This interaction also triggers a signaling process that leads to the activation of the GTPases Rac1 and Cdc42 which induce Campylobacter cell internalization (Konkel et al., 1997). It has been proposed that FlpA, another fibronectin-binding protein, and CadF act together during Campylobacter jejuni adhesion and subsequent invasion (Eucker and Konkel, 2012). Other factors such as CapA (autotransporter lipoprotein), JlpA (surface-exposed lipoprotein), Peb1 (periplasmic binding protein), Peb4 (chaperone—CadF transporter protein), and Peb3 (transport protein) seem to play a key role in the adhesion process (Ashgar et al., 2007; Jin et al., 2003; Kale et al., 2011; Min et al., 2009; Pei et al., 1998). The host cell invasion process by Campylobacter occurs as a result of the arrangement of different virulence factors, including the protein export apparatus related to flagellar system, a set of proteins involved in adhesion, invasion, and intracellular survival, capsular polysaccharide, chaperones, factors of protection against antimicrobial proteins, and apoptosis inductor (Bolton, 2015; Dasti et al., 2010). Many components of the flagellar system that are part of the protein secretion apparatus operate during cell invasion (Carrillo et al., 2004). For example, this system is responsible for carrying FlaC and Cia proteins into the host cell's cytoplasm, which are essential for colonization and invasion. The presence of

Campylobacter: An Important Food Safety Issue   399 the bile component deoxycholate stimulates Cia protein synthesis, but not their secretion, suggesting that Cia production occurs in the early colonization and secretion occurs only after adherence to the host cells (Rivera-Amill et al., 2001). The CiaB and CiaC proteins are involved in cell adhesion and invasion, respectively, while CiaI is required for intracellular survival (Bolton, 2015). Other proteins, such as HtrA, VirK, and FspA, also play important role in these processes, which display protease and chaperone activities, antimicrobial peptide resistance, and induction of apoptosis in eukaryotic cells, respectively (Bæk et al., 2011; Novik et al., 2009; Poly et al., 2007). After the internalization, Campylobacter resides within a membrane-bound compartment or vacuole, fleeing the host defense system and surviving for extended periods of time inside the epithelial cell until conditions are favorable to induce a cytotoxic response (Konkel et al., 2001; Rowe and Madden, 2014). Campylobacter produces the cytolethal distending toxin (CDT), a tripartite toxin composed of three subunits (Cdt-A, -B, and -C), which are required for the toxin to be functionally active (Asakura et al., 2008). The CdtA and CdtC subunits are responsible for toxin binding to the cell membrane and to deliver the CdtB, which is the active moiety of the Cdt ABC complex (Bolton, 2015). The CdtB subunit is translocated into the host cell cytoplasm toward the nucleus, where it is internalized, and causes cell cycle arrest in the G2/M transition phase through blocking of CDC2 kinase involved in the entry into mitosis leading to cell distension and apoptotic cell death (Dasti et al., 2010). The CDT is the primary toxin studied in Campylobacter, but is not limited to this genus and may be produced by other Gram-negative bacteria, including Escherichia coli and Salmonella (Hinenoya et al., 2014; Mezal et al., 2014). The role of CDT in Campylobacter pathogenesis in vivo remains unclear, but existing evidence indicates its importance in the colonization, invasiveness, modulation of the immune response, induction of intestinal inflammation, and gastroenteritis symptoms (Ge et al., 2008). Some Campylobacter species may produce toxins other than CDT toxin, but that has not been fully characterized (Man, 2011). Another important virulence factor related to immunological manifestations is lipooligosaccharides (LOS), a molecule composed of a core oligosaccharide and lipid A, localized on the surface of the Campylobacter cell (Bolton, 2015). This structure resembles human neuronal gangliosides, and such molecular mimicry may lead to autoimmune disorders, including GBS (Young et al., 2007). The sialylation of the LOS outer core, a process by which sialic acid groups introduce onto these molecules, increases invasive potential and reduces immunogenicity (Hermans et al., 2011). The polysaccharide capsule and O- and N-linked glycans surrounding the surface of Campylobacter cells are also important bacterial structures that facilitate survival, adherence, invasion, and evasion of the host immune system (Bolton, 2015). The capsular polysaccharides are common on cell surfaces of several foodborne pathogens and allow them to evade host immunity via several mechanisms, including resistance to phagocytosis and complement-mediated killing (Roberts, 1996). In genome sequencing of Campylobacter jejuni, the kps genes potentially involved in capsule biosynthesis (Zilbauer et al., 2008) were identified. Strains with a mutation in the kspM gene, which encodes a capsular polysaccharide

400  Chapter 13 transport protein, show a decreased ability of colonization, cell invasion, and reduction in resistance to human serum (Bolton, 2015). Regarding protein glycosylation, Campylobacter is the only bacterium known to possess both O- and N-linked systems (Jeon et al., 2010). The N-linked glycosylation system is responsible for posttranslational modification of multiple proteins, whereas the O-linked system is responsible for flagellar glycosylation (Guerry et al., 1992; Szymanski et al., 1999). Besides the above virulence factors, the iron acquisition is essential for Campylobacter survival in the host organism and the successful gut colonization, involving several membrane receptors, transporter proteins, and regulators (Bolton, 2015). This element has functioned as a cofactor for several enzymes, participates in electron transfer processes, and modulates the transcription of genes (Hermans et al., 2011). The resistance to bile salts is also fundamental for Campylobacter to support the conditions of the intestinal tract and it may be reached through the expression of the multidrug efflux systems. The efflux system CmeABC is the major operating system for this purpose, which will be discussed in more detail below. The foodborne pathogens are exposed to environmental stress conditions both outside and inside of the host organism (Young et al., 2007). The expression of stress response mechanisms by these microorganisms plays a fundamental role in their survival in various ecological niches. Campylobacter lacks many of the adaptive responses to environmental stress known and found in other foodborne pathogens (Park, 2002). They do not possess the stationary-phase sigma factor rpoS gene which encodes for the global regulator RpoS (sigma 38), which contributes to the transcription of stress response and virulence genes (Parkhill et al., 2000). However, Campylobacter can exhibit some adaptive responses against heat shock, acid tolerance, and reactive oxygen species, which allow them to survive the conditions of environmental stress (Bolton, 2015; Dasti et al., 2010; Murphy et al., 2006; Reid et al., 2008). Despite its sensibility to environmental stressors, fastidious growth requirements, and lack of many stress response mechanisms, these microorganisms can persist in the food chain and can pose a threat to the consumer (Alter and Scherer, 2006). More details on virulence factors and survival and colonization mechanisms of Campylobacter can be found in reviews of Bolton (2015), Hermans et al. (2011), Dasti et al. (2010), Zilbauer et al. (2008), and Young et al. (2007).

13.4  Antimicrobial Resistance Antimicrobial resistance in bacteria from food of animal origin has become in recent years a significant public health problem. Many of these bacteria, including Campylobacter, may be resistant to more than one type of antimicrobial (multidrug-resistant), which leads to a

Campylobacter: An Important Food Safety Issue   401 considerable reduction in the therapeutic arsenal and the constant need to develop new ways of treating infections (Možina et al., 2011; Rowe and Madden, 2014). Four main mechanisms are involved in bacterial resistance to antimicrobials: modification of the antimicrobial's target and/or its expression (i.e., DNA gyrase mutations); inability of the antimicrobial to reach its target (i.e., expression of the major outer membrane porin or MOMP); modification or inactivation of the antimicrobials (i.e., β-lactamase production); and increased expression of multidrug efflux systems (i.e., multidrug efflux pumps) (Iovine, 2013). Campylobacter presents several of these resistance mechanisms giving it resistance to the major classes of antimicrobial agents such as macrolides, quinolones, tetracyclines, β-lactams, and aminoglycosides (Wieczorek and Osek, 2013). Furthermore, these pathogens are naturally transformable and capable of acquiring and incorporating resistance genes from other microorganisms (Velàzquez et al., 1995). Resistance to macrolides and quinolones in Campylobacter has been mainly attributed to mutations in domain V of 23SrRNA gene and the gyrA subunit of DNA gyrase enzyme, respectively, which modify the target site of these antimicrobials (Payot et al., 2004; Piddock et al., 2003). Another mechanism of macrolide resistance involves an altered membrane permeability mediated by expression of the major outer membrane porin (MOMP) encoded by porA gene. Porins are outer membrane proteins from Gram-negative bacteria that form transmembrane pores and allow the passive diffusion of hydrophilic molecules including many antibiotics. The MOMP forms a small cation-selective pore in Campylobacter jejuni and Campylobacter coli, which limits the entry of the most antibiotics with a molecular weight greater than 360 kDa or negatively charged (Iovine, 2013). Regarding tetracycline, the main resistance mechanism involves the expression of a ribosomal protection protein encoded by the gene tetO, which is widely present in Campylobacter jejuni and Campylobacter coli. This protein recognizes a gap in the A site of the ribosome and binds to induce a conformational change that results in the release of the tetracycline molecule bound to the ribosome (Connell et al., 2003). On the other hand, Campylobacter resistance to β-lactams is mostly mediated by enzymes called β-lactamases that breakdown the structure of these agents (Griggs et al., 2009). In addition to this mechanism, efflux system and cation-selective MOMP also contribute to β-lactams resistance in some strains (Iovine, 2013). In general, the most of them are resistant to many β-lactams, including penicillin and cephalosporins (Wieczorek and Osek, 2013). The resistance to aminoglycosides is less frequent in Campylobacter and may be caused by antibiotic modifying enzymes, usually encoded by genes found in plasmids (Iovine, 2013; Tenover et al., 1992). Aminoglycosides resistance was first detected in Campylobacter coli and is mediated by a 3′-aminoglycoside phosphotransferase encoded by the aphA-3 gene (Lambert et al., 1985). Other genes conferring resistance to streptomycin (aadE), streptothricin (sat), and kanamycin (aphA-1 and aphA-7) have also been detected in Campylobacter (Gibreel et al., 2004; Ouellette et al., 1987; Tenover et al., 1992).

402  Chapter 13 In addition, the efflux systems confer resistance against different classes of antimicrobial agents which limit the access of these agents to their targets. Thus, it is an active way to pump these agents outside the bacterial cell, thereby preventing their intracellular accumulation required for the lethality of the cell (Kurinčič et al., 2012). The efflux systems in bacteria can be divided into five major families based on amino acid sequence homology: (1) small multidrug resistance (SMR) subfamily of drug/metabolite transporters superfamily; (2) multidrug and toxic compound extrusion subfamily of the multidrug/oligosaccharidyllipid/polysaccharide flippase superfamily; (3) resistance-nodulation-division (RND) superfamily; (4) major facilitator superfamily (MFS); and (5) superfamily of ABC-type transporters (Putman et al., 2000; Li and Nikaido, 2004). In Campylobacter, at least 14 efflux systems are present, including three of the RND superfamily (CmeB, CmeD, and Cj1373), four of the superfamily MFS, four of subfamily SMR, and one of the superfamily of ABC transporters (Zhang and Plummer, 2008). However, the efflux system CmeABC (RND) is the major system responsible for the intrinsic resistance of the bacteria to macrolides, fluoroquinolones, tetracyclines, bile salts, dyes, and detergents (Gibreel et al., 2007; Lin et al., 2002). It is composed of three components: CmeB, which is the protein located in the cytoplasmic membrane; CmeC, a protein that forms a channel in the outer membrane; and CmeA, a periplasmic fusion protein that bridges CmeB and CmeC (Lin et al., 2002; Iovine, 2013). These efflux system components are encoded by a three-gene operon located on the chromosome and regulated by CmeR, a transcriptional repressor (Lin et al., 2005). Several studies have demonstrated that the significant contribution of this efflux system in antimicrobial resistance, and also their synergetic action with other mechanisms of resistance to quinolones, macrolides, tetracycline, and ampicillin present in Campylobacter (Gibreel et al., 2007, Iovine, 2013; Lin et al., 2002; Payot et al., 2004; Pumbwe and Piddock, 2002). Furthermore, CmeABC efflux system also plays a pivotal role in the colonization of poultries by this pathogen since it is responsible for the increased resistance to bile salts (Lin et al., 2003). The methods used for evaluating antimicrobial resistance relies on the determination of the minimum inhibitory concentration or inhibition zone diameter of the antimicrobials against the bacterial isolates, which is classified as susceptible, intermediate, or resistant. These phenotypic methods include diffusion (disk, tablet, and Etest) and dilution (broth and agar dilution) methods (Ge et al., 2013; Jorgensen and Ferraro, 2009). Variations in both types of methods have been used for Campylobacter susceptibility testing (Aarestrup et al., 2008). The methods of choice are agar dilution and broth microdilution, which is recommended by the Clinical and Laboratory Standards Institute (Ge et al., 2013). However, a disk diffusion method has been standardized by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (EUCAST, 2012). Also, four automated susceptibility testing systems referred to as MicroScan Walk-Away (Siemens Healthcare Diagnostics), Phoenix (BD Diagnostics), Sensititre ARIS 2× (Trek Diagnostic Systems), and Vitek 2 (bioMérieux)

Campylobacter: An Important Food Safety Issue   403 have been used by the US Food and Drug Administration (Jorgensen and Ferraro, 2009). The criteria used to interpret the results of susceptibility testing may be of two types: clinical (clinical breakpoints) or monitoring (epidemiological cut-off values, ECOFFs) purposes. Values of clinical breakpoints depend on the antimicrobial susceptibility data generated by standardized in vitro susceptibility testing, pharmacokinetic, and pharmacodynamic information, and outcome data from well-controlled clinical efficacy trials. On the other hand, ECOFF values are based solely on antimicrobial susceptibility data of bacterial populations (Ge et al., 2013). The main factors that influence bacterial resistance are the indiscriminate use of antibiotics to treat infections in humans as well as its excessive use in veterinary medicine and as growth promoters in animal production (Alfredson and Korolik, 2007; Mor-Mur and Yuste, 2010; Iovine, 2013). Use of antimicrobial agents in poultry may lead to a selection of resistant Campylobacter strains, and some of these microorganisms can spread from food of animal origin to humans via the food processing chain (Maćkiw et al., 2012; Obeng et al., 2012). Contaminated water and direct animal contact may also contribute to spreading antimicrobial resistance. Over the years, high levels of antimicrobial resistance from Campylobacter isolated from poultry and poultry products have been reported in many studies around the world, including resistance to quinolones, tetracycline, and ampicillin (Hungaro et al., 2015; Kittl et al., 2010; Maćkiw et al., 2012; Obeng et al., 2012, Panzenhagen et al., 2016). For example, the resistance of Campylobacter jejuni and Campylobacter coli isolates from broiler and broiler meat in European Union (EU) member countries in 2014 to tetracycline was on average 54%–73.9% and to quinolones ranged from 65% to 85.8% (EFSA, 2016). On the other hand, antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolated from retail chicken in the United States in 2012 to tetracycline, quinolones, and macrolides was 50%, 22%, and 1%–12%, respectively (NARMS, 2012). Antimicrobial resistance in Campylobacter remains problematic and is a serious concern for public health, particularly concerning high levels of resistance to ciprofloxacin and tetracycline (Ge et al., 2013). Antimicrobial resistance monitoring in zoonotic and commensal bacteria in food-producing animals and foodstuffs is a prerequisite for understanding the development and diffusion of this significant problem. This control also provides relevant data on risk assessment, evaluate targeted interventions, and allow the identification of emerging or specific patterns of resistance (EFSA, 2016). Thus, multiple national surveillance programs have been established for the epidemiological monitoring of Campylobacter resistance (Ge et al., 2013). In North America, the two primary programs that measure and monitor resistance trends among foodborne pathogens, including Campylobacter, isolated from humans, animals, and retail meats are the US National Antimicrobial Resistance Monitoring System (NARMS) and the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS). The NARMS was established in 1996 as a collaboration among Centers for Disease Control and Prevention (CDC), Food and Drug Administration (FDA), US Department of Agriculture

404  Chapter 13 (USDA) (CDC, 2015a), and the CIPARS was initiated in 2002 (Public Health Agency of Canada, 2013). In the EU there are surveillance programs in place in several member countries, and the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC) analyze the data and produce summary reports on resistance prevalence and trends (EFSA, 2016). However, the establishment of monitoring programs in developing countries remains a challenge (Ge et al., 2013). More details about the antimicrobial resistance of Campylobacter, methodologies, and monitoring programs used for evaluating it may be found in reviews performed by Ge et al. (2013), Iovine (2013), and Možina et al. (2011).

13.5  Aspects and Epidemiology of Campylobacteriosis Campylobacteriosis is defined as an infection caused by Campylobacter, whose symptoms may vary from person to person and are largely indistinguishable from other bacterial gut infections. They include diarrhea with or without blood in stools, fever, vomiting, and abdominal pain. These symptoms may appear within 2–5 days after the contact with a pathogen, and the illness may last from 2 to 10 days (CDC, 2014; Young et al., 2007). Variations in the symptoms and progression to severe cases may occur due to a high genetic diversity that exists among isolates of Campylobacter (Dasti et al., 2010). As previously mentioned, the species Campylobacter jejuni and Campylobacter coli are the most common species associated with campylobacteriosis cases. However, other species with clinical relevance have been recognized, such as Campylobacter concisus, Campylobacter lari, Campylobacter upsaliensis, and Campylobacter ureolyticus (Man, 2011). Mechanisms of pathogenesis are not very well understood, but several virulence factors previously described may explain the involvement of Campylobacter in this disease and their postinfection complications. Most of the patients with campylobacteriosis recover without any specific treatment, but constant hydration is recommended due to fluid loss. Treatment with antibiotics is only recommended for severe cases of the disease or when there is a risk of progression to postinfection complications. Usually, patients who are with weakened immune systems and immunocompromised need antibiotic treatment. Antimicrobials commonly used in the treatment of this gastroenteritis are azithromycin and fluoroquinolones (CDC, 2014). Despite the selflimiting characteristic of this disease, some serious complications can occur, including GBS, Miller Fisher syndrome, reactive arthritis, endocarditis, myocarditis, irritable bowel syndrome, and diseases in the reproductive tract (Israeli et al., 2012; Kaakoush et al., 2015). Fig. 13.2 shows the stages of colonization, infection, and postinfection complications of the campylobacteriosis. The GBS is an autoimmune disease characterized by the loss of the myelin sheath and the tendon reflexes caused by an autoimmune response triggered by viral or bacterial pathogens. The immune response causes an acute inflammation of the nerves, which impairs the

Campylobacter: An Important Food Safety Issue   405

1

Colonization Diarrhea Fever Abdominal pain Sequel

2 Peripheral nervous system

GBS Macrophage

C. jejuni Axon

GM1 epitope Anti-GM1 IgG

Fig. 13.2 Stages of colonization, infection, and postinfection complications of the campylobacteriosis. (1) Campylobacter in the mucous layer of the intestine colonizes enterocytes. This infection cause the symptoms such as diarrhea, abdominal pain, and fever. (2) When the infection evolves to GBS, the human body produces anti-GM1 antibodies, and macrophages induced by LOS on Campylobacter cells. Due to the molecular mimicry of LOS to peripheral nerve ganglioside (GM1), the antibodies bind to the axon in the nervous system and leads to the blockage of the motor nerve.

conduction of the nerve stimulation of the brain to the muscles and vice versa (Wakerley and Yuki, 2015). The presence of LOS on the cell wall of Campylobacter jejuni explains such response. These structures in the cell are similar to those present in gangliosides of the nervous system which may induce the generation of IgG antibodies against these nerve cells. Thus, after infection, the individual's immune system may act on the nervous tissue compromising the myelin sheath of neurons (Hughes and Cornblath, 2005). The GBS incidence varies from 0.4 to 4.0 cases per 100,000 people annually, adolescents and young adults are the most affected group by the syndrome (Hadden and Gregson, 2001). Among the etiologic agents of GBS, Campylobacter jejuni is the most common. One-third of the GBS cases have been attributed to infection by this microorganism, although other bacteria and viruses can trigger the disease. These organisms include Mycoplasma pneumonia, Haemophilus influenza, Salmonella spp., Cytomegalovirus, Epstein-Barr virus, varicellazoster virus, influenza virus, and human immunodeficiency virus (Wakerley and Yuki, 2013; WHO, 2012). Furthermore, new findings showed that zika virus infection might also trigger GBS (Cao-Lormeau et al., 2016). The risk of developing GBS followed by a symptomatic infection by Campylobacter jejuni is about 100 times greater than the risk of developing the

406  Chapter 13 syndrome without being infected by this microorganism. Moreover, in most cases, it becomes difficult to associate the cases of GBS infection by Campylobacter since the body eliminates this bacterium within 16 days of infection and this occurs before settling neurological symptoms syndrome, which usually begins 10 days to 3 weeks after the appearance of symptoms of gastroenteritis. Thus, there may be more cases of GBS associated with infection by Campylobacter than those reported (McCarthy, 2001). The infection by this microorganism may also trigger another syndrome known as Miller Fisher syndrome which is a variation of GBS characterized by a triad of ataxia, ophthalmoplegia, and areflexia. Like in GBS, this disease may also develop after the exposure to an infectious pathogen, and Campylobacter jejuni is a common pathogen involved in the Miller Fisher syndrome (Sejvar et al., 2011). The Campylobacter infection may also evolve to reactive arthritis, which is a spondyloarthropathic disorder that can affect joints and other tissues causing inflammation of these parts of the body. Besides Campylobacter, other infectious agents may cause this disease such as Salmonella and Shigella. The risk of reactive arthritis after an infection by Campylobacter can reach up to 16% (Ajene et al., 2013). Other complications associated with Campylobacter infection such as endocarditis, myocarditis, irritable bowel syndrome, and complications of the reproductive tract has been described in the literature. The cases reported are isolated, and those findings do not show substantial evidence that there is a correlation between these complications and Campylobacter infection (Becker et al., 2007). Campylobacter is one of the leading bacterial causative agents of gastroenteritis in many countries around the world. Nevertheless, the epidemiology of this enteric infection in developed countries is remarkably different from that in developing countries (WHO, 2012). In developing countries such as in East Asia and the Pacific, South Asia, Latin America, and Caribe, Sub-Saharan Africa, Campylobacter is not the most prevalent pathogen involved in bacterial gastroenteritis. The bacteria involved in this kind of illness are Salmonella spp., Staphylococcus aureus and pathogenic E. coli (Fletcher et al., 2013; Ritter and Tondo, 2014; WHO, 2015). National surveillance programs for campylobacteriosis in developing countries do not exist or are ineffective, making it difficult to estimate the burden of this foodborne disease (WHO, 2012). The data collected about campylobacteriosis in these countries are obtained through studies on isolation of this microorganism from clinical samples, which give us a notion of the current status of the disease. Moreover, different methods used to detect this pathogen in patient's specimens influence the variation of epidemiological data among countries (Oberhelman and Taylor, 2000; Platts-Mills and Kosek, 2014). On the other hand, the annual incidence of campylobacteriosis in developed countries is estimated from 4.4 to 9.3 cases per 1000 population. However, there is a significant difference in incidence rate between countries. Thus, providing epidemiological surveillance data is pivotal to identify the strains involved in campylobacteriosis outbreaks, to detect sporadic

Campylobacter: An Important Food Safety Issue   407 cases for case-control, and to provide data that can be used in risk analysis and to assess the effectiveness of monitoring programs implemented to reduce the campylobacteriosis spread (WHO, 2012). Some countries, such as the EU, the United States, Canada, Australia, New Zealand, and Singapore have efficient epidemiological surveillance programs and consistent reports about foodborne diseases, which allow them to collect the data of outbreaks and take protective and control measures. Fig. 13.3 shows examples of campylobacteriosis incidence rates from these regions over the last few years. 180 166.3

168.2 158.6

160

152.9

151.9

Incidence rate per 100,000 population

140

120

110.0

114.1

117.2 101.6

100

93.5

80 62.8

67.0

69.0

26.6

27.6

65.9

64.8

29.3

29.1

14.3

13.8

Australia Canada EU New Zealand United States Singapore

60

40 25.9 20

0

13.0 5.2 2009

13.6 6.3 2010

14.3 7.2 2011

8.3 2012

7.4 2013

Year

Fig. 13.3 Examples of incidence rate of campylobacteriosis reported by national surveillance in Australia, Canada, EU, New Zealand, Singapore, and United States from 2009 to 2013.

Campylobacter has been the most frequent pathogen associated with foodborne outbreaks in these countries, except in the United States, where it was behind the cases of salmonellosis (CDC, 2015a; EFSA, 2015; Ministry of Health Singapore, 2014; Ministry for Primary Industries, 2015; NNDSS, 2016; Public Health Agency of Canada, 2015).

408  Chapter 13

13.6  Reservoirs and Sources of Contamination Several animals, such as poultry, cattle, sheep, swine, cats, and dogs, are hosts of Campylobacter. This pathogen may be transferred to humans by a fecal-oral route through direct contact with animals or consumption of contaminated food and water. Contact with domestic pets is a potential transmission route for campylobacteriosis (Pintar et al., 2015). Despite the lack of information on the prevalence and concentration of this pathogen in these animals, dogs and cats can be a carrier of several species of Campylobacter. These species include Campylobacter coli, Campylobacter concisus, Campylobacter fetus, Campylobacter gracilis, Campylobacter helveticus, Campylobacter jejuni, Campylobacter lari, Campylobacter mucosalis, Campylobacter showae, Campylobacter sputorum, and Campylobacter upsaliensis (Chaban et al., 2010). Wild animals, especially birds, also play an important role as reservoirs of Campylobacter, disseminating this microorganism in the environment to other animals and humans (Whiley et al., 2013). Despite the importance of these reservoirs, food-producing animals are the primary source of contamination by Campylobacter for human infections. The microorganisms in the intestine of these animals can contaminate the carcasses and meat cuts from feces during slaughtering (WHO, 2016). Moreover, the poor hygiene during milking can lead to contamination of raw milk. Poultry is the most frequent host of Campylobacter and, consequently, carcasses and meat of these animals are the primary vehicles of transmission in human campylobacteriosis. Poultry involves chicken, turkey, duck, and laying hens, of which the first one (Gallus gallus) is the predominant species used for meat production (70%–80%) (Skarp et al., 2015). Horizontal transmission of this microorganism can be considered the most likely source of contamination in poultry. These animals can get colonized by Campylobacter through the ingestion of contaminated water, contact with infected animals such as other poultry, domestic pets, wild birds, insects, as well as vehicles, equipment on the farm, old litter, and feed (Cox et al., 2012; Hazeleger et al., 2008). Campylobacter can spread quickly in flocks of poultry, mainly due to population density in breeding sheds and coprophagic behavior of animals (Humphrey et al., 2014; Man, 2011). The transmission between bird-to-bird in a farm may happen at a rate of 2.37 ± 0.295 infections per infectious bird per day, which means that in the population of 20,000 animals, 95% of this population would be infected within a week after the colonization of the first bird (van Gerwe et al., 2009). Also, it has been thought that some species of Campylobacter is a part of the poultry microbiota. However, this is still controversial, because the mechanism of colonization and infection in poultry is not entirely understood (Nyati and Nyati, 2013). Other sources of contamination on the farm include vehicle, equipment, and personnel coming into contact with poultry. On the other hand, the vertical contamination of poultry is unlikely. Some studies show that Campylobacter is not able to pass through the egg shell and contaminate its inner part. Thus, infected poultry is not capable of infecting offspring through the egg (Fonseca et al., 2014). Furthermore, chicks in most cases are only contaminated after the fourth week of life (Humphrey et al., 2007).

Campylobacter: An Important Food Safety Issue   409 The contamination of the poultry by Campylobacter into breeding farm may occur through various routes, which are difficult to set accurately. Therefore, if practices concerning biosecurity in the beginning of the production chain of poultry are inexistent or insufficient, cross-contamination between the flocks of birds is almost inevitable, especially during transport to the slaughterhouse (Ridley et al., 2011). Consequently, the infected birds may contaminate the equipments in the slaughterhouse, which will contaminate carcasses and meat during slaughter. Food remains the most common vehicle for the spread of Campylobacter in humans, which makes this microorganism one of the leading causes of foodborne diseases around the world. Among all the food related to these diseases, poultry and poultry products are believed to be a major contributor to human campylobacteriosis (Hunt et al., 2001; WHO, 2016). Also, Campylobacter has frequently been isolated from these types of food worldwide. For example, world literature surveys have shown that about 50%–58% of chicken carcasses and chicken products are contaminated by Campylobacter, but significant variations were observed in the individual findings of each study (Sahin et al., 2015; Suzuki and Yamamoto, 2009). The Campylobacter prevalence in chicken samples was 31.4% and 11.16% from official reports provided by the EU and the United States, respectively. Furthermore, campylobacteriosis outbreaks involving these foods are widespread throughout the world. For example, the consumption of undercooked chicken liver pâté was incriminated as a cause of an outbreak involving four people who ate the product in two different restaurants in Oregon, Unites States (CDC, 2015b). Another example of the outbreak that happened in a Barcelona School, Spain, affecting 75 school children, who were contaminated probably by the ingestion of undercooked poultry meat or other prepared food that have been cross-contaminated with raw poultry meat (Calciati et al., 2012). All these data reinforce that Campylobacter is globally distributed in poultry and poultry products. The contamination of meat and meat products, including poultry meat, beef, and meat of other animals, can occur during slaughter, where the bacteria present in the gut of a contaminated animal move to their carcasses, meat cuts, and viscera. In the poultry processing line, the steps where the risks of contamination of the carcasses are higher are in the stages of scalding, plucking, and evisceration. Also, water chillers are a potential hazard for cross-contamination between carcasses originating from different batches of poultry (Smith et al., 2005). The handling and processing of poultry carcasses and raw poultry products postmarketing stage at home and in public places such as retail and restaurants are also important contamination sources. Cooking poultry thoroughly before consumption ensures the destruction of Campylobacter. However, cross-contamination between raw and ready-to-eat food may occur due to poor culinary practices and hygienic habits of consumers. The method of washing raw poultry increases the risk of contamination of other food and kitchen utensils

410  Chapter 13 since the rinsing water may spread the bacteria from the chicken to other surfaces or to other food that may be close to the washing place. Moreover, storing and defrosting poultry without proper precautions may favor the contamination of other food in the refrigerator through contact with contaminated water from the meat defrosting. Thus, in order to avoid cross-contamination in the fridge, chicken must be stored below 5°C, preferably, on lower shelves of the refrigerator or in places where the dripping water coming from poultry cannot contaminate other food (FSA, 2009). Raw meat products from other food-producing animals than poultry have also been implicated in the transmission of Campylobacter. Similar to the poultry, beef, pork, and lamb meats may be contaminated during slaughter as these animals can also harbor Campylobacter in their gut (Kramer et al., 2000). Besides, the overall cleanliness of the animal in the slaughter, utensils, and equipment can be a factor of cross-contamination of the carcasses (Lake et al., 2007). Unpasteurized milk is also considered as a potential vehicle transmitting Campylobacter to humans, in which contamination occurs mainly by contact with feces and poor hygiene during milking. The incidence of Campylobacter in dairy cattle may be seasonal, with a peak occurring in the summer, while human campylobacteriosis outbreaks associated with consumption of contaminated milk arise in the fall and spring (Elangro et al., 2012). In the United States, from 2007 to 2012, the average number of outbreaks associated with unpasteurized milk was 13.5 outbreaks/year, showing that outbreaks linked to raw milk continue to pose a public health challenge (Mungai et al., 2015). The transmission of Campylobacter through contaminated fresh produce of nonanimal origin, such as raw vegetables and fruit is uncommon, but might also be important (Verhoeff-Bakkenes et al., 2011). Fruits and vegetables can become contaminated with Campylobacter during production, harvesting, processing, packaging, and distribution. Preharvest contamination sources include feces, natural or inadequately composted manure, the presence or survival of the microorganism in the soil, contaminated irrigation water, dust, and contact with domestic animals. Postharvest factors involved in the contamination of fruits and vegetables include improper hygiene practices of the equipment, utensils, and handlers; use of contaminated water to wash the foods; irregular storage (temperature and physical environment), improper packaging; and cross-contamination at the retail (Verhoeff-Bakkenes et al., 2011). Besides food, water may serve as a significant environmental reservoir for Campylobacter. Human or animal feces containing these microorganisms are the primary contamination source for water resources. Consumption of contaminated drinking water is a significant cause of campylobacteriosis outbreaks (Pitkänen, 2013). Outbreaks resulting from waterborne Campylobacter can affect thousands of individuals because of potable water demand (Miller and Mandrell, 2005). Fig. 13.4 illustrates the main routes and sources of contamination by Campylobacter.

Campylobacter: An Important Food Safety Issue   411

Fig. 13.4 Main routes and sources of contamination of Campylobacter.

13.7  Intervention and Control Strategies The control measures aiming the reduction of Campylobacter as a foodborne pathogen focus on the poultry production chain as this food is the main risk of human infection (EFSA, 2011). The measures include the implementation of biosecurity, which is a set of preventive measures to avoid the colonization of the flocks by Campylobacter. Implementation of hygiene practices at the primary production level can reduce Campylobacter contamination by more than 50% (Evans and Sayers, 2000). Hygiene practices include the use of a boot dip, change of footwear before entering in the poultry house or use of dedicated footwear, use of overshoes, use of hygiene barrier, hand washing, and sanitization of equipment since these microorganisms have to be isolated from clothes and hands of the farm personnel (EllisIversen et al., 2012; Newell et al., 2011). Reducing intestinal colonization in poultry appears to be the most efficient strategies to reduce loads of this microorganism in these animals. Vaccination of the flocks may also be a measure to control Campylobacter spread (Meunier et al., 2016). Immunization strategies have been studied to develop an accurate antiCampylobacter immune response. Some efforts have been made toward the reduction of the poultry colonization by this microorganism. Studies have shown that the vaccination using antibodies reactive against the Campylobacter jejuni could reduce this microorganism significantly in poultry. However, no commercial vaccine has been developed yet (NealMcKinney et al., 2014; Saxena et al., 2013). Bacteriocins may be used as a biocontrol measure to reduce the poultry infection of Campylobacter. Bacteriocins are short cationic antimicrobial peptides (AMPs) produced by

412  Chapter 13 many microorganisms in different environments. These substances are naturally produced by a variety of organisms and showed a successful measure to reduce Campylobacter jejuni colonization in chickens. Besides, some studies have demonstrated that Campylobacter jejuni does not show a relative resistance against bacteriocins (Hoang et al., 2011; Kaakoush et al., 2015). The use of bacteriophages and probiotics in poultry feed are also interesting and promising alternative in reducing the colonization of Campylobacter in poultry (Carvalho et al., 2010; Ghareeb et al., 2012; Nishiyama et al., 2014; Santini et al., 2010). Another approach to controlling Campylobacter colonization that could be applied in the primary production is the installation of fly screens since houseflies are a potential vehicle of Campylobacter jejuni (Shane et al., 1985). Moreover, Hald et al. (2007) demonstrated that controlling insects in the poultry house could reduce Campylobacter contamination in poultry during the seasonal peak. It has been shown that drinking water is a possible source of contamination. Some studies isolated Campylobacter jejuni strains from drinking water destined to flocks in poultry farms. Although there is no substantial evidence that poultry can get colonized by Campylobacter by ingesting water, it is recommended to treat the water used in the farm (EFSA, 2011; Trigui et al., 2015). Applying Good Manufacturing Practices during the slaughter may reduce the risk to contaminate carcasses. There are some critical points where the cross-contamination may occur in the processing chain which includes the evisceration and defeathering processes (Hue et al., 2010). The treatment of carcasses with organic acids, acidified sodium chlorite, trisodium phosphate, or ultraviolet light during processing may also help to reduce the number of Campylobacter. Moreover, freezing or irradiating the entire poultry supply would further reduce bacterial numbers by directly killing the pathogen (Lake et al., 2013). Promoting good hygiene practices by consumers may avoid Campylobacter infection since cross-contamination may occur during storage and preparation of the poultry. For instance, the defrosting poultry meat should be carried out properly and washing raw meat as well as the consumption of undercooked meat should be avoided (FSA, 2009).

13.8  Overview of Analytical Methods for Identification The monitoring of Campylobacter in food production chain is essential to identify the primary sources of contamination, to propose preventive measures, and to establish the microbiological risk of food consumption. Various analytical methods have been developed and evaluated for the isolation and enumeration of Campylobacter in food samples. However, there is no consensus about the ideal methodology, since all of these have a set of advantages and disadvantages. In general, use of culture-based conventional methods require 2 days to isolate colonies in selective media and 2 more days for species confirming tests (Castillo et al., 2008).

Campylobacter: An Important Food Safety Issue   413 As Campylobacter are fastidious organisms and require a microaerophilic environment to grow, most laboratory procedures are optimized to isolate the more common species: Campylobacter jejuni and Campylobacter coli (Huq et al., 2014). Regarding safety procedures in laboratories, Campylobacter is categorized as Biosafety Level 2 pathogens and a class II laminar flow biosafety cabinet is recommended for procedures in which splashes or infectious aerosols may be created (LQAS, 2016). All analytical methods for the identification of Campylobacter have shown the importance of appropriated sample transportation and storage for better results. Moreover, they emphasize that the pathogen being sensitive to freezing and drying, very low temperature and loss of humidity should be avoided. Food manufacturing processes such as heating, freezing, or chilling can result in sublethal injury to Campylobacter species, leading to increased sensitivity to antibiotics and lower resistance to elevated temperatures. The enrichment culture procedures allow resuscitation and recovery of injured organisms (PHE, 2014). According to the features of each analytical method for Campylobacter, they can be divided into conventional, molecular, and immunological ones, which will be discussed in detail below.

13.8.1  Culture-Based Methods The traditional methods are based on the growth of microorganisms in appropriate media and confirmation of suspect colonies by morphology and traditional biochemical tests. Some variations, especially concerning the types of culture media and incubation conditions, may occur depending on the culture method and food sample to be analyzed. 13.8.1.1  Bacteriological Analytical Manual (Food and Drug Administration) Bacteriological Analytical Manual (BAM)—Chapter 7, from Food and Drug Administration (FDA), describes a method applied for the isolation of Campylobacter species from food and water. The first step begins with preenrichment in Bolton Enrichment Broth (BEB). For samples produced or processed within 10 days, preenrich at 37°C for 4 h. However, for frozen samples or samples produced or processed more than 10 days, incubate at 30°C for 3 h and then at 37°C for 2 h. After preenrichment, move the broth to a 42°C incubator for enrichment. Keep shaking for 23–24 h. Preenrichment and enrichment should be incubated under microaerobic conditions (N2 85%, CO2 10%, O2 5%). Three methods for generating microaerobic conditions in enrichment broth can be chosen: bubbling the gas mixture through broth, shaking enrichments to incorporate the gas, or incubating in anaerobe jars with a modified atmosphere. The addition of activated charcoal or blood in media is common; reducing agents such as ferrous sulfate, sodium metabisulfite, and sodium pyruvate are also added to inhibit the oxidative stress (George et al., 1978; Hutchinson and Bolton, 1984; Solomon and Hoover, 1999). For isolation procedure, Abeyta-Hunt-Bark agar or modified charcoal cefoperazone deoxycholate agar (mCCDA) are inoculated. The plates should be placed in anaerobic jars at

414  Chapter 13 37–42°C for 24–48 h. Campylobacter colonies on agar show a thick translucent white growth spreading. Microscopically, the cells are curved, 1.5–5 μm long, usually in chains resembling zigzag shapes. For differentiation of species, biochemical tests are required (Hunt et al., 2001). 13.8.1.2  Microbiology Laboratory Guidebook (United States Department of Agriculture) Microbiology Laboratory Guidebook (MLG) (41.04), from Food Safety and Inspection Service (FSIS), United States Department of Agriculture (USDA) illustrates a method based on the qualitative direct plating and enrichment procedures for the isolation and identification of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari from poultry rinse, carcass sponge, and raw product samples. The direct plating analysis is performed in two steps: sample preparation and plating. The sample is prepared by adding a portion of the sample in buffered peptone water. One milliliter of the sample preparation is dispensed onto four CampyCefex plates, and the inoculum is spread over the entire plate. The plates are incubated for 48 ± 2 h at 42 ± 1°C, applying the appropriate microaerobic conditions. For enrichment procedures, the samples are enriched with double strength blood-free BEB (2× BF-BEB) and incubated for 48 ± 2 h at 42 ± 1°C under microaerobic conditions. After the incubation time, the enrichment samples are plated onto CampyCefex media. Typical colonies usually vary in size, and are translucent or mucoid, glistening and pink, and flat or slightly raised. Isolates are confirmed as Campylobacter jejuni, Campylobacter coli, or Campylobacter lari by microscopy (typical cellular morphology and motility) and immunological testing (LQAS, 2016). 13.8.1.3  International Organization for Standardization 10272-1 and 10272-2 International Organization for Standardization (ISO 10272-1 and 10272-2) specifies a horizontal method for the detection and enumeration of Campylobacter. Part one of this approach involves the detection method and part two the colony-count technique. In part one, the sample is preenriched in BEB and incubated under microaerobic conditions at 37°C for 4–6 h. Then, for enrichment, the broth is moved to a 42°C incubator for more 44 h. After that, the plating occurs on mCCD agar and the second medium of own choice is added. The second medium should be based on a principle different from mCCD agar to increase the chance of detecting Campylobacter species. Typical colonies are selected and purified on Columbia blood agar for confirmation by four tests: morphology/motility, oxidase, microaerobic growth at 25°C, and aerobic growth at 41.5°C. Part two, which is colony-count technique, follow the same protocol as explained above, starting with directly plating on mCCD agar (De Boer, 2009; Habib et al., 2011). The typical colonies growing on the plates of culture medium are counted, and some of them are sampled to perform the confirmatory tests. 13.8.1.4  Public Health England (FNES 15) Public Health England published the “Standard Method for Detection and Enumeration of Campylobacter species” in 2014 (FNES15). This method is based on ISO 10272-1 (2006a) and ISO 10272-2 (2006b), but it uses a single Campylobacter isolation medium, mCCDA,

Campylobacter: An Important Food Safety Issue   415 which forms grayish, flat, and moist colonies, often with a metallic sheen and with a tendency to spread. Agar plates are incubated microaerobically at 41.5°C for approximately 48 h. The number of typical colonies obtained on the selective media permits to determine the number of colony-forming units (CFU) per gram (g) or milliliter (mL) of a sample of Campylobacter species. The colonies are subsequently confirmed by morphological, biochemical, and growth property tests. Discrimination of the Campylobacter species is not carried out as part of this method (PHE, 2014). 13.8.1.5  Cape Town method The method, known as the “Cape Town Protocol” is an alternative method for the recovery and isolation of a wider variety of Campylobacter species, which may not grow in traditional culture media containing selective agents. This approach exploits the natural motility of these pathogens, which traverse a membrane filter deposited on the culture medium that acts as a selective barrier against nonmotile and larger motile competing microorganisms (Le Roux and Lastovica, 1998). The Cape Town method may be adapted for the isolation of Campylobacter from food samples, consisting of an enrichment step followed by centrifugation (low speed/time) and deposition of supernatant aliquots on a membrane of 0.45–0.85 μm pore size disposed on the surface of culture medium. The membrane is carefully removed after the absorption of the supernatant, and the inoculum is spread evenly over the surface of the agar. The incubation conditions and colonies confirmation can be performed as reported in other culture-based methods (Lynch et al., 2010). Despite the advantages of this approach, food particles can obstruct the membrane pores and hinder to pass of Campylobacter cells through of it. Also, some contaminant microorganisms such as Lactobacillus and Proteus may cross the membrane, interfering in the Campylobacter isolation (Bi et al., 2012; Lynch et al., 2010; Oakley et al., 2012). 13.8.1.6  Rapid culture-based methods Modified culture-based methods constitute a significant part of rapid methods. The use of chromogenic media facilitates the recognition of presumptive colonies of Campylobacter. The media contain enzyme substrates linked to a chromogen (color reaction). The microorganism is identified through the metabolism of a substrate (sugar or amino acid) by enzyme systems with the release of the chromogen. The result is a color change in the medium (Jasson et al., 2010). CASA agar (AES Chemunex, France) and CampyFood ID agar (bioMérieux, Portugal) are the internationally validated medium for enumeration and the most used chromogenic media for Campylobacter. Also, SimPlate Campylobacter (BioControl Systems) is a rapid enumeration of Campylobacter method, based on patented chromogenic media formulation to provide quantitative results for thermophilic species: Campylobacter jejuni and Campylobacter coli. This rapid method has been validated to provide reliable results with just a single plate, in just 48 h, up to 2 days faster

416  Chapter 13 than conventional reference culture methods. Moreover, it reduces the number of tests required for each sample, helping to reduce overall testing costs (BioControl, 2016).

13.8.2  DNA-Based Molecular Methods The DNA-based molecular methods, in particular, polymerase chain reaction (PCR) methods, are promising tools for rapid and direct detection of Campylobacter in animals used for food production and also in food, due to the specificity and the sensitivity of the methods (De Boer et al., 2015; Rudi et al., 2004). They are particularly advantageous for the detection of noncultivable organisms or those that do not grow easily using traditional cultures (Huq et al., 2014). The PCR methods and their variations aim the amplification and detection of small target fragments of the genome by specific protocols. Conventional PCR has been quite widespread and used in combination with culture-based methods in the colony-confirmation step, replacing the classic biochemical methods. It can be used to confirm the genus and distinguish Campylobacter species by using specific primers to target genes, including 16S rRNA, hipO, mapA, asp, and ceuE genes (Abubakar et al., 2007; El-Adawy et al., 2012). These genes can combine in multiplex PCR (m-PCR) protocols to confirm the genus and detect the species simultaneously (Huq et al., 2014). These methodologies, conventional and m-PCR, have been applied in routine diagnostic laboratories to detect Campylobacter in food and clinical samples. However, they are not able to quantify this pathogen in the sample. This proposal may be achieved by using real-time PCR (qPCR) methods, which can be an alternative to both detection and quantification of Campylobacter in different types of samples. The DNA extraction procedure is usually automated, and many different qPCR protocols can be applied (De Boer et al., 2015). BAX System (DuPont, Qualicon, Wilmington, DE) and iQ-Check Campylobacter (Bio-Rad, Hercules, CA) are two commercial qPCR assays available in the market for the detection of Campylobacter. The main limitation of PCR methods is that they are unable to distinguish between DNA from viable and dead cells (Rudi et al., 2005). In order to overcome this problem, intercalating dyes such as ethidium monoazide (EMA) and propidium monoazide (PMA) have been used in combination with PCR methods in viable/dead diagnostics. These dyes enter bacteria with damaged cell membranes and inhibit the amplification of DNA from dead cells (Seinige et al., 2014). The discrimination between viable and nonviable cells is an important issue in biological research (Rudi et al., 2005).

13.8.3  Immunological Methods Immunological methods based on antigen-antibody reaction have also been widely used for the detection of Campylobacter (Oyarzabal and Battie, 2012). The simplest is latex agglutination test, which has been in use for approximately 20 years for rapid identification or serotyping of cultures of Campylobacter. The principle behind this test is the use of polyclonal antibodies

Campylobacter: An Important Food Safety Issue   417 covering latex particles, which react against flagellar or outer membrane proteins from several Campylobacter species, including Campylobacter jejuni, Campylobacter coli, and Campylobacter lari (Gharst et al., 2013). The enzyme-linked immunosorbent assay (ELISA or EIA) and its variations are the most popular immunoassay used for the detection of pathogens in foods and stool. They are typically designed as a “sandwich” assay, in which an antibody bound to a solid matrix is used to capture the bacterium and a second antibody conjugated to an enzyme then binds to the bacterium (Oyarzabal and Battie, 2012). An example of a commercial automated EIA system is the VIDAS Campylobacter (bioMerieux, Marcy l'Etoile, France), which has undergone several validations and has been used by several countries around the world (Liu et al., 2009; Reiter et al., 2010). Other nonautomated EIA assays are commercially available for clinical samples, such as the Premier CAMPY microplate EIA, the ImmunoCard STAT! CAMPY (Meridian Bioscience, Cincinnati, OH), the ProSpecT Campylobacter assay (Remel Inc., Lenexa, KS), and the Ridascreen Campylobacter (R-Biopharm AG, Germany) (Bessede et al., 2011; Granato et al., 2010; Tribble et al., 2008). The lateral flow immunochromatographic method is a modified EIA packaged in a simple device, in which the antigens present in the sample bind to the antibodies conjugated to colored particles that in turn bind to immobilized antibodies during its migration on a device, producing a visible line on the capture zone. To ensure a working test, the sample migrates further until it reaches the control zone, where excess conjugate is bound to produce a second visible line on the membrane. Two clear lines on the membrane are a positive result. A single line in the control zone is a negative result (Oyarzabal and Battie, 2012). Immunocapture of Campylobacter cells from food and water samples has been successfully used in combination with PCR and other methods for the identification of this pathogen. This approach utilizes magnetic spheres coated with antibodies to capture target bacteria from a variety of matrices, which are separated from the matrix and other bacteria by the application of a magnetic field (Morales-Rayas et al., 2008).

13.8.4  Typing Methods The typing of Campylobacter species is fundamental to conduct of the epidemiological studies and maybe performed using different methodologies. Among typing methods, pulsedfield gel electrophoresis (PFGE) and multilocus sequence typing (MLST) are the most used for this purpose (Rowe and Madden, 2014). The PFGE is a molecular method based on the digestion of genomic DNA with restriction enzymes and subsequent electrophoretic separation of the generated fragments in an agarose matrix using a pulsed electric field (Taboada et al., 2013). In the MLST method, short DNA fragments are sequenced within seven housekeeping genes (asp, glnA, gltA, glyA, pgm, uncA, and tkt). Sequenced PCR products for each of the seven loci are assigned an allele number based on a comparison of alleles in a database. The range of seven allele numbers is then assigned a sequence type. Sequence types that share four or more alleles belong to the same clonal complex or lineage

418  Chapter 13 (Dingle et al., 2001). Some World Wide Web sites such as PulseNet and PubMLST have been established for the storage and exchange of data from these typing methods. These global databases can be used to identify and characterize outbreaks, as well as to direct decision making in epidemiologic surveillance (Dingle et al., 2001; Taboada et al., 2013).

13.9  Concluding remarks Campylobacteriosis is one of the most important foodborne diseases notified in the world. The consumption of contaminated poultry and poultry products has been reported as the major cause of this disease. Campylobacter jejuni and Campylobacter coli are the species in the Campylobacter genus more isolated from food and responsible for the most of foodborne outbreaks. However, other species with clinical relevance have also been recognized as foodborne pathogens in the past few years. Campylobacter is a vast and diverse group of bacteria widely distributed in the environment, which may live as commensals in the gut of a wide range of wild, domesticated, and food-producing animals. Although these microorganisms are fastidious regarding the environmental and nutritional conditions, they have developed strategies to persist and cause disease in humans and animals. Over the past few years, many advances have been made regarding the biology and pathogenicity of Campylobacter. These advances also include identification of reservoirs and sources of contamination, development of analytical methods, and ways to prevent and control Campylobacter from spreading in the food processing chain. However, many challenges still have to be overcome to obtain effective control of this microorganism and, thus reduce the campylobacteriosis outbreaks. These include an elucidation of the mechanisms used by this microorganism to survive under environmental stress as well as the differences in the colonization/infection between humans and animals. In addition, there is a need for a review of analytical methods to expand the range of identification of species, since most of them are targeted to Campylobacter jejuni and Campylobacter coli. Another concern is the lack of surveillance programs on campylobacteriosis and consistent reporting on Campylobacter in developing countries. Therefore, efforts to monitor and control this pathogen should be intensified worldwide. There are different ways to control Campylobacter along the food processing chain as described in this chapter. Nevertheless, the implementation of effective control measures to reduce Campylobacter spreading is still deficient.

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430  Chapter 13 Zhang, Q., Plummer, P., 2008. Mechanisms of antibiotic resistance in Campylobacter. In: Nachamkin, I., Szymanski, C., Blaser, M. (Eds.), Campylobacter. third ed. ASM Press, Washington, DC, pp. 263–276. https://doi.org/10.1128/9781555815554.ch14. Zilbauer, M., Dorrell, N., Wren, B.W., Bajaj-Elliott, M., 2008. Campylobacter jejuni-mediated disease pathogenesis: an update. Trans. R. Soc. Trop. Med. Hyg. 102 (2), 123–129. https://doi.org/10.1016/j. trstmh.2007.09.019.

Further Reading Blaser, M.J., Engberg, J., 2008. Clinical aspects of Campylobacter jejuni and Campylobacter coli infections. In: Nachamkin, I., Szymanski, C.M., Blaser, M.J. (Eds.), Campylobacter. third ed. ASM Press, Washington, DC, pp. 99–121. https://doi.org/10.1128/9781555815554.ch6. Lomovskaya, O., Bostian, K.A., 2006. Practical applications and feasibility of efflux pump inhibitors in the clinic—a vision for applied use. Biochem. Pharmacol. 71 (7), 910–918. https://doi.org/10.1016/j. bcp.2005.12.008. Pagès, J.M., Amaral, L., 2009. Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of gram-negative bacteria. Biochim. Biophys. Acta (BBA) Proteins Proteomics 1794 (5), 826–833. https://doi.org/10.1016/j.bbapap.2008.12.011. Tenover, F.C., Gilbert, T., O’Hara, P., 1989. Nucleotide sequence of a novel kanamycin resistance gene, aphA-7, from Campylobacter jejuni and comparison to other kanamycin phosphotransferase genes. Plasmid 22 (1), 52–58. https://doi.org/10.1016/0147-619x(89)90035-8. USDA—United States Department of Agriculture, 2011. Isolation and identification of Campylobacter jejuni/coli/ lari from poultry rinse, sponge and raw product samples (MLG 41.04). Available at http://www.fsis.usda.gov/ wps/wcm/connect/0273bc3d-2363-45b3-befb-1190c25f3c8b/MLG-41.pdf?MOD=AJPERES. (accessed May 24, 2016).

CHAPTE R 14

Food Contamination: From Food Degradation to Food-Borne Diseases Antonietta M. Gatti*, Stefano Montanari†

Health, Law and Science, Geneve, Switzerland †Nanodiagnostics, Modena, Italy

*

14.1 Introduction This chapter shows a few types of food found to be contaminated by inorganic particulate matter that is nonbiodegradable and is often nonbiocompatible for the human body: pollution that can be ingested along with vegetables or fruit grown under the fallout of a polluting source. However, similar pollution can be generated, for instance, by the combustion processes of waste incineration, cement production, foundries, home heating, car traffic, etc. By means of investigations carried out with an Environmental Scanning Electron Microscope (ESEM, Quanta, FEI, The Netherlands) and a Field Emission Gun Environmental Scanning Electron Microscope (FEG-ESEM, Quanta 250, FEI, The Netherlands), we identified foreign bodies that have an atomic density higher than that of the surrounding biological matrix. That particulate matter cannot be digested or anyhow used by human metabolism and, in any case, has no nutritive property. Thanks to an X-ray microprobe of an Energy Dispersive System (EDS by EDAX, USA) coupled to the FEG-ESEM, the chemical composition of these debris can be detected. This type of instrumentation can analyze the food samples, namely, a biological matrix which is nonelectrically conductive as an Electron Microscope working modality needs, without needing any process of Carbon or Gold coating. So there is no risk to detect/create artifacts. All the foreign bodies identified were actually present in the food before the analysis. These analyses can detect any type of inorganic contamination even when it is nanosized (lower limit 10 nm), whatever the matrix it is contained in. The contamination identified can have different origins: from the already mentioned fallout of inorganic dust to the feed given to the animals whose meat we eat, from engineered particles added on purpose to metal debris lost by cutting or grinding instruments used to prepare the ingredients of certain products. The environmental pollution due to organic pesticides is not described, with the exception of the particles used as their carriers. Also, the (nonbiodegradable and nonbiocompatible) Food Safety and Preservation. https://doi.org/10.1016/B978-0-12-814956-0.00014-7 © 2018 Elsevier Inc. All rights reserved.

431

432  Chapter 14 contamination of charcoal dust in smoked and grilled meat is not considered either. The results of our investigations will show different types of contamination and a tentative explanation will be advanced about that contamination, considering the chemical composition and the characteristics of the debris and the industrial process the food underwent. For instance, in the case of salami, the crushing of frozen meat with stainless steel blades can induce the release of stainless steel wear debris that enters the meat mixture contaminating it. Other industrial processes like milling can induce the inclusion of wear debris in bread and biscuits. Industrial additions of thickeners, dyers, emulsifiers, stabilizers, etc. can detrimentally interfere with the healthiness of the product, resulting in nonbiodegradable components remaining included in the digestive tract. If nanosized particles are added (Nanofood), they are very likely to pass into the blood circulation and be dispersed throughout the body as nonbiodegradable foreign bodies (Nemmar et al., 2002; Ballestri et al., 2001; Gatti et al., 2002). The interactions of this particulate matter can develop pathologies. (Gatti, 2004; Sabbioni et al., 2004; Lucarelli et al., 2004; Hansen et al., 2006). Direct observations of pathological human and animal tissues of the digestive system affected by pathologies like cancer demonstrate the physical presence of those foreign bodies. (Gatti and Montanari, 2008) Some examples of stomach cancer containing nanoparticles of titanium dioxide, or colon cancer with stainless steel or silver nanoparticles, or ulcers of the rectum with silicate particles are presented. When proper investigation is carried out, these chemical compositions can be traced in food or in the environment contamination where the patient lives.

14.2  Food Contamination Our life is strictly and obviously linked to food (“we are what we eat” said, though somewhat misunderstood, the German philosopher Ludwig Feuerbach), but what we eat, particularly now, can be contaminated and represent a risk for our life. We need safe and healthy food to ensure our survival, but the increase of the industrial/environmental contamination represents a real risk for humans and animals. In a nutshell, this describes one of the most important aspects of the scenario where man lives. Man can exist, live, multiply only if his food is healthy and contains what he actually needs. In the industrialized countries, as well as in the third-world countries where the most modern technologies have not yet arrived, food could not meet its essential features. This chapter deals with something going beyond the now largely known risks coming from pesticides and the “normal” additives used in agriculture and the raising of cattle, and discusses the risks linked to the inorganic environmental and industrial contamination, and how serious the pathologies it induces can be.

Food Contamination: From Food Degradation to Food-Borne Diseases  433 Row food (in contrast with a manipulated one) can be contaminated as well as industrialized food. Of course, it is contaminated in a different way, since, in the two cases, pollution is chemically and morphologically different. The chapter will highlight and analyze some of the most frequently encountered pollutions. The investigations presented here are carried out on fresh samples (i.e., not chemically treated) or samples fixed in 4% formalin and subsequently dehydrated in ascending concentration alcohols. The sections or fragments of the samples are then placed on an aluminum stub covered with an adhesive carbon disc. The set is placed inside the chamber of an FEG ESEM (QUANTA 200, FEI, the Netherlands) equipped with an EDS (EDAX, USA) (Gatti and Montanari, 2008).

14.2.1  Vegetables and Fruit According to current practice, in order to protect vegetables and fruit from pests, crops are treated with pesticides. Those chemicals exert their toxicity not only on the parasites, but also on the plant itself, and residues can be involuntary ingested by animals and humans and enter into the food chain. It is only obvious that residues of those toxic chemicals, mostly organic molecules, once dispersed in the air, can contaminate vegetables, fruit, and green grass grown under the follow out of the pollution, which, if not correctly washed and cleaned and in any case when that poison has reached their interior, can in turn contaminate who eats them, that is, humans and animals. However, while they are exposed to bacteria, fungi, and other pests, they can be contaminated also by natural dust polluting the air, a dust that can be composed of silicates of natural origin: volcanoes, desert sand, and rock erosion. In addition to that, particularly, but far from exclusively, in industrial areas, there is also the contamination coming from the chimneys dispersing in the environment fumes mixed with micro- and nanosized particulate matter. Vegetables grown under that fallout are contaminated by products whose biocompatibility, as a matter of fact, was never properly checked. If those contaminants are oily or acidic, especially when containing Sulfur or Nitrogen, they can visibly interact with the leaves causing damage. However, the sole presence of particulate matter can also represent a risk for humans and animals. In all cases, we eat something that is not what our body needs and, even worse, is what is harmful to it. Fig. 14.1(A–C) shows what we found in a homemade vegetable soup whose main ingredient was a cauliflower cultivated on the slope of Mt. Etna (Sicily, Italy), an active volcano then erupting. The cauliflower was full of fine basalt dust that three accurate washings could not eliminate because of the roughness of the vegetable surface that allowed the dust to penetrate it. The soup had been cooked 1000 km away from the place where the contamination had occurred and only FEGESEM-EDS investigation, plus a wide database of standard materials, could discover the nature and origin of the pollution.

434  Chapter 14 (A)

(B)

Si

Al

O C 100.0 µm

Ca Na

K P

CI

Ti

Fe

0.80 1.60 2.40 3.20 4.00 4.80 5.60 6.40 7.20

(C)

C

Fe

O

Mg Si Na Al

P

S

Ti

K Ca

Mn

0.80 1.60 2.40 3.20 4.00 4.80 5.60 6.40 7.20

Fig. 14.1 The image (A) shows particulate matter (white debris) that we identified in a cauliflower soup. The white debris are composed of silicon-aluminum-calcium-iron (basalt) or iron-titanium-siliconaluminum-potassium (B, C).

In a number of other samples, we found and keep finding particulate residues emitted from industrial chimneys that deposited on the exterior of all kinds of vegetables. Particulate pollutants are scattered all over the environment according to a few variables including their size, the height of the stack, and the wind direction. In the north of Italy, at the mouth of the river Po, there is a power plant that produced energy by burning heavy oils, that is, the residues of oil distillation contaminated with metals. The local Prosecutor’s office asked us to analyze some vegetables grown under the fallout of the soot without letting us know where the specimens had been collected.

Food Contamination: From Food Degradation to Food-Borne Diseases  435 Among the different samples, we were quite surprised to find some of the vegetables completely covered with greasy dust, while others were virtually clean, without any particular debris on them. When the codes identifying each sample were disclosed, we discovered that we had analyzed lettuce and other vegetable leaves grown in the open air and others in a greenhouse. The leaves grown in the greenhouse, and because of that not exposed to the fallout, did not show the presence of the oily soot in turn containing solid particles. Figs. 14.2(A, B) and 14.3(A–H) show the two types of samples: the former, that is, the one grown inside the greenhouse, is free from dust and the EDS spectrum shows only the natural elemental content: carbon-oxygen-calcium-potassium. Fig. 14.3 shows different areas of contaminated lettuce with particles of titanium (B), lead (D), bismuth-potassium (F), iron-chromium-manganese-zinc-potassium-calcium (H). (A)

(B)

C

O

K Ca

Mg 1.0 mm

P

S

Cl

0.90 1.80 2.70 3.60 4.50 5.40

Fig. 14.2 (A, B) Image of a portion of lettuce with its natural chemical components (carbon, oxygen, potassium, calcium, magnesium, phosphorus, chlorine, and sulfur).

Lichens and tomatoes were also given to us to be analyzed and the chemical composition of the particles identified in all vegetables (lettuce, lichens, tomatoes, etc.) was compared with the environmental pollutants detected in the air and with the ash from the combustion of heavy oil collected inside the power plant. As clearly resulted from our electron-microscope observation, the particles found on the surface of the vegetables were the same as those present in the ash, though not necessarily those found in the oil. It must be understood that combustion breaks the molecules of the fuel into atoms and those atoms recombine in the atmosphere to form solid particles whose composition is a random combination of those elements. Just to give a simple example, it is like having a mosaic of images made of colored tiles. If the mosaic is broken and the tiles are allowed to combine without any direction, the

(A)

(B)

Ti

C O Al Mg Si 20.0 µm

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Ca

Fe

0.70 1.40 2.10 2.80 3.50 4.20 4.90 5.60 6.30 7.00

(D)

C

O

Pb

K

P 20.0 µm

(E)

Pb

Ca

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.0012.00 13.00

(F)

C

Bi

K

O

Bi

Ca 50.0 µm

2.00

4.00

6.00

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8.00

10.00

Fe

12.00

14.00

(H)

C O

Zn Fe Si S Mg P 20.0 µm

K

Ca

Mn Cr

Zn

1.10 2.10 3.10 4.10 5.10 6.10 7.10 8.10 9.10

Fig. 14.3 (A–H) Images of some samples of lettuce grown under environmental pollution, showing the presence of debris. The EDS spectra (B, D, F, H) identify titanium (B), lead (D), bismuth-potassium (F), iron-chromium-manganese-zinc-potassium-calcium (H).

Food Contamination: From Food Degradation to Food-Borne Diseases  437 result will appear very different from the original but the tiles will be the same. Very often, the combinations detectable in the environment are very peculiar and, originating by chance, do not belong to any known material. They are a sort of “fingerprint” making it easier or, in the best of circumstances, possible to trace the source of the pollution. What follows are a few examples of how a source of contamination of vegetables can be traced. The image of Fig. 14.4. shows particulate matter contamination on the skin of a peach taken in the area of Modena, Italy. The chemical compositions of the debris (based on siliconaluminum-potassium-calcium-iron combinations) reveal that the contamination was generated by ceramic factories present in great number in the area of Sassuolo, a few kilometers south of Modena. (A)

(B)

C Mn

Si O Fe

AI 50.0 µm

P

K Ca

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

(C) Ti

C O Mg

Si AI

K Ca

Fe

0.80 1.60 2.40 3.20 4.00 4.80 5.60 6.40 7.20

Fig. 14.4 The image shows debris detected on the peach’s skin. The 4 micron sized particle (A) is composed mostly of manganese-silicon-Iron (B), while the small particle (C) is composed of titanium-silicon. Similar pollution was identified in the air around ceramic tiles factories around Sassuolo (an industrial village only for ceramic products), Italy.

438  Chapter 14 Nowadays, pesticides that use silver inorganic nanoparticles are rather common. Silver exerts a toxic effect on parasites, but in that form is not biodegradable. Owing to their extremely small size, those nanoparticles cannot only adhere to the surface of the vegetable, but also enter inside it through its roots, pass through the stomata, or clog them. It is long known that ingesting silver can cause a disease called argyria: The skin turns blue or bluish-gray by the interaction of silver with proteins. To date, there is no therapy against that apparently irreversible condition. However, silver nanoparticles also share all the features of other inorganic, nonbiodegradable foreign bodies of the same size and, when ingested along with the polluted food, part of them is captured by tissues and organs with all the already described consequences. We found hay contaminated by silver particles in a farm (Gatti and Montanari, 2008, pp. 241) we visited because of the occurrence of a severe neurological disease shown by a cow. In that circumstance, we could analyze the cow’s feed as we wanted to check if there was a detectable correlation between the animal’s diet and its pathology. To be able to do that, we should have examined at least the cow’s brain, something that was not possible to do. Our suspicion was that those metal particles could have reached the brain, thus interfering with its physiology. However, owing to our not being able to complete our study, we could not find any link. What is sure, though, is that Silver nanoparticles had entered the food chain and it is not unlikely that those foreign bodies were present in the animal’s tissues destined to become food for man. In a study carried out for a university thesis (Sola, 2008), we analyzed some aromatic plants grown in a vegetable garden located in downtown Parma, Italy, a town with about 150,000 inhabitants. All the plants (rosemary, sage, laurel, parsley, etc.) showed the sharp presence of micro- and nanodust, occasionally with unheard of chemical compositions. So, in that case, leaves, having never been treated with pesticides, were supposed to be healthy and were eaten with confidence, but they were actually heavily polluted by urban dust. During our research, we had the possibility to analyze a vineyard producing a famous wine, where the plants were grown under the fallout of a cement plant burning waste (used tires and petroleum coke, i.e., a carbonaceous solid waste coming from oil refinery coker units or other cracking processes). Fig. 14.5 shows a contamination on a wild grape grown in the vicinity of Pederobba, Italy. On the grape’s skin, we could find copper sulfate (pesticide, Fig. 14.5C and D), which comes from the vineyards located in the surroundings, and calcium-silicon-aluminum-sulfur-iron from the emissions of the stack of the cement plant. In an agricultural area close to Bologna, Italy, we spotted a great number of malformed ears of corn and flowers of chamomile (Fig. 14.6A–C). The analyses performed on the root system and in the stems showed the presence of submicronic debris containing, among other elements, bismuth coming from the soil and adsorbed by the plants. These small entities, foreign to the plants, can destabilize their metabolism by interfering

Food Contamination: From Food Degradation to Food-Borne Diseases  439 (A)

(B)

Ca

Counts 2.8k 2.4k 2.0k 1.6k 1.2k

O 0.8k C

0.4k 400.0 µm

Si AI S 1.00

(C) Counts

2.00

Fe 3.00

4.00

5.00

6.00

7.00

(D)

S

4.0k 3.5k 3.0k 2.5k 2.0k 1.5k 1.0k C

O 0.5k 50.0 µm

Cu

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Fig. 14.5 (A–D). Image of the environmental contamination of the grape from a cement plant (A, B) and a drop of copper sulfate deposited on a grapefruit leaf from a pesticide.

with their DNA and altering their genetic heritage. Malformations are the most obvious consequence. We discovered that the pollutants come from a biomass plant whose digestate is used as a soil improver. It is likely that the plants submitted to the anaerobic process aimed at generating gas were contaminated and those nonbiodegradable tiny pollutants contaminated in turn the soil and the plants grown on it. A similar result can occur when watering plants with water containing toxic particles. Our nano-ecotoxicological experiments on plants purposefully contaminated with nanoparticles by mixing them with the soil they were growing in led to results that showed how those small entities can induce showy modifications (Vittori et al., 2013; Degrassi et al., 2012; Vittori et al., 2012). We had no opportunity to check if there were changes in the nutritional content. What is sure is that animals and humans feeding on those plants ingest that pollution with partially unknown effects on their health.

440  Chapter 14 (A)

(B)

20.0 µm

(C)

C 4.8k 4.2k 3.6k

Bi

3.0k 2.4k

O 1.8k 1.2k

K

0.5k

2.00

Bi

Ca 4.00

6.00

8.00

10.00

12.00

14.00 keV

Fig. 14.6 (A–C) The image shows a malformed chamomile: (A) a new flower is grown through the pseudanthium, (B) ESEM image of the stem containing foreign bodies pulled from the contaminated soil, and (C) containing bismuth.

14.2.2 Fish Similar pollution problems can occur to fish and shellfish. When rivers and sea are polluted by industrial or urban wastewater, the possibility is high that fish and shellfish ingest those pollutants. The problems with Mercury and the people feeding on them, for example, have been known for decades and what was called Minamata disease is the most tragic example (Harada, 1995). Figs. 14.7–14.9 show examples of pollution respectively identified in Italy in the internal organs of a river fish, of an anchovy fished in the Adriatic Sea and in a clam grown in an aquaculture area at the mouth of the river Po. As can be seen, all these fish contain bodies foreign to their organisms coming from water contamination, a contamination reaching people who eat them. It is necessary to distinguish among the dangerousness of different particulate matter. In our experience on more than 4000 cases of pathological tissues, heavy metals are more reactive in

(A)

(B) C

W

O

50.0 µm

W

P CI S

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0011.0012.0013.00

(C)

C

Zr

O

S CI 2.00

Zr 4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

Fig. 14.7 (A–C) Image of (A) debris identified in internal organs of a catfish caught in the river Po in the vicinity of a small concentration of metallurgical industries. Debris of tungsten and zirconium or zirconium oxide are visible (B, C). (A)

(B)

C

Co

Cr Nb W O S CI Ca AI 10.0 µm

Mn Fe

W

Nb

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

Fig. 14.8 (A, B) The image shows particulate matter identified in the internal organs of an anchovy fished in the sea at the mouth of the river Po. The debris is composed of cobalt-chrome-manganese-iron-tungsten-niobium-sulfur-chlorine.

442  Chapter 14 (A)

(B)

Si

Counts 5.6k 4.9k

Ca

O

4.2k 3.5k 2.8k

AI

2.1k C 1.4k 0.7k 20.0 mm

Mg Fe 1.00

P

S

2.00

K 3.00

Fe 4.00

5.00

6.00

7.00

Fig. 14.9 (A, B) The image shows the internal part of a clam grown in an aquaculture in the Adriatic Sea. It contains submicron-sized particles (silicon-calcium-aluminum-iron-manganese-potassium) that the shellfish retained by filtering seawater.

the body than ceramic materials, and the smaller the particles, the more dangerous they are due to the easier chance they have to penetrate tissues and even cells (Gambardella et al., 2013).

14.2.3 Bread A few years ago, while analyzing some pathological human tissues affected by colon cancer, we identified many micro- and nanosized foreign bodies. The systematic presence of those debris induced us to look for the origin of this pollution. In our opinion, the most likely responsible was food, so, as a start, we investigated bread, as it is a very common, daily aliment, and some other products made with flour, be they artisan or industrially made. The article we published (Gatti et al., 2009a) considered 135 specimens of different kinds of wheat-based products (86 specimens of bread and 49 of different biscuits) randomly collected from 14 different countries, and in about 40% of the samples we found particulate contamination. It was somewhat surprising to find debris of cobalt-tungsten in a loaf of bread made in Tierra del Fuego (Argentina) where there are no industries, virtually no car traffic, and villages are small and far apart. The explanation was easy to find: the flour comes from elsewhere, mainly from the USA. The industrial crushing machine tools have a hard metal coating of cobalt-tungsten, adopted to limit the wear of the tools but, though limited, wear is still there and, inevitably, the small waste end in flour. Of course, the ingestion of bread, biscuits, and other derivatives of that contaminated flour transfers those pollutants to our digestive system. Very tiny particles can adhere to and interact with the mucosa cells causing an inflammation as is characteristic of foreign bodies.

Food Contamination: From Food Degradation to Food-Borne Diseases  443 Though the majority of them are eliminated with feces, some stick to the digestive tract walls, with the smaller ones being able to enter the blood circulation. The analyses performed on the surface of a cracker (Mulino Bianco, Italy) revealed the presence of aluminum-copper (Fig. 14.10). In this case, we advanced the hypothesis that the contamination was due to the industrial process of lamination with a tool made of that same alloy. (A)

Counts

(B)

AI

14k 12k 10k 8k 6k 4k

C O Cu 200.0 µm

Cu

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 keV

Fig. 14.10 Image of a metallic debris identified on a cracker’s surface (A). The EDS spectrum (B) shows that is composed of aluminum-copper, a metallic debris that is eaten with the cracker.

14.2.4 Meat In this section, we are presenting two examples of meat contamination, mostly by heavy metal particles. The first example is about the industrial hamburgers that represent one of the foods of choice for many teenagers (Fig. 14.11A and B). We bought our samples at a restaurant belonging to a fast-food chain and analyzed them with the usual method. What we detected was the presence of metallic debris that, in addition to being devoid of nutritional properties not being any of those elements bioavailable, can also induce the inflammatory reaction typical of foreign bodies that size. Among these particles, we found debris of lead-bismuth (Fig. 14.11A and B). Fig. 14.12(A–D) shows a wide area full of stainless steel debris of submicrometric and nanometric size. The analysis of those particles revealed that the contamination comes from two different types of alloys (probably cutting metallic tools), namely, two types of stainless steels containing iron-chromium and iron-chromium-nickel. Fig. 14.13 shows a selection of the chemical compositions found in the small group of hamburgers we analyzed. Numerous particles not originally belonging to meat were identified: aluminum, aluminum-zirconium, and gold-copper. Of course, as expected, among metallic particles we identified also debris composed of calcium-phosphorus, the basic composition of bone.

444  Chapter 14 (A)

(B)

C 4.5k 4.0k 3.5k 3.0k 2.5k

Bi Pb

2.0k 1.5k

O Mg

1.0k

P Si

0.5kNa AI 10.0 µm

K Ca

2.00

Bi Pb

4.00

6.00

8.00

10.00 12.00 14.00

keV

Fig. 14.11 (A, B) The image shows many debris identified in the meat of an industrial hamburger. They are composed of lead-bismuth-potassium-calcium, etc.

(A)

(B)

C

Fe

1.8k 1.6k 1.4k 1.2k 1.0k 0.8k

Cr

0.6k 0.4k

O Fe

0.2k 50.0 µm

(C)

1.00

Ni

P Si S CI K 2.00

3.00

4.00

5.00

6.00

7.00

(D)

Fe

Counts

8.00

2.8k 2.4k 2.0k 1.6k

C

1.2k

Cr

0.8k

S P CI K

0.4k O 10.0 µm

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

Fig. 14.12 (A–D) The images show numerous debris of two different types of stainless steels, namely, iron-chromium-nickel and iron-chromium.

Food Contamination: From Food Degradation to Food-Borne Diseases  445 Once in the body, particles of iron-chromium-nickel alloy can corrode and release ions of the three elements that are toxic according to the classical toxicity (Kirkpatrick et al., 2002). C

Counts 4.5k

P Ca

C

5.6k

4.0k

4.9k

3.5k

4.2k

3.0k

3.5k

2.5k

2.8k

2.0k

O AI 2.1k Zr P 1.4k Mg CI Na S K Ti 0.1k

(A)

2.00

Counts

4.00

O

1.5k 1.0k

6.00

0.5k

Zr

Fe 8.00

10.00

12.00

14.00

16.00

keV

AI

Mg Na

(B)

1.00

SCI K 2.00

3.00

4.00

5.00

6.00

7.00

8.00

C

2.1k

C

8.1k

1.8k

7.2k 6.3k

1.5k

5.4k

1.2k

4.5k 0.9k

3.1k

0.6k

2.0k

O

O

0.3k

PS 1.00

(C)

2.00

Cu

Fe

K 3.00

4.00

5.00

6.00

Au Na P 0.0kCu CI K

1.2k

7.00

8.00

9.00

keV

Cu

Au

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

(D)

keV

Fig. 14.13 (A–D) The images show four EDS spectra of different debris identified in the meat of a single hamburger. We found particles of aluminum-zirconia (A), aluminum (C), gold-copper (D). The spectrum of image C shows a debris of bone.

Another example is related to an animal disease that in the 1980s alerted all the world health organizations: the so-called Mad Cow disease or, to be more accurate, bovine spongiform encephalopathy (BSE). During the supposed epidemic, for precautionary reasons, consumption of bovine meat, especially entrails, bone marrow, and brain, was banned. The prohibition concerned not only Great Britain, where the problem had first come to prominence, but also the rest of Europe. At that time, we collaborated with the Veterinary Laboratories Agency of Scotland, so we had the possibility to analyze brain samples of cows affected by that disease. What we found in the brain was something unexpected. For two reasons: (1) we did not suspect that some tiny particles could reach the brain and the blood-brain barrier was really inefficient against the particulate matter; (2) we did not suspect that the animals received such “waste” feed. Fig. 14.14(A–F) shows some examples of the mostly metallic debris we found in the cows’ brains: nickel (B), mercury-silver-copper (D), lead-titanium-chromium (F).

446  Chapter 14 (A)

(B)

Ni

C Ni

O S 10.0 µm

CI Fe

0.90 1.80 2.70 3.60 4.50 5.40 6.30 7.20 8.10 9.00 keV

(D)

(C) Hg

C

Ag Hg O

50.0 µm

(E)

Cu 2.00

4.00

6.00

8.00

10.00

12.00

(F)

C

O

Pb Si AI

2.0 µm

14.00

Ti

Cr

Pb

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.0010.00 11.00 12.00

Fig. 14.14 (A–F) Images of the debris found in the brain samples of cows affected by BSE. The particles were composed of nickel (B), mercury-silver-copper (D), lead-titanium-chromium (F).

Those pieces of evidence demonstrate that the dispersion of very tiny particles, inhaled or ingested, can have a wide, total dispersion in the body. If ingested with food, they can contaminate the intestine and at least those with the tiniest size can cross the intestinal wall and reach the blood circulation. As already mentioned, the blood can transport them throughout all the body, to every internal organ, and they can also reach the brain where they can interact with the nervous functions.

Food Contamination: From Food Degradation to Food-Borne Diseases  447 Finding an explanation to these important presences is not easy, but we found a possible answer. At the time of the “epidemic” (though it was just a simultaneous adoption of some precautionary procedures), most breeders had for years fed their cattle with the so-called “meat and bone meal (MBM),” a product that is the result of the rendering industry through which waste animal tissues coming from slaughterhouses, restaurants, and butcher shops and including also expired meat from stores were reduced to powder. That product can include fatty tissue, bones, offal, and entire carcasses of animals, no matter how they had died. Such a food cannot be swallowed as such and, to make it usable, oil is added in order to obtain a homogenous, fluid, ingestible mixture. Instead of adding edible oils, though, some breeders used costless waste oil from internal combustion engines, oil that is full of tiny metallic wear debris. It is by no means surprising that those particles, once ingested, reached the brain. Being metallic, these debris are electrically conductive. So, besides triggering the usual inflammatory reaction typical of foreign bodies, they can interfere with the local electric physiological field and disturb, altering the normal electrical conduction. The conduction of the electrical signal along the nerve can be deflected by the presence of the metallic conductive particles. This can explain the behavior of the cattle, their tremors, and difficulty with balance. This could also explain why none of the people who kept eating bovine meat in spite of prohibition (and in Italy they were many) ever showed any pathological reaction apparently linked with BSE. The presence of prions, as a rule mentioned in connection with the BSE, could be considered as the biological answer to the presence of those metallic debris or as the no more biodegradable aggregate formed by the debris, their oily coating, and the biological proteins. Whatever the opinion, there are a couple of indisputable facts: all brains of the BSE-affected bovines we analyzed contained metal foreign bodies. Owing to of their metallic nature, those foreign bodies are electrically conductive and, in our specimens, were contained in an organ whose function is mainly electric. Anyone who has seen the behavior of a BSE-affected cow realizes that the problem is fundamentally neurological. The examples we showed make it evident that malnutrition of animals and/or bad meat processing can contaminate the food obtained and represent a risk for health. Feeding cattle with natural, pollution-free feed makes meat safe for humans.

14.2.5 Milk As a sort of vicious chain, animal milk can be polluted if the dairy animals are fed with polluted feed. As is only obvious, milk containing non-biodegradable and nonbiocompatible foreign bodies can start to contaminate infants just at birth. Similarly, a human mother exposed to an environmental contamination can also have her milk polluted. Mothers living in highly polluted areas or eating polluted food can feed their baby with milk containing the same foreign bodies as the environment. Fig. 14.15 shows the contamination of a sample of human milk of a mother living in a much industrialized, polluted area of Sicily, Italy. The drops of milk examined contained many

448  Chapter 14 different debris of palladium-rhodium-gold-copper-iron-nickel and zinc. Such a composition cannot be found in any handbook of material, has no industrial application, and is just the result of a random combustion of materials containing many different elements. (A)

(B)

C

Pd Rh Au

O Cu

Cu Ca

10.0 µm

Au

Fe Ni Zn

Rh Pd

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

keV

Fig. 14.15 (A, B) Image of a debris found in a sample of human milk of a mother exposed to a particular environmental pollution. It is a debris of a precious material composed of Palladium, Rhutenium, Gold, and Copper. It is obvious that the mother had an exposure to this pollution probably in a particular working place.

In an artificial milk sample for babies (Fig. 14.16), we found particles composed of aluminum-manganese-iron-silicon. That contamination is probably the result of an industrial process to obtain the synthetic milk sample whose actual origin can be identified only by an accurate inspection of the factory. (A)

(B)

AI

C

O Si

5.0 µm

Mn Fe

1.00 2.00 3.00 4.00 5.00 6.00 7.00 keV

Fig. 14.16 (A, B) Image of a debris found in an artificial milk sample. The particle is composed of aluminum-manganese-iron-silicon.

Food Contamination: From Food Degradation to Food-Borne Diseases  449

14.3  Disease Induced by Contaminated Food Unfortunately, foods containing inorganic, nonbiodegradable debris, which cannot be digested by our organism and have no nutritive value, but, on the contrary, can represent a risk for our health, are growing more and more common. As a general rule, physiological barriers (lung, colon, brain, etc.) stop micrometric foreign particles to safeguard our body from the invasion (Gatti and Montanari, 2015). However, they are inefficient in case of particulate matter with particularly small sizes (below 2.5 μm). When a thorn pricks the skin and remains trapped in the tissue, a biological reaction is triggered. First, redness appears; then, if the thorn is not removed, a granuloma is formed. Macrophages and giant cells, in an attempt to destroy the foreign body (without success, if that body is not degradable), are the main responsibility of the reaction. Submicronic and nanoparticles activate this biological mechanism only partially. Owing to their tiny size, they can cross virtually all physiological barriers and interact directly with the cells, with their cytoplasm and their nuclear content. They can also interact with the DNA (Gatti and Montanari, 2008; Gatti et al., 2009b), damaging it, and a damaged DNA can induce a metabolic disequilibrium as well as a carcinogenic reaction. In addition, it must be pointed out that often the solid, inorganic, and nonbiodegradable particles are endocrine disruptors.

14.3.1 Stomach We carried out investigations in the tissues of pathological cases affected by forms of stomach cancer or heavy inflammation to verify the possible presence of foreign bodies. In all circumstances, our observations showed the presence of particulate matter that might have been ingested with polluted food. Nevertheless, it must not be forgotten that particles entered through inhalation/respiration or otherwise can reach the stomach the way they can with any other organ. One case attracted our attention. An elderly patient (a 73-year old) was hospitalized for stomach cancer and the surgical pathological tissue observed under FEG ESEM showed the presence of nanotitania, namely, nanotechnological particles of titanium dioxide (titanium dioxide is a food additive approved by official agencies, including the US Food and Drug Administration, also in the form of nanoparticles, not only molecules). Fig. 14.17 shows the titania nanoparticles identified in the surgical samples. Among the so-called nanofoods that the industry proposes to consumers, be it legally allowed or legally ignored, there are a few of them like chocolate, mayonnaise, cheese, cereals, and yogurt sometimes containing titanium dioxide particles, Just to mention one case, the addition of titania nanoparticles to chocolate is made because it gives the product a smooth, creamy texture and prevents the separation of fat (cocoa butter) from cocoa, thus forming an unsightly, superficial, white layer with the aging of chocolate. Titania cannot be digested or anyhow used or degraded by our organism, since they are crystalline lattices, with all the already mentioned consequences.

450  Chapter 14 (A)

(B)

2.0 µm

10.0 µm

Ti

(C)

C

O Si P

CI

0.60 1.20 1.80 2.40 3.00 3.60 4.20 4.80 5.40

Fig. 14.17 (A, B) The images show a cluster of titania nanoparticles at different magnification. The EDS spectrum C shows that the particles are composed of titanium and silicon. These entities are not biodegradable.

In another case, we found a 50-μm sized ball of zirconia (zirconium oxide) in a stomach specimen, an unusually bulky presence. The patient, affected by stomach cancer, was a soldier who had served in peace-keeping missions during the Balkan war. Among the subjects we checked, we had many soldiers contaminated by zirconia, since that is a material used to make the shelter of tanks. Once the tanks are hit by depleted uranium weapons, as already described, their shelters are aerosolized and the pollutants can be dispersed in the air or it can fall on vegetables, fruit, etc. In another case of stomach carcinoma, we found stainless steel (iron-chromium-nickel) (Fig. 14.18A and B) particles and debris of antimony-chlorine (Fig. 14.19A–C). Even if we had some explanations for the steel findings and for that see what has been said for meat, we have no explanation for antimony chloride.

Food Contamination: From Food Degradation to Food-Borne Diseases  451 (A)

(B) Fe

Counts

C 1.5k 1.2k

Cr

0.9kO

Fe

0.6k 0.3k 50.0 µm

Ni

P Si S CI

Ca

Ni

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

Fig. 14.18 (A, B) The image shows numerous stainless steel debris in a surgical sample of stomach cancer.

(A)

(B)

50.0 µm

50.0 µm

(C)

C Sb

O CI P 0.80 1.60 2.40 3.20 4.00 4.80 5.60

Fig. 14.19 (A–C) The image shows four antimony-chlorine debris in a surgical sample of stomach cancer.

452  Chapter 14

14.3.2 Colon In our investigations on intestine samples, in particular those of colon affected by Crohn’s disease or carcinoma, we found numerous foreign bodies. In one of those cases, in a frame of 300 × 300 μm, we found debris with 15 different chemical compositions: the whole alimentary history and its related pollution was conserved in that tissue. In those cases, all foreign bodies contributed to an inflammatory reaction that became chronic and evolved into cancer, and in all circumstances inside the granulomas of a tissue affected by Crohn’s disease we found foreign bodies. Close to the ileocecal valve we found debris Fig. 14.20 containing lead-chlorine-aluminum. Also, this combination is very unusual and we could not guess a possible source, probably it is the result of a casual combustion. Namely it could be part of the emission of an incineration plant.

(A)

(B) C

Pb O Al 10.0 µm

CI

2.00

Pb 4.00

6.00

8.00

10.00 12.00 14.00

Fig. 14.20 (A, B) The image shows some submicronic debris found in a surgical sample affected by ileocecal carcinoma. The chemical composition of these particles: lead-chlorine-aluminum is very unusual, namely it is not known an alloy of lead, chlorine, aluminum in the handbook of materials.

Much more probable was the identification of the source of stainless steel particles in the colon cancer of a very young patient (Fig. 14.21). As we have already mentioned, stainless steel debris in food could be due to the wear of grinding/cutting machines for seeds or meat. In a case of a patient affected by Chron's disease we found nonbiocompatible metallic debris of ironmanganese-silicon and zinc (Fig. 14.22). We do not know the origin of this contamination, but we hope that the feeding of animals happens under logic and safe rules: healthy food means healthy meat for humans.

Food Contamination: From Food Degradation to Food-Borne Diseases  453 (A)

(B)

Fe

7,1 5,7 4,2 KCnt

Cr

2,8 1,4 C

Si

Ni

0,0 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00

20.0 µm

(C)

Pt

2,3

O

C 1,8 1,4 KCnt 0,9 0,5

O Na

Si Al

Ca

Fe

0,0 0,00 1,00 2,00 3,00 4,00 5,00 6,00 7,00

Fig. 14.21 (A–C) The images show numerous debris deeply embedded in colon villi. The sample affected by cancer showed the presence of stainless steel and platinum debris.

14.3.3 Rectum We analyzed some pathological samples affected by ulcerous recto-colitis and in all samples we found many debris, that we supposed to have been ingested with food and the physical presence of non biocomaptible and nonbiodegradble “foreign bodies” in the tissue, inducing an inflammatory reaction, do not allow the tissue to cicatrize and recover. These pieces of evidence suggested to us to hypothesize that the ingestion of a safe food not containing such nonbiodegradable and nonbiocompatible particles probably could help to recover better than many pharmaceutical treatments. The case we are showing is (Fig. 14.23) that of a rectum carcinoma containing Silver nanoparticles. We do not know the origin of that debris, since silver is used in toothpaste and in toothbrushes, can be found in meat and in vegetables, enters the composition of a number of industrial products and applications (filters for drinking water, washing machines, fridges, air conditioners, fabrics, etc.), and is even voluntarily ingested in colloidal form.

(B)

(A) C

Fe Si Mn

O P 50.0 µm

1.00

Cl S

2.00

Ca 3.00

4.00

5.00

6.00

7.00 8.00 9.00

(D)

(C)

C

Zn

O

Zn

P Cl Si S Al

Ca K

Ba

Fe

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0011.00

20.0 µm

Fig. 14.22 (A, C) The images show debris deeply embedded in a sigma tissue affected by Crohn’s disease. The particles are composed of iron-silicon-manganese (B) and zinc (D). (A)

Ag

(B)

C O 10.0 µm

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Fig. 14.23 (A, B) The image shows some nanosized debris found in a rectum. The particles are composed of silver. It is a noble metal.

Food Contamination: From Food Degradation to Food-Borne Diseases  455

14.4 Conclusions All the internal mucous tissues of the digestive system we analyzed because they were affected by some pathology showed the presence of foreign bodies: the higher the concentration, the more serious the illness. Our conclusion is that the ingestion of food that cannot be degraded by our metabolic functions into its primary, usable components must not be eaten. It is necessary to recover the best aspects of food, eating only what is necessary and usable by our organism and, what is of the utmost importance, introducing food that our organism can recognize. Continuing to eat “unknown” things a fortiori when polluted, we put our health at risk. Probably all the alimentary intolerances we see nowadays with increasing frequency can be due, at least in part, to these foreign bodies. Recently it was demonstrated that a damage to the microbiota (bacterial flora of the guts) that these foreign bodies can induce can have an influence on the brain inducing neurological pathologies (Sekirov et al., 2010; Li et al., 2017). In our opinion, it is high time that farmers, breeders, and industrialists are not only aware of the problem but also become more prudent. Also, even more urgent is that lawmakers legislate fairly and in step with scientific knowledge, having public health as their sole interest.

References Ballestri, M., Baraldi, A., Gatti, A.M., Furci, L., Bagni, A., Loria, P., Rapanà, R., Carulli, N., Albertazzi, A., 2001. Liver and kidney foreign bodies granulomatosis in a patient with maloocclusion, bruxism, and worn dental prostheses. Gastroenterology 121, 1234–1238. Degrassi, G., Bertani, I., Devescovi, G., Fabrizi, A., Gatti, A.M., Venturi, V., 2012. Response of plant-bacteria interaction models to nanoparticles. EQA Environ. Qual. 8, 39–50. Gambardella, C., Aluigi, M.G., Ramoino, P., Diaspro, A., Bianchini, P., Gatti, A.M., Rottigni, M., Tagliafierro, G., Falugi, C., 2013. Developmental abnormalities and cholinesterase activity alteration in sea urchin embryos and larvae obtained from sperms exposed to engineered nanoparticles. Aquat. Toxicol. 130–131, 77–85. Gatti, A.M., Ballestri, M., Bagni, A., 2002. Granulomatosis associated to porcelain wear debris. Am. J. Dent. 15 (6), 369–372. Gatti, A.M., 2004. Biocompatibility of micro- and nano-particles in the colon (part II). Biomaterials 25 (3), 385–392. Gatti, AM, Montanari, S, 2008, “Nanopathology: The health impact of nanoparticles” book, ed. by PanStanford Publishing Pty Ltd, Singapore, pp. 1-298. Gatti, A.M., Tossini, D., Gambarelli, A., Montanari, S., Capitani, F., 2009a. Environmental scanning electron microscope and energy dispersive system investigation of inorganic micro- and nano-particles in bread and biscuits. Critic. Rev. Food Nutri. 49 (3), 275–282. Gatti, A.M., Quaglino, D., Sighinolfi, G.L., 2009b. A morphological approach to monitor the nanoparticle-cell interaction international. J. Imaging 2 (S09), 2–21. Gatti, A.M., Montanari, S., 2015. Case Studies in Nanotoxicology and Particle Toxicology. Elsevier, USA1–260. Hansen, T., Clermont, G., Alves, A., Eloy, R., Brochhausen, C., Boutrand, J.P., Gatti, A.M., Kirkpatrick, J., 2006. Biological tolerance of different materials in bulk and nanoparticulate form in a rat model: Sarcoma development by nanoparticles. J. R. Soc. Interface 3, 767–775. Harada, M., 1995. Minamata disease: methylmercury poisoning in Japan caused by environmental pollution. Crit. Rev. Toxicol. 25 (1), 1–24.

456  Chapter 14 Kirkpatrick, C.J., Barth, S., Gerdes, T., Krump-Konvalinkova, V., Peters, K., 2002. Pathomechanisms of impaired wound healing by metallic corrosion products. Mund Kiefer Gesichtschir. 6 (3), 183–190. Li, Q., Han, Y., Belle, A., Hagerman, R.J., 2017. The gut microbiota and autism Spectrum disorders. Front. Cell. Neurosci. 11, 120. Lucarelli, M., Gatti, A.M., Savarino, G., Quattroni, P., Martinelli, L., Monari, E., Boraschi, D., 2004. Innate defence function of macrophages can be biased by nano-sized ceramic and metallic particles. Cytokin Netw. 15 (4), 339–346. Nemmar, A., Hoet, P.H.M., Vanquickenborne, B., Dinsdale, D., Thomeer, M., Hoylaerts, M.F., Vanbilloen, H., Mortelmans, L., Nemery, B., 2002. Passage of inhaled particles into the blood circulation. Humans Circulation 105, 411–414. Sabbioni, E., Gatti, A.M., Hartung, T., 2004. Pathology of new diseases induced by nanomaterials and in vitro toxicology research. Pathol. Int. 50 (S), 141–148. Sekirov, I., Russell, S.L., Caetano, L., Antunes, M., Brett Finlay, B., 2010. Gut microbiota in health and disease. Physiol. Rev. 90 (3), 859–904. Sola, F., 2008/09. “Una metodica micro-spettroscopica per la rilevazione di contaminanti inorganici all’interno di vaccini approvati per uso umano” Thesis of University of Parma. Vittori, L., Carbone, S., Gatti, A.M., Fabrizi, A., Vianello, G., 2012. Toxicological effects of engineered nanoparticles on earth worms (lombricusrubellus) in short exposure. EQA Environ. Qual. 8, 51–60. Vittori, L., Carbone, S., Gatti, A.M., Vianello, G., Nannipieri, P., 2013. Toxicity of metal oxide (CeO2, Fe3O4, SnO2) engineered nanoparticles on soil microbial biomass and their distribution in soil. Soil Biol. Biochem. 60 (5), 87–94.

CHAPTE R 15

A Review on the Implications of Interaction Between Human Pathogenic Bacteria and the Host on Food Quality and Disease Rishi Mahajan⁎, Shalini Chandel†, Gunjan Goel⁎

Jaypee University of Information Technology, Solan, India †Directorate of Mushroom Research, Solan, India

*

15.1  Human Pathogenic Enteric Bacteria and Their Association With Fresh Agricultural Products Studies suggest that increasing health awareness has resulted in a significant increase in the demand for ready-to-eat fresh produce. However, several reports indicate the emergence and outbreaks of food-borne illness that have been found to be closely associated with fresh fruits and vegetables; therefore, researchers across the globe have been focusing on identifying microbial contamination of fresh produce (Tomasi et al., 2015; Mir et al., 2018). Until the advent of this century, much research was focused on studying the interaction of enteric pathogens with human and animal hosts and food-borne illness was believed to be result of postharvest microbial contaminations. Recently, the notion has been significantly questioned in the scientific community and, therefore, much research in today’s scenario is focused on the ecology of enteric pathogens on plant surfaces. Previously, enteric pathogens were known to colonize human or animal digestive tract, an environment that provides protection from external environmental factors and also provided a variety of easily accessible nutrients. However, recently documented reports suggest that enteric pathogens colonize the phyllosphere and the rhizosphere of plants. In the present section, we shed light on the most important and classical examples of enteric pathogens that are known to be associated with food-borne illnesses of fresh fruits and vegetables.

15.1.1  Escherichia coli Consumption of contaminated foods, especially raw or undercooked ground meat products and raw milk has been known to be associated with the transmission of E. coli O157:H7 into humans. Other sources of infection include fecal contamination of water and other foods, Food Safety and Preservation. https://doi.org/10.1016/B978-0-12-814956-0.00015-9 © 2018 Elsevier Inc. All rights reserved.

457

458  Chapter 15 as well as cross-contamination during food preparation (from contaminated surfaces and kitchen utensils). E. coli O157:H7 has been considered as a serious pathogen because of its high pathogenicity, its ability to infect the host even at very low infective doses, its ability to survive harsh and deleterious environmental conditions, especially frozen conditions (Tilden Jr et al., 1996; Franz et al., 2008; van Elsas et al., 2011; Semenov et al., 2009; Yao et al., 2013; Wang et al., 2014) and what make it even a more serious and dangerous pathogen is its ability to transfer the virulence genes into nonpathogenic E. coli strains (Herold et al., 2004). The bacterial pathogen causes hemolytic uremic syndrome that can lead to death, particularly in children, elderly especially the ones who are immunocompromised. Of serious concern is the fact that raw fruits and vegetables, especially fresh-cut leafy greens, are more and more being recognized as the important vehicles for the transmission of E. coli O157:H7 (Fremaux et al., 2008). It is pertinent to mention here that E. coli O157:H7 was identified as the causal agent of the largest outbreak of bacterial enteric disease in recent times, with more than 6000 cases epidemiologically linked to contaminated radish sprouts in Japan in 1996 (National Institute of Health Infection Disease Control Division, 1998; Watanabe et al., 1999). Recently in 2013, consumption of contaminated, sprouted fenugreek seeds resulted in an outbreak of hemolytic-uremic syndrome (HUS) and hemorrhagic colitis (HC) that started in Germany and subsequently spread throughout Europe and North America. The causative agent was an enterohemorrhagic E. coli (EHEC) strain of serotype O104:H4 (European Food Safety Association, 2013). Bielaszewska et al. (2011) and Mellmann et al. (2011) reported that the strain possessed a combination of virulence factors from both Shiga toxin (Stx)producing E. coli (STEC) and enteroaggregative E. coli (EAEC) strains. Information on E. coli contamination of fresh produce has been compiled and is presented in Table 15.1.

15.1.2  Salmonella enterica A review on the literature of Salmonella enterica shows that the occurrences of S. enterica in fresh produce have been associated with surface waters, used commonly for irrigation purposes in agricultural fields. Documented literature also suggests that S. enterica is the cause for most frequent outbreaks from fresh produce (Barak and Schroeder, 2012). S. enterica serovar Typhimurium DT104 has been reported in cases where contaminated lettuce was consumed (Horby et al., 2003; Takkinen et al., 2005). The survival of the enteric pathogen on fresh produce has been studied in great detail over the last decade. Findings suggest that S. enterica has abilities to survive desiccation stress (Davies and Wray, 1995). Studies have shown that small populations of S. enterica can survive on crop plants and eventually increase to infectious dose levels once favorable conditions are restored. These characteristics of the pathogen make it a serious contaminant associated with fresh produce (details are presented in Table 15.1). A comprehensive analysis of the documented literature suggests that the reported cases of S. enterica outbreak on fresh produce are more on fruits as compared with leaves (Barak and Schroeder, 2012). Reports suggest that S. enterica

Human Pathogenic Bacteria and Host on Food Quality and Disease   459 Table 15.1: Human pathogenic bacteria and their association with fresh agricultural produce Pathogen

Contaminated Agricultural Produce

E. coli O157:H7

Spinach, radish, alfalfa, cabbage, celery, coriander, cress sprouts

E. coli O157 CDC E. coli O104:H4 E. coli O26 E. coli O145 S. enterica

Clostridium difficile Bacillus cereus Campylobacter spp. Listeria monocytogenes Cronobacter spp.

References

Jay et al. (2007), Jay-Russell et al. (2010), Nathan (1997), and Zepeda-Lopez et al. (1995) Leafy greens California Leafy Green Handler Marketing Board (2012) Fenugreek Buchholz et al. (2011) Clover sprouts http://www.cdc.gov/ecoli/2012/ O26-02-12/ Lettuce Taylor et al. (2013) Alfalfa, mung, clover, tomato, cantaloupe, Rimhanen-Finne et al. (2011), beet leaves, cabbage, cauliflower, chili, Mohle-Boetani et al. (2009), and green onion, parsley, pepper, spinach Orozco et al. (2008) Ready-to-eat salads; potatoes, onion, Bakri et al. (2009) and Al Saif mushroom, carrot, radish, cucumber and Brazier (1996) Ready-to-eat vegetables, potato Chon (2016) and Luo et al. (2016) Leafy vegetables, lettuce, parsley, pepper, Park and Sanders (1992) and prepacked salads, spinach Salaheen et al. (2016) Cucumber, cabbage, carrot, tomato, lettuce Ajayeoba et al. (2016) Chinese cabbage, cucumber, carrot, lettuce, Chen et al. (2016) and Vojkovska strawberry, cabbage et al. (2016)

lacks biochemical assimilatory machinery for sucrose utilization (Lin, 1996), one of the main sugars present in leaf (Lindow and Brandl, 2003). The two most severe serotypes of S. enterica Typhi and Paratyphi A, which cause typhoid and paratyphoid fever, are responsible for hundreds of thousands of deaths worldwide every year. Results show that serovars have not evolved independently and recombination plays a key role in the genomic evolution, diversification, and ecological adaptation of lineages of S. enterica (Didelot et al., 2011).

15.1.3  Clostridium difficile Clostridium difficile infection can often result in severe diarrhea. C. difficile a spore-forming, Gram-positive anaerobe produces two toxins, viz., toxin A (an enterotoxin) and toxin B (a cytotoxin), which can cause gastrointestinal disease, especially in immunosuppresed patients (Nitzan et al., 2013). A review of the documented literature shows that C. difficile has been reported from both animals and humans (Keessen et al., 2011; Songer, 2010). Reports on the occurrence of C. difficile in ready-to-eat salads and vegetables have been previously reported by several researchers (documented in Table 15.1). Recent studies have shown that the pathogen has potential of transmission through food supply. Eckert et al. (2013) assessed the prevalence of ready-to-eat raw vegetables contaminated with C. difficile and observed that ready-to-eat salads were contaminated. In another recent study, the prevalence of

460  Chapter 15 C. difficile was observed in ready-to-eat foods, indicating food as a reservoir for the pathogen (Rahimi et al., 2015). Soil, fertilizers (manure), water, and processing environments could be the various possible sources of contamination of C. difficile in fresh produce (Weese, 2010). The spore-forming abilities of C. difficile allows the bacterium to survive environmentally unfavorable conditions, thereby facilitating its transmission.

15.1.4  Bacillus cereus B. cereus is a group of free-living bacteria and some strains can cause food-borne illnesses in humans. B. cereus is one of the major pathogens associated with raw vegetables worldwide (Park et al., 2013). The two types of gastrointestinal disease (emesis and diarrhea) are caused by B. cereus (Ki Kim et al., 2009; Arslan et al., 2014). Ingestion of the toxin cereulide secreted by B. cereus causes an emetic type of illness (Takeno et al., 2012), whereas consumption of food contaminated with diarrheal toxin secreted by B. cereus causes diarrhea (Kim et al., 2011). Since B. cereus is a soil deviling microbe, its wide occurrence in vegetables and other agricultural fresh food is not surprising. Compiled information on the occurrence of B. cereus on fresh produce is presented in Table 15.1. Occurrence of B. cereus in fresh vegetables depends significantly on whether or not the crops were fertilized with human or animal wastes or irrigated with water containing such wastes. A review on the recent literature indicates occurrence of B. cereus in a wide variety of foods (Dzieciol et al., 2013; Li et al., 2016). Recent studies carried out by Kim et al. (2011) and Hwang and Park (2015) have shed light on the molecular characterization of enterotoxin genes in B. cereus. Their findings show that four different enterotoxins, viz., hemolysin BL (Hbl), nonhemolytic enterotoxin (Nhe), cytolysin K (CytK), and enterotoxin FM (EntFM), are hypothesized to play key roles in disease.

15.1.5  Campylobacter spp. Recent documented literature suggests that cross-contamination from fertilizers, soil, and irrigation water is among the prime reasons for Campylobacter spp. contamination in fresh produce (information on the occurrence of Campylobacter spp. on fresh produce is presented in Table 15.1). The pathogen has resulted in a number of food-borne outbreaks associated with raw fruits and vegetables (Khalid et al., 2015; Tang et al., 2016). Chai et al. (2009) also reported Campylobacter spp. contamination in vegetable farms. One of the major concerns associated with Campylobacter spp. contamination (especially C. jejuni) is that of multidrug resistance especially toward quinolones and erythromycin (Ge et al., 2013). Studies carried out by Sheppard et al. (2013) highlight the molecular mechanisms involved in the pathogenicity. Their findings indicate that C. coli became progressively adapted to the agricultural niche via genomic introgression with C. jejuni. Recent studies by Pascoe et al. (2015) have shown that the mechanism of biofilm production in Campylobacter spp. has

Human Pathogenic Bacteria and Host on Food Quality and Disease   461 considerably evolved and is a result of different genetic backgrounds. These evolved biofilm formation mechanisms are believed to be responsible for the organism’s survival and dispersal in agricultural environments. Pearson et al. (2015) have recently carried out studies on C. coli and C. jejuni isolates in agricultural and nonagricultural systems to evaluate whether phylogenetic relatedness or sharing of environmental niches affects the distribution and dissemination of type II CRISPR (clustered regularly interspaced short palindromic repeats)Cas (CRISPR-associated) system.

15.1.6  Listeria monocytogenes Among the six known species of Listeria (L. monocytogenes, L. ivanovit, L. seeligeri, L. innocua, L. welshimeri, and L. grayi), L. monocytogenes is considered to be the most deleterious food-borne pathogen. Its detection in vegetables, fruits, and dairy products has been widely reported (Kasalica et al., 2011; Ajayeoba et al., 2016). The pathogen is a Gram-positive, facultative anaerobe that is a nonsporulating motile bacterium capable of causing listeriosis (Bayoub et al., 2010). Recent studies have suggested the involvement of previously uncharacterized cellobiose PTS system in central nervous system infections (Grad and Fortune, 2016). The occurrence of the pathogen in ready-to-eat vegetables has been a matter of serious concern (details in Table 15.1), considering the high demand of such products in the market. The primary source of contamination of agricultural fresh produce has been associated with the contaminated water from sewage sludge for the purpose of irrigation (Oranusi and Olorunfemi, 2011). A comprehensive detailed overview has been recently documented in a review on the fresh farm produces as a source of pathogens by Mritunjay and Kumar (2015). They highlight that a major source of contamination of fresh agricultural produce is because of the use of contaminated water (used for sprinkling purpose in order to keep the vegetables fresh) and contaminated containers (used for transportation purposes). Recent report by Stea et al. (2015) highlights the prevalence and diversity of L. monocytogenes, in an urban and a rural municipal source. The study documents important findings that can go a long way in order to understand the ecology and occurrence of the pathogen under agriculturally diverse environments.

15.1.7  Cronobacter spp. Cronobacter spp. was formerly described in the literature as Enterobacter sakazakii. The recent literature based on whole-genome sequencing and multilocus sequence typing (MLST) targeting 7 genes (atpD, fusA, glnS, gltB, gyrB, infB, ppsA) has resulted in the identification of seven species in the Cronobacter genus viz. C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, C. dublinensis, C. universalis, and C. condiment (Forsythe et al., 2014). Studies on the virulence factors associated with the pathogenicity of C. sakazakii have been recently published from our laboratory (Singh et al., 2016). The authors highlight that in addition to

462  Chapter 15 different virulence factors viz. outer membrane protein A (ompA), plasmid-associated genes such as filamentous hemagglutinin (fhaBC), Cronobacter plasminogen activator (cpa), and genes responsible for iron acquisition (eitCBAD and iucABD/iutA), several biophysical growth factors such as the formation of biofilms and resistance to various environmental stresses also contribute to the pathogenic potential of this pathogen. Recent studies carried out by Chen et al. (2016) have focused on analyzing a large number of vegetables in an attempt to find the source for this pathogen. Similar studies have been carried out in our laboratory that have been focusing on the isolation of C. sakazakii from a wide variety of food sources (for details please refer to Singh et al., 2015a, b). From our laboratory studies, it is evident that out of the 219 food samples have been evaluated, a total of 45 Cronobacter spp. were isolated. Cronobacter spp. in a food sample category of herbs and spices accounted for 34.3% of total samples, whereas 26% were from vegetables and fruits. In another recent study, Vojkovska et al. (2016) detected Cronobacter spp. in vegetables, fruit, and environmental samples collected from local farms and supermarkets in the Czech Republic. They further reported that environmental isolates of Cronobacter spp. create the capsule more often than the isolates of clinical origin. The capsule formulation facilitates enhanced desiccation resistance of the bacterium and increases its ability to attach to surfaces and create biofilms.

15.2  Entry of Human Pathogenic Bacteria into the Food Chain: Tracking the Point of Origin A review on the literature, in an attempt to track the point of origin of entry of human pathogenic, enteric bacteria into the food chain (with special reference to fresh produce) shows that the entire process can be divided into two broad categories: first, the point of entry of enteric pathogen is at the site where the agricultural produce is being raised. The contaminating sources include water (used commonly for irrigation purposes) and raw or inadequately amended manure. Second, the enteric pathogen can enter the food chain during postharvest processing. In the following section, we review the potential sources of contamination of enteric pathogens in fresh agricultural produce.

15.2.1  The Potential Role of Water in the Contamination of Fresh Agricultural Produce As a general practice in agricultural farms, groundwater is used for irrigation purposes. However, it is well-established and a well-known fact that irrigation water could be a potential source of enteric pathogen contamination, since it is often contaminated by effluents from municipal waste (Pachepsky et al., 2011). The problem is highly reported in underdeveloped and developing countries. A recent report by Akinde et al. (2016) highlights that fresh vegetables could be an easily available transmission vehicle for human pathogens, because of poor irrigation water quality at vegetable farms in southwestern Nigeria.

Human Pathogenic Bacteria and Host on Food Quality and Disease   463 Contamination of agricultural produce in fields irrigated with contaminated waters is easily possible when the produce is in close contact with the soil matrix. Contaminated water can lead to the colonization of enteric pathogens into aerial tissues and root systems of fruit and vegetable plants (Martínez-Vaz et al., 2014). The consumption of raw agricultural produce by humans can eventually lead to the transmission of enteric pathogens and therefore has been identified as the most likely cause of disease outbreak as discussed in detail earlier, in Section 15.2. Contamination of irrigation waters in agricultural fields as a result of municipal waste is only one of the possible means of transmission of enteric pathogens. Water can also be contaminated by livestock (cattle or sheep) defecation, especially if they use rivers for drinking or as crossing points. Runoff from animal pastures into artificial or natural water bodies can also create havoc. A review of the documented literature in this context shows interesting reports and reviews on the contamination of water sources by E. coli O157 (Quilliam et al., 2011). Recently, Hamm et al. (2016) documented a first report on cattle being an important reservoir of an unusual, highly virulent EHEC O104:H4 strain. As discussed previously in Section 15.2.1, the pathogen resulted in a serious outbreak in early May 2011. The development of quick and reliable methods for the detection of enteric pathogens in water samples is the need of the hour. In this regard, recently researchers have been focusing on developing accurate and less time-consuming methodologies. Recently, Banting et al. (2016) described a most probable number (MPN)-qPCR assay for molecular-based detection of Campylobacter spp. especially in irrigation water samples. Henao-Herreño et al. (2017) evaluated Salmonella contamination in Bogotá River water that was being used for irrigation purposes for lettuce, broccoli, and cabbage. Based on the Salmonella concentration, the authors developed a qPCR-based quantitative microbial risk assessment model. Their results emphasized the presence of wastewater treatment, before Bogotá River water is used for irrigation purposes.

15.2.2  The Potential Role of Noncomposted or Improperly Composted Manure in the Contamination of Agricultural Fresh Produce Organic fertilizer inputs in agricultural fields in the present scenario has significant environmental benefits over the use of chemical fertilizers and the practice has gained much importance. Organic matters such as animal manure, sewage sludge, and food wastes are decomposed using the anaerobic digestion process which has been reported to be an effective measure in controlling enteric pathogens (Horan et al., 2004). However, it has been observed that there are reports on the occurrence of food-borne pathogenic bacteria on crops, which were grown in soil that contained contaminated manures. A recent study in this regard carried out by Biswas et al. (2016) suggests that there exists significant variations in the survival of pathogens (the authors carried out their study with three enteric pathogens viz. E. coli, Salmonella spp., and Listeria monocytogenes) with temperature and environmental conditions, that is, liquid dairy manure in anaerobic and limited aerobic storage conditions.

464  Chapter 15 Another important aspect of enteric pathogens in manure, which eventually contaminate the fresh produce, is the ability of these pathogens to be able to develop resistance against antibiotics. A review of the documented literature shows that there is a significant rise in antibiotic-resistant bacteria in animal feces, which can enter into the food chain since the animal manure is extensively used in farmlands (da Costa et al., 2013). A recently documented report by Takemura et al. (2016) indicates that because of extensive use of veterinary antibiotics, there is high persistence and survival of antibiotic-resistant bacteria in livestock manure. Similar studies on the prevalence and persistence of potentially pathogenic and antibiotic-resistant bacteria during anaerobic digestion treatment of cattle manure have been reported by Resende et al. (2014). Considering the above problems of persistence and survival of enteric pathogens in noncomposted or improperly composted manure, we must (a) understand the factors the can help in the reduction of enteric pathogens in manure and (b) develop biotechnologies for improvement of dairy manure treatment. The physical character of the manure (i.e., liquid manure, slurry manure, or solid manure) can significantly influence the survival of enteric pathogens, which in turn depends on the farm management practices and also on the livestock. In manure which is in slurry or liquid form, survival of the enteric pathogens is high because of the presence of favorable moisture and alkaline pH (Cools et al., 2001). In case of solid/ dried manure, the temperature is an important factor that determines the persistence of enteric pathogens in the manure. Recent reports published by Park et al., 2016, specifically focus on the role of temperature in the survival of manure-borne generic E. coli, E. coli O157:H7, and fecal coliform in soils. Erickson (2016) carried out studies on the survival of Salmonella or E. coli O157:H7 during the holding of manure-based compost mixtures at sublethal temperatures (20–40°C). They simultaneously interrogated the influence of carbon amendment to the compost mixtures. An environmental concern which has recently received much attention is that agricultural facilities having storage of large quantities of manure especially in dried form, may serve as a source of airborne contamination of leafy greens being cultivated in nearby fields. Dehydrated animal manure, results in the generation of dust-like particles that can be small enough to become readily airborne (Berry et al., 2015). Research in regard has been initiated and Oni et al. (2015) has recently documented a report that focuses on the characterization of parameters that focus on the survival of S. enterica in or on dust particles of dried turkey manure and litter that could be aerosolized during handling and survive on leafy greens in the fields. Biotechnologies for the reduction of survival and persistence of enteric pathogen in manure are highly advocated. Anaerobic digestion of cattle manure is one of the most environmentally favorable biotechnologies for managing enteric pathogen microbial load (Manyi-Loh et al., 2013). Recently Manyi-Loh et al. (2014) documented a 1-log reduction of E. coli and Campylobacter spp. (i.e., 90% decay rate) as opposed to a 2-log reduction of Salmonella spp. that occurred between day 9 and 14, but a similar 1-log reduction of these cells during the rest of the process indicating a 90%–99% kill rate was achieved in mesophilic

Human Pathogenic Bacteria and Host on Food Quality and Disease   465 anaerobic digestion. A detailed review on the use of anaerobic digestion technology for reducing the persistence and survival of enteric pathogens has been recently documented in a review by Manyi-Loh et al. (2016).

15.2.3  Enteric Pathogen Can Enter the Food Chain During Postharvest Processing Contamination of fresh produce during the postharvest processing is one of the most significant points of entry of enteric pathogens into food chain. The contamination can be due to injury to the plants during the harvesting or during storage and transportation of the fresh produce. Mechanical means to harvest the fresh produce at times result in injury to the plant tissue. Such injuries are an open invitation for the enteric pathogens to colonize the plants, since the nutrients available are easily accessible to the bacterium. Similar problems arise during storage and transportation of fresh produce. Washing of the fresh produce, is a common practice, however, the presence of pathogenic bacterium in contaminated water can easily result in entry of the bacterium into the agricultural produce. Good manufacturing practices (GMPs) can play a significant role in preventing the risk of contamination. A recent review article by Gil et al. (2015), highlights the strategies that can be employed to prevent microbial contamination in fresh leafy vegetables during pre- and postharvest processes.

15.3  Interaction Between Enteric Pathogens and Plant Hosts Previously, it had been believed that enteric bacterial pathogenesis on humans was defined by their ecological niche. However, reports on the outbreak of disease that have been caused as a result of consumption of contaminated fresh produce have challenged the traditional view point (recent reports are discussed in Sections 15.2 and 15.3). It is now a well-established fact that enteric pathogens colonize plant tissues and thereby uses the plant host as a transmission vehicle to gain eventually entry into the human host. In an attempt to understand the dynamics of the interaction between enteric pathogens and plant hosts, we must first understand (a) fitness of plant surfaces as host, (b) factors influencing the survival and growth of enteric pathogens on fresh produce, and (c) molecular/genomic capabilities of enteric pathogens that allows them to use plants as vehicles for the transmission. These aspects have been discussed in the following sections.

15.3.1  Enteric Pathogens in Plant Habitats The enteric pathogens survival in the plant habitat is subject to its point of contact with the plant tissue. Ideally, the pathogenic bacteria can interact with the plant host at three points, viz. in the rhizosphere, at the leaf surface, that is, plyllosphere and finally in the spermosphere (the zone surrounding the seeds). In the following sections, we discuss the fitness of these three regions as host for enteric pathogens.

466  Chapter 15 15.3.1.1  Plant rhizosphere as a habitat for enteric pathogens The rhizosphere has been long considered as a hot spot for microbial interactions because of the influence of the below-ground system of the plants, which are known to release large amounts of nutrient-rich root exudates. In the rhizosphere, diverse microbial communities both beneficial and deleterious coexist and interact (Mahajan and Shirkot, 2014). More light on this has been shed because of the recent developments in metagenomics (Hirsch and Mauchline, 2012). Recent studies using metagenomic analysis on the rhizosphere microbiome show evidence for plant species-specific microbiomes (İnceoğlu et al., 2012) and existence of plant genotype-specific rhizosphere microbiomes (Weinert et al., 2011). Recent studies carried out in field-grown potato rhizosphere show that the rhizosphere microbiome is affected by the growth stage of the plant (İnceoğlu et al., 2012). Organic materials (e.g., farm-yard manure and slurry) have been considered as the most economically viable option for improving soil quality (Semenov et al., 2009). With the increasing demand of organic fertilizer application in fields, it is often observed that immature manure is often used in the farms. This problem often results in enteric bacterial contamination of fruits and vegetables (as discussed in detail in Section 15.3.2). Several reports on the persistence and survival of enteric pathogens in organic manure have been documented. Recently, Yao et al. (2015) studied the survival of E. coli O157:H7 in different organic fertilizers (vermicompost, pig manure, chicken manure, peat, and oil residue). Raw fruits and vegetables, especially cut-leafy greens grown in soils amended with enteric pathogen-contaminated organic fertilizers are highly prone to food-borne contamination, since the below-ground plant system is in direct contact with pathogens. The primary step involved is the attraction followed by colonization. It is believed that rhizobacteria and enteric pathogens both preferentially colonize root tips and/or at the root base where lateral roots emerge (Jablasone et al., 2005; Cooley et al., 2003). The associations might correlate with the fact that the nutrient-rich rhizosphere is commonly available for both pathogenic and nonpathogenic microbes. Distinct and localized spatial patterns of sucrose, amino acids, and nitrate abundance have also been mapped in the rhizosphere (DeAngelis et al., 2005; Jaeger et al., 1999). S. enterica is an enteric human pathogen that has been reported by Barak and Schroeder (2012) to colonize crop plants as secondary hosts. Similar findings have also been reported in lettuce by Klerks et al. (2007). It is believed that the movement of S. enterica in lettuce rhizosphere is because of the chemotaxis toward the sugar compounds in lettuce root exudates. In a detailed review by Barak and Schroeder (2012), the authors highlight that enteric pathogens especially S. enteric prefer to colonize specific leafy greens radicchio and endive compared with lettuce. The researchers are of the opinion that this specificity is primarily due to the difference in nature of root exudates. Following colonization, the survival of the enteric pathogens in plant rhizosphere is believed to be favored by the nutrient-rich rhizosphere. In the rhizosphere, the enteric pathogens are challenged by environmentally

Human Pathogenic Bacteria and Host on Food Quality and Disease   467 unfavorable conditions; therefore they tend to colonize the internal regions of the plant tissue. A recent report has shown that E. coli O157:H7 endophytically colonize spinach and lettuce plants Wright et al. (2013). High-resolution microscopic examination and O-antigen labeling have shown that the food-borne enteric pathogen colonization occurred within the apoplast, between the plant cells. 15.3.1.2  Phyllosphere as a habitat for enteric pathogens The above-ground regions of the plant are physically more easily accessible to the enteric pathogens. Soil splashing, irrigation, and insect transmission (as discussed in Section 15.3) are easy modes available for the transmission of pathogens. The phyllosphere accounts for the aerial parts of plants, which are dominated by leaves. A recent review by Vorholt (2012) on the microflora associated with the phyllosphere shows that the global bacterial population present in the phyllosphere could comprise up to 1026 cells. In spite of the large leaf surface area offered to the microbes, their colonization is subject to several environmental challenges such as limited nutrient availability, high ultraviolet radiation, and fluctuating water availability. It is surprising that in spite of such stringent environmental pressure, the microbial populations survive on the phyllosphere. Several documented reports on the occurrence of enteric pathogens on the leaf surface have been documented. Recently, Han and Micallef (2016) reported S. enterica colonization on tomato plant surface. Their findings have shown that tomato surface compounds and exudates play an important role in colonization by the enteric pathogen. Documentation of previous reports indicates that S. enterica colonization on phyllosphere is a result of chemotaxis. Furthermore, the colonization by the enteric pathogen in phyllosphere is highly concentrated in type 1 trichomes, which happen to be an area on the phyllosphere in the genus Solanum where maximum exudates are synthesized and accumulated (Barak et al., 2011). In an attempt to understand the risk associated with irrigation water as a potential source of enteric pathogen contamination especially in the phyllosphere, Wood et al. (2010) studied the survival behavior of enteric pathogen (E. coli). They introduced the enteric pathogen into agricultural systems during irrigation in the spinach phyllosphere and observed declines in their culturable E. coli populations on spinach leaf surface under open environmental conditions. The possible reason associated could be the ability of enteric pathogenic bacteria to colonize the internal regions of the phyllosphere. Recent documented literature have indicated that enteric pathogens are also known to internalize the leafy green phyllosphere tissue, in order to avoid the harsh environmental conditions, as discussed earlier in this section. Erickson et al. (2014) reported internal colonization by E. coli O157:H7 into the spinach tissue. They reported that the mobilization of enteric pathogen cells into the leaf surface and its further survival were not influenced by virulence factors of Shiga toxin since they used Shiga toxin-negative E. coli O157:H7 isolates in their study.

468  Chapter 15 15.3.1.3  Spermosphere as a habitat for enteric pathogens Spermosphere is an extremely small zone (2–12 mm) around the seed, where interactions between soil, microbial communities, and germinating seeds take place (Schiltz et al., 2015). The spermosphere has a very short-lived interface, that is, only during the seed germination, yet it is highly significant. This is the first point of contact between the plant and the microbial community in the soil, which can be beneficial microbes, plant pathogenic microbes, or enteric pathogens (in the context of the present book chapter). These associations are known to influence the future microflora in the rhizosphere, which in turn influences the future plant growth and yield (Singh et al., 2011). Once the seed imbibes, it is believed that a number of compounds are exuded into the spermosphere (Schiltz et al., 2015). Several reports are well documented in literature that report on the characterization of the exudates during germination. A few recent reports by da Silva Lima et al. (2014), Scarafoni et al. (2013), and Kamilova et al. (2006) have shown that the exudates during germination process include (a) carbohydrates, viz. arabinose, fructose, galactose, glucose, maltose, mannose, lactose, raffinose, rhamnose, ribose, sorbose, sucrose, xylose; (b) amino acids viz. alanine, glutamic acid, glutamine, glycine, homoserine, leucine/isoleucine, methionine, phenylalanine, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine; (c) organic acids, viz., acetic acid, citric acid, formic acid, malonic acid, oxalic acid, succinic acid; (d) fatty acids viz. hexadecanoic acid, octadecanoic acid isomers, tridecanoic acid; (e) proteins, viz., chitinases, cysteine-rich protein, galactosidases, glycosyl hydrolases; (f) secondary metabolites, viz., phenolic derivatives, steroids, terpenoids. As a result, the microbial communities in the soil are believed to be attracted toward the spermosphere because of the chemotaxis toward the exudates. Enteric pathogens as earlier discussed (in Section 15.1) are known to be associated with food-associated disease outbreaks, which are especially similar to contaminated seeds. Harris et al. (2003) reported that sprouted-seed-related disease outbreaks were linked to seeds contaminated with S. enterica. Much recently in 2013, sprouted fenugreek seeds were contaminated with E. coli (EHEC) strain of serotype O104:H4 (European Food Safety Association, 2013). Their consumption resulted in an outbreak of HUS and HC. Reports on epidemiological investigation suggested that the seed contamination with STEC O104:H4 occurred more than a year before the seeds were used for sprout production. Recently, Knödler et al. (2016) carried out long-term survival studies and observed that in none of the strains tested cultivable cells were found without enrichment on contaminated seeds after more than 24 weeks of storage, thereby suggesting that contamination previous to the distribution of fenugreek seeds from the importer was less likely. Much recently, researchers have been attempting to understand the changes that occur in the composition of seed and early root exudates once the seeds are contaminated with S. enterica. Kwan et al. (2015) in this context carried out studies using S. enterica-contaminated alfalfa

Human Pathogenic Bacteria and Host on Food Quality and Disease   469 seeds as a model system. The authors concluded that individual amino acids are important, but not essential, for S. enterica growth in the spermosphere. The protein surveys carried out by the researchers revealed that central carbon metabolites serve as essential intermediates for cellular biosynthesis and therefore to achieve dramatic reductions in bacterial growth in spermospheres, central metabolic networks need to be targeted in future studies.

15.3.2  Survival and Growth of Enteric Bacterial Pathogens on Fresh Produce For enteric pathogenic bacteria to survive on fresh produce, it must possess certain characteristic features that allow them (a) to form synergistic/antagonistic relation with the existing microbiota, (b) to separate themselves from the existing microbiota in space, (c) to evade plant defense, just like certain phytopathogens, and (d) formation of specialized biological networks, that is, biofilm formation. 15.3.2.1  Synergistic/antagonistic relation of enteric bacteria with the existing microbiota The existing microbiota on the plant may be plant beneficial or pathogenic to the host plant. Therefore, the enteric pathogenic bacteria must be in synergism/antagonistic relation with either of them or both. Recent research in this regard indicates that the presence of plant pathogenic bacteria would idly favor the colonization by enteric bacteria; therefore the two groups of bacterium are believed to act in synergism. The two probable reasons are (a) because of the injury to the plants, the entry for the enteric pathogenic bacteria into the plant is facilitated and (b) because of the damaged plant tissue, the wide variety of nutrients are easily accessible to the enteric pathogens (Campbell et al., 2001, Wu et al., 2000). Enteric pathogens synergism or antagonism with the host plants microbiota is also believed to be dependent on the iron acquisition systems with enteric pathogens in the plant habitat (Brandl, 2006). This mechanism of iron acquisition is believed to be carried out by siderophores (lowmolecular-weight molecules that bind ferric iron with an extremely high affinity). In the case of enteric bacterial pathogens, it is hypothesized that the siderophores act in a similar manner as that observed in the case of plant pathogens. The iron-acquiring ability of siderophore assists the pathogenic microbe to chelate iron and thereby increase its possibilities to colonize the host plant. 15.3.2.2  Ability of enteric bacteria to colonize internal plant tissue Enteric bacteria causing food-borne diseases have the ability to colonize the internal plant tissues, in a manner similar to the one adopted by beneficial or pathogenic microbes residing in the phyllosphere/rhizosphere. In an attempt to avoid the harsh environmental conditions, enteric bacterial pathogens are known to gain entry within the plant tissue. Similar to phytopathogens, enteric pathogenic bacteria can preferentially attach to the cut surfaces and natural openings such as the stomata that provides not only a protective niche, but also a source of nutrients into the apoplastic fluid. Documented literature is testament to the

470  Chapter 15 endophytic colonization of pathogens such as Salmonella and E. coli O157:H7 into the cracks of the developing lateral roots of seedlings, which is especially relevant for sprouted seeds such as alfalfa, mung bean sprouts, and spinach (Klerks et al., 2007; Warriner et al., 2003; Dong et al., 2003; Cooley et al., 2003; Erickson et al., 2014). Many researchers have reported internal colonization by E. coli O157:H7 into tissues. Warriner and Namvar (2010) based on their review of the documented literature suggested that under preharvest conditions and natural environmental conditions there is significantly low frequency of internal colonization by enteric pathogens. The possible reason being the fact that tissue injury is very frequent. However, with the modernization of agricultural systems and the use of modern machinery in farms, the green leafy vegetables and other crops can sustain injury which can facilitate the entry of enteric pathogens and thus can cause food-borne disease outbreaks. 15.3.2.3  The ability of enteric bacterial pathogens to evade plant defense mechanisms Plant defense system has been studied in great deal over the last decade, in an attempt to understand the response of plants against phytopathogens (Esposito et al., 2008; Mahajan and Shirkot 2014). Upon infection plants respond by a hypersensitive response, coupled with secretion of plant hormones (salicylic acid, jasmonic acid, and ethylene). This activates the PR genes that trigger the systemic acquired resistance (SAR) defense. With the continuous evolution of phytopathogens, certain strategies have been developed to avoid detection by plant defenses. Enteric pathogenic bacteria such as Salmonella have devised similar methods as that of phytopathogens to avoid detection. They are known to avoid detection by alteration of their LPS and their lack of outer structures, such as the ability of Salmonella to shed flagella, facilitates its unnoticed entry into the plant system. 15.3.2.4  Specialized abilities of enteric bacteria to form biofilms The bacterial cells that colonize the phyllosphere are subjected to adverse environmental conditions (as discussed previously). The bacteria can secrete exopolysaccharides, which can contribute to the formation of a specialized matrix-like structure referred to as biofilm. Researchers since the advent of 21st century have been carrying out studies on the ability of enteric pathogenic bacteria to form biofilm on agricultural fresh produce. Fett (2000) studied biofilm formation by Salmonella and E. coli O157:H7 on different plant parts of broccoli, cloves, and afalfa. Several well-documented reports of biofilm formation on leaf surfaces of Chinese cabbage, spinach, lettuce, and other green leafy vegetables are available (Méric et al., 2013). Recent studies carried out by Méric et al. (2013) show that plant-associated E. coli when compared with strains from other sources had a greater ability to form biofilms. Moreover, significant differences were also observed in the utilization of common carbon sources between strains of E. coli isolated from spinach and rocket salad in comparison to E. coli strains isolated from the mammalian counterpart. Recent work being carried out in our laboratory on Cronobacter spp. shows the biofilm-forming ability on different biotic and abiotic surfaces (Singh et al., 2017).

Human Pathogenic Bacteria and Host on Food Quality and Disease   471 Quorum sensing (QS) mechanism is a well-known mechanism for bacterial cell-to-cell communication and several documented reports suggest that QS is essential for biofilm formation, which is often used as a strategy by phytopathogens to colonize the plant hosts. A review of the documented literature shows that Hughes and Sperandio (2008) reviewed the presence of the mechanism as part of plants’ defense system, in which the plants secrete hormones that mimic bacterial QS signals and as a result causing confusion in the signaling pathogenic bacterium. It would be of interest to investigate that whether on similar lines food-borne pathogenic enteric bacteria could be prevented to form biofilms on fresh produce by plant hosts using a similar mechanism.

15.3.3  Molecular Capabilities of Enteric Pathogens That Allow Them to Use Plants as Vehicles for the Transmission As discussed in the above sections, the survival and growth of enteric pathogenic bacteria relies essentially on its ability to colonize plant host. A recent review on enteric pathogenplant interactions, Martínez-Vaz et al. (2014) highlight the importance of genes involved in the cell surface structures, virulence, motility, and biofilm formation. Previous reports highlight the presence and role of virulence genes such as intimin and stx in E. coli O157:H7. Kyle et al. (2010) tracked the expression of these genes on lettuce leaves. Molecular interaction of enteric bacteria with green leafy vegetables is mediated by a number of diverse genes. In E. coli, genes involved in Curli formation (csgA) and flagella biosynthesis (fliN) have been reported. Similarly regulator of biofilm formation through the production of colanic acid (ybiM) has also been reported by researchers to play an important role in biofilm formation (Fink et al., 2012; Hou et al., 2012). In S. enteric genes involved in stress response regulator and biofilm modulation (ycfR), cellulose biosynthesis (bcsA), adhesin expressed from pathogenicity island-3 (misL), and response regulator involved in biofilm formation (sirA) are few of the important documented genes involved in attachment and biofilm formation (Fink et al., 2012; Hou et al., 2012; Kroupitski et al., 2013; Salazar et al., 2013). Transcriptomic analysis of enteric bacterial association with agricultural fresh produce has been recently carried out by researchers in an attempt to understand transcriptional responses triggered by the association of these organisms with plant tissues. Transcriptional responses of the E. coli strains K-12 and O157:H7 associated with intact lettuce leaves has been recently studied in detail by Fink et al. (2012). The authors reported that genes involved in the formation of biofilms and curli fibers were expressed at high levels. A time-dependent experiment studying the transcriptional responses of phyllosphere-associated K-12 and O157:H7 over 3 days showed that adaptation to the leaf environment was characterized by an overall decrease in the expression of genes mediating cellular energy and metabolism, especially those involved in the synthesis of ribosomal RNA and iron homeostasis. The findings thus supported the hypothesis that enteric pathogens survive and propagate in leafy

472  Chapter 15 greens by inducing physiological responses that allowed them to cope with the scarcity of food sources encountered on vegetable surfaces. Studies on the molecular events triggered by the association of enteric pathogens with green leafy vegetables have improved our understanding of the mechanisms by which these organisms survive outside their normal host (Martínez-Vaz et al., 2014).

15.4  Future Research Prospects and Conclusion In today’s scenario, where global population is on an all time high, there is an ever-increasing pressure on the food industry to deliver. In order to feed the growing population, it is essential to have ready-to-eat food available, with the prerequisite being that the quality of food is not compromised. Contamination of fresh vegetables with enteric pathogens has reached concerning proportions in recent years. The slogan of “farm to fork” can be truly realized if appropriate diagnostic tests are available to keep a check on the enteric pathogenic contaminants. As researchers make new discoveries it is essential that the implications of the basic and fundamental research reach the consumer. Recent technological advancements allow us to track the course of contamination; however, it is worthwhile to mention that these technological advancements are put to use before there is any disease outbreak. Today the scientific community has advanced understanding of food-borne enteric bacterial contaminations in fresh produce. However, the presence of cross-domain pathogens continues to challenge human health. Bacterial evolution poses a challenge in managing food-borne diseases because of the horizontal gene transfer. Another challenge being faced by researchers is the viable but nonculturable state, which the enteric pathogens enter while they interact with the foods at different stages of the food production chain. The use of highly sensitive and rapid diagnostic methodologies, backed by molecular biology tools for analysis of the global transcriptional changes that occur in enteric food-borne pathogens while they interact with agricultural fresh produce is the need of the hour.

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Further Reading Sapers, G.M., Gorny, J.R., Yousef, A.E. (Eds.), 2005. Microbiology of Fruits and Vegetables. CRC Press, Boca Raton, FL.

CHAPTE R 16

Novel Strategies for the Reduction of Microbial Degradation of Foods Tuba Dilmaçünal, Hakan Kuleaşan Süleyman Demirel University, Isparta, Turkey

16.1 Introduction Food is any substance, whether processed, semiprocessed, or raw which is intended for human consumption including chewing gum and any substance that has been used in the preparation or treatment of food but excluding cosmetics, tobacco, and substances used only as drugs (Alum et al., 2016). The importance of food can never be overemphasized (Alum et al., 2016). Vegetables and fruits are fundamental sources of water-soluble vitamins (Vitamin C and group B Vitamins), Provitamin A, phytosterols, dietary fibers, minerals, phytochemicals (Di Cagno et al., 2013), phenolic compounds, anthocyanins, and flavonols (Birmpa et al., 2013) in the human diet. Scientific evidences encouraged the consumption of vegetables and fruits to prevent chronic pathologies such as hypertension, coronary heart diseases, the risk of stroke (Di Cagno et al., 2013), and certain types of cancers (Birmpa et al., 2013). The 2010 Dietary Guidelines for Americans also recommended to fill one-half of food plates with fruits and vegetables (Chang et al., 2016). Unfortunately, the daily intake of vegetables and fruits is estimated to be lower than the doses (400 g, excluding potatoes and other starchy tubers) recommended by the World Health Organization and Food and Agriculture Organization (FAO) (Di Cagno et al., 2013). There may be many different reasons of the insufficient fruit and vegetable intake. The spoilage and losses occurred in pre and/or postharvest (harvesting, precooling, cleaning, disinfecting, sorting and grading, processing, packaging, storing, and transportation) conditions, because of the unconscious or wrong practices, may be one of the reasons for the low consumption of fresh fruits and vegetables. The FAO of the United Nations estimates that roughly one-third of all the edible food produced for human consumption is wasted or lost from the food supply chain, or about 1.3 billion metric tons per year. An analysis of the FAO’s food balance sheets for 2007 suggests that food

Food Safety and Preservation. https://doi.org/10.1016/B978-0-12-814956-0.00016-0 © 2018 Elsevier Inc. All rights reserved.

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482  Chapter 16 waste in North America and Europe is roughly 95–115 kg/capita/year compared with 6–11 kg/ capita/year in South/Southeast Asia and sub-Saharan Africa (Buzby and Hyman, 2012). Sales of fresh produce have significantly been increased during the last decade, as consumers become increasingly concerned with healthy food and nutrition (Birmpa et al., 2013). Although the consumption of fresh produce is beneficial for optimal health, these foods may be associated with food-borne illness (Alegre et al., 2012). Fruits and vegetables often contain a great diversity of microbial flora and are frequently involved in food-borne outbreaks (Alexandre et al., 2011). Fruit tissue is rich in nutrients and high in moisture which favors growth of the bacteria. Proliferation of bacteria in fruit tissue can reach harmful levels, causing food poisoning and degradation of fruit organoleptic quality such as appearance, taste, and odor (Alwi and Ali, 2014). Mesophilic microorganisms, coliforms, yeasts, and molds are the commonly found bacterial population in those products, which are responsible for quality degradation and safety compromise (Alexandre et al., 2011). Since fruits and vegetables are mainly consumed uncooked or minimally processed (such as in ready-to-eat salads), microbiological safety becomes a very important issue to minimize consumers’ risks (Alexandre et al., 2011). Food spoilage is both a sustainability and a commercial issue because visible mold and undesirable odors lead to consumer rejection, which in turn causes significant economic losses and food waste (Arango et al., 2016). Worldwide, postharvest losses have been estimated at 50% and much of this is due to fungal and bacterial infections. Molds are ubiquitous biological agents that are able to colonize foods because of their potential to synthesize a wide diversity of hydrolytic enzymes. They cause pathologic disorders in plants bringing considerable economic losses to food producers (da Cruz Cabral et al., 2013). In the Middle East, many types of vegetables are eaten raw in salads or used as garnishes in appetizers, and also increasingly in traditional dishes because of their perceived healthy attributes. Yet, they have been in recent years a major contributor to food-borne illnesses in other parts of the world. In the United States, leafy greens top the list of 10 riskiest foods regulated by the Food and Drug Administration accounting for almost 40% of food-borne outbreaks based on data derived from the Centers for Disease Control and Prevention (FaourKlingbeil et al., 2016). The food-borne diseases are a serious and global problem. The World Health Organization (WHO) estimates that worldwide food-borne and waterborne diarrheal diseases taken together kill about 2.2 million people annually. Fresh produce has been increasingly implicated as the vehicle of transmission and became second leading cause of food-borne illnesses, which cost for instance the US economy $6.9 billion of loss in productivity and medical expenses. Bacillus cereus spores and vegetative forms are frequently found in fresh vegetables, berries, cereals, and fruits (Aponiene et al., 2015).

Novel Strategies for the Reduction of Microbial Degradation of Foods  483 Outbreaks of food-borne illness related to the consumption of fresh and minimally processed fruit, primarily due to Escherichia coli O157:H7 and Salmonella, have increased dramatically since the 1970s (Alegre et al., 2012). Outbreaks of E. coli O157:H7 infection have been linked to the consumption of fresh fruits and vegetables such as alfalfa and radish sprouts, different lettuce varieties, carrots, spinach, unpasteurized apple cider, berries, and melon (Abadias et al., 2012). Gastrointestinal diseases are responsible for 25 million infections each year, with diarrheal diseases as second leading cause of death in the world. In fact, food-borne diseases along with waterborne diseases are responsible for the death of approximately 1.8 million people every year, most of whom are children, and accountable for economic losses in food industry, health systems, tourism, and also for the consumer. Sets of norms and rules have been developed over the years, such as HACCP (Hazard Analysis and Critical Control Points), in order to guarantee safe food for the consumer (Baptista et al., 2016).

16.2  Different Ways of Contamination and Spoilage Codex Alimentarius defines a contaminant as follows: “Any substance not intentionally added to food, which is present in such food as a result of the production (including operations carried out in crop husbandry, animal husbandry, and veterinary medicine), manufacture, processing, preparation, treatment, packing, packaging, transport or holding of such food, or as a result of environmental contamination. The term does not include insect fragments, rodent hairs, and other extraneous matter” (Codex Alimentarius, 2015). Contamination of food and feed may pose a risk to human (and/or animal health). Moreover, in some cases they may also have a negative impact on the quality of the food or feed. Food and feed can become contaminated by various causes and processes (Codex Alimentarius, 2015). Numerous pests and diseases attack food crops around the world; most of them are related to pathogenic fungal diseases (da Cruz Cabral et al., 2013). Most of the food products contain high levels of nutrients or a high water activity (aw); therefore they are particularly susceptible of microbial spoilage which results in a deterioration of their organoleptic characteristics, and may even risk the health of immunocompromised individuals (Ferrentino and Spilimbergo, 2011). Life-threatening outbreaks of food-borne bacteria have been an issue for more than a century (Alwi and Ali, 2014). Fresh fruits and vegetables are increasingly being recognized as important vehicles for the transmission of food-borne diseases worldwide. A recent survey of food-borne disease outbreaks in the United States during 1998–2008 showed that vegetables and fruit are among the top four food commodities that cause outbreak-related illnesses (Carter et al., 2015).

484  Chapter 16 Sources of microbial contamination on fruits and vegetables during production include animal and human feces, contaminated manure, inorganic amendments, irrigation water, water used for pesticide application or other agricultural purposes, and contaminated dust (Birmpa et al., 2013). Within the food production chain, fruits and vegetables may be contaminated at different stages since they are exposed to bacteria, parasites, viruses, and fungi/molds through multiple sources including insects, irrigation water or rain, soil, air, manure-based fertilizers, manual handling by workers during the harvest and packaging process, food-processing facilities, and transportation (Arango et al., 2016). Bacteria contamination on fruit can be due to various sources such as contaminated irrigation water, improperly processed manure, and unhygienic handling procedures (Alwi and Ali, 2014). All these factors directly influence their mode of decay and the time to reach the end of shelf life (Arango et al., 2016). Apart from quality concerns related to plant disease, dispersal of enteric human pathogens (i.e., verotoxin producing strains of E. coli, Salmonella spp., Campylobacter spp., Yersinia enterocolitica, Norovirus) via foods consumed fresh or after minimal preparation has become an important global food safety issue (El-Mogy and Alsanius, 2012). New technologies for processing and preserving foods create new routes for environmental contamination and proliferation of microorganisms. With the advent of minimal processing, vegetables have increasingly become one of the food types that can cause food-borne outbreaks because, while most food-processing techniques stabilize the products and extend their shelf life, minimal processing increases their perishability. Moreover, most minimally processed foods are consumed raw, representing a potential microbiological safety problem, especially when processed under unsatisfactory sanitary conditions (de Sousa et al., 2012). Food safety is an increasingly important public health issue (El-Mogy and Alsanius, 2012). A fundamental need when fruit and vegetables are handled is sanitation of harvest bins, wash solutions, rotary brushes, belts, grading, and other processing equipment. All sanitizers that inactivate pathogen propagules can accomplish this if used properly. However, pathogens survive if they reside within the wounds on the host or if they are present as incipient, latent, or quiescent infections within host tissue. Even very high rates of sanitizers fail to control these pathogens once infection has occurred, although some gases may be exceptions (Feliziani et al., 2016). A wide range of produce items has been implicated in outbreaks of human illness worldwide and certain commodities are more frequently linked to these outbreaks; for example, leafy greens, such as lettuce and spinach, and fresh herbs, such as parsley and basil, are wellrecognized potential sources of bacterial infections. The United States has experienced several large high-profile multistate outbreaks attributable to leafy vegetables, including the 2006 outbreak of E. coli O157:H7 infection, which was linked to the consumption of bagged spinach and resulted in almost 200 cases of food poisoning and three deaths. In 2007,

Novel Strategies for the Reduction of Microbial Degradation of Foods  485 a microbiological study of fresh herbs sold at retail in the United Kingdom uncovered an international outbreak of Salmonella infection linked to contaminated basil from Israel that affected at least 51 individuals from England, Wales, Scotland, Denmark, the Netherlands, and the United States (Denis et al., 2016). Recalls due to cantaloupe contamination with Salmonella and Listeria have recently heightened public concern to the microbiological safety of this fruit. The first documented outbreak of salmonellosis in the United States associated with cantaloupe was reported in 1990. Subsequent outbreaks of Salmonella infections in the United States, Canada, and Australia have also been associated with cantaloupe consumption. These outbreaks highlighted the need for effective decontamination methods to ensure the safety of fresh cantaloupes (Chen et al., 2012). The contamination of fresh fruits with human pathogens can occur at several points during growing, harvesting, processing, and handling, and although pH is thought to be a limiting factor, the growth of E. coli, Salmonella, Listeria innocua, and Listeria monocytogenes has been previously reported on, for example, fresh-cut apples and peaches (Alegre et al., 2012). Hepatitis A virus (HAV) is the leading cause of acute viral hepatitis, which may occasionally be fatal. Thus, it constitutes a serious concern for public health authorities. Transmission of these viruses occurs not only by the fecal-oral route, primarily through direct person-toperson contact, but they are also efficiently transmitted by the ingestion of contaminated drinking water or contaminated food. The foods most likely to be contaminated by HAV are leafy vegetables, fruits, shellfish, and ready-to-eat foods (i.e., those with no lethality step prior to consumption). Leafy vegetables are often consumed raw or mildly heated (e.g., blanched) and thus may become vehicles for viral transmission if contamination occurs anywhere from farm to fork (Bozkurt et al., 2015).

16.3  Microorganismal Species Causing Deterioration The Codex definition of a contaminant implicitly includes naturally occurring toxicants including toxic metabolites of certain microfungi that are not intentionally added to food and feed (mycotoxins) (Codex Standard, 2010). Codex Alimentarius listed contaminants are as follows: mycotoxins (aflatoxins, total; aflatoxin M1; deoxynivalenol (DON); fumonisins; ochratoxin A (OTA); patulin), metals (arsenic, cadmium, lead, mercury, methylmercury, tin), radionuclides, and others (acrylonitrile, chloropropanols, hydrocyanic acid; melamine, vinylchloride monomer) (Codex Alimentarius, 2015). Organization for Economic Cooperation and Development (OECD, 2011) reported that suitable pathogens are those that are likely to be found in the commodity, which thereby

486  Chapter 16 becomes a potential vehicle for its transmission to consumers. Food-borne microbial pathogens have been classified into following categories based on hazard: (1) severe hazards (Brucella melitensis, Brucella abortus, Brucella suis, and Mycobacterium bovis; Clostridium botulinum types A, B, E, and F; Salmonella typhi, Salmonella paratyphi A, B, and C; Salmonella sendai, and Salmonella choleraesuis; Shigella dysenteriae; Vibrio cholerae); (2) moderate hazards with potentially extensive spread (β-hemolytic Streptococcus (groups A, C, and G), toxigenic and pathogenic E. coli, Salmonella typhimurium, and other Salmonella serovars, Shigella flexneri, Shigella boydii, and Shigella sonnei); (3) moderate hazards with limited spread (B. cereus, Campylobacter fetus subsp. Jejuni, Clostridium perfringens type A, Staphylococcus aureus, Vibrio parahaemolyticus, and Y. enterocolitica); and (4) other pathogens (Aeromonas and Legionella) considered (OECD, 2011). Fruits and vegetables are highly susceptible to fungal spoilage, both in the field and during postharvest storage. Significant genera include Pythium, Phytophthora, Fusarium, Penicillium, Alternaria, Botrytis, Geotrichum, Sclerotinia, and Rhizoctonia spp. Fungal growth on fresh fruits and vegetables is responsible for food spoilage and numerous plant diseases, which lead to significant economic losses. Mold growth depends on abiotic factors such as pH, aw, solute concentration, temperature, atmosphere, time, etc. However, conditions of temperature and aw are the main variables that determine the development of fungi. Grain crops are also vulnerable to fungal contamination, with Aspergillus, Penicillium, Fusarium, and Alternaria being the most frequent genera. In this matrix, molds are responsible for off-flavor formation and contribute to heating and loss in dry matter in grains by utilizing carbohydrates as an energy source, degradation of lipids and proteins, production of volatile metabolites, and production of allergenic compounds. This causes a reduction in the quality of animal feed and seed. These events can take place even before the fungal growth is evident (da Cruz Cabral et al., 2013). Pathogens identified as hazards on fresh vegetables include Shigella spp., L. monocytogenes, S. aureus, Aeromonas hydrophila and the spore formers B. cereus, C. botulinum, and C. perfringens. However, the one simplicated in most outbreaks involving fresh fruits and vegetables are Salmonella, E. coli O157:H7 with reported doses as low as 10 cells and 2–2000 cells, respectively. Among the pathogens Norovirus is also of greatest concern that are associated with fresh produce outbreaks and the high likelihood of inflicting illnesses is attributed to its low infectious doses of 10–100 viral particles (Faour-Klingbeil et al., 2016). The presence and growth of pathogenic microorganisms (bacteria, mold, viruses, and fungi) in food may cause spoilage and result in the reduction in its quality and quantity (Edziri et al., 2012). Many bacteria including Bacillus, Salmonella, Listeria, Staphylococcus, and

Novel Strategies for the Reduction of Microbial Degradation of Foods  487 Escherichia are capable of adhering to and forming a biofilm on different surfaces; however, investigations on the adhering to and formation of biofilm on the surface of fresh vegetables are limited. When spoilage and pathogenic microorganisms come in contact with produce in the fruit and vegetable production environment, they can rapidly attach and strongly adhere themselves. Some pathogens can also form biofilms on fruit and vegetable surfaces (Bilek and Turantaş, 2013). Spices and dried vegetable seasonings are potential sources of bacterial contamination of foods. However, little is known about lactic acid bacteria (LAB) in spices and dried vegetables, even though certain LAB may cause food spoilage. The LAB identified were predominantly Weissella spp. (61%) and Pediococcus spp. (15%) with Weissella confusa, Weissella cibaria, Weissella paramesenteroides, Pediococcus acidilactici, and Pediococcus pentosaceus being the species identified. Other species identified belonged to the genera of Enterococcus spp. (8%), Leuconostoc spp. (6%), and Lactobacillus spp. (2%). Among the LAB identified, Leuconostoc citreum, Leuconostoc mesenteroides, and W. confusa have been associated with food spoilage. The findings revealed that spices and dried vegetables are potential sources of LAB contamination in the food industry (Säde et al., 2016). Pathogens have been associated with fresh produce, with leafy vegetables estimated to have the highest risk among them, and with the bacterial pathogens E. coli O157 and Salmonella spp. as the most prevalent pathogens on leafy vegetables (Van Haute et al., 2015). E. coli serotype O157:H7 is a bacterium that can cause severe food-borne disease and a number of outbreaks have been reported in many countries around the world. Studies on the control of E. coli O157:H7 have largely focused on the use of chemical preservatives (El-Mogy and Alsanius, 2012). Salmonellosis is an infectious food-borne disease caused by enterobacteria belonging to the genus Salmonella. The pathogenesis includes a set of clinical symptoms mainly manifested as acute gastroenteritis. Not all species, strains, or serotypes are recognized as potential pathogens. The current taxonomic classification of Salmonella has simplified the spectrum by grouping all strains (pathogenic or not) in only two species: Salmonella enterica and Salmonella bongori. The latter is not pathogenic for humans. S. enterica is divided into six subspecies: enterica, salamae, arizonae, diarizonae, indica, and houtenae, also known as subspecies I, II, IIIa, IIIb, IV, and VI, respectively (Chalón et al., 2012). Molds can grow on a great variety of substrates and a wide range of pH, aw, and temperature. Molds can develop at the field level. It has been reported that 25% of agricultural products are contaminated with mycotoxins. But molds can also develop during storage of raw products, and subsequent transport and sale causing considerable economic losses annually for food manufacturers and consumers alike. It is very difficult to assess losses attributable to molds. In the baking industry, these losses varied between 1% and 3% of products depending on

488  Chapter 16 season, type of product, and method of processing. Another estimate from a bakery in the United States was 5% losses. Even assuming only 1% losses, molds could be spoiling over 23,000 tons of bread worth nearly £20 million in the United Kingdom every year. Throughout Western Europe the annual losses could be around 225,000 tons of bread worth £242 million. More generally, losses of food to fungal spoilage in Australia must be in excess of $10,000,000 per annum: losses in damp tropical climate and countries with less developed technology remain staggering (Dao and Dantigny, 2011). Botrytis cinerea is the causal agent of gray mold, which can attack strawberry leaves, stems, flowers, and fruits. This disease is currently primarily controlled by using of synthetic fungicides. However, there is increasing concern about the human health and environmental contamination risks associated with fungicide residues. In addition, the extensive use of these chemicals in commercial packinghouses has led to the proliferation of resistant strains of some pathogens. These problems have encouraged researchers to explore alternatives to synthetic fungicides for control of postharvest diseases of fruits, including gray mold decay of strawberries (El-Mogy and Alsanius, 2012). Toxoplasma gondii, Cryptosporidium spp., and Giardia intestinalis are protozoan parasites, which have been recently highlighted as emerging food-borne pathogens by the FAO of the United Nations and the World Health Organization. Their emergence is favored among others, by change in eating habits (i.e., preference for fresh or minimally processed foods), the global trade of foodstuff, changes in the food production systems, and the increased number of sensitive people. Contamination by pathogens can occur at any stage of the food processing from primary production to consumers. In the context of food safety growing concerns, this implies that the food industry has to constantly monitor and control its production chain from the field to the fork (Hohweyer et al., 2016). The OTA is the most important mycotoxin contaminants of foodstuffs and beverages due to its potent immunotoxic, teratogenic, nephrotoxic, and genotoxic properties to humans. The OTA has been classified as a possible human carcinogen (group 2B) by the International Agency of Research on Cancer. Notably, Aspergillus ochraceus and Penicillium verrucosum were considered to be the main cause of OTA in tropical and temperate regions, respectively. However, apart from Penicillium nordicum and P. verrucosum, the genus Aspergillus is considered as the most important species responsible for the presence of OTA in food. This genus is reported to have more than 100 species, with a complex taxonomy under continuous revision. The OTA is a common contaminant of a wide range of food and food products, which include cereals and its derived products, grapes, cocoa beans, spices, nuts, olives, grapes, beans, coffee beans, and figs. After cereals, grapes and its derived products are the second most contaminated products with OTA (Zhang et al., 2016).

Novel Strategies for the Reduction of Microbial Degradation of Foods  489 Rhodotorula is a common environmental yeast found in air, soil, lakes, ocean water, milk, and fruit juice. Rhodotorula species, part of the Basidiomycota phylum, colonize plants, humans, and other mammals. The genus Rhodotorula includes eight species, of which Rhodotorula mucilaginosa, Rhodotorula glutinis, and Rhodotorula minuta are known to cause disease in humans. Rhodotorula produces pink to red colonies and blastoconidia that are unicellular lacks pseudohyphae and hyphae. Several authors have isolated Rhodotorula in different ecosystems and environments as well as described infections in animals. Rhodotorula spp. have been recognized as emerging yeast pathogens in humans in the last two decades. While no cases of Rhodotorula infection were reported in the medical literature before 1985, there has been an increase in infections after that time, most likely because of the wider use of intensive treatments and central venous catheters (Wirth and Goldani, 2012). R. glutinis isolated from apple surface is shown in Fig. 16.1.

Fig. 16.1 Rhodotorula glutinis (×1000, photo taken by Hakan Kuleaşan).

Saccharomyces cerevisiae, a nonspore forming yeast, also known as brewer’s yeast or baker’s yeast, is a common colonizer of the human respiratory, gastrointestinal, and urinary tracts and is generally considered as a benign organism (Pillai et al., 2014). There has not been much interest in the research on the possible undesirable effects of its consumption. This is mainly because S. cerevisiae has always been considered a safe microorganism for nutritional use (“GRAS,” generally regarded as safe) without considering its undesirable facets (Llopis et al., 2014). Its role as a clinically important invasive pathogen is not well known. However, several cases of invasive diseases in the setting of chronic underlying diseases like malignancy, HIV/ AIDS, or of bone marrow transplantation have been reported (Pillai et al., 2014). S. cerevisiae isolated from grape juice is shown in Fig. 16.2.

490  Chapter 16

Fig. 16.2 Saccharomyces cerevisiae (×1000, photo taken by Hakan Kuleaşan).

Aspergillus is a diverse genus with high economic and social impact. Species occur worldwide in various habitats and they are known to spoil food, produce mycotoxins, and are frequently reported as human and animal pathogens. Furthermore, many species are used in biotechnology for the production of various metabolites such as antibiotics, organic acids, medicines, or enzymes, or as agents in many food fermentations. The classification and identification of Aspergillus has been based on phenotypic characters but in the last decades it was strongly influenced by molecular and chemotaxonomic characterization (Samson et al., 2014). The Aspergillus glaucum which is proliferated on a cheese is shown in Fig. 16.3.

Fig. 16.3 Aspergillus glaucum proliferated on a cheese (photo taken by Hakan Kuleaşan).

Novel Strategies for the Reduction of Microbial Degradation of Foods  491

16.4  The Contaminants Causing Poisoning in Humans Food-borne diseases are a serious and global problem. Food-borne illness is defined by the World Health Organization as “diseases, usually either infectious or toxic in nature, caused by agents that enter the body through the ingestion of food” (FSA-Food Standards Agency, 2011). Food-borne diseases have become a widespread and serious public health problem worldwide. Most of these diseases are caused by pathogenic microbial infections that enter the food chain at some point from farm to fork and are generally considered a serious health problem for young, elderly, and immunocompromised individuals (Chakchouk-Mtibaa et al., 2014). Among common food-borne bacterial pathogens, Salmonella, Shiga toxinproducing E. coli (STEC), and Listeria are considered the top etiologic agents associated with hospitalizations and mortality. Several studies suggest that enteric pathogens have sufficient fitness to survive on plants (Carter et al., 2015). Selective food-borne disease outbreaks, detected in some foods, occurred between 2005 and 2014 is given in Table 16.1. Table 16.1: The selected food-borne disease outbreaks in last decade Year

Place

Agent

Food

2005 2006

South Wales North America

E. coli O157 E. coli O157:H7

Meat Spinach

Infected Death 157 199

1 3

2008 2008 2011

Canada United States Germany

Listeria Salmonella E. coli O104:H4

>50 >200 3842

20 8 53

2011

United States

Listeria

Cold cuts Peanuts Fenugreek sprouts Cantaloupe

147

33

2014

Denmark

Listeria

Meat Products

>37

15

Source Pennington (2009) Centers for Disease Control and Prevention (2006) Greenber and Elliott (2009) Scientific American (2009) Burger (2012) Centers for Disease Control and Prevention (2012) Food Safety News (2014)

Food-borne pathogens impose over $15.5 billion (2013 dollars) economic burden on the US public each year. Just five pathogens cause 90% of this burden. Estimates of economic burden per case vary greatly, ranging from $202 for Cyclospora cayetanensis to $3.3 million for Vibrio vulnificus. In all, 15 pathogens cause 95% or more of the food-borne illnesses, hospitalizations, and deaths in the United States for which a specific pathogen cause can be identified. They are Campylobacter spp., C. perfringens, Cryptosporidium spp., C. cayetanensis, L. monocytogenes, norovirus, Salmonella nontyphoidal species, Shigella spp., STEC O157, STEC non-O157, T. gondii, V. vulnificus, V. parahaemolyticus, Vibrio other noncholera species, and Y. enterocolitica. A total of 84% of the economic burden from these 15 pathogens is due to deaths. This reflects both the importance the public places in preventing deaths and the fact that the measure of economic burden used for nonfatal illnesses (medical costs + productivity loss) is a conservative measure of willingness to pay to prevent nonfatal illness (Hoffmann et al., 2015).

492  Chapter 16 The reportedly held rationale that increased consumption of fresh vegetables is actually the reason for the increased number of food-borne illnesses has been challenged in a American Society for Microbiology report stating that the proportion of outbreaks due to leafy greens has increased beyond what can be explained by increased consumption. This led us to focus on the primary production stages on farms and subsequent processing as the main contamination sources, although no doubt coupled with enhanced epidemiological and surveillance programs and the expanded interaction of the local and international markets of fresh produce (Faour-Klingbeil et al., 2016). Fresh-cut fruits are susceptible to microbial contamination in any phase of the production or distribution, due to the destruction of natural protective barriers and their high water and nutrient contents. In addition, they are neither heat treated nor contain added preservatives. As a result, they may be a vehicle for the transmission of microbial pathogens causing health problems. Fruits may contain various microorganisms that are present in nature or acquired/ gained during harvest, processing operations, or even during the handling by workers or consumers. If the initial load of microorganisms is high and/or the preparing operations are inadequate, some microorganisms will survive and subsequently will grow and cause spoilage and possibly illness if the surviving microorganisms are virulent (Graça et al., 2015). Apart from causing diseases in plants, many species of Fusarium, Aspergillus, Penicillium, and Alternaria can also synthesize mycotoxins (da Cruz Cabral et al., 2013). Mycotoxins are toxic metabolites produced by fungi, particularly by saprophytic molds growing on agricultural products. These cause not only economic losses but also pose health hazards to humans and animals (Bhatnagar-Mathur et al., 2015). These compounds are hazardous to animal and human health as they can be lethal, carcinogenic, mutagenic, teratogenic, immunosuppressant, or may mimic estrogens (da Cruz Cabral et al., 2013) with harmful effects on humans, livestock, and poultry (Bhatnagar-Mathur et al., 2015). Their activity depends on the type of toxin and their concentration in the food. Concern about these chemical hazards has been increasing due to the wide range of food types that may be affected and the variability in the severity of symptoms caused (da Cruz Cabral et al., 2013). Aflatoxin was first identified in 1960 following a severe outbreak of a disease called “Turkey‘X’ Disease” in the United Kingdom that killed over 100,000 turkey birds (Bhatnagar-Mathur et al., 2015). The presence of mycotoxins in food is associated with fungal inoculum on predisposed substrates. Mycotoxins can be produced before and after harvest and levels may increase during postharvest handling and storage. Thus, prevention of fungal growth is an effective means of preventing mycotoxin accumulation. Mycotoxins may reach consumers either by direct contamination of plant materials or products thereof, or by “carry over” of mycotoxins and their metabolites into animal tissues, milk, and eggs after intake of contaminated feed. Furthermore, this hazard remains in processed food because these metabolites are not

Novel Strategies for the Reduction of Microbial Degradation of Foods  493 removed by normal industrial processing, and the risk could increase if moldy fruits or plants are used in processed by-products (da Cruz Cabral et al., 2013). Most of the food-borne infections so far reported were caused by pathogenic bacteria, including E. coli O157:H7 and Salmonella species (Chakchouk-Mtibaa et al., 2014) and Listeria spp. which are a public health concern (Bilek and Turantaş, 2013). E. coli O157:H7 and S. enterica serovars Typhimurium have commonly been reported in a wide variety of raw meats, poultry and dairy products, vegetables, and water. Enterohemorrhagic E. coli O157:H7 is a causative agent of hemorrhagic colitis and hemolytic uremic syndromes. Salmonella species are a leading cause of food-borne bacterial illnesses in humans. In nature, S. typhimurium may survive several years in the soil. This pathogen is known to cause food poisoning, resulting in gastroenteritis in humans and other mammals. S. typhimurium infects the host by penetrating the intestinal mucosa and migrating to the spleen and liver where it causes systematic diseases (Chakchouk-Mtibaa et al., 2014). L. monocytogenes is a pathogenic bacterium that relatively frequently contaminates food products, in particular cheese and ready-to-eat meat-containing food products. Depending of the host’s susceptibility, it can cause mild gastroenteritis or severe infections of the blood stream and/or the central nervous system, and abortion. This bacterium is capable of surviving in broad range of temperatures during food production and storage, and contamination with this microorganism is of primary concern in processed food products (Šrajer Gajdošik et al., 2013). Pathogenic Gram-positive bacteria Bacillus subtilis and L. monocytogenes, as well as Gram-negative E. coli and Y. enterocolitica, are important microorganism involved in food spoilage and human food-borne infections. These bacteria are also often used as model microorganisms when bacterial food contamination is experimentally investigated (Šrajer Gajdošik et al., 2013). Human noroviruses (NoVs) and hepatitis A virus (HAV) are currently recognized as the most important human food-borne pathogens with regard to the number of outbreaks and people affected in the Western world. Human NoVs are one of the most frequent causes of nonbacterial gastroenteritis worldwide and HAV is the main cause of human enteric hepatitis. These viruses are mainly transmitted via the fecal-oral route either by person-to-person contact or by ingestion of contaminated water and food, particularly shellfish, soft fruits, and vegetables (Fraisse et al., 2011). The World Health Organization estimates that worldwide food-borne and waterborne diarrhoeal diseases taken together kill about 2.2 million people annually (FSA-Food Standards Agency, 2011). In fact, the food-borne outbreaks caused by E. coli and Salmonella isolated from fruits and vegetables resulted with 727 cases/6 deaths and 2288 cases/3 deaths, respectively, between 2006 and 2010 in the United States. In recent years, foodborne outbreaks

494  Chapter 16 caused by fruits and vegetables have shown an increasing trend (Bilek and Turantaş, 2013). Food Standarts Agency, (2011) estimated that each year in the United Kingdom around a million people suffer from food-borne illness, around 20,000 people receive hospital treatment due to food-borne illness, around 500 die from food-borne illness, and it costs nearly £1.5 billion. The annual cost of food-borne disease in the United Kingdom was estimated, in 2008, to be approximately £1.5 billion (FSA-Food Standards Agency, 2011). Most of the gastrointestinal tract infections, with the exception of typhoid fever, should not be treated with antibiotics unless affecting patients with underlying illnesses or prolonged and complicated febrile cases. However, the general trend in the last few years was the use of antibiotics even for mild Salmonella-caused gastroenteritis. This fact leads to an everincreasing number of multiresistant isolates of Salmonellae. Furthermore, since Salmonella are zoonotic, the observed resistance is also due to the use of antibiotics in farm animals. Integrons class I and the so-called Salmonella genomic island 1, a chromosomal multidrug-resistant region, play a crucial role in the spread of classic antibiotic resistance (Chalón et al., 2012). Protozoa are excreted in high quantities in the environment by infected hosts under an environmentally resistant form, i.e., the oocyst for T. gondii and Cryptosporidium spp., and the cyst for G. intestinalis, that can contaminate waters and soils. Consequently, these three parasites are associated with many waterborne outbreaks with more than 30,000 human cases over the world these last 15 years and Cryptosporidium was involved in 60% of them between 2004 and 2010. Fecally contaminated waters can also present a risk of contamination for vegetables and mollusks during primary production (Hohweyer et al., 2016). Since food-borne illnesses are a great risk factor in terms of human health and affect the economy in a negative way, effective measures need to be taken to protect and maintain the safety of foods. In this sense, growers and marketers as well as consumers must be conscious and careful. Food Standards Agency (FSA, 2011) reported the benefits of reducing food-borne disease as • • •

reduced morbidity, mortality, and demands on health-care services, reduction in absences from education, or loss of productivity at work, increased consumer confidence in food safety (FSA, 2011).

16.5  Chemical and Natural Ways to Prevent Contamination of Food and Agricultural Products In the fruit industry, postharvest losses are 5%–10% when postharvest fungicides are used. Without fungicides, losses of 50% or higher have occurred in some years. For example, in a 1993 test to assess the decay potential of stone fruit, an average of 52.8% (range 15%–100%) of the fruit decayed during the ripening of eight collections that had not been treated with postharvest fungicides (Dao and Dantigny, 2011).

Novel Strategies for the Reduction of Microbial Degradation of Foods  495 The major part of vegetables and fruits is consumed fresh or as industrially minimally processed, which include canned, dried, juice, paste, salad, sauce, and soup preparations. Cooking and pasteurization as well as the addition of chemical preservatives are the main technology options that guarantee safe vegetables and fruits, but would bring about a number of not always desirable changes in their physical characteristics and chemical composition (Di Cagno et al., 2013). Fresh produce and minimally processed vegetables are widely consumed worldwide as they are important natural sources of essential nutrients. For the modern consumer, these products are necessary to maintain a healthy diet, and their fresh and nutritional status is largely recognized. However, despite the increased awareness of food safety issues, the occurrence of food-borne disease outbreaks related to these products is constantly increasing with several pathogenic bacteria (Meireles et al., 2016). Bacterial pathogens are frequently responsible for both food spoilage and food-borne illnesses that cause enormous commercial and health damage around the world. Outbreak of food-borne diseases has always been a severe health risk in developing countries. However, food-borne diseases are still a problem in industrial countries, too. Consequently, protection against spoilage as well as prevention of food-borne diseases is a task of enormous economic and social importance (Šrajer Gajdošik et al., 2013). Populations of human pathogens are composed of single cells, so their control is expressed as a reduction in colony forming units. In contrast, fungal postharvest pathogens exist as discrete propagules and later they become an interconnected fungal mass deep within the host. Their control is best quantified as a reduction in the percentage of infected individual pieces of produce (Feliziani et al., 2016). Inhibition of fungal growth in crops, fresh fruits, and vegetables is thus necessary to reduce the risk to human and animal health. However, it is important to note that partial inhibition of fungal growth, such as reduction in fungal growth rate, could enhance mycotoxin production as a response of the mold to stress (da Cruz Cabral et al., 2013). Synthetic chemical fungicides have been used to reduce postharvest fungal spoilage, but because of problems regarding toxicity, fungicide resistance, and negative impact on both the environment and human health, alternative measures for disease control are increasingly demanded. In general, decay control methods that are alternatives to conventional synthetic fungicides can be classified as physical, chemical, or biological (Fagundes et al., 2013). During the last 5 years, occurrences between 3.3% and 77.8%, and 4.3% and 75% were described, respectively, for Cryptosporidium and Giardia (oo)cysts on different leafy green vegetables. At the moment only one study showed the presence of T. gondii oocysts in vegetables. Conventional decontamination treatments applied in minimally

496  Chapter 16 processed vegetable industries are known to be inefficient on protozoan (oo)cysts. Indeed, Cryptosporidium and Giardia (oo)cysts were recently identified, respectively, in 5.9% and 1.8% of ready-to-eat leafy greens samples in Canada (Hohweyer et al., 2016). Sanitation after harvest is critically important for all fresh products, where it can reduce spoilage losses by 50% or more. This occurs primarily by the sanitation of wash water, produce surfaces, equipment, and storage rooms rather than direct control of infections of the decay pathogens within the produce. Sanitizers differ in two fundamental aspects from postharvest fungicides. Sanitizers cause rapid mortality of pathogens they come in contact with and do not deposit a persistent antimicrobial residue in treated products. In contrast, most fungicides are primarily fungistatic in action, arresting pathogen growth when present in sufficient concentration, and many confer persistent protection from decay often after long treatment. Many move systemically within the host tissue, while all sanitizers do not (Feliziani et al., 2016). The most popular sanitizer is chlorine (hypochlorite) (Feliziani et al., 2016). However, some disinfection methods for fresh produce involve the use of chemical agents such as chlorine solutions, which can leave some residues in the product and might not be completely effective to inactivate all of the microbial loads (Bermúdez-Aguirre et al., 2013). Moreover, chlorine (in chlorinated water) reacts with organic materials and can produce harmful by-products such as chloramines and trihalomethanes as well as the chlorine being inactivated and rendered inert in the presence of organics. In addition, several researchers have discovered that if produce is chemically or hot water sanitized and then recontaminated during subsequent processing steps, microbes are capable of increasing to prewash levels or higher (Chen et al., 2012). Due to the risks posed by the use of chlorine in the food industry, the use of these compounds is forbidden in European countries such as the Netherlands, Sweden, Germany, and Belgium (Bilek and Turantaş, 2013). Therefore, more effective sanitation technologies are required for the food industry to reduce contamination and prevent cross-contamination to produce safer and higher quality fruit and vegetable products (Chen et al., 2012). Actually, there is a trend in eliminating chlorine-based compounds from the decontamination and disinfection process and applying innovative and emerging technologies in the food industry (Bilek and Turantaş 2013). A number of alternative sanitizers have been proposed or in minor use in washing or the storage of fresh produce such as chlorine dioxide, ozone, ethanol, hydrogen peroxide, organic acids, and electrolyzed water. Organic acids differ from the others in that rapid mortality only occurs at relatively high concentrations and their action is primarily biostatic. All are nonselectively toxic, so their risk of injury to treated products should be rigorously evaluated before adoption. All of these compounds are approved for use in some food contact roles (Feliziani et al., 2016).

Novel Strategies for the Reduction of Microbial Degradation of Foods  497 The exploration of naturally occurring antimicrobials for food preservation receives increasing attention due to consumer awareness of natural food products and a growing concern of microbial resistance toward conventional preservatives (Edziri et al., 2012). Meireles et al. (2016) classified the alternative disinfection methods to chlorine for use in the fresh-cut industry. According to researchers, disinfection methods can be classified into four groups. First group is biological-based methods (bacteriocins, bacteriophages, enzymes, and phytochemicals), second group is chemical-based methods (calcium lactate, chlorine dioxide, copper compounds, electrolyzed oxidizing water, hydrogen peroxide, ozone, quaternary ammonium compounds, sodium bicarbonate, and weak organic acids), third group is physical-based methods (ionizing irradiation, membrane filtration, steam jet-injection, temperature, ultrasounds, and ultraviolet light), and the last group is the combination of all the disinfection methods (physical-chemical, chemical-chemical, chemical-biological, and biological-biological methods).

16.5.1  Chemical Ways to Prevent Contamination of Food and Agricultural Products Physical and chemical treatments are used in food processing to eliminate or reduce the population of pathogenic and spoilage microorganisms (Chen et al., 2012). The most common disinfecting agent is chlorine applied as a spray or dip. Sanitation may be followed by treatment with one or more fungicides, which deposit a residue in the product that inhibits decay pathogens that infect later or escaped the action of the sanitizers. Sanitizers are also widely employed to minimize contamination of produce with pathogens of human health concern (Feliziani et al., 2016). Vegetables are washed typically with water that generally contains free chlorine from approximately 0–30 ppm. The chlorine and chlorinated compounds have already been used for several decades and these compounds are still the most widely used sanitizers in the food industry (Bilek and Turantaş, 2013). However, washing cantaloupes with chlorinated water can only achieve c.1–2 log10 CFU/mL reductions (Chen et al., 2012). Washing in disinfectant solutions can be done to enhance the removal of microorganisms from the produce, although the main motivation is to avoid cross-contamination via water. In general, chemical oxidants, including peracetic acid, are much more effective for the inactivation of bacterial pathogens in wash water than for removal of these pathogens from fresh produce. In addition, once cross-contamination has occurred, rewashing the newly infected lettuce in disinfectant solutions is unable to remove completely the newly attached E. coli O157, even shortly after the contamination event. Therefore, the primary purpose of washing produce in disinfectant solutions seems to be avoiding cross-contamination via wash water. Furthermore, microbial contamination of produce should be avoided as much as possible by respecting good agricultural and manufacturing practices during the production and processing of fresh produce (Van Haute et al., 2015).

498  Chapter 16 Gray mold (B. cinerea) is one of the most major fungal pathogens in paprika. Generally, gamma irradiation over 1 kGy is effective for the control of fungal pathogens; however, a significant change in fruit quality (physical properties) on paprika was shown from gamma irradiation at over 0.6 kGy (P 170,000 tons. c Period: 01/01/2001–31/12/2003; number of samples: 157; volume of analyzed oil: >41,000 tons.

Relevance and Legal Frame in Novel Food Preservation Approaches  531 For quality reasons, oil palm fruits are processed within a few days and preferably within 24 h after harvesting and chemical crop protection is not required, which explains the absence of detectable pesticide levels in the crude palm oil samples. Palm kernels and coconuts (or copra after drying) are stored and collected before centralized processing; chemical crop protection is not applied in these supply chains. Organic (oil or hexane) soluble preservation agents, such as endosulfan, will concentrate in the oil phase during milling; the level of preservation agents in crude oil will be higher than the level in the seed. Theoretically, an oil seed with a pesticide level at MRL may result in a crude oil with a pesticide level higher than the seed MRL. In principle, the oil-dissolved preservation agents may be removed by refining in several ways: •

•

• •

Physically during neutralization: the pesticide (e.g., dichlorvos) dissolves in the alkaline solution (perhaps followed by ester or phosphate hydrolysis) and is removed with the soap stock. Physically during deodorization: the relatively volatile preservation agents (pesticides with molecular weight below that of free fatty acids) transfer from the oil to the lowpressure steam bubbles and are removed bysteam strippling. Physicochemically: the pesticide (e.g., pirimiphosmethyl) adsorbs by acid-base interaction onto the bleaching earth. Chemically at high temperature (during deodorization): some preservation agents decompose and are subsequently removed by steam stripping. Other preservation agents (e.g., malathion) undergo a transesterification reaction with the triacylglycerols. As a result, ethyl alkanoates are formed and a diacylglycerol becomes attached to the pesticide. This reversible reaction occurs simultaneously with the removal of the pesticide by stripping. It is therefore highly unlikely that a significant fraction of the pesticide will be bound in this way.

Roszko et al. (2012) have studied the presence of preservation agents in several coldpressed vegetable oils (pumpkin seed oil, linaire oil, poppy seed oil, hempseed oil, borage oil, sesame seed oil, evening star oil, and rapeseed oil) used as flavoring agents or regarded as possessing extraordinary nutritional value. Concentrations of pesticide residues found in the studied cold-pressed vegetable oil samples are given in Table 17.3. The results are quite surprising. Five out of eight composite oil samples studied showed detectable amounts of pesticide residues. Mostly OPPs insecticide residues (pirimiphosmethyl, chlorpyrifos-methyl, DDVP) were found. The DDT isomers and trifluralin herbicide were found in pumpkin seed oil. The DDT is listed in the Stockholm persistent organic pollutant (POP) list and its presence in oil samples is most likely related to the environmental POPs background level. The highest total concentration of preservation agents was

532  Chapter 17 Table 17.3: Pesticide residues found in the studied vegetable oil samples (Roszko et al., 2012) Oil Sample

Compound

Concentration (mg/kg)

Poppy seed Pumpkin seed Linaire Borage Evening star

DDVP DDT Pirimiphosmethyl Chlorpyrifos-methyl Pirimiphosmethyl

0.032 0.02 1.170 0.026 0.059

Compound

Concentration (mg/kg)

Pirimiphosmethyl Trifluralin Pirimiphosmethyl

0.179 0.137 0.054

Note: Mean value of two parallel determinations for the composite oil sample.

found in the linaire oil sample, with pirimiphosmethyl concentration at 1.170 mg/kg level. The EU legislation does not recognize any MRLs for the determined compound/ investigated oil combinations. Nevertheless, the concentrations are very high when compared with MRLs defined for oil seeds (0.05 mg/kg for sum of DDT isomers, 0.05 mg/kg for pirimiphosmethyl, 0.01 mg/kg for DDVP, 0.1 mg/kg for trifluralin, 0.05 mg/kg for chlorpyrifos-methyl). Those MRLs were exceeded in the majority of the tested samples. Pesticide residue levels found in the oil samples are probably among the most unexpected results. The OPPs insecticides are commonly used in agriculture for the protection of oil producing crops. Most probably this fact is responsible for the presence of those compounds in cold-pressed vegetable oils. Most of OPPs are fat soluble and might be easily stored in oil seeds. What’s more, fats—as energy reserves for oil seeds—may be not subject to metabolic pathways that could affect OPPs. This hypothesis might also explain the very high concentrations of OPPs determined in the samples. Persistence of OPPs insecticides in vegetable oils was also reported previously (Cabras et al., 1997). Due to specific flavors and other characteristics of cold-pressed vegetable oils, they are not as widely consumed as refined vegetable oils [except for virgin olive oil (VOO)]. Refined vegetable oils probably have significantly lower concentrations of various contaminants due to high temperature and/or pressure treatment. Those suppositions are also confirmed by several literature data (Škrbić and Ðurišić-Mladenović, 2007; Duijn, 2008; Lacoste et al., 2005). According to data reported by oil producers, OPPs insecticides are commonly found in vegetable oils at relatively high concentrations. Results of this study seem to confirm that data (Duijn, 2008; Lacoste et al., 2005; Duijn and den Dekker, 2010).

17.5  Olives, Olive Oil, and Preservation Agents 17.5.1  Olives and Preservation Agents The olive (Olea europaea L.) was cultivated traditionally for oil production and table olives. Mediterranean basin have >97% of the global olive production and Spain being the world’s

Relevance and Legal Frame in Novel Food Preservation Approaches  533 leading producer (7,870,000 tons) followed by Italy, Greece, Turkey, Morocco, and Tunisia. In 2013 olive production exceeded 20,000,000 tons (FAO, 2015). Olives are grown in >11 million hectares of land in the world, spread across the 5 continents, 2 hemispheres, and 47 countries where olive oil is currently produced (IOC, 2016). Annually, olive growers lose part of the production because of the attack of pests such as Dacus oleae, Prays oleae, Saissetia oleae, Hylesinus olivine, and Phloeotribus scarabaeoides. In Mediterranean countries, the olive production loss translates into huge markup of lost revenue per year and to confront the loss large amounts of preservation agents are used. Persistent preservation agents negatively affect human health and thereby the detection of pesticide residues in olives and olive oil is of great importance.

17.5.2  Pest Control The annual cost of olive pest control exceeds 100 million Euros worldwide, 50% of which corresponds to preservation agents’ use, not including the cost of the adverse side effects of preservation agents’ use (Amvrazi and Albanis, 2008). There are 2–4 significant pests associated with olive plantations plus a further 10 or so of secondary or localized importance (including fungi and other problems). The main pests cited by Cirio (1997) are Bactrocera oleae (olive fly), P. oleae, S. oleae, and Capnodium elaeophilum. To these should be added Cycloconium oleaginum, which Guerrero (1997) cites as a widespread problem in Spain. The presence and seriousness of these pests, fungi, etc., depends partly on prevailing environmental conditions (temperature, humidity), and partly on practices such as cultivation, pruning, and irrigation (Cirio, 1997). At least 40 species of “useful” insects are believed to be parasites of P. oleae and a further 20 of olive fly and S. oleae (Cirio, 1997). Practices which adversely affect these beneficial species may increase pest problems. Olive fly is the most important pest. It is much more a problem in more humid, frost-free areas, where it can decimate the olive crop leading to reduced oil quality largely. However, in dry, high-altitude areas, the presence of olive fly tends to be much less and control measures may not be necessary on a regular basis. Olive fly is normally treated with dimethoate sprays, either by the farmer or through largescale aerial spraying. According to Guerrero (1997), typical quantities applied are 1.5 L of 40% dimethoate per hectare for terrestrial application or 0.5 L per hectare for aerial application. Alternative control systems are being developed, such as mass-trapping using baits, but these are more expensive and labor intensive. In some areas, chemical control measures against olive fly only started to be taken relatively recently while in some areas no measures are taken; as a result, olive oil may be of inferior quality, especially in years when this pest is widespread (Al Ibrahem et al., 2010).

534  Chapter 17 Prays are also treated with dimethoate and malathion, although there are many other suitable agents. Many farmers treat Prays as a serious matter, of course, even though the impact of this pest on the production is often minimal, according to some authors (Pajarón, 1997). Guerrero (1997) points out that dimethoate is a broad-spectrum agent which eliminates numerous different types of insect. It is therefore seen as a useful way of “cleansing” an olive plantation of potentially damaging pests, even though it eliminates various beneficial species (e.g., predators of olive fly) at the same time. For the treatment of S. oleae, Guerrero (1997) cites five different preservation agents which can be used (e.g., methidathion at a dosage of 0.1%–0.15%). Many other preservation agents are used in olive plantations, including fenthion, triclorfon, etc. Traditional pest control products, such as copper, lime, white oils, Bordeaux mixture (copper sulfate and lime), are still used in some areas. In more traditional plantations, preservation agents use is low or nonexistent, or limited to traditional products, as mentioned above. For example, in La Vera in Cáceres (Spain) it is estimated that 80% of which use centrifugal systems. Currently, olive oil is consumed in over 160 countries (IOC, 2016). The importance of VOO is mainly attributed both to its high content of oleic acid in a balanced composition of PUFA and its richness in phenolic compounds, which act as natural antioxidants and may contribute to the prevention of several human diseases (Bendini et al., 2007). All these positive characteristics have increased the demand for this commodity throughout the world. In order to satisfy the increasing demand and provide new alternatives to consumers, other countries such as China, Iran, Turkey, Australia, and Argentina are starting to produce olive oil. Taking the data for 2012 as a yardstick, olive oil production amounts to 3.1 million tons; it represents a 1.7% share of total output of edible animal fats and vegetable oil (184 × 106 tons). These figures highlight the economic importance of the olive oil sector and its influential position in the international arena in terms of production and consumption (Cox et al., 1996). Crop protection products in general and herbicides in particular are essential for the subsistence of modern agriculture, therefore some residues can persist until the harvest stage, thus contaminating the olives picked up from the soil. The maximum residues limits (MRLs) for pesticide residues in olive oil and olives were established by EU, the Codex Alimentarius Commission of the Food and Agriculture, Organization of the United Nations (FAO), and the WHO. EU Regulation (EC) 396/2005 establishes MRLs for some of preservation agents’ residues in olives for oil production. Currently, there are no harmonized MRLs established for pesticide residues in olive oil yet (Gilbert-López et al., 2010). Currently, maximum residue level (MRL) is assessed by the monitoring pesticide residues in olive oil (product obtained only by mechanic extraction from olives and is suitable to be consumed directly without more treatments) whose consumption has been increased due to its beneficial effects on human health. An extensive amount of works have been carried out for the determination of pesticide residues in olive oil by making use of available many new improved methods (García et al., 2007). However, extent studies of preservation agents’ residues in olive oil are few and generally refer a certain classes of preservation agents with OPPs insecticides

536  Chapter 17 that have been found mostly in olive oil (Botitsi et al., 2004; Cunha et al., 2007; Dugo et al., 2005; Hiskia et al., 1998; Lentza-Rizos and Avramides, 1991; Rastrelli et al., 2002; Tsatsakis et al., 2003; Tsoutsi et al., 2006). In recent works, it was determined by multiresidue method applications in real samples that a large number of pesticide residues could be accumulated in olive oils. The registered data of endosulfan occurrence in the 22% of a total number of 338 Greek olive oil samples (Lentza-Rizos et al., 2001) as well as the recently registered detection of endosulfan residues in Spanish olive oils with detection rates of 58%–100% (Ferrer et al., 2005; Guardia-Rubio et al., 2006; Sánchez et al., 2006; Yagüe et al., 2005) consist that the analysis of pesticide residues is of great importance since this insecticide is not allowed for use in olive cultivations any more. On the other hand, recent analysis pointed the contamination of olives with herbicides by determining positive detections of simazine, terbuthylazine, diuron, and oxyfluorfen herbicides residues in high amounts in surveyed Spanish olive oils (Aramendia et al., 2006; Ballesteros et al., 2006; Guardia-Rubio et al., 2006). Olive oil production is characterized by significant amounts of residues, both solids and liquids, and their management is a challenge for the olive oil mill workers from economic and environmental perspectives. 17.5.3.1  Olive oil extraction systems The olive oil production processes can be subdivided in two main phases: • •

Preparation of a homogeneous paste. Oil extraction and purification.

In the first phase, olives are processed by means of grinding and mixing pulp and olive stone, followed by a heating process to further break down the olive cells and to create large oil droplets. In the second phase, oil is extracted by a press or a decanter. Water and solids are thus separated from the oil and further centrifuged to recover residual oil. Oil is purified through clarification by sedimentation or filtration by vibrating screens. Olive mill wastewater (OMW) streams are also clarified before disposal. Residual solids from the purification step are mixed with those coming from the extraction step. In modern olive mills, extraction from the olive paste is based on the principles of: 1. Pressing (traditional or classical system). 2. Centrifugation (continuous system): • Process using decanter with three exits. • Process using decanter with two exits. 3. Stone-removing process. 4. Percolation (selective filtering). 5. Chemical separation. 6. Electrophoresis.

Relevance and Legal Frame in Novel Food Preservation Approaches  537 The last four methods are hardly used. In the pressing system and continuous process using decanter with three exits the waste obtained is composed of a liquid fraction (liquid waste product, 3-OMW) and a solid fraction (solid waste product); in the olive oil mills using decanter with two exits, olive oil mill wastewater is a slurry waste (2-OMW). Pressing system as well as the three- and two-exit centrifugal systems are represented schematically in Fig. 17.3.

Fig. 17.3 Olive oil production systems (press and continuous using decanter with three and two exits).

Quantity and quality of the produced liquid and solid wastes are strongly influenced by the oil extraction method. The traditional press system produces the “strongest” wastewater (P-OMW), with concentrations of the order of 100–200 g COD/L (Ben Sassi et al., 2006). The extraction process using decanter with three exits produces wastewater (3-OMW) with slightly lower concentration (Ben Sassi et al., 2006; Sayadi and Ellouz, 1995; Mebirovk et al., 2007; Azzama et al., 2015). According to the latest developments in olive oil production, the great emission of OMW can practically be reduced to nil by transferring it to the spent olive residues. This two-exit technology is considered to be very promising but, in fact, it simply transfers the problem of disposing the olive oil mill waste from the mill to the oil refineries, where the spent olive residues, prior to oil solvent extraction, must be dried which require considerably higher energy than for traditional or continuous oil production processes (Borja et al., 2006).

17.5.4  Preservation Agents in Olive Oil Irrigation introduction with the improvement and development of olives production techniques has tremendously boosted olive oil production in the last few years. High

538  Chapter 17 production has led to the development of new systems for olive harvest and transport and also modern olive oil extraction systems (Nieto et al., 2009a,b). The use of mechanical machines (sweepers, blowers) that facilitate the collection of olives fallen to the ground is the frequent harvesting practice. Nevertheless, this collection form augments the amount of earth accompanying the olives to the mill and thus olives and final oil qualities are seriously declined. Also, phytosanitary products as insecticides, fungicides, and herbicides, are widely used to control pests, diseases, and weeds. It should be noted that each of the active ingredients has a safety period (minimum number of days that should elapse between the final application and harvest). Moreover, nondegraded chemical products (pesticide not degraded during the safety period) can be persist and pollute not just the water but also the soil at harvest time, contaminating the olives that fall on the ground. Also, some of the active ingredients of the preservation agents are degraded through hydrolytic degradation or photolytic degradation mediated by sunlight. As a result, harmless or harmful residues (phytosanitary residues) can be retained by the ground or leach into the surface water or groundwater (Nieto et al., 2009a,b). The quality of olive oil is linked to the quality of the raw material, which must be free of any defects such as surface blemishes, scars, punctures, or other damage caused by pests (Kırış and Velioglu, 2016). Results of surveyed olive oils from conventional cultivations (n = 90), obtained by Amvrazi and Albanis (2009), are presented in Table 17.4. Overall, no residues were detected in 10% of the samples tested for target preservation agents whereas 20 of the 35 pesticides tested were detected in the rest of the samples. No detectable residues of the herbicides tested were found in surveyed samples. Four samples were found to contain endosulfan and two chlorpyrifos residues. The number of different pesticide residues in a sample ranged from 0 to 7 and the median, mean, and mode values were equal to 3 for different preservation agents in a sample. In these counts, metabolites and parent compound were measured as one pesticide. Furthermore, 31.1% of surveyed samples contained only organophosphates, 31.1% endosulfan and organophosphates whereas in 12.2% endosulfan together with organophosphates and pyrethroid residues were detected. In the same study, realized by Amvrazi and Albanis (2009), overall five types of olive oils from conventional cultivations were analyzed for pesticide residues: extravirgin olive oil (EVOO), VOO, and lampante olive oil from individual growers and commercially packed EVOO and ordinary olive oil. Mean concentrations and numbers of positive detections of the preservation agents’ residues detected in samples per type of olive oil are given in Table 17.5. Also, Table 17.6 illustrates high preservation agents’ frequency in olive oil in the Mediterranean region through the olive oil production process (Amvrazi and Albanis, 2009).

Relevance and Legal Frame in Novel Food Preservation Approaches  539 Table 17.4: Pesticide residues detected in the 90 conventional olive oil samples which were collected directly from olive mills and commercial centers (Amvrazi and Albanis, 2009) Pesticide Omethoate Dimethoate Total dimethoate Diazinon Parathion-methyl Malathion Fenthion Fenthion sulfoxide Fenthion sulfone Total fenthion Chlorpyrifos Quinalphos Methidathion Ethion Azinphos methyl α-Endosulfan β-Endosulfan Endosulfan sulfate Total endosulfan λ-Cyhalothrin α-Cypermethrin Fenvalerate I Fenvalerate II Fenvalerate (sum of isomers) Deltamethrin

Mean Value (μg/kg)

Concentration Range (μg/kg)

Positive Samples (No.)a

Samples Exceed MRL (No.)

MRLsb (μg/kg)

23.2 14.8 21.6 4.0 11.6 – 102.7 54.7 44.5 166.6 24.8 – 11.0 24.2 247.4 10.0 8.4 21.4

10.2–45.1 5.0–90.9 5.0–90.9 3.3–4.8 4.9–28.8 23.4 4.6–767.0 10.2–206.6 14.2–130.7 10.1–997.6 10.4–51.6 29.1 4.9–19.3 1.6–82.1 56.4–438.4 5.0–24.0 5.9–12.8 5.7–52.7

16 (3)c 59 (20) 60 (19) 7 (3) 10 (4) 1 65 (2) 64 (4) 44 (20) 67 (2) 22 (11) 1 12 (3) 13 (2) 5 (3) 30 (15) 30 (21) 46 (4)

– – 0 0 0 0 – – – 0 2 0 0 0 0 0 – –

200 2000 2000 20 200 500 – – – 2000 50 50 1000 100 500 – – –

26.8 16.8 33.6 BQLc BQL BQL

5.7–56.5 11.1–19.5 18.3–48.9 BQL BQL BQL

46 (4) 18 (6) 2 4 (4) 4 (4) 4 (4)

4 0 0 – – –

50 20 50 – – 20

45.2

43.3–47.6

5 (2)

0

100

a

MRLs established by European Union for the commodity of olives. Number (no.) of samples were the residue was detected. In parentheses are shown the number of samples that were positive and below method quantification limit. c BQL, below quantification limit. b

17.5.5  Effect of Olive Oil Extraction Process in Preservation Agents’ Residues Level Industrial processing may alter pesticide residues when compared with raw crops via chemical and biochemical reactions (hydrolysis, oxidation, microbial degradation, etc.) and physicochemical processes (volatilization, absorption, etc.). Thus, processing factors of preservation agents in industrial processes depend on pesticide nature as well as the composition of crops under process. Fruit processing (e.g., washing, peeling, and cooking) is known to reduce and/or decompose pesticide residues in final products (Zabik et al., 2000; Rasmussen et al., 2003; Kontou et al., 2004; Stıpán et al., 2005; Balinova et al., 2006; Poulsen et al., 2007).

EVOO-Individual Growers (n = 45) Pesticide

C (μg/kg)a

Omethoate Dimethoate Total dimethoate Diazinon Parathion-methyl Malathion Fenthion Fenthion sulfoxide Fenthion sulfone Total fenthion Chlorpyrifos Quinalphos Methidathion Ethion Azinphos methyl α-Endosulfan β-Endosulfan Endosulfan sulfate Total endosulfan λ-Cyhalothrin α-Cypermethrin Fenvalerate Deltamethrin Sample without pesticide residues

21.5 11.7 18.4 4.0 N.D. N.D. 158.4 62.7 32.6 236.7 19.6 29.1 12.1 34.5 31.4 11.0 8.2 22.3 28.1 16.7 N.D. N.D. N.D. 3

Positive Samplesb 11 (1) 32 (5) 33 (5) 2 0 0 30 31 (1) 27 (6) 32 4 1 4 7 4 (3) 11.0 5 28 28 8 0 0 0

EVOO-Commercial (n = 21) C (μg/kg)a 45.1 3.5 6.2 4.0 11.1 N.D. 24.2 33.7 6 60.7 11.8 N.D. 6.7 2.4 N.D. 6.7 7.7 28.1 32.9 18.1 48.9 BQL 45.2 1

Positive Samplesb 1 17 (13) 17 2 2 0 19 (1) 19 (2) 8 (8) 19 2 0 4 (1) 1 0 2 2 6 6 2 1 2 (2) 3

VOO (n = 15) C (μg/kg)a 10.2 13.6 14.9 N.D. 6.8 N.D. 47.3 48.1 30.5 110.4 51.3 N.D. 6.6 3.3 438.4 6.7 12.8 13.9 16.6 14.4 N.D. N.D. N.D. 4

Positive Samplesb 1 6 (1) 6 0 2 0 9 8 (1) 6 (1) 9 2 0 1 1 1 1 1 7 7 1 0 0 0

Ordinary Olive Oil (n = 6) C (μg/kg)a 7.9 6.2 14.1 N.D. 4.9 N.D. 11.6 34.1 6.0 42.0 25.9 N.D. N.D. 1.8 N.D. 8.0 6.0 10.3 24.3 17.5 18.3 BQL BQL 0

Positive Samplesb 3 (2) 3 (1) 3 (1) 0 1 0 6 (1) 5 (0) 2 (2) 6 (1) 2 0 0 1 0 1 1 1 1 1 1 2 (2) 2 (2)

EVOO = extra virgin olive oil, VOO = virgin olive oil, N.D. = not detected, BQL = below quantification limit. a Mean values given are the statistical means computed by replacing the missing values of not detected residues with zero and values of BQL detections with the 0.5* value of the method limit of quantification. b Number (no.) of samples were the residue was detected. In parentheses are shown the number of samples that were positive and below method quantification limit.

540  Chapter 17

Table 17.5: Mean concentrations and positive detections of the pesticide residues detected in samples classified by type of olive oil (Amvrazi and Albanis, 2009)

Relevance and Legal Frame in Novel Food Preservation Approaches  541 Table 17.6: Chemical structure and physicochemical properties of main pesticides in olive oil

Omethoate (C5H12NO4PS): CAS number: 1113-02-6 Pv (mmHg) = 2.48 × 10−5 (20°C) Solubility in water (mg/L) = 1 × 106 log Kow: −0.74a

Fenthion (C10H15O3PS2): CAS number: 55-38-9 Pv (mmHg) = 1.05 × 10−5 (25°C) Solubility in water (mg/L) = 7.5 (20°C) log Kow: 4.09

Chlorpyrifos (C9H11Cl3NO3PS): CAS number: 2921-88-2 Pv (mmHg) = 2.03 × 10−5 (25°C) Solubility in water (mg/L) = 1.12 (24°C) log Kow: 4.09

α-Endosulfan C9H6Cl6O3S): CAS number: 959-98-8 Pv (mmHg) = 3.00 × 10−6 (25°C) Solubility in water (mg/L) = 0.51 (24°C) log Kow: 3.83

Dimethoate (C5H12NO3PS2): CAS number: 60-51-5 Pv (mmHg) = 8.25 × 10−6 (25°C) Solubility in water (mg/L) = 25 × 103 log Kow: −0.74

Fenthion sulfoxide (C10H15O4PS2): CAS number: 3761-41-9 Pv (mmHg) = 5.51 × 10−6 (25°C) Solubility in water (mg/L) = 3.72 log Kow: 1.92

Diazinon (C12H21N2O3PS): CAS number: 333-41-5 Pv (mmHg) = 9.01 × 10−5 (25°C) Solubility in water (mg/L) = 40 (20°C) log Kow: 3.81

Fenthion sulfone (C10H15O5PS2): CAS number: 3761-42-0 Pv (mmHg) = –b Solubility in water (mg/L) = – log Kow: 2.17

Azinphos methyl (C10H12N3O3PS2): CAS number: 86-50-0 Pv (mmHg) = 1.60 × 10−6 (25°C) Solubility in water (mg/L) = 20.9 (20°C) log Kow: 2.75

β-Endosulfan C9H6Cl6O3S): CAS number: 33213-65-9 Pv (mmHg) = 6.00 × 10−7 (25°C) Solubility in water (mg/L) = 0.45 (20°C) log Kow: 3.83

Endosulfan sulfate (C9H6C6O4S): CAS number: 1031-07-8 Pv (mmHg) = – Solubility in water (mg/L) = 0.48 (20°C) log Kow: 3.66 Continued

542  Chapter 17 Table 17.6  Chemical structure and physicochemical properties of main pesticides in olive oil—cont’d

Deltamethrin (C22H19Br2NO3): CAS number: 52918-63-5 Pv (mmHg) = 1.50 × 10−8 (25°C) Solubility in water (mg/L) = 0.002 (24°C) log Kow: 6.20

Cyhalothrin (C23H19ClF3NO3): CAS number: 68085-85-8 Pv (mmHg) = 1.59 × 10−9 (25°C) Solubility in water (mg/L) = 0.0008 (20°C) log Kow: 7.00

a

–, Not found. Log Kow: Octanol-water partition coefficient. Modified from Amvrazi and Albanis (2008).

b

In addition, low-processing factors of certain preservation agents have been reported in juice and wine-making processes as well as in jam preparation processes (Tsiropoulos et al., 1999; Christensen et al., 2003; Fernández et al., 2005a,b; Ruediger et al., 2005). However, in some cases, residue levels may increase in the final product as in the production of dry fruit (e.g., resins and prunes) (Lentza-Rizos et al., 2006; Cabras et al., 1998) and unrefined vegetable oil (Ferriera and Tainha, 1983; Cabras et al., 1993, 2000; Leandri et al., 1993; Cabras et al., 1997; Guardia-Rubio et al., 2006; Amvrazi, 2007) due to concentration factors of raw commodities in the process of the final product. Works on the behavior of pesticide residues during technological transformation of olives to olive oil are scarce. These few works have studied the effect of washing of olives and olive oil on pesticide residues. According to these findings, the effect of olive washing on preservation agents is limited, and the decrease in residues is not correlated with pesticide water solubility. Pesticide residue processing factors have been estimated for several insecticides (azinphos methyl, diazinon, dimethoate, methidathion, parathion, parathion-methyl, quinalphos, fenthion, acephate, buprofezin, phosphamidon, formothion, and deltamethrin) (Ferriera and Tainha, 1983; Cabras et al., 1993, 2000; Leandri et al., 1993; Cabras et al., 1997). However, in the latter studies, the olive oil extraction processes were not always specified, the oil separation was usually performed with no water addition (as in pressure and two-phase centrifugation systems), or are dealt with high concentrations of pesticide residues in the olives processed. In any agro-food industry process, an initial conditioning step is used to separate and remove solid particles and soil residues. Usually, washing step is employed to eliminate soil and pesticide residues. Table 17.7 summarizes the obtained total pesticide concentrations in olive samples (before and after being washed) and wastewater samples from the washing devices. Although total concentrations of preservation agents are reported, the main contribution was due to herbicides. Differences in the behavior of olives collected from the ground after they

Relevance and Legal Frame in Novel Food Preservation Approaches  543 Table 17.7: Total pesticide concentration for olives and wastewater samples (Guardia-Rubio et al., 2007) Total Pesticide Concentration

Ground olives

Tree olives

No. Sample

Not Washed Olives (mg/kg)

Washed Olives (mg/kg)

Washing Water (μg/L)

1 2 3 4 5 6 7 8 9 10

0.699 0.632 0.433 0.310 0.400 0.094 0.003 0.097 0.012 0.005

0.057 0.038 0.038 0.105 0.027 0.021 0.009 0.040 0.086 NDa

26.45 4.05 34.20 18.19 12.14 0.44 0.26 0.44 3.08 1.18

a

ND: Not detected.

have fallen down (ground olives) and those collected directly from the olive tree (tree olives) were observed. Ground olives showed higher residue levels than tree olives and washing greatly decreased concentrations in the former (Guardia-Rubio et al., 2007). Cabras et al. (1997) suggested that, in general, only pesticide residues sorbed to the dust may be removed by washing, which could explain the differences observed in the samples analyzed. In this sense, Kırış and Velioglu (2016) determined the fall in preservation agents’ residues levels in olives treated by ozone and potable water and the transference of preservation agents to olive oil. Precisely, to eliminate diflubenzuron, triflumuron, imidacloprid, λ-cyhalothrin, α-cypermethrin, β-cyfluthrin, deltamethrin, dimethoate, and chlorpyrifos olives are washed using potable water and ozonated water for several times, with the aim to determine the transference ratios of these preservation agents during olive oil extraction process. In general, washing operations with ozonated water and potable water offered a significant reduction in pesticide residues from olives with the exclusion of 2-min ozonated water washing in λ-cyhalothrin, α-cypermethrin, and deltamethrin. This fact can be explicated by the “The demand of ozone by the medium.” Ozone is an oxidant and reacted virtually with all organic and inorganic compounds. Residual ozone is a term used to indicate the detectable ozone concentration in the medium after its application. The effectiveness of treatment by ozone depends on the concentration of ozone used and on the residual ozone concentration detected after treatment. The application conditions determined the stability of ozone and the effectiveness of ozone in the degradation of organic substance in the medium. Fresh water has a lowest ozone demand in comparison with other contaminated water. Impurities in water (e.g., organic or minerals) react with ozone and increase the ozone consumption, in other

544  Chapter 17 words ozone demand (Karaca and Velioglu, 2007). When pure water is treated with ozone the ozone activity remains in water after 20 min; but in the case of contaminated water, residual ozone may remain only for a few minutes (2–3 min) (Suslow, 2004). Usually, prolonging the application time of ozonated water increased the pesticide elimination ratios but no significant change was detected on potable water. From the different treatments realized, the highest removal percentages (after 5 min of ozonated water washing) in pesticide content were 38%, 50%, 55%, and 61% for chlorpyrifos, β-cyfluthrin, α-cypermethrin, and imidacloprid, respectively. Even though, 5 min of ozonated water washing significantly decreased the amount of preservation agents in olives, the pesticide residual levels detected were still higher than the national MRLs. This could be explained considering the high oil content and thick surface of the olives, as well as the high pesticide contents on the olives. Kusvuran et al. (2012) and Wu et al. (2007a) established that the effective removal of pesticide residues depended on the chemical properties of the preservation agents and the matrices where they are the pesticides. The reactivities of preservation agents can be assessed by means of the energy of the highest occupied molecular orbital of the chemicals (EPA, 2001; Wu et al., 2007b, 2009). Ikeura et al. (2011) informed about the lower reduction of residual fenitrothion in cherry tomatoes considering that the dissolved ozone and hydroxyl radicals could not penetrate through the thick pericarp of the cherry tomatoes to reach the sarcocarp and were not activated by contact with the pericarp. In their work, the percentage of fenitrothion eliminated from strawberries was higher than that from cherry tomatoes. The authors of the earlier work affirmed that the rougher and larger surface area of strawberries compared with cherry tomatoes led to more efficient contact with the ozone, thereby improving the pesticide removal in the sarcocarp. As indicated previously, among the treatments realized, the highest reductions in pesticide concentration, 38%, 50%, 55%, and 61% for chlorpyrifos, β-cyfluthrin, α-cypermethrin, and imidacloprid, respectively, were reached after 5 min using ozonated water for washing. Similar result was obtained by Wu et al. (2007b) in the degradation of parathion-methyl, parathion, diazinon, and cypermethrin on vegetable surfaces (Brassica rapa). They described that ozone most effectively removed cypermethrin (>60%), of the four preservation agents examined. Most synthetic pyrethroids (cypermethrin) are composed of several stereoisomers because of the presence of multiple asymmetric carbons, often in a cyclopropane ring (Spurlock and Lee, 2008). In diflubenzuron and triflumuron preservation agents, minimum elimination rates were observed after 5 min of ozonated water washing (Kırış and Velioglu, 2016). In these preservation agents the phenyl ring, alkyl chain, and double bonds were somehow protected from degradation by ozone. Similar result was observed by Karaca et al. (2012) for iprodione on table grape berries stored in an ozone atmosphere. They determined that storing in

Relevance and Legal Frame in Novel Food Preservation Approaches  545 an ozone atmosphere has enhanced the reduction rates of fenhexamid, cyprodinil, and pyrimethanil, but not those of boscalid or iprodione. The ozone resistance of boscalid is due to the aromatic and heteroaromatic rings stability in the structure of this fungicide.

17.5.6  Impact of Washing on Olive Oil Production The processing factor can be determined as the ratio between the pesticide content found in industrial final product to pesticide content in raw agricultural product. A processing factor >1 means an increase in the pesticide residue concentration during industrial processing and a processing factor 1 were registered with the exception of dimethoate in control group. This indicates that pesticide residue concentrations in olive oil were higher than that in olives, the low olive oil yield obtained by extraction was the reason. In the above study, after washing treatment, dimethoate had the lowest concentration and triflumuron had the highest concentration in olive oil. Moreover, imidacloprid did not register in the olive oil obtained.

546  Chapter 17 Table 17.9: Effect of washing treatment: % pesticide transfer in the olive oil (n = 3)a (Kırış and Velioglu, 2016) Application Ozone Washing

Water Washing

Pesticide

Control

2 min

5 min

2 min

5 min

Diflubenzuron Imidacloprid Triflumuron λ-Cyhalothrin β-Cyfluthrin α-Cypermethrin Deltamethrin Chlorpyrifos Dimethoate

14.9 ± 0.30 – 17.8 ± 1.69 13.8 ± 1.89 9.5 ± 0.77 10.9 ± 1.74 9.5 ± 1.07 12.3 ± 0.92 6.1 ± 1.24

18.7 ± 1.81 – 24.6 ± 3.54 16.2 ± 1.97 14.9 ± 2.11 12.8 ± 1.45 12.4 ± 2.09 18.2 ± 1.93 9.1 ± 1.15

13.9 ± 1.69 – 19.9 ± 3.04 13.0 ± 1.61 12.5 ± 0.76 11.9 ± 1.18 12.9 ± 1.27 17.1 ± 0.55 8.9 ± 1.32

16.4 ± 0.69 – 19.9 ± 0.77 15.3 ± 0.89 11.4 ± 0.40 10.9 ± 1.29 13.6 ± 2.57 12.8 ± 1.26 8.2 ± 1.38

16.3 ± 0.38 – 19.8 ± 1.00 11.9 ± 1.02 10.7 ± 0.22 10.4 ± 1.42 12.4 ± 0.24 14.2 ± 0.69 10.0 ± 0.80

a

Percentage transferred: processing factor × oil yield (%). Oil yield (%) = (mass “kg” of oil obtained × 100)/mass “kg” of olives processed.

Preservation agents with an octanol-water partition coefficient, log Kow > 3, usually concentrate in oils. Imidacloprid and dimethoate have values (0.57 and 0.704, respectively) of log Kow 30% (Table 17.12). The photochemical degradation of oxifluorfen, diflufenican, phosmet, alpha-cypermethrin, and deltamethrin was detected only in the experiments with operation times of 150 min. On the other hand, all chemical compounds studied were reduced in content after being submitted to UV light (Katagi, 2004). Also, photochemical degradation experiments performed with EVOO without mixing any preservation agents (only with the residual preservation agents detected), recorded the photochemical degradation yields of trichlorfon equal to 96% at 16 min and 98% at 30 min (T = 20°C). Other experiments with oil mixed with onlyterbuthylazine registered values of photodegradation yields (T = 20°C) equal to 38% and 55% at 16 and 30 min, respectively. Although some degradation products of the preservation agents can be less toxic and harmless, it is not uncommon for them to be more toxic than the parent preservation agents. Indeed, the evaluation of the degradation products is important from the standpoint of human health and environmental protection (Cunha et al., 2007). Toxic effects associated with exposure of phosmet are related to its irreversible inhibition of the acetylcholinesterase enzyme, which causes acute effects in humans (Pope, 1999). In recent years, strong research effort has been made to identify the products arising from photodegradation of phosmet. Tanabe et al. (1974) irradiated phosmet in diethyl ether and have identified N-methylphthalimide and N-methoxymethylphthalimide as its main degradation products. Photodegradation of diuron by the Photo-Fenton or TiO2 system have recently been studied with the aim of reducing the diuron concentration in water (Malato et al., 2002). Total mineralization (i.e., complete disappearance of TOC) can be achieved after a long irradiation (>200 min). However, it appears that 90% of the initial TOC could be mineralized in approximately 125 and 159 min, respectively. The carbaryl photodegradation products in the absence and presence of the AgY zeolites were quantified using the gas chromatographymass spectrometry (GC-MS) technique. In the absence of the catalysts, only α-naphthol was produced after a solution of carbaryl was irradiated for up to 12 h. However, in the presence of the AgY catalysts, α-naphthol, and phthalic acid were the major photodegradation products that were produced (Kanan, 2001). The degradation curve of chlorpyrifos, with a half-life of 13.3 min, indicates the complete degradation of the parent compound within 120 min of irradiation. The GC-MS identification of chlorpyrifos photodegradation products suggested the formation of only one product, that is, chlorpyrifos-oxon A1. The degradation curve of malathion was similar to that of chlorpyrifos, with the calculated half-life of 11.6 min. The complete degradation of the starting compound was achieved within 60 min of irradiation. The GC-MS chromatograms of irradiated samples indicate the formation

Relevance and Legal Frame in Novel Food Preservation Approaches  553 of several photoinduced by-products. The majority of these belong to a family of bute(a) ne diethyl esters. The other three identified photoproducts belong to the family of toxic compounds, all members of the phosphate ester group: phosphorodithioic O,O,S-trimethyl ester (C4) and phosphorothioic O,O,S-trimethyl ester (C1) as major compounds and diethyl (dimethoxyphosphoryl) succinate in traces (C9) (Bavcon-Kralj et al., 2007). Photochemical degradation of endosulfan in controlled aqueous systems was performed by UV light irradiation at λ = 254 nm. The photolysis of (alpha + beta: 2 + 1) endosulfan, alpha-endosulfan, and beta-endosulfan were first-order kinetics. The observed rate constants determined from linear least-squares analysis of the data were 1 × 10−4 s−1, 1 × 10−4 s−1, and 2 × 10−5 s−1, respectively, and the calculated quantum yields were 1, 1, and 1.6, respectively. Preliminary differential pulse polarographic analysis indicated the possible endosulfan photochemical degradation pathway. This degradation route involves the formation of the endosulfan diol, its transformation to endosulfan ether, and finally the ether’s complete degradation by observing the potential shifts (Barcelo-Quintal et al., 2008). In relationship to the operation time, the largest decreases, except pirimiphosmethyl, malathion, and endosulfan I, were recorded after an operation time equal to 150 min. With regard to operating temperature, maximum reductions have been detected within a temperature range of 15–20°C. This can be explained considering that increasing the temperature decreases the viscosity of olive oil (oxygen solubility in olive oil is almost refractory to temperature change, varying  white > black > blue tea extracts (Lage et al., 2013). Li et al. (2014) reported that addition of GTE, grape seed extract (proanthocyanidins, OPC), gingko leaf extract into gelatin film is an ideal choice to be developed into active food packaging for food preservation.

18.5.5  Grape Seed Extract (GSE) Antimicrobial properties of GSE have been evaluated against L. monocytogenes, S. typhimurium, S. aureus, B. cereus, Enterobacter sakazakii, E. coli O157:H7, Aeromonas hydrophila, and other foodborne pathogens, both in vitro and to a limited extent in foods. Phenolic compounds extracted from defatted grape seed extract have demonstrated inhibitory effects on S. aureus and E. coli. The GSE showed the inhibition of S. aureus after 48 h and the inhibition of A. hydrophila after 1 h. minimum inhibitory concentration of GSE for antilisterial activity was determined as 0.26 mg GAE/L. Furthermore, GSE (1%) when combined with nisin (6400 IU/mL) inhibited L. monocytogenes populations to undetectable levels (minimum detection limit was 100 CFU/g) in full fat turkey frankfurter formulations (21% fat) when stored at 4°C and 10°C (Perumalla and Hettiarachchy, 2011). Lastly, GSEs containing abundant polymeric tannins and monomeric flavonoids such as catechin and epicatechin also show good antioxidant activities, and it has been reported that the oxidation resistance of OPC, active constituent of grape seed extract, is 50 times stronger than that of vitamin E and 20 times stronger than that of vitamin C (Li et al., 2014).

18.5.6 Thyme Plant extracts as an alternative to chemical or synthetic antimicrobials inhibited lipid oxidation and have antimicrobial effects against foodborne pathogens (Perumalla and Hettiarachchy, 2011). Among the most effective EOs, thyme and oregano EOs have been reported to posseses better antimicrobial potential for meat applications, which could be ascribed to the presence of phenolic compounds, particularly thymol and carvacrol (Emiroğlu et al., 2010). Thyme oil has excellent activity against a broad spectrum of microorganisms, including Gram-positive

586  Chapter 18 pathogenic bacteria of L. monocytogenes and S. aureus, Gram-negative bacteria of E. coli O157:H7 and Salmonella, yeasts, and molds (Wu et al., 2014). Regarding Gram-positive bacteria, although S. aureus and S. epidermidis belong to the same genus, thyme preparations only presented activity against S. epidermidis. The most pronounced effect was observed for Gram-negative bacteria, with the order E. coli >  P. vulgaris, P. aeruginosa > E. aerogenes = E. sakazakii (Martins et al., 2005). According to Espitia et al. (2014), films had antimicrobial activity, with thyme essential oil (TEO) being stronger than apple skin polyphenols (ASP) against L. monocytogenes. Considering significant film properties, optimal formulation was 6.07% ASP and 3.1% TEO. Therefore, optimal açai-pectin films have potential food preservation application. The antibacterial effects of oregano EO and TEO exhibited antibacterial activity against Salmonella enteritidis, S. typhimurium, S. aureus, methicillinresistant S. aureus, E. coli, and B. cereus (Boskovic et al., 2015). Most of the natural compounds mentioned above are used in edible films. Edible films are made up of molecular interactions between the polymers used in the film, including covalent bonds (i.e., disulfide interactions and cross-linking), ionic bonds, hydrogen bonds, and electrostatic interactions. These interactions generate films that are rigid and brittle, providing less protection to the food (Palma et al., 2016). The most frequent antimicrobials incorporated into food packaging films are organic acids (e.g., sorbic, benzoic, citric, and propionic acids), enzymes (e.g., lysozyme), bacteriocins (e.g., nisin), polysaccharides (e.g., chitosan), EOs (e.g., bergamot) among others and they can be effective in reducing levels of pathogenic organisms such as E. coli O157:H7, L. monocytogenes, S. thyphimurium, and S. aureus (Rocha et al., 2014) (Table 18.3). Biodegradable films made of polysaccharides, proteins, and lipids have currently a variety of advantages over synthetic materials, such as biodegradability, edibility, biocompatibility, and environmental-friendly properties. These films are loaded with many functional ingredients, such as antioxidants, antimicrobial agents, flavors, spices, and colorants which improve the functionality of the packaging materials via adding novel or extra functions (Hafsa et al., 2016). Other alternatives that have been employed with the objective of improving the properties of the edible films are the chemical and/or physical modification of the starch (Gutierrez et al., 2016). Properties of these films are listed below: 1. Polysaccharide-based films usually show relatively good mechanical properties and oxygen barrier, but their hydrophilic nature results in poor water vapor barrier and moisture resistance (Rodrigues et al., 2016). These films have advantages over the direct application of antibacterial agents on food, because edible films can be designed to slow antimicrobial diffusion to the surface of food. Therefore, smaller amounts of antimicrobials agents would be needed in edible films to achieve a target shelf life, compared with direct application on the food surface (Mohamed et al., 2013).

The Current Approaches and Challenges of Biopreservation  587 2. Lipids may be associated with a hydrophilic material either by laminating a hydrocolloid film with a lipid layer or by forming an emulsion. Although emulsion-based films are usually less effective against water permeation than bilayer films because of the nonhomogeneous distribution of lipids. They have the advantages to present better tensile properties and require a single step to manufacture and application against one step per layer for bilayer and multilayer films. For emulsion films, the smaller and more homogeneously distributed the lipid droplets are, the better is the film performance in terms of barrier to water vapor (Rodrigues et al., 2016). 3. Protein- and polysaccharide-based films generally have good mechanical properties and are good barriers to gases, but not to water vapor. Contrastingly, lipid-based films have good water barrier properties but form brittle films. The combination of proteins and lipids is thus a way of developing composite edible films matching the requirements for use as food packaging (Otoni et al., 2016). Prakash et al. (2014) recommended Boswellia carterii EO as plant-based preservative in view of its antifungal, antiaflatoxigenic, antioxidant activity, and efficacy in food system. Spices play an important role in the edible film packaging, mainly because of their superior antioxidant and antimicrobial properties. Minimum inhibitory concentration mixture of Syzygium aromaticum (clove) and Cinnamomum cassia (cinnamon) was supplemented with optimized edible film mixture and improvement in antioxidant and antibacterial properties of edible films were observed by Mohan et al. (2016). Avila-Sosa et al. (2012) determined that chitosan films exhibited better antifungal effectiveness than amaranth films and chitosan and amaranth films had a significant increase of lag phase.

18.6  Legal Practices Ensuring food safety to protect public health and promote economic development remains a significant challenge for many countries worldwide. Foodborne outbreaks reported in international literature are various. Recently, listeriosis (L. monocytogenes) linked to melon caused more deaths (October 2011 30 deaths; December 2011 33 deaths; February 2012 10 deaths) in the United States (CDC, 2012). It was observed that 2025 outbreaks were associated with seafood in the EU in 2007 (FAO, 2010). A series of precautions have been taken to prevent foodborne illness. Council Directive 70/524/EEC (Council Directive, 1970) on feed additives is based on three main principles: 1. premarket authorization, 2. the positive-list principle. 3. thorough risk assessment of the effects of a particular strain on human and animal health as well as on the environment (Calo-Mata et al., 2008).

588  Chapter 18 In 1988, the FDA recognized nisin as a GRAS for use in pasteurized cheese spreads, and in 1969, a joint FAO/WHO expert committee on food additives concluded that nisin was safe for use as a food additive for a narrow range of foods. Currently, nisin is a permitted food additive in >50 countries, including the United States and some European countries (CaloMata et al., 2008). It also used for fish and seafood product packaging in the form of coated or impregnated film (Ghanbari et al., 2013). The Codex Alimentarius Commission (2009) released a statement in which zero tolerance was adopted in RTE foods that support L. monocytogenes growth. and the Committee exclude from the criteria RTE foods where a limited potential growth over a specified shelf life is ensured (Takahashi et al., 2011). Enterococci (E. faecium) were approved as probiotics (Council Directive 70/524/EEC) (CaloMata et al., 2008). The RTE foods are classified according to consumer risk. First group of RTE foods include those foods which can support the growth of L. monocytogenes throughout the stated shelf life. These foods are milk, dairy products (butter and cream), soft unripened cheeses (>50% moisture) (cottage cheese and ricotta cheese), cooked crustaceans (shrimp and crab), smoked seafood (smoked finfish and mollusks), raw seafood (sushi or sashimi), many vegetables (such as broccoli, cabbage, and salad greens), nonacidic fruit (such as melon, watermelon, and papaya), and some deli-type salads and sandwiches. However, L. monocytogenes does not grow when the pH of the food is less than or equal to 4.4; or the water activity of the food is less than or equal to 0.92; or the food is frozen. These foods are fish, ice cream, other frozen dairy products, processed cheese (cheese foods, spreads, slices), cultured milk products (yogurt, sour cream, buttermilk), hard cheeses (