Trending Topics in Escherichia coli Research: The Latin American Perspective [2 ed.] 3031298810, 9783031298813

Table of contents :
Foreword
Preface
Contents
Contributors
Chapter 1: WHO Critical Priority Escherichia coli in Latin America: A One Health Challenge for a Post-Pandemic World
1.1 Introduction
1.1.1 Critical Priority E. coli in Brazil
1.1.2 Critical Priority E. coli in Argentina
1.1.3 Critical Priority E. coli in Uruguay
1.1.4 Critical Priority E. coli in Chile
1.1.5 Critical Priority E. coli in Ecuador
1.1.6 Critical Priority E. coli in Bolivia
1.1.7 Critical Priority E. coli in Peru
1.2 Conclusions
References
Untitled
Untitled
Chapter 2: Recent Progress on Enterotoxigenic E. coli (ETEC) and Antibiotic Resistance in Pathogenic E. coli
2.1 General Concepts About ETEC
2.1.1 ETEC Is a Major Human Pathogen
2.1.2 ETEC Can Be a Food and Waterborne Pathogen
2.1.3 Animal ETEC
2.2 Recent Discoveries in Virulence and Pathogenesis
2.3 Epidemiology of ETEC in Latin America
2.4 Deciphering ETEC Evolution and Dissemination Through Genomics
2.5 The Microbiome and ETEC
2.6 ETEC Vaccines
2.7 Updates on Antibiotic Resistance in Intestinal and Extraintestinal E. coli
2.7.1 Antibiotic Resistance in Diarrheagenic E. coli (DEC)
2.7.2 Antibiotic Resistance in Extraintestinal E. coli (ExPEC)
References
Chapter 3: New Concepts on Domestic and Wild Reservoirs and Transmission of E. coli and Its Environment
3.1 Introduction
3.2 Animal Reservoirs of Several Pathotypes Described in the Last Years
3.2.1 Small Ruminants
3.2.2 Other Ungulates
3.2.3 Pets
3.2.4 Birds
3.2.5 Rodents
3.2.6 Other Reports from Wild Animals
3.3 E. coli Outside the Host
3.3.1 E. coli in the Aquatic Environment: Fecal Source or Adaptation?
3.3.2 E. coli in Soil
3.3.3 E. coli in the Soil of Urban Areas
3.3.4 STEC in the Environment of Farm
3.4 E. coli as a Source of Antimicrobial Resistance
3.4.1 Antimicrobial Resistance Crisis and Commensal E. coli
3.4.2 AMR in the Environment
3.4.3 AMR in the Food
3.4.4 Directionality of Domestic Animals and Humans
References
Chapter 4: New Molecular Mechanisms of Virulence and Pathogenesis in E. coli
4.1 Introduction
4.2 The Type III Secretion System (T3SS) in E. coli
4.2.1 Architecture of the Type III Secretion System
4.2.2 Hierarchical Substrate Secretion: Molecular Switches
4.3 Type III Secretion System as Targets of Anti-virulence Agents for Pathogenic E. coli
4.4 Role of the Type VI Secretion System (T6SS) in E. coli
4.4.1 Structure of the T6SS
4.4.2 E. coli T6SS Effectors
4.5 Virulence Response of Pathogenic E. coli to the Microbiota
4.5.1 Regulation of E. coli Virulence Factors by the Microbiota
4.5.2 Modulation of E. coli Pathogenesis by Bacterial Proteases
4.5.3 Interbacterial Competition Using the T6SS
4.6 Role of Bacterial Cell Surface Structures in E. coli Biofilm Formation
4.6.1 Levels of Regulation in the Expression of Colonization Factors
4.6.1.1 Regulation at the Pre-transcriptional Level: Phase Variation
4.6.1.2 Regulation at the Transcriptional Level: Regulators and Effectors
4.6.1.3 Regulation at the Post-transcriptional Level
4.6.1.4 Regulation at the Translational Level
4.6.1.5 Regulation at the Post-translational Level
4.7 Cross-Communication Mechanisms Between Pathogenic E. coli and Cell-Host
4.7.1 Functionality and Effect of Bacterial OMVs
4.7.2 STEC and AIEC as Pathogenicity Models of Cross-Communication Mediated by OMVs
4.8 Conclusions
References
Chapter 5: Bovine Reservoir of STEC and EPEC: Advances and New Contributions
5.1 Shiga Toxin-Producing Escherichia coli (STEC)
5.2 Enteropathogenic Escherichia coli (EPEC)
5.3 Bovine Reservoir
5.4 Prevalence of STEC in Bovines: The Latin American Perspective
5.4.1 STEC in Bovines from Argentina
5.4.2 STEC Prevalence in Bovines from Brazil
5.4.3 STEC in Chilean Cattle
5.4.4 STEC in Cattle from Colombia
5.4.5 STEC Prevalence in Bovine Beef in Paraguay
5.4.6 STEC Prevalence in Dairy Cattle in Uruguay
5.4.7 EPEC Prevalence in Bovines: Latin America
5.5 Serotypes, Virulence Factors, and Resistance Mechanisms in STEC and EPEC Isolated from Cattle
5.6 Antibiotic Resistance
5.7 Biofilm Formation
5.8 Prevention and Control
5.8.1 Animal Diet
5.8.2 Feed Additive
5.8.3 Immunization and Bacteriophage Therapy
5.8.4 Inhibition of STEC Biofilm
References
Chapter 6: Phages and Escherichia coli
6.1 Brief and General Description of Phages
6.2 Natural Interactions Between Phages and Bacteria
6.3 Phages and STEC
6.3.1 Phages That Encode Shiga Toxins (Stx Phages)
6.3.2 Stx Phages from STEC Strains of Latin America
6.3.3 Stx Phage and HUS Development: The Forgotten Piece
6.4 Bacteriophages as Therapeutic Agents: Advantages and Disadvantages
6.5 Phage Biocontrol/Therapy Against Escherichia coli Pathotypes in Latin America
6.5.1 Phages Against Enteropathogenic E. coli
6.5.2 Phages Against Shiga Toxin-Producing E. coli
6.5.3 Phages Against Uropathogenic E. coli
6.5.4 Phages Tested Against Biofilms Formed by E. coli
6.6 Conclusions
References
Chapter 7: Insights into Animal Carriage and Pathogen Surveillance in Latin America: The Case of STEC and APEC
7.1 General Concepts
7.2 General Concepts of Shiga Toxin-Producing E. coli (STEC)
7.3 STEC in Animals
7.4 STEC in Animals in Latin American Countries
7.4.1 Chile
7.4.2 Argentina
7.4.3 Brazil
7.5 Foodborne Infection Surveillance in Latin America
7.6 Avian Pathogenic Escherichia coli (APEC)
7.7 APEC in Latin American Countries
7.8 Current Situation in Brazil
7.9 Perspectives and Control
References
Chapter 8: Shiga Toxin and Its Effect on the Central Nervous System
8.1 Introduction
8.2 Predictors of Neurological Deficits as Worst Risk Factors in Typical HUS
8.3 Deleterious Action of Stx in Neurons
8.4 BBB Functional Loss by Endothelial Cells
8.5 Cerebrospinal Fluid-Brain Barrier Impairment: Involvement of AQP4
8.6 Involvement of Glial Cells in STEC-HUS Encephalopathy
8.6.1 Reactive Astrocytes Triggering Neuroinflammation by STEC Toxins
8.6.2 Heat or LPS Modulate the Microglial Response to Stx
8.6.3 Oligodendrocytes Are Oxidative and Proinflammatory Targets
8.7 Current Pharmacological Treatments Used for STEC-HUS Encephalopathy
8.7.1 Steroid Pulse Therapy
8.7.2 Immunoglobulin G (IgG) Immunoadsorption
8.7.3 Complement Factor Binding Antibody
8.8 Perspectives on Stx-Produced Encephalopathy Treatment
8.9 Conclusion
References
Chapter 9: Relevance of Escherichia coli in Fresh Produce Safety
9.1 Introduction
9.2 Relevance of Total and Fecal Coliforms and E. coli Detection in Produce
9.2.1 Total and Fecal Coliforms
9.2.2 Isolation and Identification of E. coli (Generic and Pathogenic) in Fresh Produce
9.3 Colonization and Internalization of E. coli
9.3.1 Adherence
9.3.2 Mechanisms of Colonization
9.3.3 Internalization
9.3.4 Biofilms
9.3.5 Other Mechanisms
9.4 Presence of Generic and Diarrheagenic E. coli in Fresh Produce in Latin America
9.5 Sources of Contamination During the Preharvest and Postharvest of Fresh Vegetable Production
9.5.1 Preharvest Factors
9.5.2 Postharvest Factors
9.6 Preventive Measures as Safety Mitigation Strategies in Fresh Produce
9.7 Antimicrobial Alternatives for Postharvest Use in Fresh Vegetables
9.8 Conclusion
References
Chapter 10: Quantitative Microbial Risk Assessment of Hemolytic Uremic Syndrome due to Beef Consumption: Impact of Interventions to Reduce the Presence of Shiga Toxin-Producing Escherichia coli
10.1 Introduction
10.2 Quantitative Microbial Risk Assessment (QMRA)
10.3 Antimicrobial Effect of Different Interventions Against STEC on Beef Under Controlled Experimental Conditions
10.4 Antimicrobial Effect of Different Interventions Against STEC on Beef Carcasses in Commercial Abattoirs
10.5 Analysis of Scenarios to Reduce the Probability of Acquiring HUS Associated with Beef Consumption
References
Chapter 11: An Updated Overview on the Resistance and Virulence of UPEC
11.1 General Characteristics of UPEC
11.2 UPEC Virulence Factors and Pathogenicity
11.2.1 Interaction of UPEC Host Cells
11.3 Mechanisms of Resistance to the Main Antibiotics Used in Clinical Practice Against UPEC
11.3.1 β-Lactam Resistance
11.3.2 Aminoglycoside Resistance
11.3.3 Quinolone Resistance
11.3.4 Sulfamethoxazole-Trimethoprim Resistance
11.3.5 Polymyxin Resistance
11.4 Epidemiology of the Antimicrobial Resistance of UPEC in Latin America
11.5 Alternative Therapeutics and Prevention Against UPEC Strains Causing UTIs
11.5.1 Vaccines
11.5.2 Plant Extracts
11.5.3 Probiotics
11.5.4 Phage Therapy
11.5.5 Additional Approaches
References
Chapter 12: Interactions of Pathogenic Escherichia coli with Gut Microbiota
12.1 Microbiota in Health and Disease
12.1.1 Crosstalk Between Gut Microbiota and Enteric Pathogens
12.1.1.1 Nutrients Availability
12.1.1.2 Mucus Barrier
12.1.2 Co-infections
12.1.3 Omics Tools for a More Comprehensive View of the Molecular and Physiological Events Underlying Diarrheal Disease
12.1.4 Microbiota Changes During DEC Infections
12.1.4.1 Interactions Between Gut Microbiota and STEC
12.1.4.2 Gut Microbiota in STEC-Infected Patients
References
Chapter 13: Emergence of Hybrid Escherichia coli Strains
13.1 Hybrid Diarrheagenic E. coli Pathotypes Associated with Human Intestinal Infections
13.2 Hybrid Extraintestinal Pathogenic E. coli: One Pathogen, Two Diseases
13.3 Research Perspectives in the Area
13.4 Potential Clinical Implications of Hybrids (More Severe Clinical Cases?)
13.5 Conclusions
References
Chapter 14: Genomic Analysis of Pathogenic Escherichia coli Strains in Latin America
14.1 Complexity of the Pathogenic E. coli Diagnosis and Surveillance
14.2 Moving from Conventional Methodologies to Whole-Genome Sequencing: A Brief Tour
14.3 General Approaches for the Implementation of Whole-Genome Sequencing in Latin America
14.4 The E. coli Whole-Genome Sequencing and Data Analysis in Practice
14.5 Driving Toward E. coli Genomic Surveillance in Latin America
14.5.1 Global Framework for Genomic Epidemiology
14.5.2 Specific Strategies for Whole-Genome Sequencing Analysis Applied Worldwide for E. coli Surveillance and Outbreak Investigation
14.5.3 Advances to Tackle the E. coli Genomic Surveillance in Latin America
14.6 Genomics for Identification and Characterization of Virulence Factors and Antimicrobial Resistance
14.7 Omics for Culture-Independent Subtyping of STEC and Outbreak Investigations
14.8 Conclusions
References
Chapter 15: Therapeutic Options for Diarrheagenic Escherichia coli
15.1 Historical Perspective
15.2 Diarrheagenic Escherichia coli Characteristics and Clinical Description
15.2.1 Enteropathogenic E. coli
15.2.2 Enterotoxigenic E. coli
15.2.3 Enteroaggregative E. coli
15.2.4 Shiga Toxin-Producing E. coli/Enterohemorrhagic E. coli
15.3 Diarrheal Features: Prevention and Management
15.3.1 Dehydrating Diarrhea
15.3.2 Non-STEC Bloody Diarrhea
15.3.3 Bloody Diarrhea/Dysentery and HUS Associated with STEC
15.3.4 Extraintestinal Infections
15.4 Alternative Options for Treatment
15.4.1 Antibodies
15.4.2 Bacteriophage Therapy
15.5 Remarks and Research Perspectives
References
Index

Citation preview

Alfredo G. Torres   Editor

Trending Topics in Escherichia coli Research The Latin American Perspective Second Edition

Trending Topics in Escherichia coli Research

Alfredo G. Torres Editor

Trending Topics in Escherichia coli Research The Latin American Perspective Second Edition

Editor Alfredo G. Torres Department of Microbiology and Immunology University of Texas Medical Branch Galveston, TX, USA

ISBN 978-3-031-29881-3    ISBN 978-3-031-29882-0 (eBook) https://doi.org/10.1007/978-3-031-29882-0 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2016, 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Foreword

The knowledge of the biology of the Escherichia coli species began at the end of the nineteenth century when the species was first described and named Bacterium coli commune by Theodor Escherich. Some strains belonging to this species were soon identified by the same discoverer as potential cause of urinary tract and intestinal infections in the subsequent 10 years, shortly before the species was renamed after him. The knowledge of the organism lagged until after World War II when W. H. Ewing, from the communicable diseases center of Atlanta in the USA, observed that “In a large number of otherwise unexpected epidemics of diarrheal disease of infants, certain Escherichia coli serotypes are recovered”. This observation raised a certain interest, and in the subsequent years, the reports of E. coli infections associated with both intestinal and extraintestinal disease accumulated in the low- and high-income countries, respectively, and the studies on the mechanisms of interaction between these bacteria and the human host hinted at the extraordinary plasticity of this species in deploying adhesion and invasion mechanisms as well as in producing toxins. In the 1980s, the identification of a new pathogenic group of E. coli, Shiga toxin-­ producing E. coli (STEC), was determined to be the cause of severe forms of gastro-­ intestinal infections to humans and as the cause of hemolytic uremic syndrome (HUS) in childhood, mostly preceded by diarrhea. This discovery boosted again the microbiologists to study the organism and to decide to look deeper into the evolution of the pathogenic strains belonging to the E. coli species, gave pediatricians treating children with HUS better tools to characterize their patients and study the epidemiology and pathophysiology of the STEC infections and STEC-induced hemolytic uremic syndrome (STEC-HUS), and stimulated researchers from various backgrounds and all over the world to unravel the habits of these intriguing pathogens and their relation to disease. It was with the diffusion of polymerase chain reaction and, later, of genomics that the understanding of how diverse and plastic this species was made a quantum leap.

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As a matter of fact, the quick and sensitive detection of the genes associated to virulence allowed an easier attribution of the E. coli strains isolated from the different forms of illness to the different pathogenic types and permitted a deeper knowledge of their circulation into their natural reservoirs, eventually shedding light on their potential to expose and cause disease to humans and animals. At the same time, the ability to delve into the phylogenesis brought the researchers to describe the dynamics that led the different E. coli pathotypes and serotypes to diversify into subpopulations with more potential to harm and cause disease. The ability to completely sequence the genome of the pathogenic E. coli strains permitted to determine the size of the core genome of the E. coli species, which is largely used as a base for the strain typing, and to define the accessory genome, conveying all the determinants linked to pathogenicity and conferring traits favoring the survival and persistence of the species into countless niches. One of the latest discoveries concerning the extreme plasticity of the E. coli species is related with its talent for spreading mobile genetic elements conveying virulence genes and generating cross-pathotype or hybrid strains. The first example of a hybrid pathotype with a shuffled virulence repertoire was the Shiga toxin-producing enteroaggregative E. coli (Stx-producing EAEC). Such a hybrid combination of virulence features was first identified in an STEC strain of serogroup O111 associated with a small outbreak of hemolytic uremic syndrome (HUS) in France at the beginning of the 1990s and followed 20 years later by the infamous O104:H4 strain that caused in 2011 the largest outbreak occurred in Europe and the most severe STEC epidemic ever reported. Retrospectively, similar strains of other serogroups have been described in several sporadic cases and small outbreaks of infections in Japan, Italy, and Northern Ireland. The emergence of the Stx-producing EAEC seems to be linked to the acquisition of an Stx-converting phage by an enteroaggregative E. coli strain, which is largely supported by the high capacity of bacteriophages to infect E. coli and spread their load of transduced genes, including the Stx-coding ones. Whole genome sequencing allowed the researchers to discover that, beside the Stx-producing EAEC, hybrid combinations of virulence determinants can be found in E. coli strains belonging all the E. coli pathotypes. Of public health impact are the Stx-producing ETEC and the STEC with the virulence genes repertoire of extraintestinal pathogenic E. coli, often isolated from severe forms of HUS with bacteremia, to mention a few of these. The emergence of STEC strains with virulence genes of other E. coli pathotypes, raise the point of how STEC, are to be identified. As a matter of fact, the ability of Stx-phages to move between strains together with their wide diffusion in the reservoirs and the environment makes any E. coli potentially an STEC and suggests that this pathotype could be considered an association of two organisms, a pathogenic E. coli and an Stx-phage. Even though more than 130 years have passed since its discovery, the species E. coli continues to represent a public health problem around the world with new groups of pathogenic strains continuously emerging. Their extreme genomic plasticity generates new variants and pathotypes that prompt researchers to carry out new studies and research to understand the extraordinary adaptability of this species.

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This book represents the most up-to-date collection of ideas and data produced by scientists from Latin America and will certainly be the starting point for further new knowledge to be produced by the global scientific community. Stefano Morabito European Reference Laboratory for E. coli Microbiological food safety and food borne diseases unit. Food safety, Nutrition, and Veterinary Public Health Department Istituto Superiore di Sanità Roma, Italy Nicole van de Kar Department of Pediatric Nephrology, Radboud Institute for Molecular Life Sciences, Amalia Children’s Hospital Radboud University Medical Center Nijmegen, The Netherlands January 24, 2023

Preface

“You are the Petri dish to Escherichia coli, and it could be your friend or your foe”.

Pathogenic Escherichia coli infections remain a major global public health issue in Latin America and around the globe, and despite efforts to combat human and animal infections, these bacteria remain associated with disease and significant morbidity. In the Americas, the need to understand this pathogen and to tackle all the challenges that E. coli infections posed to the medical, veterinary, research communities and population at large resulted in the establishment of the Latin American Coalition for E. coli Research (LACER). This regional scientific group was created in 2009 to promote and expand the research efforts in the Americas using the One Health model, to support and expand quality science within Latin American counties, to prepare the next generation of scientist-physicians and research investigators, and to work together with the community to translate regional scientific findings into products improving the well-being of the population suffering from E. coli infections. By 2023, the LACER group has grown to become a multidisciplinary network of more than 70 research groups in Latino America and the USA, working on different aspects of pathogenic E. coli, including, but not restricted to, epidemiology, pathogenesis, vaccine and therapeutic design and testing, public health, surveillance, and clinical bacterial isolation and treatment. One of the key missions of the LACER community is to disseminate their scientific findings in the form of manuscripts, invited commentaries, social media post, books, and interviews. As part of the efforts to bring the knowledge about these bacteria to the students, researchers, and the community in general, the first book by the LACER network was published in 2010 and entitled Pathogenic Escherichia coli in Latin America. The central theme of this book was to present the current understanding of the Latin American research community about pathogenic E. coli, featuring comprehensive analysis of the most common categories of E. coli associated with diarrheal illness, as well as outlining prospects for future research in the region. The second book was published in 2016 and entitled Escherichia coli in the Americas. The theme of this book included a series of chapters written by Latin American experts, covering basic concepts regarding the different categories of ix

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E. coli, including their environmental niche, virulence mechanisms, host reservoir, and disease outcomes, as well as diagnosis, vaccine development, and treatment. This second book was written to target trainees and students learning about the basic and clinical aspects of E. coli pathogenesis, but also providing information to experts around the globe who wish to learn more about this pathogen and the public health impact these bacteria is having in the Americas. As the LACER network gained maturity with several consolidated research groups who are driving novel research projects in the Latin American region, as well as the presence of new investigators initiating their independent research careers and laboratories, it was decided that it was time again to publish a book displaying the talent of all these scientists and their recent research findings. This third book is entitled Trending Topics in Escherichia coli Research. The Latin American Perspective, and it represents a collection of chapters that have unique characteristics: they are written by a multidisciplinary group of experts, and they all emphasize research progress performed in the Latin American region. Many of the topics included in this book are novel because they represent new areas of research in the region including priority areas such as the One Health challenge to understand E. coli isolated in the Americas as well as the need to monitor emergence of antibiotic resistant strains. The other chapters present recent progress on understanding virulence of different pathogenic E. coli strains and their association with human or animal infections or their presence in the environment. More specialized chapters included in the book discussed the importance of phages in the lifestyle of E. coli or the role of Shiga toxin and its effect on the central nervous system. Other topics discussed the interactions of these bacteria with fresh produce and beef consumption and the need of microbial risk assessments to predict food contamination and increase prevention. Finally, the book also includes new areas of research emerging in the Latin American region, such as the need to understand the association of E. coli with intestinal microbiota and the emergence of hybrid pathogenic E. coli strains. All these research advances required complete analysis of the E. coli genomes to understand their pathogenic profiles and to help in the development of novel therapeutic approaches. As we continue advancing the goals of the LACER network for the benefit of the Latin American community, we must keep in mind that the best approach to tackle the challenges that we have in this region is to use One Health principles, because they can help us assess the interdisciplinary interaction and interdependence between health and wellbeing in a constantly changing environment. We must continue using these principles to encourage sustainable collaborative partnerships and to promote optimal health for people, animals, plants, and the environment of the Latin American region, information that can be exported to the whole world. In Latin America, despite the concept still being discussed among health professionals and educators, we have been incorporating several of the One Health initiatives in the LACER goals. LACER has become a network that is recognized by microbiological and other scientific societies in Latin America and by E. coli investigators worldwide as a group that has created a model that combines unique talent, expertise, and

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collaborative attitudes that makes the group stronger together than apart. It is my hope that this book motivates students and trainees to join LACER and become the next generation of researchers in Latin America. Galveston, TX, USA January 12, 2023

Alfredo G. Torres

Contents

1 W  HO Critical Priority Escherichia coli in Latin America: A One Health Challenge for a Post-Pandemic World��������������������������    1 Nilton Lincopan, Danny Fuentes-Castillo, Maria Espinoza-Muñoz, Fernando Gonzales-Zubiate, Edgar Gonzales-Escalante, Lenin Maturrano, Rafael Vignoli, Jose Di Conza, and Gabriel Gutkind 2 Recent  Progress on Enterotoxigenic E. coli (ETEC) and Antibiotic Resistance in Pathogenic E. coli������������������������������������   33 Enrique Joffré, Jeannete Zurita, Carla Calderon Toledo, and Sergio Gutiérrez-Cortez 3 New  Concepts on Domestic and Wild Reservoirs and Transmission of E. coli and Its Environment ��������������������������������   55 Adriana Bentancor, Ximena Blanco Crivelli, Claudia Piccini, and Gabriel Trueba 4 New  Molecular Mechanisms of Virulence and Pathogenesis in E. coli����������������������������������������������������������������������������������������������������   79 Fernando Navarro-García, Antonio Serapio-Palacios, Bertha González-­Pedrajo, Mariano Larzábal, Nora Molina, and Roberto Vidal 5 Bovine  Reservoir of STEC and EPEC: Advances and New Contributions����������������������������������������������������������������������������  107 Nora Lía Padola, Vinicius Castro, Analía Etcheverría, Eduardo Figueiredo, Rosa Guillén, and Ana Umpiérrez 6 Phages and Escherichia coli��������������������������������������������������������������������  129 Paula M. A. Lucchesi, Leticia V. Bentancor, Alejandra Krüger, Edgar González-Villalobos, and José Molina-López

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7 Insights  into Animal Carriage and Pathogen Surveillance in Latin America: The Case of STEC and APEC ��������������������������������  149 Nicolás Galarce, Fernando Sánchez, Indira Kudva, Erika N. Biernbaum, Terezinha Knöbl, and André B. S. Saidenberg 8 Shiga  Toxin and Its Effect on the Central Nervous System ����������������  177 Alipio Pinto, Ana Beatriz Celi, and Jorge Goldstein 9 Relevance of Escherichia coli in Fresh Produce Safety������������������������  205 Juan J. Luna-Guevara, Magaly Toro, Christian Carchi-Carbo, Juan L. Silva, and M. Lorena Luna-Guevara 10 Quantitative  Microbial Risk Assessment of Hemolytic Uremic Syndrome due to Beef Consumption: Impact of Interventions to Reduce the Presence of Shiga Toxin-­Producing Escherichia coli��������������������������������������������  229 Victoria Brusa, Mariana Cap, Gerardo Leotta, Marcelo Signorini, and Sergio Vaudagna 11 An  Updated Overview on the Resistance and Virulence of UPEC������  249 Edwin Barrios-Villa, Luciana Robino Picón, Rodolfo Bernal Reynaga, and Margarita María de la Paz Arenas-Hernández 12 I nteractions of Pathogenic Escherichia coli with Gut Microbiota��������������������������������������������������������������������������������  277 Elizabeth Miliwebsky, María Ángela Jure, Mauricio J. Farfan, and Marina Sandra Palermo 13 Emergence of Hybrid Escherichia coli Strains��������������������������������������  295 Tânia Aparecida Tardelli Gomes, Ana Carolina de Mello Santos, Rodrigo Tavanelli Hernandes, Monica Yurley Arias-Guerrero, Ana Elvira Farfán-García, and Oscar G. Gómez-Duarte 14 G  enomic Analysis of Pathogenic Escherichia coli Strains in Latin America��������������������������������������������������������������������������������������  317 Isabel Chinen, Carolina Carbonari, Natalie Weiler Gustafson, Cindy Fabiola Hernández Pérez, Bruna Fuga, and Narjol González-Escalona 15 Therapeutic  Options for Diarrheagenic Escherichia coli ��������������������  339 Alejandro Balestracci, Daniela Luz, Flavia Sacerdoti, Maria Marta Amaral, Oscar G. Gómez-Duarte, and Roxane Maria Fontes Piazza Index������������������������������������������������������������������������������������������������������������������  361

Contributors

Maria  Marta  Amaral  Universidad de Buenos Aires, Facultad de Ciencias Médicas, Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia, Buenos Aires, Argentina CONICET  – Universidad de Buenos Aires. Instituto de Fisiología y Biofísica Bernardo Houssay (IFIBIO Houssay), Buenos Aires, Argentina Margarita  María  de  la  Paz  Arenas-Hernández  Posgrado en Microbiología, Centro de Investigación en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Puebla, Puebla, Mexico Monica Yurley Arias-Guerrero  Instituto de Investigaciones Masira, Facultad de Ciencias Médicas y de la Salud, Universidad de Santander, Bucaramanga, Colombia Alejandro Balestracci  Unidad de Nefrología, Hospital General de Niños Pedro de Elizalde, Ciudad Autónoma de Buenos Aires, Argentina Edwin Barrios-Villa  Universidad de Sonora, Departamento de Ciencias Químico Biológicas y Agropecuarias, Caborca, Sonora, Mexico Adriana  Bentancor  Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Microbiología, CABA, Argentina Leticia  V.  Bentancor  Instituto de Estudios para el Desarrollo Productivo y la Innovación (IDEPI), Universidad Nacional de José C. Paz (UNPaz), José C. Paz, Pcia, Buenos Aires, Argentina Rodolfo  Bernal  Reynaga  Unidad de Investigaciones en Salud Pública “Dra. Kaethe Willms,” Facultad de Ciencias Químico-Biológicas, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa, Mexico Erika N. Biernbaum  Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, USA Oak Ridge Institute for Science and Education, Oak Ridge, TN, USA xv

xvi

Contributors

Ximena  Blanco  Crivelli  Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Microbiología, CABA, Argentina Victoria  Brusa  IGEVET  – Instituto de Genética Veterinaria “Ing. Fernando N.  Dulout” (UNLP-CONICET LA PLATA), Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, Buenos Aires, Argentina Carla Calderon Toledo  Unidad de Microbiología Ambiental, Instituto de Biología Molecular y Biotecnología (IBMB), Carrera de Biología, Universidad Mayor de San Andrés, La Paz, Bolivia Mariana Cap  Instituto Tecnología de Alimentos, INTA, Buenos Aires, Argentina Instituto de Ciencia y Tecnología de Sistemas Alimentarios Sustentables (UEDD INTA-CONICET), Buenos Aires, Argentina Carolina Carbonari  Servicio Fisiopatogenia, Instituto Nacional de Enfermedades Infecciosas – ANLIS “Dr. Carlos G. Malbrán”, Buenos Aires, Argentina Christian Carchi-Carbo  Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile Vinicius  Castro  Department of Biological Science, University of Lethbridge (ULETH), Lethbridge, AB, Canada Ana  Beatriz  Celi  Universidad de Buenos Aires, Facultad de Medicina, Departamento de Ciencias Fisiológicas, Buenos Aires, Argentina Universidad de Buenos Aires-CONICET, Instituto de Fisiología y Biofísica “Houssay” (IFIBIO), Laboratorio de Neurofisiopatología, Buenos Aires, Argentina Isabel  Chinen  Servicio Fisiopatogenia, Instituto Nacional de Enfermedades Infecciosas – ANLIS “Dr. Carlos G. Malbrán”, Buenos Aires, Argentina Jose Di Conza  Facultad de Farmacia y Bioquímica, Instituto de Investigaciones en Bacteriologia y Virología Molecular, Universidad de Buenos Aires, Buenos Aires, Argentina Maria  Espinoza-Muñoz  Hospital Cochabamba, Bolivia

Seguro

Social

Universitario,

Analía Etcheverría  Universidad Nacional del Centro de la Provincia de Buenos Aires, Facultad Ciencias Veterinarias, Departamento Sanidad Animal y Medicina Preventiva- CIVETAN, Tandil, Buenos Aires, Argentina Mauricio  J.  Farfan  Departamento de Pediatría y Cirugía Infantil, Facultad de Medicina, Universidad de Chile, Santiago, Chile Ana  Elvira  Farfán-García  Instituto de Investigaciones Masira, Facultad de Ciencias Médicas y de la Salud, Universidad de Santander, Bucaramanga, Colombia Eduardo  Figueiredo  Departamento de Alimentos e Nutrição, Universidade Federal de Mato Grosso, Cuiabá, Mato Grosso, Brazil

Contributors

xvii

Danny  Fuentes-Castillo  Departamento de Patología y Medicina Preventiva, Facultad de Ciencias Veterinarias, Universidad de Concepción, Chillán, Chile Bruna Fuga  Departamento Microbiologia, Instituto de Ciências Biomédicas, São Paulo, Brazil Nicolás Galarce  Escuela de Medicina Veterinaria, Facultad de Ciencias de la Vida, Universidad Andrés Bello, Santiago, Chile Jorge  Goldstein  Universidad de Buenos Aires, Facultad de Medicina, Departamento de Ciencias Fisiológicas, Buenos Aires, Argentina Universidad de Buenos Aires-CONICET, Instituto de Fisiología y Biofísica “Houssay” (IFIBIO), Laboratorio de Neurofisiopatología, Buenos Aires, Argentina Tânia  Aparecida  Tardelli  Gomes  Laboratório Experimental de Patogenicidade de Enterobactérias, Disciplina de Microbiologia, Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil Oscar  G.  Gómez-Duarte  International Enteric Vaccine Research Program, Division of Pediatric Infectious Diseases, Department of Pediatrics, State University of New York at Buffalo, Buffalo, NY, USA Division of Pediatric Infectious Diseases, Department of Pediatrics, State University of New York (SUNY) at Buffalo, Buffalo, NY, USA Edgar  Gonzales-Escalante  Facultad de Medicina, Universidad de Piura, Lima, Peru Fernando  Gonzales-Zubiate  Escuela de Ciencias Biológicas e Ingeniería, Universidad Yachay Tech, San Miguel de Urcuquí, Ecuador Narjol  González-Escalona  Division of Microbiology, Office of Regulatory Science, Food and Drug Administration, Silver Spring, MD, USA Bertha  González-Pedrajo  Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico Edgar González-Villalobos  Laboratorio de Patogenicidad Bacteriana, Unidad de Hemato-oncología e investigación, Hospital Infantil de México “Federico Gómez”, Facultad de Medicina UNAM, México City, México Rosa  Guillén  Instituto de Investigaciones en Ciencias de la Salud, Universidad Nacional de Asunción, San Lorenzo, Paraguay Sergio  Gutiérrez-Cortez  Unidad de Microbiología Ambiental, Instituto de Biología Molecular y Biotecnología (IBMB), Carrera de Biología, Universidad Mayor de San Andrés, La Paz, Bolivia Gabriel Gutkind  Facultad de Farmacia y Bioquímica, Instituto de Investigaciones en Bacteriologia y Virología Molecular, Universidad de Buenos Aires, Buenos Aires, Argentina

xviii

Contributors

Rodrigo Tavanelli Hernandes  Departamento de Ciências Químicas e Biológicas (Setor de Microbiologia e Imunologia), Instituto de Biociências, Universidade Estadual Paulista (UNESP), Botucatu, SP, Brazil Cindy Fabiola Hernández Pérez  Centro Nacional de Referencia de Plaguicidas y Contaminantes – SENASICA, Ciudad de México, Mexico Enrique  Joffré  Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden María  Ángela  Jure  Cátedra de Bacteriología. Instituto de Microbiología Luis C. Verna. Fac. de Bioquímica, Qca y Fcia. Universidad Nacional de Tucumán, San Miguel de Tucumán, Argentina Terezinha  Knöbl  Department of Pathology, School of Veterinary Medicine and Animal Science – University of São Paulo (FMVZ-USP), São Paulo, SP, Brazil Alejandra  Krüger  Universidad Nacional del Centro de la Provincia de Buenos Aires (UNCPBA), Facultad de Ciencias Veterinarias, CISAPA, Tandil, Pcia. Buenos Aires, Argentina Centro de Investigación Veterinaria de Tandil (CIVETAN), UNCPBA-­CICPBA-­ CONICET, Tandil, Pcia. Buenos Aires, Argentina Indira Kudva  Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, USA Mariano  Larzábal  Instituto de Agrobiotecnología y Biología Molecular INTA-­ CONICET, Hurlingham, Buenos Aires, Argentina Gerardo  Leotta  Instituto de Ciencia y Tecnología de Sistemas Alimentarios Sustentables (UEDD INTA-CONICET), Buenos Aires, Argentina Nilton  Lincopan  Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil Paula M. A. Lucchesi  Universidad Nacional del Centro de la Provincia de Buenos Aires (UNCPBA), Facultad de Ciencias Veterinarias, CISAPA, Tandil, Pcia. Buenos Aires, Argentina Centro de Investigación Veterinaria de Tandil (CIVETAN), UNCPBA-­CICPBA-­ CONICET, Tandil, Pcia. Buenos Aires, Argentina Juan  J.  Luna-Guevara  College of Food Engineering, Faculty of Chemical Engineering, Meritorious Autonomous University of Puebla, Puebla, Mexico M.  Lorena  Luna-Guevara  College of Food Engineering, Faculty of Chemical Engineering, Meritorious Autonomous University of Puebla, Puebla, Mexico Daniela Luz  Laboratório de Bacteriologia, Instituto Butantan, São Paulo, SP, Brazil

Contributors

xix

Lenin Maturrano  Facultad de Medicina Veterinaria, Universidad Nacional Mayor de San Marcos, Lima, Peru Elizabeth  Miliwebsky  Servicio Fisiopatogenia, Departamento de Bacteriología, Instituto Nacional de Enfermedades Infecciosas” Carlos G.  Malbrán”, CABA, Argentina Nora Molina  Centro Universitario de Estudios Microbiológicos y Parasitológicos, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, Buenos Aires, Argentina José Molina-López  Laboratorio de Patogenicidad Bacteriana, Unidad de Hemato-­ oncología e investigación, Hospital Infantil de México “Federico Gómez”, Facultad de Medicina UNAM, México City, México Fernando Navarro-García  Department of Cell Biology, Centro de Investigación y de Estudios Avanzados, Mexico City, Mexico Nora  Lía  Padola  Universidad Nacional del Centro de la Provincia de Buenos Aires, Facultad Ciencias Veterinarias, Departamento Sanidad Animal y Medicina Preventiva- CIVETAN, Tandil, Buenos Aires, Argentina Marina Sandra Palermo  Instituto de Medicina Experimental (IMEX) CONICET-­ Academia Nacional de Medicina, CABA, Argentina Roxane  Maria  Fontes  Piazza  Laboratório de Bacteriologia, Instituto Butantan, São Paulo, SP, Brazil Claudia  Piccini  Departamento de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay Alipio Pinto  Universidad de Buenos Aires, Facultad de Medicina, Departamento de Ciencias Fisiológicas, Buenos Aires, Argentina Universidad de Buenos Aires-CONICET, Instituto de Fisiología y Biofísica “Houssay” (IFIBIO), Laboratorio de Neurofisiopatología, Buenos Aires, Argentina Luciana  Robino  Picón  Universidad de la Republica, Facultad de Medicina, Instituto de Higiene, Departamento de Bacteriologia y Virologia, Montevideo, Uruguay Flavia  Sacerdoti  Universidad de Buenos Aires, Facultad de Ciencias Médicas, Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia, Buenos Aires, Argentina CONICET  – Universidad de Buenos Aires. Instituto de Fisiología y Biofísica Bernardo Houssay (IFIBIO Houssay), Buenos Aires, Argentina André B. S. Saidenberg  Department of Pathology, School of Veterinary Medicine and Animal Science – University of São Paulo (FMVZ-USP), São Paulo, SP, Brazil

xx

Contributors

Fernando  Sánchez  Departamento de Medicina Preventiva Animal, Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Santiago, Chile Ana Carolina de Mello Santos  Laboratório Experimental de Patogenicidade de Enterobactérias, Disciplina de Microbiologia, Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil Antonio  Serapio-Palacios  Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada Marcelo  Signorini  Instituto de Investigación de la Cadena Láctea (IdICaL) (CONICET-INTA), EEA Rafaela, INTA, Santa Fe, Argentina Juan  L.  Silva  Department of Food Science and Technology, Mississippi State University, Starkville, MS, USA Magaly Toro  Joint Institute for Food Safety and Applied Nutrition, University of Maryland, College Park, MD, USA Institute of Nutrition and Food Technology (INTA), University of Chile, Santiago, Chile Gabriel  Trueba  Instituto de Microbiología, Colegio de Ciencias Biológicas y Ambientales, Universidad San Francisco de Quito, Vía Interoceánica y Diego de Robles, Cumbayá, Quito, Ecuador Ana  Umpiérrez  Departamento de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay Sergio  Vaudagna  Instituto Tecnología de Alimentos, INTA, Buenos Aires, Argentina Instituto de Ciencia y Tecnología de Sistemas Alimentarios Sustentables (UEDD INTA-CONICET), Buenos Aires, Argentina Roberto  Vidal  Instituto de Ciencias Biomédicas, Universidad de Chile, Santiago, Chile Rafael Vignoli  Departamento de Bacteriología y Virología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay Natalie  Weiler  Gustafson  Laboratorio Central de Salud Pública, Ministerio de Salud Pública y Bienestar Social, Asunción, Paraguay Jeannete  Zurita  Unidad de investigaciones en Biomedicina. Zurita y Zurita Laboratorios, Facultad de Medicina, Pontificia Universidad Católica del Ecuador, Quito, Ecuador

Chapter 1

WHO Critical Priority Escherichia coli in Latin America: A One Health Challenge for a Post-Pandemic World Nilton Lincopan, Danny Fuentes-Castillo, Maria Espinoza-Muñoz, Fernando Gonzales-Zubiate, Edgar Gonzales-Escalante, Lenin Maturrano, Rafael Vignoli, Jose Di Conza, and Gabriel Gutkind Chapter Summary  The dissemination of carbapenem-resistant and third generation cephalosporin-resistant pathogens, classified as priority pathogens by the World Health Organization (WHO), is a critical issue that is no longer restricted to hospital settings. In this regard, in Escherichia coli, resistance to these last resort β-lactam antibiotics is mediated by the production of carbapenemases and extended-­spectrum beta-lactamases (ESBLs). In South American countries, several types of ESBLs have been detected, including TEM-, SHV-, and CTX-M-type variants (e.g., ­CTX-­M-­2, -3, -8, -9, -14, -15, -27, -55, -65). However, CTX-M has been the most N. Lincopan (*) Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil e-mail: [email protected] D. Fuentes-Castillo Departamento de Patología y Medicina Preventiva, Facultad de Ciencias Veterinarias, Universidad de Concepción, Chillán, Chile M. Espinoza-Muñoz Hospital Seguro Social Universitario, Cochabamba, Bolivia F. Gonzales-Zubiate Escuela de Ciencias Biológicas e Ingeniería, Universidad Yachay Tech, San Miguel de Urcuquí, Ecuador E. Gonzales-Escalante Facultad de Medicina, Universidad de Piura, Lima, Peru L. Maturrano Facultad de Medicina Veterinaria, Universidad Nacional Mayor de San Marcos, Lima, Peru R. Vignoli Departamento de Bacteriología y Virología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay J. Di Conza · G. Gutkind Facultad de Farmacia y Bioquímica, Instituto de Investigaciones en Bacteriologia y Virología Molecular, Universidad de Buenos Aires, Buenos Aires, Argentina © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 A. G. Torres (ed.), Trending Topics in Escherichia coli Research, https://doi.org/10.1007/978-3-031-29882-0_1

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widespread ESBL group. Endemic status of CTX-M-positive E. coli strains in the region is associated with the successful expansion of international clones belonging to sequence types (STs) ST2, ST10, ST38, ST44, ST58, ST90, ST115, ST131, ST155, ST167, ST224, ST354, ST410, ST457, ST517, ST648, and ST711, which are shared between human and animal (pets, wildlife, and food-producing) hosts, and polluted aquatic environments. Regarding carbapenem resistance, the increased frequency of reports on carbapenemases in Latin America shows that they have successfully spread, becoming endemic in some countries, such as Argentina, Colombia, and Brazil. In this respect, carbapenem-resistant E. coli belonging to global ST10, ST48, ST90, ST131, ST155, ST167, ST224, ST349, ST354, ST457, ST502, ST648, ST730, and ST744 clones have been isolated from humans, in association with the production of KPC-2 or NDM-1 carbapenemases. After the COVID-19 pandemic, a major concern has been the appearance of more virulent and resistant E. coli strains. The convergence of wide resistome and virulome is contributing to the persistence and rapid spread of international high-risk clones of critical priority E. coli at the human-animal-environmental interface, which must be considered a One Health challenge for a post-pandemic scenario. Here, we present updated information on the status of WHO critical priority E. coli in Latin America.

1.1 Introduction The dissemination of oximino-cephalosporins and/or carbapenem-resistant E. coli is a major public health concern worldwide, no longer restricted to hospital settings. E. coli and other clinically relevant Gram-negative bacteria have developed several mechanisms of resistance, including the production of β-lactamases, efflux pumps, and porin mutations. However, most clinical and epidemiologically relevant resistance mechanisms are the production of extended-spectrum β-lactamases (ESBLs) and carbapenemases, whose genes rapidly spread by horizontal gene transfer. In 2017, the WHO published the global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics (Tacconelli et  al. 2018). In this list, ESBL-producing and carbapenem-resistant E. coli are included as a critical priority. Historically, oximino-cephalosporins became available for clinical use in South America in the early 1980s. In 1988 and 1989, first ESBLs were reported in Argentina and Chile, in clinical Klebsiella pneumoniae strains carrying blaSHV-2 and blaSHV-5 genes, respectively. In 1992, production of CTX-M-2 ESBL was reported in Salmonella enterica serovar Typhimurium in Argentina. Currently, CTX-M has been the most widespread ESBL group in this region, and although ESBLs in human samples are most studied, ESBLs in food, livestock, companion animal, and environmental samples have been prevalent. CTX-M-producing E. coli clones ST131 and ST10 are the most widespread clones found in South America, and migratory species of birds have been identified as reservoirs. On the other hand, CTX-M-2 and CTX-M-8 have been identified in E. coli from poultry and chicken meat samples,

1  WHO Critical Priority Escherichia coli in Latin America: A One Health…

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supporting that ESBL-producing E. coli strains have also adapted to the agribusiness sector (Fig. 1.1). At the end of the 1980s, imipenem was the first carbapenem available for clinical use in Latin American countries, and although surveillance studies began to report imipenem resistance, almost 20  years after its introduction, the first report of a member of the Enterobacterales order producing a carbapenemase was found related

Fig. 1.1 Distribution and sources of extended-spectrum beta-lactamase (ESBL)- and/or carbapenemase-­producing Escherichia coli in South America

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to production of the IMP-1 metallo-beta-lactamase (MBL) by a clinical K. pneumoniae strain in Brazil. Nowadays, production of KPC-2 serine-carbapenemase has been the main mechanism of carbapenem resistance in E. coli, with NDM-1 rapidly emerging among isolates from Argentina and Brazil (García-Betancur et al., 2021). Therefore, the rapid spread of critical priority pathogens in South America is worrying, considering the dimension of the continent, diversity of international trade, livestock production, and human travel.

1.1.1 Critical Priority E. coli in Brazil Resistance to third generation cephalosporins is a particularly significant issue in Brazil. Although occurrence of ESBL producers was early documented in 1997, in private and public tertiary hospitals located in Rio de Janeiro and São Paulo (Sampaio and Gales 2016), the first molecular studies were published in 2000 (Bonnet et  al. 2000), evidencing predominance of CTX-M ESBL variants, and describing the emergence of the novel CTX-M-8 enzyme, in strains other than E. coli, from Rio de Janeiro. Currently, CTX-M enzymes are the most prevalent among E. coli strains, becoming endemic in clinical settings (Table 1.1). In 2001, two blaCTX-M genes encoding β-lactamases of pI 7.9 and 8.2 were implicated in resistance to broad-spectrum cephalosporins. While the blaCTX-M-9 gene was observed in the E. coli strain Rio-7, a novel CTX-M-encoding gene, designated blaCTX-M-16, was identified in the E. coli strain Rio-6 (Bonnet et al. 2001). Interestingly, both strains were isolated in 1996, whereas a study published in 2012, revealed that CTX-M-2- and CTX-M-59-producing E. coli were already associated with human infections in 2000. While E. coli strains producing CTX-M-14 and CTX-M-15 were isolated in 2006 (Cergole-Novella et  al. 2010), the novel CTX-M-8 emerged in E. coli in 2008, 8  years after its first identification in 1996 (Bonnet et  al. 2000; Peirano et al. 2011). After 2016, CTX-24-, CTX-M-27-, and CTX-M-55-positive E. coli strains were isolated from human infections, denoting an epidemiological change, following a global trend (Table 1.1). Among human isolates of CTX-M-producing E. coli circulating in Brazil, virulent clones belonging to global ST131, ST410, ST127, and ST354 have been identified. In this regard, the emergence of the ST131 C1-M27 high-risk extraintestinal pathogenic lineage is of critical concern (Soncini et al. 2022). In fact, this lineage has been recently identified in sea food and vegetable samples (Fernandes et  al. 2020; Lopes et al. 2022). Dissemination of CTX-M E. coli beyond hospital walls is another public health concern in Brazil. In this respect, CTX-M-8 E. coli were initially identified in livestock in 2010 (Aizawa et al. 2014). However, the main problem has been the occurrence of CTX-M-2-, CTX-M-8-, CTX-M-15-, and CTX-M-55-producing E. coli in poultry, since Brazil is the world’s largest poultry exporter and third largest producer (https://brazilianfarmers.com/category/discover/poultry/). Strikingly, international lineages of CTX-M-positive E. coli belonging to ST58, ST90, ST131, ST155,

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Table 1.1  Characteristics of WHO critical priority E. coli circulating at the human-animal-food-­ environmental interface in Brazil and Argentina Country Brazil

Source Human Human

ESBL Carbapenemase MLST CTX-M-9, – CTX-M-16 CTX-M-2 –

Human

CTX-M-59

ST34

Human

CTX-M-2

ST533

Human

SHV-5

ST528

Human

CTX-M-2

Human

SHV-5

ST62, ST127, ST167, ST359, ST362, ST405, ST652 –

Human

CTX-M-14, CTX-M-15

–

Human

CTX-M-15

ST998

Human

–

Human

CTX-M-15

ST131, ST410

Human

CTX-M-8

–

Human

CTX-M-15 KPC-2

ST502

Human

CTX-M-14

ST127

Human

CTX-M-2, CTX-M-15

ST354

Human

KPC-2

ST90

Human

CTX-M-2, 14, 15 –

KPC-2

ST648

Human

–

NDM-1

–

Human

–

NDM-1

ST707

Human

–

KPC-2

ST744

KPC-2

–

Year 1996

References Bonnet et al. (2001) 2000 Queiroz et al. (2012) 2000 Queiroz et al. (2012) 2001 Minarini et al. (2007) 2001 Minarini et al. (2007) 2003– Berman et al. 2008 (2014)

2005

Dropa et al. (2015) 2006 Cergole-­ Novella et al. (2010) 2006 Berman et al. (2014) 2008 Carvalho-­ Assef et al. (2010) 2008– Peirano et al. 2009 (2011) 2008– Peirano et al. 2009 (2011) – Almeida et al. (2012) 2009 Berman et al. (2014) 2015 Conceição-­ Neto et al. (2017) 2013 Fuga et al. (2022) 2013 Fuga et al. (2022) 2013 Campos et al. (2015) 2014 Fuga et al. (2022) 2014 Dalmolin et al. (2017) (continued)

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Table 1.1 (continued) Country

Source Human

ESBL –

Carbapenemase MLST KPC-2 ST167

Human

–

KPC-2

ST224

Human

–

KPC-2

ST457

Human

CTX-M-24 KPC-2

ST354

Human

–

ST155

Human

ST354, ST131

Human

CTX-M-24, CTX-M-27 CTX-M-8, CTX-M-55 – NDM-1

Human

CTX-M-15 KPC-2

ST648

Human

–

NDM-1

ST48, ST167

KPC-2

2509

Human

Human Food-­ producing animal (buffalo) Food-­ producing animal (chicken) Food-­ producing animal (chicken) Food-­ producing animal (swine) Food-­ producing animal (Turkey)

NDM-1

ST117 ST744, ST349

Year 2015

References Conceição-­ Neto et al. (2017) 2015 Fuga et al. (2022) 2015 Fuga et al. (2022) 2016 Dias et al. (2022) 2016 Fuga et al. (2022) 2016– Soncini et al. 2019 (2022) 2017 Fernandes et al. (2018a) 2017 Fuga et al. (2022) 2017 Fuga et al. (2021) 2018 Fuga et al. (2022) 2019 Fuga et al. (2022) 2010 Aizawa et al. (2014)

CTX-M-8

ST224, ST2179, ST2308

CTX-M-2

ST93, ST155, ST2309

2011– Ferreira et al. 2012 (2014a)

CTX-M-8

ST155, ST1011, ST2197, ST2929 ST224, ST410, ST1284

2012

Ferreira et al. (2014b)

2012

Silva et al. (2016)

–

2012

da Silva et al. (2017)

CTX-M-15

CTX-M-2

(continued)

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Table 1.1 (continued) Country

Source Food-­ producing animal (broiler) Food-­ producing animal (calf/ cow) Food-­ producing animal (cattle) Food-­ producing animal (poultry) Food-­ producing animal (poultry) Food-­ producing animal (poultry) Food-­ producing animal (poultry)

ESBL Carbapenemase MLST Year References CTX-M-55 ST457, ST453, 2013– Cunha et al. ST117, ST1706 2016 (2017)

Food-­ producing animal (poultry) Companion animal (horse) Companion animal (dog, cat) Companion animal (dog) Companion animal (dog)

CTX-M-15

ST90

2014

Sartori et al. (2017)

CTX-M-2

ST443

2014

Palmeira et al. (2018)

CTX-M-2

ST57, ST1158, ST2369, ST10064

2019

Dos Anjos et al. (2022)

CTX-M-8

ST106, ST117, 2019 ST345, ST2179

Dos Anjos et al. (2022)

CTX-M-15

ST224

2019

Dos Anjos et al. (2022)

CTX-M-55

2019

Dos Anjos et al. (2022)

SHV-12

ST58, ST93, ST131,ST155, ST162, ST224, ST295,ST366, ST1485, ST2607 ST93

2019

Dos Anjos et al. (2022)

CTX-M-15

ST2179

2012

Leigue et al. (2015)

CTX-M-2

ST371, ST457, ST155, ST457

2012

Melo et al. (2018)

CTX-M-8

ST58, ST372, ST2541 ST10, ST3395

2012

Melo et al. (2018) Melo et al. (2018)

CTX-M-9

2012

(continued)

N. Lincopan et al.

8 Table 1.1 (continued) Country

Source Companion animal (dog) Companion animal (dog) Companion animal (horse) Companion (cat) Companion animal (cat) Companion animal (dog) Synanthropic animal (pigeon) Wildlife (wild bird) Wildlife (fish) Wildlife (fish) Wildlife (vulture, owl, coati) Wildlife (coati) Wildlife (coati) Wildlife (vulture) Wildlife (vulture) Wildlife (penguin) Wildlife (seabirds) Wildlife (frigatebirds) Wildlife (frigatebirds)

ESBL Carbapenemase MLST CTX-M-15 ST90

Year 2012

CTX-M-16

ST3267

2012

CTX-M-8

ST711

2012

CTX-M-8

ST224

2015

CTX-M-2, CTX-M-15 CTX-M-15 KPC-2

ST648

2017

ST648

2017

CTX-M-8

ST359

2012

CTX-M-8

–

CTX-M-2

ST744

CTX-M-55

ST746

CTX-M-55

ST212, ST744

2010– Borges et al. 2012 (2017b) 2016 Sellera et al. (2018a) 2016 Sellera et al. (2018a) 2017– De Carvalho 2018 et al. (2020)

CTX-M-2

ST58

CTX-M-15

ST1251

CTX-M-2

ST1158

CTX-M-14

ST38

–

NDM-1

ST156

CTX-M-8

ST131

SHV-12

ST177

CTX-M-55

ST11350

References Melo et al. (2018) Melo et al. (2018) Fernandes et al. (2018b) Silva et al. (2018) Fernandes et al. (2018c) Sellera et al. (2018b) Borges et al. (2017a)

2017– 2018 2017– 2018 2017– 2018 2017– 2018 2018

De Carvalho et al. (2020) De Carvalho et al. (2020) De Carvalho et al. (2020) De Carvalho et al. (2020) Wink et al. (2022) 2018– Ewbank 2019 et al. (2022a) 2018– Ewbank 2019 et al. (2022a) 2018– Ewbank 2019 et al. (2022a) (continued)

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Table 1.1 (continued) Country

Source Wildlife (merganser)

ESBL CTX-M-8

Carbapenemase MLST ST58

Year 2019

Wildlife (frigatebird) Wildlife (whale) Captive animal (giant anteater) Captive animal (elephant) Food (meat)

CTX-M-2

ST648

2020

ST162

2020

CTX-M-65

ST156

2017

CTX-M-8

ST410

2018

CTX-M-2

–

2006

Food (chicken carcasses) Food (chicken meat) Food (chicken meat) Food (chicken meat) Food (sea food) Food (vegetable) Food (sea food) Food (chicken and pork meat) Food (chicken meat) Food (vegetable) Environment (hospital wastewater)

CTX-M-15

–

CTX-M-2

–

2011

Casella et al. (2015)

CTX-M-8

–

2013

Casella et al. (2015)

CTX-M-15

ST345

2014

Casella et al. (2017)

CTX-M-27

ST38, ST131

2016

CTX-M-15 CTX-M-14

ST38, ST648, ST14012 ST4012

CTX-M-55

ST117

CTX-M-44

–

–

Iark et al. (2018)

CTX-M-27 KPC-2

ST131

2019

CTX-M

–

2008

Lopes et al. (2022) Chagas et al. (2011)

–

NDM-1

References Fuentes-­ Castillo et al. (2021) Ewbank et al. (2022b) Sellera et al. (2022) Rueda Furlan et al. (2019) Furlan et al. (2020)

Warren et al. (2008) 2010– Botelho et al. 2011 (2015)

Fernandes et al. (2020) 2016 Lopes et al. (2021a) 2016– Bueris et al. 2017 (2022) 2016– Soncini et al. 2019 (2022)

(continued)

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N. Lincopan et al.

Table 1.1 (continued) Country

Source ESBL Carbapenemase MLST Environment CTX-M-15 ST4401 (sewage) Environment CTX-M-8 ST4445, (sewage) ST4402, ST4403 Environment CTX-M-2, – (wastewater) CTX-M-5, CTX-M-8, CTX-M-9, CTX-M-­ 15,SHV-12 – Environment CTX-M-2, CTX-M-3, (superficial CTX-M-8, water) CTX-M-9, CTX-M-15, Environment CTX-M-15 ST617 (urban lake) Environment CTX-M-8 ST58 (mangrove) ST131 Environment CTX-M-15 (agricultural soil) ST131 Argentina Human CTX-M-2, – CTX-M-14, CTX-M-15 Human – KPC-2 – Human

CTX-M-15 KPC-2

Human

CTX-M-2, KPC-2, KPC/ ST10, ST131 CTX-M-15 OXA-439, KPC-2/ OXA-163, IMP-8, VIM-­1,NDM-1 CTX-M-2, – – CTXM-­9/14, CTXM-­8/25, CTXM-­1/15, PER-2 – NDM-1 –

Human

Human

ST131

Year 2009 2009

References Dropa et al. (2016) Dropa et al. (2016)

2012– Conte et al. 2013 (2017)

2012– Conte et al. 2013 (2017)

2012– Nascimento 2013 et al. (2017) 2017 Sacramento et al. (2018) 2019 Lopes et al. (2021b) 2010

Rincón Cruz et al. (2013)

2010

Anchordoqui et al. (2015) 2011– De Belder 2014 et al. (2018) 2012, Sanz et al. 2014– (2022) 2017

2012– Faccone 2018 et al. (2020)

2014

Martino et al. (2019) (continued)

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Table 1.1 (continued) Country

Source Human

ESBL CTXM-­9/14

Carbapenemase MLST KPC-2 ST131

Human

–

IMP-8

–

Human

–

NDM-5

–

Human

–

KPC-2

ST730

Human

CTX-M-14 OXA-232

Food-­ producing animal (poultry) Food-­ producing animal (swine)

CTX-M-2, – CTX-M-14

CTXM-­8/25, CTXM-­1/15, CTX-M-2, CTXM-­9/14, PER-2 CTX-M-2, CTX-M-14, CTX-M-15 CTX-M-14 CTX-M-2 CTX-M-2

Companion animal (dog, cat) Wildlife (kelp gulls) Food (minced meat) Environment CTX-M-2 (wastewater) Environment CTX-M (lake, sediment/ water)

–

Year References 2015– Figueroa-­ 2016 Espinosa et al. (2019) 2016 Elena et al. (2018) 2018 Costa et al. (2021) 2019 Álvarez et al. (2022) 2020 Garcia et al. (2022) 2014 Dominguez et al. (2017, 2018)

‘

ST95, ST131

2017

Faccone et al. (2019)

–

–

2014

Rumi et al. (2019)

–

–

2012

–

–

–

–

–

–

Liakopoulos et al. (2016) 2017– Rumi et al. 2018 (2021b) 2018

Ghiglione et al. (2019, 2020) 2021– Gonzalez 2022 et al. (2022)

ST224, and ST410 seem to have adapted to the production chain of chicken and other foods. Indeed, since 2006, CTX-M-2-, CTX-M-8-, CTX-M-14, CTX-M-15-, and CTX-M-55-positive E. coli strains of ST38, ST345, ST117, ST648, or ST4012 have been increasingly identified in chicken and pork meat and vegetables (Table 1.1). Companion animals and wildlife have also been colonized or infected by ESBL-­ producing E. coli. Since 2012, surveillance studies have documented the occurrence

12

N. Lincopan et al.

of E. coli producing CTX-M-2, CTX-M-8, and CTX-M-15 belonging to ST10, ST58, ST90, ST224, and ST648 in dogs, cats, and horses, whereas those CTX-M variants, CTX-M-14 and CTX-M-55 ESBLs, have been identified among E. coli of ST38, ST58, ST131, ST410, and ST648 isolated from samples collected in wild animals. To monitor the impact of antimicrobial resistance on the environment, the role of wildlife as bioindicators or sentinels has begun to be studied, because most Brazilian urban areas with aquatic environments have been anthropogenically impacted. As such, E. coli strains producing CTX-M-2, CTX-M-8, or CTX-M-15 have been isolated from sewage, wastewater, and surface water. Of note, E. coli CTX-M-8/ST58 and CTX-M-15/ST13 have been isolated from mangrove and agricultural soil samples. The increased frequency of reports on carbapenemases in this country shows that they have successfully spread. While KPC-2 has an endemic status, production of NDM-1 is becoming frequent. From the first description, in 2010, of KPC-2  in E. coli (D’Alincourt Carvalho-Assef et al. 2010), lineages related to ST90, ST167, ST224, ST354, ST457, ST649, and ST744 have acquired the blaKPC-2 gene (Table  1.1), producing severe infections in hospital settings. Specifically, E. coli KPC-2/ST648 has persisted since 2013, and recently it has expanded to veterinary medicine, causing the first case of fatal infection in a dog (Sellera et  al. 2018b). Moreover, E. coli ST131 co-producing KPC-2 and CTX-M-27 has been detected in fresh vegetables. In human medicine, NDM-1 metallo-β-lactamases have been successfully incorporated by E. coli lineages of ST48, ST155, ST167, ST349, ST707, and ST744, from 2013 (Table 1.1). Of concern, NDM-1-producing E. coli belonging to ST156 and ST162 have been identified in marine wildlife, representing an emerging ecological threat to marine ecosystems, since anthropogenic pollution and infectious diseases have been the most notorious threats for vulnerable and endangered species.

1.1.2 Critical Priority E. coli in Argentina Even if resistance of third generation cephalosporins (TGC) was originally due to the presence of TEM and SHV mutants with an extended spectrum activity, or de-­ repression of chromosomal AmpCs, CTX-M-2 was (after emerging in different Salmonella serotypes) the first and most prevalent ESBL for decades, in different microorganisms such as E. coli (Rossi et  al. 1995; Radice et  al. 1994). A prior manuscript summarizes early dissemination reports in different meetings previously unpublished (Radice et al. 2002). A 1-month-period prospective study conducted in 2000 from public hospitals in Buenos Aires clearly displayed CTX-M-2 as the most prevalent ESBL produced by E. coli and other Enterobacterales (Quinteros et al. 2003). The same conclusion was obtained after analyzing consecutive isolates from the same year in a single hospital in Posadas, Misiones (Quiroga et al. 2008). Since then and assuming the endemic presence of this enzyme, no interventions were aimed at preventing emergence and

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dissemination of other ESBLS (and their accompanying resistance) until a multicenter survey, carried out in 2010, with 15 hospitals from three regions, showed an increased prevalence of ESBL-producing enterobacteria, due to an accumulation of CTX-M-2 still in circulation in addition to CTX-M-15 and to a lesser extent CTX-­ M-­14 emergence, which was observed in E. coli isolates (some belonging already to ST131) (Sennati et al. 2012). Marchisio et al. found that outpatient urine cultures in two health institutions from Santa Fe city rendered only a single TGC-resistant E. coli isolate (1.6%) due to the presence of plasmidic CMY-2 (Marchisio et  al. 2015). A previous multicenter prospective study conducted for 3 months of 2009 detected 0.9% of pAmpC enzymes in enterobacteria, with CMY-2-producing E. coli as the most prevalent (Cejas et al. 2012). A prospective study of TGC-resistant E. coli isolates recovered from January 2013 to March 2015 at the Hospital Regional de Ushuaia, Tierra del Fuego province, displayed CTX-M-1/15 (54%) as the most prevalent ESBL followed by CTX-­ M-­9/14 (25%) and CTX-M-2 (17%) (Gramundi et  al. 2022). A similar study including all clinically relevant Enterobacterales isolated in December 2012 and April 2013, from infected patients in four health institutions at Santa Fe city, revealed that TGC resistance in the recovered E. coli (n = 16) was due to CTX-M-2 (n = 5), CTX-M-9/14 (n = 4), CTX-M-1/15 (n = 3), CMY-2 (n = 3), and PER-2 (n = 1) (Marchisio et al. 2021). Even if no resistance mechanisms are analyzed, electronic reports from the national surveillance program show almost TGC resistance levels in E. coli (18% in 2010 to 20 or 23.4% in 2021) (Vigilancia Nacional de la Resistencia a los Antimicrobianos Argentina, Tendencia 2010–2021  – Red WHONET, visited 2022-12-01 (http://antimicrobianos.com.ar/category/resistencia/whonet/analisis-­ de-­ram/). From the same report, TGC resistance among community-onset E. coli urinary tract infections was 8% in 2020, with strong differences accordingly to age and sex (15% in elder males). Regarding carbapenemases, after the initial isolation of KPC-positive Citrobacter freundii and K. pneumoniae in early 2007, an electronic emergency report was transmitted by the national reference center (ANLIS), reinforced in June 2010, after an 800% increase was observed as compared with the number of isolates submitted for confirmation from the previous year (Alerta Epidemiológica, 2010, http://antimicrobianos.com.ar/category/alerta/, visited 2022-12-01). The in  vivo horizontal dissemination of blaKPC-2 was promptly documented between E. coli and K. pneumoniae from a single patient (Anchordoqui et  al. 2015). Even though KPC-­ producing E. coli are not frequent, they were described in a prospective study from Argentina’s patients (2011–2014), including some from the high-risk ST131 clone (De Belder et al. 2018). By 2013, NDM-1 MBLs had emerged among Enterobacterales (Alerta Epidemiológica, 2013, http://antimicrobianos.com.ar/category/alerta/, visited 2022-12-01). A few years later, a new variant, NDM-5, was described in an ExPEC recovered from an old female patient (Costa et al. 2021), and this enzyme is currently becoming more frequently isolated. A retrospective study on 71 carbapenemase-­producing ExPEC across Argentina was conducted (Sanz et  al.

14

N. Lincopan et al.

2022), where a plethora of combined mechanisms were described. More relevant combinations of beta-lactamases are highlighted in (Table 1.1). A recent comparison of nosocomial resistance rates modifications comparing isolates before (2018) and during the COVID-19 (2021) pandemic showed significant increase of non-susceptible rates to imipenem (1.1% vs 2.3%), meropenem (MER, 1.0% vs 1.9%), and ciprofloxacin (CIP, 38.5% vs 41.7%). However, some antibiotics showed a small decrease: gentamicin (GEN, 16.8% vs 15.2%), trimethoprim/sulfamethoxazole (TMS, 47.4% vs 44.5%), and colistin (COL, 1.3% vs 0.8%); or no modification: tigecycline (TIG, 22.6%), piperacillin-tazobactam (PTZ, 16.4%), and amikacin (AKN, 1.8%) (Lucero et al. 2022). ESBL-positive nosocomial E. coli were highly resistant to CIP (83.6%) and TMS (69.7%), whereas resistance to GEN (34.6%) and PTZ (33.9%) was less observed, as well as low resistance rates to fosfomycin (FOS, 5.5%), AKN (4.4%), COL (2.2%), and TIG (0.6%). Among carbapenem-resistant E. coli (CREC), 63.5% displayed a MER MIC greater than or equal to 16 mg/L which are not suitable for synergy. These strains were highly resistant to TMS (60.0%), CIP (57.3%), GEN (45.4%), and AKN (31.9%), whereas the low resistance level to FOS (7.5%), COL (2.0%), and TIG (0%) indicated that these drugs were still suitable (in combination) for treatment of the infections produced by E. coli. According to the information from WHONET, 26% of CREC produced KPC enzyme, 21% MBL, 3% OXA-48 like, 1% MBL+ KPC, and the remaining 49% of strains were not completely characterized (some of them were ESBL and impermeable). Trend of carbapenemases was varying between both pre-COVID-19 (2018) and COVID-19 (2021): 59.5% KPC and 35.7% MBL versus 45.5% KPC and 46.5% MBL.  OXA-48-type increased from 0% to 7.4%. Lastly, among mcr-1-positive E. coli of this period, 41.8% produced ESBL (most of them CTX-M) and only 2 isolates carbapenemases (1 KPC and 1 NDM). Regarding animals and environment ecosystems, in a systematic review and meta-analysis recently conducted (Prack McCormick et  al. 2022), antimicrobial resistance to all agents evaluated was observed for E. coli isolates in food-producing animals, except for carbapenems and glycylcyclines. MCR-1-producing E. coli was described in most of niches analyzed in Argentina recovered from gulls, chicken, pigs, dogs, aquatic environment, and sediment of a lake (Table 1.1); whereas identical IncI2 plasmids have been reported in some of these mcr-1-positive E. coli (Quiroga et al. 2019). The mcr-1.5 variant was detected in plasmids of E. coli isolates recovered from human samples, from healthy chicken on commercial farms, and from diarrheic piglets and healthy fattening pigs (Dominguez et al. 2019). E. coli recovered from local companion animals display resistance patterns similar to local human strains (Rumi et al. 2021a). E. coli was one of the most frequently isolated species in both cats and dogs. Interestingly, the values found for cefotaxime (CTX)-resistant E. coli were like those reported in human isolates from Argentinian hospitals (18.3%). In this country, resistance to third generation cephalosporins in pet isolates is due to ESBL and plasmid-mediated AmpC enzymes (Rumi et  al.

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2019), with predominance of CTX-M-2, CTX-M-14, CTX-M-15, and CMY-2 betalactamases. In a systematic review of the status of ESBLs in E. coli conducted in South America, Argentina was one of the countries with the second highest research contributions (Bastidas-Caldes et  al. 2022a). Therefore, since ESBLs producing E. coli are widely distributed at the human-animal-food-­environmental interface across Argentina, there is a need to increment studies to strengthen multi-sectoral antimicrobial resistance research and surveillance.

1.1.3 Critical Priority E. coli in Uruguay The first report of ESBL-producing E. coli in Uruguay comes from isolates obtained from children admitted to the pediatric hospital with diarrhea between 1991 and 1993. Of 68 EPEC isolates obtained between October 1990 and April 1993, 13 (19.1%) carried blaPER-2 (Vignoli et  al. 2005). Subsequent studies, however, have shown that the importance of Diarrheagenic E. coli (DAC) as a reservoir of ESBL-­ encoding genes would not be a problem today (Peirano et al. 2018; Mota et al. 2020). Regarding extra-intestinal E. coli clinical isolates, the main ESBL detected in both adults and pediatric population were CTX-M-15 (55–60%), followed by CTX-­ M-­2 (15–20%) and CTX-M-9 (12–15%) (Vignoli et  al. 2016; García-Fulgueiras et al. 2011; Garcia-Fulgueiras et al. 2017). Comparable results were found in stool samples from patients admitted to the ICU (Bado et al. 2016). As of 2017, sporadic cases of clinical isolates of E. coli carrying ESBL (CTX-M-15) or class C plasmid β-lactamase pAmpC (CMY-2), co-carriers of the colistin resistance gene mcr-1, have been reported (Papa-Ezdra et  al. 2020).The high-risk clones related to this resistance belonged to the sequence types of ST 131, ST405, and ST10 (Vignoli et al. 2016; Bado et al. 2016; Papa-Ezdra et al. 2020). For critical priority E. coli in animals, resistance to oximino-cephalosporins in bovine feces has been detected in 1–2% of the animals studied, finding the ESBL CTX-M-14 and CTX-M-15 (Umpiérrez et  al. 2017; Cóppola et  al. 2020). In the case of poultry, resistance has been reported in 17.4% of the animals studied, with the most frequently resistant mechanisms detected being CMY-2, followed by CTX-M-2, CTX-M-8, SHV2, and CTX-M-55 (Cóppola et  al. 2020) (Fig.  1.1). Interestingly, some of these enzymes, such as CTX-M-8, CMY-2, and CTX-M-55, were detected in 1-day-old chicks imported from Brazil, which suggests a source of resistance entry into the country (Coppola et al. 2022). Finally, the highest levels of resistance were observed in pig breeding, where resistance to oximino-cephalosporins was detected in the feces of 72% of the animals studied. In this population, the main β-lactams detected were CTX-M-8, followed by CMY-2, SHV-12, and to a lesser extent CTX-M-14 and CTX-M-15 (Cóppola et al. 2020).

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N. Lincopan et al.

1.1.4 Critical Priority E. coli in Chile Although WHO critical priority E. coli and its impact are widely recognized in Chile, reports of these pathogens are still scarce. Epidemiological surveillance in the community is conducted by the Public Health Institute (Instituto de Salud Pública, ISP) of Chile, which works as the reference center for the confirmation of resistant pathogens sent by hospitals throughout the country. On the other hand, E. coli carrying ESBL or carbapenemases detected in domestic animals, wildlife, food, vegetables, and water are not reported to the ISP, leading to a lack of knowledge on dissemination through the different transmission routes. In human medicine, detection of ESBL- or carbapenemase-producing E. coli became relevant after the 2000s. The first reports using phenotypic tests made it possible to establish that there were ESBL-producing E. coli causing septicemia in cancer patients (Rabagliati et al. 2009). Then, investigations using molecular analyses showed that CTX-M-­ type enzymes were the main β-lactamases in E. coli isolated from clinical samples (Wozniak et  al. 2012); and specifically, enzymes from the CTX-M-1, CTX-M-2, and CTX-M-9 groups and SHV-like were carried (García et al. 2011; Elgorriaga-­ Islas et  al. 2012; Wozniak et  al. 2012; Álvarez et  al. 2018; Pavez et  al. 2019) (Fig. 1.1). Regarding carbapenemases, KPC-2-producing E. coli belonging to the ST378 lineage has been reported among a multispecies outbreak of carbapenem-resistant bacteria in a patient admitted to a hospital coronary care unit (Wozniak et al. 2021). Currently, hospitals in Chile must notify to the ISP the presence of carbapenem-­ resistant bacteria; these records show that E. coli carry carbapenemases of KPC, NDM, and VIM types among nosocomial infections. Furthermore, the co-­production of KPC + VIM has already been found in E. coli. However, carbapenemases are more common in other species as Klebsiella spp., Acinetobacter spp., and Pseudomonas aeruginosa (ISP 2022). In domestic animals, a study reports the presence of CTX-M-1 and CTX-M-14 and PER-2 β-lactamases in E. coli isolated from hospitalized cats and dogs (Moreno et  al. 2008). Subsequently, a study described the presence of E. coli carrying enzymes of groups CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25 in dogs from the Chacabuco province in the Metropolitan Region of central Chile (Benavides et al. 2021). The same study reports E. coli harboring β-lactamases of the groups CTX-M-1 and CTX-M-2, and SHV-like among livestock samples (cattle, pigs, sheep, and chickens). Sporadically, ESBL-producing E. coli causing urinary tract infections in dogs and horses are detected in the Veterinary Microbiology Laboratory of the University of Concepción in Chillán. Until now, no carbapenemase-­ producing E. coli has been reported in Chilean domestic animals. In wildlife, studies on ESBL-producing E. coli began to be conducted in 2009 in migratory birds (Hernandez et  al. 2013), detecting CTX-M-1, CTX-M-15, CTX-­ M-­ 3, and CTX-M-14 enzymes among E. coli isolated from fecal samples of Franklin’s gulls in Con-Con and Talcahuano coasts. In the same species, but in Antofagasta, northern Chile, another study reports the presence of E. coli harboring

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enzymes of the CTX-M-2, CTX-M-3, CTX-M-15, and CTX-M-22 groups (Báez et al. 2015) (Fig. 1.1). Both studies reported ESBL genes in various lineages including the high-risk clones ST10 and ST131. Wildlife rescue and rehabilitation centers have been important components of epidemiological surveillance on WHO critical priority pathogens in wild animals (Fuentes-Castillo et  al. 2019, 2020). In fact, CTX-M-8-producing E. coli were detected in two owl species (rufous-legged owl and Magellanic horned owl). Subsequently, international clones of E. coli ST162, ST602, ST1196, and ST1485 carrying CTX-M-14, CTX-M-55, or CTX-M-65 ESBL were detected in Andean condors admitted at two wildlife rehabilitation centers. Carbapenemase-producing E. coli in wild birds were reported in Franklin’s gulls (Ahlstrom et al. 2022). In this regard NDM-5 MBL was detected in E. coli strains belonging to ST345, ST744, and ST1178. In the center of the country, E. coli harboring CTX-M genes were detected in the Claro and Lontue rivers, in Maule Region, a large area with agricultural activity (Díaz-Gavidia et  al. 2021). The same study revealed the presence of CTX-M-­ producing E. coli in parsley and lettuce ready for human consume. In contrast, studies that looked for STEC in samples from cattle and pigs found no association of E. coli strains carrying Shiga toxin with the presence of ESBL or carbapenemase (Galarce et al. 2021; Sánchez et al. 2021).

1.1.5 Critical Priority E. coli in Ecuador In Ecuador, the first report of ESBL-producing E. coli was in 2009, from the screening of samples collected from patients with intra-abdominal infections. In this study, the presence of E. coli expressing CTX-M-3 and CMY-2 β-lactamases was reported (Hawser et al. 2012). Nowadays, Ecuador is the country with the second highest number of human detections of ESBLs-producing E. coli. The high occurrence of ESBL producers has been associated with a high prevalence of CTX-M-1, CTX-­ M-­15, and CTX-M-55 variants, with CTX-M-15 being most common in clinical isolates (Bastidas-Caldes et al. 2022a; Delgado et al. 2016; Chiluisa-Guacho et al. 2018; Hawser et al. 2012; Nordberg et al. 2013; Ortega-Paredes et al. 2016, 2020a; Soria Segarra et al. 2018; Zurita et al. 2019) (Fig. 1.1). In healthy carriers, CTX-­ M-­55 has been a prevalent variant (Bastidas-Caldes et  al. 2022a; Calderón et  al. 2022; Salinas et al. 2021). In a study performed between 2013 and 2014, in three high-complexity teaching hospitals in Quito, analysis of ESBL-producing E. coli from bloodstream infections revealed the emergence of the CTX-M-15-producing E. coli ST131-B2 clone, representing an important public-health problem, because this multi-resistant clone is considered highly virulent, and a vehicle for the propagation of antimicrobial resistance genes (Zurita et al. 2019). Other epidemiologically valuable information of this study was the description of CTX-M-15-positive E. coli ST10, ST23, ST46, ST168, ST354, ST405, and ST6548, as well as CTX-­ M-­14-producing E. coli strains belonging to ST14, ST23, and ST405. In this study,

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the authors reported the emergence of the CTX-M-27/ST131 global E. coli clone (Zurita et al. 2019). Poultry products have been the most important source of animal protein (CONAVE, 2021, https://conave.org/informacion-­sector-­avicola-­publico/), and because of the overuse of antibiotics in their breeding, they are perfect reservoirs of antibiotic-resistant microorganisms. In the case of food-producing animals, most of the studies have presented data of E. coli from poultry (Hedman et al. 2019; Ortega-­ Paredes et al. 2020a; Vinueza-Burgos et al. 2019). In this respect, a study on chicken samples and carcasses collected from farms and markets, between 2017 and 2018, in Quito, showed the occurrence and predominance of CTX-M-55 and CTX-M-65 among ESBL-producing E. coli strains (Ortega-Paredes et al. 2020a). In companion animals, E. coli isolates producing CTX-M-type ESBLs have been more prevalent in canines (Albán et al. 2020; Bastidas-Caldes et al. 2022a; Ortega-­ Paredes et  al. 2019; Salinas et  al. 2021). Interestingly, a surveillance study conducted in Quito revealed that E. coli strains from children and domestic animals shared the same blaCTX-M allelic variants, whereas the most prevalent ESBL genes were CTX-M-55 and CTX-M-65, which were found in isolates from children, dogs, and chickens (Salinas et al. 2021) (Fig. 1.1). In food, the occurrence of epidemic clones of E. coli ST131, ST162, ST410, and ST744, producing CTX-M-8, CTX-M-14, CTX-M-15, CTX-M-24, CTX-M-55, or CTX-M-65 ESBLs, in ready-to-eat street food samples has alerted that street food is a possible way to spread harmful multidrug-resistant E. coli strains in the community (Zurita et al. 2020). Moreover, reports in the city of Ambato and Riobamba showed the presence of E. coli expressing SHV and CMY genes in ready-to-eat street food samples and fresh vegetables (Barragán-Fonseca et al. 2022; Tubón et al. 2022). Of public health concern has been the detection of ESBL-producing E. coli in alfalfa, leaf lettuce, and parsley/cilantro samples, in a municipal market in Quito, in 2015 (Ortega-Paredes et al. 2018). In this regard, the hyperepidemic CTX-M-15-­ producing E. coli clone ST410-A was reported for the first time in fresh vegetables, alerting regarding the health risk that this could pose, since vegetables and fruits are usually consumed raw. In the same study, the authors identify CTX-M-15-producing E. coli strains belonging to ST44 in leaf lettuce, alfalfa, and parsley/cilantro samples (Ortega-Paredes et  al. 2018). Noteworthy, a recent investigation by these authors also confirmed the presence of CTX-M-15-producing E. coli ST44 in the polluted Machángara River, where most of the wastewater of Quito city is discharged directly through sewage drainage (Ortega-Paredes et  al. 2020b). Additionally, water samples collected from points with domestic and industrial activities, and animal rearing, irrigation, domestic, and recreational purposes were positive for CTX-M-15-producing E. coli of ST10, ST46, ST162, ST167, ST457, ST1140, and ST1711, and CTX-M-18-, CTX-M-29-, and CTX-M-65-producing E. coli of ST10, ST362, and ST394, respectively, highlighting the high potential of polluted urban rivers as sites of emergence and sources of spread of critical priority E. coli. Furthermore, predominance of CTX-M-15 in E. coli isolates supports the establishment of this variant in the city, and its aquatic dissemination through the sewage to the environment (Ortega-Paredes et al. 2020b). Moreover, CTX-M-55,

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CTX-M-65, and CTX-M-15 have been confirmed in produce and irrigation water across different provinces of Ecuador (Bastidas-Caldes et al. 2022b; Montero et al. 2021). Regarding carbapenemase-producing E. coli, during a surveillance study of colonized patients, first KPC-positive E. coli strains were identified in 2016, from inguinal and perineal swab cultures performed in ICU patients, in Guayaquil (Soria-­ Segarra et al. 2020).

1.1.6 Critical Priority E. coli in Bolivia In Bolivia, E. coli is one of the most clinically important pathogens, and during the last decade an important dissemination of CTX-M-type ESBL has been observed (Fig. 1.1), despite being a country with very few published data, restricted to clinical settings. The first description of E. coli strains producing the CTX-M-2 ESBL was reported in 2002, in fecal samples of healthy children from the Bolivian Chaco, denoting the role that commensal E. coli isolates could play as potential reservoirs of these clinically relevant resistance mechanisms (Pallecchi et  al. 2004). Three years later, a survey carried out among members of the same healthy population revealed that fecal carriage of E. coli strains resistant to broad-spectrum cephalosporins was remarkably increased compared to that observed in the same settings in 2002, with the emergence of CTX-M-15-producing E. coli being documented (Pallecchi et  al. 2007). Another survey conducted in 2011, in the same setting, reported a relentless increase of resistance to ciprofloxacin and broad-spectrum cephalosporins, with occurrence of CTX-M-1-, CTX-M-2-, and CTX-M-9-type producing E. coli (Bartoloni et al. 2013). CTX-M-2 was replaced by CTX-M-15 and CTX-M-65. In 2015, it was reported that widespread dissemination of CTX-­ M-­65 was related to the polyclonal spreading of an IncI1 sequence type 71 (ST71) epidemic plasmid (Riccobono et al. 2015). Analysis of clinical isolates recovered between 2010 and 2014, from culture of urinary tract infections, resulted in the identification of CTX-M-15-positive E. coli belonging to the international ST131 (Bartoloni et al. 2016). In Cochabamba, a molecular survey that includes clinical samples recovered from different health centers, during 2012–2013, reported a high prevalence of the CTX-M-1 group among E. coli isolates (Saba Villarroel et al. 2017). More recently, emergence of CTX-M-8-positive E. coli and occurrence of CTX-M-1 and CTX-­ M-­9 producers was reported in school-age children from Indigenous communities of the Chaco, where the consumption of antibiotics is limited (Boncompagni et al. 2022). Regarding aquatic environments, the presence of CTX-M-3-producing E. coli was reported in the Choqueyapu River in La Paz, from a sample collected in 2013. Interestingly, although ST648, ST410, and ST162 were identified, only E. coli of ST162 carried the blaCTX-M-3 ESBL gene, indicating the possibility of antibiotic resistance transfer from the environment to the community (Guzman-Otazo et al. 2019).

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1.1.7 Critical Priority E. coli in Peru In the same way as in other Latin American countries, in Peru, CTX-M-type ESBL producers have been predominant, mainly CTX-M-15 (Medina-Pizzali et al. 2022; Yauri-Condor et al. 2020; Alcedo et al. 2022; Palma et al. 2017; Benavides et al. 2022; Ymaña et al. 2022) (Fig. 1.1). The first E. coli strains producing CTX-M-15 or CTX-M-2 were isolated, in 2002, from fecal swab specimens taken from healthy children living in Yurimaguas and Moyobamba (Pallecchi et al. 2004). Later, a survey conducted in 2005, among the same members of a healthy population of children, revealed the emergence of CTX-M-14 and CTX-M-24 (CTX-M-9 group) in commensal E. coli strains (Pallecchi et al. 2007). Moreover, E. coli isolates causing bacteremia in children from Lima carried a variety of ESBL-encoding genes, including blaSHV-12, blaCTX-M-15, blaCTX-M-2, and blaCTX-M-65. However, predominance of blaCTX-M-15 was confirmed (Palma et al. 2017). In urinary tract infections, a prevalence near to 55.0% of ESBL production, associated with CTX-M among E. coli isolates has been observed in public hospitals (Marcos-Carbajal et al. 2021), including the Peruvian jungle departments (León-Luna et al. 2021). In animal hosts, ESBL-producing E. coli have been isolated in fecal samples collected from wild common vampire bats and livestock near Lima, where molecular analyses revealed that most of this resistance resulted from the production of CTX-­ M-­15, with CTX-M-14-, CTX-M-15-, and CTX-M-26-postive E. coli belonging to ST2, ST356, ST466, and ST779 being isolated from cows; CTX-M-15 producers of ST2, ST422, ST472, ST721, ST305, and ST716 from bats; and CTX-M-14- and CTX-M-15-producing clones ST2, ST21, ST716, ST849, ST850, and ST851 recovered from pigs (Benavides et al. 2018). A more recent survey revealed the occurrence of CTX-M-3, CTX-M-14, CTX-M-15, CTX-M-55, and CTX-M-65 among commensal E. coli ST10, ST117, ST155, ST156, ST162, ST167, ST410, ST602, ST617, ST648, ST744, ST1049, ST1485, and ST2197 isolated from vampire bats and livestock (Benavides et al. 2022). Strikingly, CTX-M E. coli of ST2 and ST17 are shared between wild birds and livestock (Benavides et al. 2018, 2022). CTX-M-­ producing E. coli has also been isolated in poultry farms in Ica, highlighting a critical need for effective policy development and antimicrobial stewardship interventions in poultry production (Dávalos-Almeyda et al. 2022). In companion animals, CXT-­ M-­8-producing E. coli ST5259 has been identified in feces collected from dog (Medina-Pizzali et al. 2022). In environmental settings, in a study performed between 2016 and 2017, a CTX-­ M-­3-producing E. coli was recovered from drinking water sample obtained from a rural Andean household from Cajamarca (Larson et al. 2019). More recently, CTX-­ M-­55 E. coli ST227 was also isolated from drinking water (Medina-Pizzali et al. 2022). Regarding carbapenemases, in humans, occurrence of these enzymes in E. coli has been restricted to identification of NDM-1  in a ST155 clone isolated from a urine culture of an elderly patient with pancreatic cancer, in 2017 (Tamariz et al. 2018). Worryingly, the blaKPC-3 gene was identified in one market chicken isolate of E. coli ST10, in 2018, in Lima (Murray et al. 2021).

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1.2 Conclusions E. coli resistant to broad-spectrum cephalosporins and carbapenems antibiotics, classified as a critical priority by the WHO, have successfully disseminated beyond the hospital settings, and are now being identified in the environment, and in pets, food-producing, and wild animals, in South America. Molecular surveillance has significantly improved the ability to investigate the spread and emergence of clones at the human-animal-food-environment-animal interface. In this regard, studies conducted in Latin American countries have confirmed the occurrence of CTX-M (2, 3, 8, 9, 14, 15, 27, 55, 65)-producing E. coli strains belonging to international sequence types (STs) ST2, ST10, ST38, ST44, ST58, ST90, ST131, ST155, ST162, ST167, ST224, ST354, ST362, ST394, ST405, ST410, ST457, ST602, and ST648. They have been identified from humans, animals, and plant sources in Argentina, Bolivia, Brazil, Chile, Ecuador, Peru, and Uruguay. On the other hand, E. coli producing KPC-2 or NDM-1 carbapenemases, belonging to the ST10, ST48, ST90, ST131, ST155, ST167, ST354, ST457, ST502, ST648, ST730, and ST744 have disseminated in humans, in Argentina, Brazil, and Peru, whereas E. coli ST156, ST162, ST345, ST648, ST744, and ST1178 producing KPC-2, NDM-1, or NDM-5 have emerged in animal infections in Chile and Brazil. Anthropogenic actions and accelerated urbanization seem to contribute to this phenomenon, where endangered species such as iconic birds (e.g., Andean condor), whales, and marine turtles are now colonized or infected by WHO critical priority pathogens genomically related to hospital bacteria. The rapid spread of critical priority pathogens in South America is worrying, considering the dimension of the continent, diversity of international trade, livestock production, and human travel, becoming a challenge within a One Health perspective that must be monitored, in the current post-pandemic scenario.

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Peirano V, Bianco MN, Navarro A, Schelotto F, Varela G (2018) Diarrheagenic Escherichia coli associated with acute gastroenteritis in children from Soriano, Uruguay. Can J Infect Dis Med Microbiol 2018:8387218 Prack McCormick B, Quiroga MP, Álvarez VE, Centrón D, Tittonel P (2022) Antimicrobial resistance dissemination associated with intensive animal production practices in Argentina: a systematic review and meta-analysis. Rev Argent Microbiol:S0325-7541(22)00058-X Queiroz ML, Antunes P, Mourão J, Merquior VL, Machado E, Peixe LV (2012) Characterization of extended-spectrum beta-lactamases, antimicrobial resistance genes, and plasmid content in Escherichia coli isolates from different sources in Rio de Janeiro, Brazil. Diagn Microbiol Infect Dis 74(1):91–94 Quinteros M, Radice M, Gardella N, Rodriguez MM, Costa N, Korbenfeld D, Couto E, Gutkind G, Microbiology Study Group (2003) Extended-spectrum beta-lactamases in Enterobacteriaceae in Buenos Aires, Argentina, public hospitals. Antimicrob Agents Chemother 47(9):2864–2867 Quiroga M, Caceres MG, Stefanuk R, Villalba V, Rodriguez MM, Radice M, Gutkind G, Vergara M (2008) Characterization of extended-spectrum beta-lactamases in clinical isolates of Klebsiella pneumoniae and Escherichia coli from Posadas, Misiones, Argentina. J Chemother 20(1):130–133 Quiroga C, Nastro M, Di Conza J (2019) Current scenario of plasmid-mediated colistin resistance in Latin America. Rev Argent Microbiol 51(1):93–100 Rabagliati BR, Fuentes LG, Orellana UE, Oporto CJ, Domínguez MI, Benítez GR, Aedo CI, Ramos G, Garrido SM, García CP (2009) Etiología de episodios de neutropenia febril en pacientes adultos con cancer hematológico y de órganos sólidos en el Hospital Clínico Universidad Católica, Santiago-Chile. Rev Chil Infectol 26(2):106–113 Radice M, Rossi A, Venuta M, Lopardo H, Gutkind G (1994) XVI Congreso Chileno de Microbiologia, p. 31 Radice M, Power P, Di Conza J, Gutkind G (2002) Early dissemination of CTX-M-derived enzymes in South America. Antimicrob Agents Chemother 46(2):602–604 Riccobono E, Di Pilato V, Di Maggio T, Revollo C, Bartoloni A, Pallecchi L, Rossolini GM (2015) Characterization of IncI1 sequence type 71 epidemic plasmid lineage responsible for the recent dissemination of CTX-M-65 extended-spectrum β-lactamase in the Bolivian Chaco region. Antimicrob Agents Chemother 59(9):5340–5347 Rincón Cruz G, Radice M, Sennati S, Pallecchi L, Rossolini GM, Gutkind G, Di Conza JA (2013) Prevalence of plasmid-mediated quinolone resistance determinants among oxyimino-­cephalosporin-resistant Enterobacteriaceae in Argentina. Mem Inst Oswaldo Cruz 108(7):924–927 Rossi A, Lopardo H, Woloj M, Picandet AM, Mariño M, Galds M, Radice M, Gutkind G (1995) Non-typhoid Salmonella spp. resistant to cefotaxime. J Antimicrob Chemother 36(4):697–702 Rueda Furlan JP, Moura Q, Lima Gonzalez IH, Locosque Ramos P, Lincopan N, Guedes Stehling E (2019) Draft genome sequence of a multidrug-resistant CTX-M-65-producing Escherichia coli ST156 colonizing a giant anteater (Myrmecophaga tridactyla) in a zoo. J Glob Antimicrob Resist. 17:19–20 Rumi MV, Mas J, Elena A, Cerdeira L, Muñoz ME, Lincopan N, Gentilini ÉR, Di Conza J, Gutkind G (2019) Co-occurrence of clinically relevant β-lactamases and MCR-1 encoding genes in Escherichia coli from companion animals in Argentina. Vet Microbiol 230:228–234 Rumi MV, Nuske E, Mas J, Argüello A, Gutkind G, Di Conza J (2021a) Antimicrobial resistance in bacterial isolates from companion animals in Buenos Aires, Argentina: 2011–2017 retrospective study. Zoonoses Public Health 68(5):516–526 Rumi MV, Crespi E, Broglio A, Ghiglione B, Figueroa Espinosa R, Nuske E, Gutkind G, Ambros L, Albarellos G, Di Conza J, Bentancor A (2021b) Marcadores de resistencia en aislamientos de E. coli obtenidos de muestras de carne picada de Tierra del Fuego. XXV Congreso Latinoamericano de Microbiología. ALAM 2021. Paraguay. 25–28 agosto 2021

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Saba Villarroel PM, Gutkind GO, Di Conza JA, Radice MA (2017) First survey on antibiotic resistance markers in Enterobacteriaceae in Cochabamba, Bolivia. Rev Argent Microbiol 49(1):50–54 Sacramento AG, Fernandes MR, Sellera FP, Muñoz ME, Vivas R, Dolabella SS, Lincopan N (2018) Genomic analysis of MCR-1 and CTX-M-8 co-producing Escherichia coli ST58 isolated from a polluted mangrove ecosystem in Brazil. J Glob Antimicrob Resist. 15:288–289 Salinas L, Loayza F, Cárdenas P, Saraiva C, Johnson TJ, Amato H, Graham JP, Trueba G (2021) Environmental spread of extended spectrum beta-lactamase (ESBL) producing Escherichia coli and ESBL genes among children and domestic animals in Ecuador. Environ Health Perspect 129(2):27007 Sampaio JL, Gales AC (2016) Antimicrobial resistance in Enterobacteriaceae in Brazil: focus on β-lactams and polymyxins. Braz J Microbiol 47 Suppl 1(Suppl 1):31–37 Sánchez F, Fuenzalida V, Ramos R, Escobar B, Neira V, Borie C, Lapierre L, López P, Venegas L, Dettleff P, Johnson T, Fuentes-Castillo D, Lincopan N, Galarce N (2021) Genomic features and antimicrobial resistance patterns of Shiga toxin-producing Escherichia coli strains isolated from food in Chile. Zoonoses Public Health 68(3):226–238 Sanz MB, De Belder D, de Mendieta JM, Faccone D, Poklepovich T, Lucero C, Rapoport M, Campos J, Tuduri E, Saavedra MO, Van der Ploeg C, Rogé A, Carbapenemases-ExPECGroup, Pasteran F, Corso A, Rosato AE, Gomez SA (2022) Carbapenemase-producing extraintestinal pathogenic Escherichia coli from Argentina: clonal diversity and predominance of hyperepidemic clones CC10 and CC131. Front Microbiol 13:830209 Sartori L, Fernandes MR, Ienne S, de Souza TA, Gregory L, Cerdeira L, Lincopan N (2017) Draft genome sequences of two fluoroquinolone-resistant CTX-M-15-producing Escherichia coli ST90 (ST23 complex) isolated from a calf and a dairy cow in South America. J Glob Antimicrob Resist 11:145–147. https://doi.org/10.1016/j.jgar.2017.10.009 Sellera FP, Fernandes MR, Moura Q, Carvalho MPN, Lincopan N (2018a) Extended-spectrum-­ β-lactamase (CTX-M)-producing Escherichia coli in wild fishes from a polluted area in the Atlantic Coast of South America. Mar Pollut Bull 135:183–186 Sellera FP, Fernandes MR, Ruiz R, Falleiros ACM, Rodrigues FP, Cerdeira L, Lincopan N (2018b) Identification of KPC-2-producing Escherichia coli in a companion animal: a new challenge for veterinary clinicians. J Antimicrob Chemother 73(8):2259–2261 Sellera FP, Cardoso B, Fuentes-Castillo D, Esposito F, Sano E, Fontana H, Fuga B, Goldberg DW, Seabra LAV, Antonelli M, Sandri S, Kolesnikovas CKM, Lincopan N (2022) Genomic analysis of a highly virulent NDM-1-producing Escherichia coli ST162 infecting a Pygmy Sperm Whale (Kogia breviceps) in South America. Front Microbiol 13:915375 Sennati S, Santella G, Di Conza J, Pallecchi L, Pino M, Ghiglione B, Rossolini GM, Radice M, Gutkind G (2012) Changing epidemiology of extended-spectrum β-lactamases in Argentina: emergence of CTX-M-15. Antimicrob Agents Chemother 56(11):6003–6005 Silva KC, Moreno M, Cabrera C, Spira B, Cerdeira L, Lincopan N, Moreno AM (2016) First characterization of CTX-M-15-producing Escherichia coli strains belonging to sequence type (ST) 410, ST224, and ST1284 from commercial swine in South America. Antimicrob Agents Chemother 60(4):2505–2508 Soncini JGM, Cerdeira L, Sano E, Koga VL, Tizura AT, Tano ZN, Nakazato G, Kobayashi RKT, Aires CAM, Lincopan N, Vespero EC (2022) Genomic insights of high-risk clones of ESBL-­ producing Escherichia coli isolated from community infections and commercial meat in southern Brazil. Sci Rep 12(1):9354 Soria Segarra C, Soria Baquero E, Cartelle Gestal M (2018) High prevalence of CTX-M-1-like enzymes in urinary isolates of Escherichia coli in Guayaquil, Ecuador. Microb Drug Resist 24(4):393–402 Soria-Segarra C, Soria-Segarra C, Catagua-González A, Gutiérrez-Fernández J (2020) Carbapenemase-producing Enterobacteriaceae in intensive care units in Ecuador: results from a multicenter study. J Infect Public Health 13(1):80–88

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Tacconelli E, Carrara E, Savoldi A, Harbarth S, Mendelson M, Monnet DL, Pulcini C, Kahlmeter G, Kluytmans J, Carmeli Y, Ouellette M, Outterson K, Patel J, Cavaleri M, Cox EM, Houchens CR, Grayson ML, Hansen P, Singh N, Theuretzbacher U, Magrini N, WHO Pathogens Priority List Working Group (2018) Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis 18(3):318–327 Tamariz J, Llanos C, Seas C, Montenegro P, Lagos J, Fernandes MR, Cerdeira L, Lincopan N (2018) Draft genome sequence of the first New Delhi metallo-β-lactamase (NDM-1)-producing Escherichia coli strain isolated in Peru. Genome Announc 6(13):e00199–e00118 Tubón J, Barragán-Fonseca G, Lalaleo L, Calero-Cáceres W (2022) Data on antibiograms and resistance genes of Enterobacterales isolated from ready-to-eat street food of Ambato, Ecuador. F1000Res 11:669 Umpiérrez A, Bado I, Oliver M, Acquistapace S, Etcheverría A, Padola NL, Vignoli R, Zunino P (2017) Zoonotic potential and antibiotic resistance of Escherichia coli in neonatal calves in Uruguay. Microbes Environ 32(3):275–282 Vignoli R, Varela G, Mota MI, Cordeiro NF, Power P, Ingold E, Gadea P, Sirok A, Schelotto F, Ayala JA, Gutkind G (2005) Enteropathogenic Escherichia coli strains carrying genes encoding the PER-2 and TEM-116 extended-spectrum beta-lactamases isolated from children with diarrhea in Uruguay. J Clin Microbiol 43(6):2940–2943 Vignoli R, García-Fulgueiras V, Cordeiro NF, Bado I, Seija V, Aguerrebere P, Laguna G, Araújo L, Bazet C, Gutkind G, Chabalgoity A (2016) Extended-spectrum β-lactamases, transferable quinolone-resistance and Virulo typing in extra intestinal E. coli in Uruguay. J Infect Dev Ctries 10(1):43–52 Vinueza-Burgos C, Ortega-Paredes D, Narváez C, De Zutter L, Zurita J (2019) Characterization of cefotaxime resistant Escherichia coli isolated from broiler farms in Ecuador. PLoS One 14(4):e0207567 Warren RE, Ensor VM, O'Neill P, Butler V, Taylor J, Nye K, Harvey M, Livermore DM, Woodford N, Hawkey PM (2008) Imported chicken meat as a potential source of quinolone-resistant Escherichia coli producing extended-spectrum beta-lactamases in the UK.  J Antimicrob Chemother 61(3):504–508 Wink PL, Lima-Morales D, Meurer R, Barth AL (2022) Escherichia coli carrying blaNDM-1 obtained from a migratory penguin (Spheniscus magellanicus) in the Brazilian seacoast. Braz J Microbiol 53(1):499–502 Wozniak A, Villagra NA, Undabarrena A, Gallardo N, Keller N, Moraga M, Román JC, Mora GC, García P (2012) Porin alterations present in non-carbapenemase-producing Enterobacteriaceae with high and intermediate levels of carbapenem resistance in Chile. J Med Microbiol 61(Pt 9):1270–1279 Wozniak A, Figueroa C, Moya-Flores F, Guggiana P, Castillo C, Rivas L, Munita JM, García PC (2021) A multispecies outbreak of carbapenem-resistant bacteria harboring the blaKPC gene in a non-classical transposon element. BMC Microbiol 21(1):107 Yauri-Condor K, Zavaleta Apestegui M, Sevilla-Andrade CR, Sara JP, Villoslado Espinoza C, Taboada WV, Gonzales-Escalante E (2020) Extended-spectrum beta-lactamase-producing Enterobacterales carrying the mcr-1 gene in Lima, Peru. Rev Peru Med Exp Salud Publica 37(4):711–715 Ymaña B, Luque N, Ruiz J, Pons MJ (2022) Worrying levels of antimicrobial resistance in Gram-­ negative bacteria isolated from cell phones and uniforms of Peruvian intensive care unit workers. Trans R Soc Trop Med Hyg 116(7):676–678 Zurita J, Solís MB, Ortega-Paredes D, Barba P, Paz Y Miño A, Sevillano G (2019) High prevalence of B2-ST131 clonal group among extended-spectrum β-lactamase-producing Escherichia coli isolated from bloodstream infections in Quito, Ecuador. J Glob Antimicrob Resist 19:216–221 Zurita J, Yánez F, Sevillano G, Ortega-Paredes D, Paz Y, Miño A (2020) Ready-to-eat street food: a potential source for dissemination of multidrug-resistant Escherichia coli epidemic clones in Quito, Ecuador. Lett Appl Microbiol 70(3):203–209

Chapter 2

Recent Progress on Enterotoxigenic E. coli (ETEC) and Antibiotic Resistance in Pathogenic E. coli Enrique Joffré, Jeannete Zurita, Carla Calderon Toledo, and Sergio Gutiérrez-Cortez Chapter Summary Enterotoxigenic Escherichia coli (ETEC) is one of the leading bacterial causes of diarrhea, particularly among children in low-resource settings, travelers, and military personnel from high-income countries. Due to the lack of safe water and sanitation in low- and middle-income countries (LMICs) in Latin America, ETEC is endemic in the region and has a wider range of reservoirs that include One Health-defined settings such as the environment, farm animals, and crops. In the last 10 years, the significant advance in next generation sequencing (NGS) technologies has allowed the study of hundreds of ETEC genomes and revealed that part of the success of ETEC is the large gene and genomic diversity, but also conserved combinations of virulence genes packed in conjugable plasmids and the bacterial chromosome. Advances in knowledge of new virulent genes, microbial pathogenesis, host-pathogen, and microbiome interactions have contributed to the understanding of the various aspects of ETEC infections, as well as in identifying alternative targets for treatment development. Currently, several vaccines are being developed, and the search for targets in ETEC pangenomes will benefit the development of future vaccines with broad coverage, particularly when antibiotic resistance in E. coli and ETEC strains is on the rise.

E. Joffré (*) Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden e-mail: [email protected] J. Zurita Unidad de investigaciones en Biomedicina, Zurita y Zurita Laboratorios, Facultad de Medicina, Pontificia Universidad Católica del Ecuador, Quito, Ecuador C. Calderon Toledo · S. Gutiérrez-Cortez Unidad de Microbiología Ambiental, Instituto de Biología Molecular y Biotecnología (IBMB), Carrera de Biología, Universidad Mayor de San Andrés, La Paz, Bolivia © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 A. G. Torres (ed.), Trending Topics in Escherichia coli Research, https://doi.org/10.1007/978-3-031-29882-0_2

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2.1 General Concepts About ETEC The gastrointestinal tract is home to an extraordinary diversity of bacterial species, including Escherichia coli (E. coli). E. coli was initially thought to be the predominant commensal bacterial species in the human gut; however, it was later confirmed that it only comprises 0.1% of the total intestinal microbiota, which is dominated by obligate anaerobic bacteria (Tenaillon et al. 2010). E. coli is a facultative anaerobic Gram-negative bacterium that is innocuous; however, given its heterogeneity and versatile nature, this microorganism can also cause disease through the expression of virulence genes acquired by horizontal gene transfer. Pathogenic strains of E. coli are usually classified into pathotypes and are associated with significant diarrheal and extraintestinal diseases (Croxen et al. 2013). Among the pathotypes of E. coli, enterotoxigenic E. coli (ETEC) alone accounts for millions of diarrheal episodes and is one of the major agents of moderate to severe infantile diarrhea in LMICs (Qadri et al. 2005). The first reports of ETEC date back to De’s observations in 1956 (De, Bhattacharya, and Sarkar 1956), which were further explored by Sack’s research group in 1971 (Bradley Sack et al. 1971; Gorbach et al. 1971; Sack 2011). In Latin America, one of the first studies on the prevalence of ETEC in pediatric populations was published in 1997 (Evans et al. 1977). One of the main characteristics of ETEC isolates is the production of a heat-labile toxin (LT), which is similar in structure and function to the cholera toxin (Gill and Richardson 1980). Furthermore, the expression of a heat-stable toxin (ST, whose variants were discovered in humans (STh) and pigs (STp)) is also considered a hallmark of this E. coli pathovar. Currently, the presence of either or both toxins serves as a molecular marker for the identification of ETEC (Qadri et al. 2005; Jesser and Levy 2020). In addition to the classical LT and ST toxins, there is growing interest in the contribution of other potential genetic products that are important for the pathogenesis of ETEC, including EAST-1, EatA, ClyA, and LeoA (Veilleux and Dubreuil 2006; Patel et al. 2004; Ludwig et al. 2004; Fleckenstein et al. 2000). On the other hand, colonization factors (CF) or colonization surface antigens (CS) have been intensively characterized due to their relationship with the adhesion events necessary to initiate ETEC infection. To date, at least 25 types of CFs/CS have been identified, which are diverse in their morphology, serotype, primary structure, and host receptor binding properties (Madhavan and Sakellaris 2015). Another aspect that stresses the importance of ETEC CFs is related to their potential as vaccine target candidates. A clear example of this aspect is the results of the recent Global Enteric Multicenter Study (GEMS), which concluded that vaccines directed to major CF in ST-encoding ETEC could potentially prevent up to 66% of moderate to severe diarrhea in children from developing countries (Vidal et al. 2019).

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2.1.1 ETEC Is a Major Human Pathogen Diarrheal diseases are a topic of great concern, especially in developing countries, and several studies have assessed the contribution of ETEC to this public health problem. A systematic review and meta-analysis of 130 studies comprising 29 years of research (1990–2019) suggest that there is a moderate to high association between ETEC and human diarrheal episodes (Baker et al. 2021). Worldwide data show that diarrheal diseases represent the fourth cause of death in children under 5 years of age (Perin et  al. 2022). From a variety of pathogens, the Global Burden Disease (GBD) report identified ETEC, along with Shigella, as the leading causes of mortality associated with diarrhea (Mortality and Collaborators 2016; Khalil et al. 2019). On the other hand, in adults, ETEC is a common etiological agent of traveler’s diarrhea with an overall rate of 44% of bacterial cases (Olson et al. 2019).

2.1.2 ETEC Can Be a Food and Waterborne Pathogen ETEC is a food and waterborne pathogen that can survive in the environment and cope with harsh conditions. The ETEC pathotype has been repeatedly found in drinking water and rivers, including vegetables irrigated with contaminated water (Gonzales-Siles and Sjöling 2016). Studies in the La Paz River basin in Bolivia identified ETEC as the most frequent pathogen detected in soil and vegetable samples (Poma, Mamani, and Iñiguez 2016; Guzman-Otazo et al. 2019) The isolated ETEC colonies showed different profiles of toxin genes, LT + STh being the most prevalent, while STp was detected only in vegetable samples (Poma, Mamani, and Iñiguez 2016). In a study conducted in Mexico that evaluated the presence of diarrheagenic E. coli (DEC) in water samples collected from irrigation systems, rivers, and other water bodies, ETEC bacteria represented 6.8% of all DEC isolates. The presence and persistence of ETEC bacteria in the water bodies used in irrigation systems may represent an important source of human infection. The presence of ETEC bacteria has been reported in contaminated food. A study that evaluated the presence of multidrug-resistant E. coli in cooked and fresh vegetables in Mexico reported that ETEC strains were present in 8% of all Nopalito (Mexican Cactus) samples (a Cactaceae used as a fresh green vegetable in Mexico) (Canizalez-Roman et al. 2019). A total of 21 ETEC strains were isolated and characterized, and all of them exhibited resistance at least to six antibiotics; remarkably, 7 strains exhibited resistance to 11 antibiotics (Canizalez-Roman et  al. 2019). Similarly, another study conducted in Mexico that analyzed the presence of multidrug-­resistant E. coli in fresh cheese samples found that 5% of all samples were positive for ETEC and that all isolated strains were resistant to at least five antibiotics (de la Rosa-Hernández et al. 2018). ETEC strains were also detected in fresh carrot juice (Torres-Vitela et al. 2013) and bean sprouts (28); and in both studies, all ETEC isolates were positive for the ST marker.

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2.1.3 Animal ETEC ETEC is the most common cause of E. coli diarrhea in farm animals, causing neonatal diarrhea in piglets, calves, sheep, and dogs and postweaning diarrhea (PWD) in piglets. The diarrheal episodes are caused by the expression of enterotoxins such as LT (LTI and LTII), ST (Sta and STb), and/or enteroaggregative heat-stable toxin 1 (EAST1), as well as a different set of colonization factors (F5, F6, F17, F18, and F41) (Dubreuil, Isaacson, and Schifferli 2016). LT is a heterogeneous toxin, with two described subtypes: LTI and LTII. LTI can be divided into LT1h (human) and LTIp (porcine), while LTII consists of three antigenic variants, LTIIa, LTIIb, and LTIIc, and they differ in their B subunit, which binds to various gangliosides in the intestine. The colonization factors F5 (K99), F6 (987P), F7, and F41 are associated with ETEC strains causing neonatal diarrhea, while F18 is often associated with PWD and F4 (also designated K88) with both disease (da Silva et al. 2001).

2.2 Recent Discoveries in Virulence and Pathogenesis ETEC is a heterogeneous pathogen capable of sensing different environmental stimuli in the host and modulating the gene expression of the diverse set of virulence genes to cause infection (Crofts et al. 2018). For example, bile salts – an abundant host factor produced by the liver, secreted by the gallbladder into the duodenum – have been shown to induce gene expression and translation of plasmid encoded genes of the CS5 operon, cexE, the type 1 secretion system (aatPABCD) and an AraC-like transcriptional activator CsvR (coli surface virulence factor regulator) in ETEC strains with global distribution (Nicklasson et al. 2012; Joffre et al. 2019). Bile salts have also been associated with the induction of ETEC self-aggregation (biofilm) and attachment to epithelial cells, which was demonstrated to be mediated by bile-induced CF CS5 while bacterial motility was reduced. In another study, bile exposure induced STp toxin secretion, particularly isolates that harbor the STa5 variant, which have been shown to be associated with disease in adults and travelers (Joffré et al. 2016). The same study also found that bile negatively regulated the expression of the STh variant STa3/4 which is found more often in strains infecting indigenous children than adults (Joffré et  al. 2016). Haycocks et  al. (2015) have investigated the role of glucose and salts, the two main components of oral rehydration therapy used to counteract electrolytes lost due to the secretion of ETEC toxins during infection. The authors demonstrated that CRP and H-NS control the toxins expression of ETEC H10407 that respond to glucose and salts. High osmolarity (salts) has been shown to induce enterotoxin expression by relieving H-NS repression, while glucose inhibited estA2 (STh) but not estA1 (STp) expression. H-NS can also prevent CRP regulation, which has been shown to activate estA2 expression and indirectly suppress eltAB expression, and although the pathways are unknown, it is speculated H-NS involvement (Haycocks et al. 2015). When ST secretion and

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gene expression were tested in the presence of glucose in 47 clinical ETEC from endemic regions, both STp and STh genes were significantly negatively regulated (Joffré et al. 2016), suggesting more complex regulatory mechanisms and strain-­ specific responses modulating ETEC virulence. During the transit of ETEC through the small intestine, the bacteria encounter an alkaline flush of bicarbonate. Gonzales et al. (2013) demonstrated that CRP can also function as a positive regulator of LT secretion in vivo and in vivo and that toxin secretion was enhanced during exposure to alkaline conditions (pH 9). By examining the transcriptomic response of ETEC H10407 directly in infected stool samples, it was found that LT and CFA/I genes were downregulated and speculated that stool samples could represent the environment of the large intestine which is characterized by low oxygen conditions. Further analysis of toxin gene expression in a knockout of the fumarate and nitrate reductase (FNR) transcription factor (known to have a role in the regulation of low oxygen genes) and identification of binding sites have revealed direct repression of the promoters LT, STh, and STp by FNR. FNR has also been shown to regulate the ETEC biofilm which is enhanced under anaerobic conditions (Crofts et al. 2018). Iron is needed for bacterial survival, but high concentrations can be quite toxic (Murdoch and Skaar 2022). In ETEC expressing CS6, iron at 0.2 mM enhanced CS6 expression and epithelial bacterial adhesion, but higher concentrations resulted in opposite results (Bhakat et al. 2021). These studies suggest that these host factors are signal molecules for ETEC to stabilize infection in the small intestine. Besides classical virulence factors, ETEC can also contain a large group of non-­ classical virulence factors, and in recent years, its contribution to the disease has been widely investigated. In a study (Abd El Ghany et al. 2021), the role of the aatC and cexE genes in mouse colonization was investigated. Mutants of these genes inoculated orally in mice demonstrated impaired bacterial colonization and provide evidence of the role of cexE in the pathogenesis of ETEC. CexE is a well-conserved protein among ETEC strains and a homolog of the dispersin protein (aap gene) in EAEC (Abd El Ghany et al. 2021) that was highly induced during exposure to bile (Joffre et al. 2019). For optimal toxin delivery, the TolC efflux protein and intimate contact with the host cell mediated by the EtpA adhesive (Zhu et al. 2018) were found to be essential. In ETEC, the highly conserved chromosomally encoded type 1 pili, which is also conserved in other strains of E. coli, are used for optimal engagement with the intestinal epithelium (Sheikh et  al. 2017). Identification of new adhesins such as CS23 (Del Canto et al. 2012), closely related to CS13 pili, CS26 (Nada et al. 2011), and CS30 (von Mentzer et al. 2017; von Mentzer et al. 2020) expanded the repertoire of virulence factors that play an important role in bacterial colonization, as well as target noncanonical antigens for use in the development of the ETEC vaccine (Kuhlmann et al. 2021).

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2.3 Epidemiology of ETEC in Latin America ETEC is a major pathogen in pediatric and adult populations in developing nations in Latin America and Caribbean Islands (Vidal, Chamorro, and Girón 2016). As such, several ETEC studies have focused on this part of the world, where poor sanitation is a common condition. Vidal et al. (Vidal, Chamorro, and Girón 2016) have published a comprehensive review of ETEC epidemiology in Latin America, including the first studies and advances up to 2016. In this section, we will build on this information to include recent publications on this topic. After the 2010 earthquake in Chile, an outbreak of gastroenteritis was studied in the city of Atacama, where ETEC was associated with 36% of the cases (Díaz et al. 2012). Further characterization of 39 isolates from patients with gastroenteritis in Atacama during this outbreak showed that the STh toxin profile was present in 38.5% of the ETEC isolates, 89.7% of them carried at least one CF gene, and CS21 was the most frequent (Montero et al. 2017). A large-scale comparative genomic analysis of ETEC isolates obtained from different geographical locations, including the Atacama outbreak, showed that most of the circulating ETEC isolates (88%) in Chile belong to phylogroup A, which represents a deviation from global genomic trends (Rasko et al. 2019). This same study established that the ETEC L6 lineage was present at a higher rate (21.6% vs 1.5%) than in previous studies (von Mentzer et  al. 2014). Another natural disaster in Latin America revealed that, in flooded areas, the risk of ST-ETEC infection was higher at the beginning of the climatological event of La Niña in the city of Iquitos in the Loreto province of Peru (Colston et al. 2020). In Colombia, a large prospective multicenter age-matched case-control study conducted in children under 5 years of age in Bucaramanga found that 2.4% of the attributable fraction for moderate to severe diarrhea was related to ETEC (Farfán-García et al. 2020). In Mexico, a comprehensive review of epidemiological studies carried out in different cities showed that ETEC-associated diarrhea can range from 9 to 41%, depending on the location and age of the groups involved (Ríos-Muñiz et  al. 2019). And finally, taking into account data from Bolivia, El Salvador, Guatemala, Haiti, Honduras, and Nicaragua, Anderson and colleagues (Anderson et al. 2019) reported that the morbidity and stunting rate of ETEC were the highest (comparable to some African nations) among these LMICs in Latin America. The travel-related diarrhea (TD) is a common condition, especially affecting adults who visit endemic ETEC regions, such as Latin American countries. A prospective study involving foreign adults visiting Cusco, Peru, found ETEC in 11% (N = 230) of patients who attended a local physician (Jennings et al. 2017). Other studies of TD reported a prevalence ranging from 19% to 33% (Zaidi and Wine 2015; Olson et  al. 2019). However, military personnel deployed in Honduras revealed that 37.5% of TD cases were attributable to ETEC (Walters et al. 2020). A more comprehensive review by Jiang and Dupont (2017) reported that 42% (out of 1389 patients) of TD cases from studies conducted in Latin America between 2010 and 2016 were diagnosed with ETEC infections (Jiang and DuPont 2017).

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2.4 Deciphering ETEC Evolution and Dissemination Through Genomics Advances in whole-genome sequencing have resulted in a significant reduction in cost, and developed countries have incorporated this tool as part of their routine in diagnostic and public health microbiology. In LMICs in Latin America, the genomic era has not fully arrived due to the lack of infrastructure and capacity. However, international collaborations with Latin American researchers have resulted in case studies that contributed to the understanding of diarrheal disease and advanced vaccine development. Several examples of fruitful studies incorporating genomics by Latin American and international researchers have been made to understand the diversity, transmission, and targets of ETEC. Earlier studies using MLST on 1019 ETEC isolates from endemic regions uncovered 42 different lineages, 15 representing 78% of the strains, and suggested well-established globally distributed ETEC lineages (Steinsland et al. 2010). Sahl et al. (2015) employed WGS to study the clonal diversity of ETEC within a single subject. The authors showed not only a large phylogenomic diversity of ETEC isolates from an individual patient, but also different virulence factors and suggest that ETEC diarrhea is largely caused by several highly virulent clones. Later, von Mentzer et al. (2014) performed whole genome sequencing of 365 well-­ characterized clinical ETEC with worldwide distribution and collected over a period of 30 years, including ETEC isolates from Argentina, Bolivia, Guatemala, Mexico, and Venezuela. This study identified 21 stable lineages, of which 5 (L1-L5) were major and included the most prevalent virulence profiles described in the literature, with stable combinations of the bacterial chromosome and plasmids with improved fitness and transmissibility. Although most ETEC strains harbor colonization factors, 15–50% apparently lack known CF or adhesins (Del Canto et al. 2011). Chilean researchers (Del Canto et al. 2017) have analyzed 35 draft genomes of ETEC isolates without known CF (CF negative) to look for novel colonization factors. They focused on adhesins assembled by the chaperone-usher (CU) mechanism, which is found in 17 of the 25 described CFs. This study revealed 10 new pili loci belonging to 4 CU families (β, γ2, κ and π) with functional structures (Del Canto et al. 2017). Furthermore, WGS in 125 ETEC isolates from Chile allowed the study of genetic diversity, gene content, and CF/toxin profiles among isolates from different Chilean regions (Rasko et  al. 2019). Interestingly, an important overrepresentation of the E. coli phylogroup A and lineage L6 was identified among Chilean isolates. These results were significantly different from reports from other countries and suggest that these specific clones have been successfully expanded within Chilean territory (Rasko et al. 2019). A collaborative study between the USA and Chile (Hazen et al. 2019) conducted a genome comparison of a population of ST-only ETEC isolates encoding CS6 and CFA/I. This group of strains was reported by the GEMS (Vidal et  al. 2019) study as the most prevalent CF profiles significantly associated with moderate to severe diarrhea in children under 5 years of age. Of 1194 ETEC isolates, 269 unique ETEC isolates encoding CFA/I or CS6 were whole genome

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sequenced, revealing that this population was genomically diverse with representatives of the phylogroups A, B1, and D of E. coli, and with a large diversity of plasmid replicons. However, a considerable proportion of the genomes belonged to ST2332 and ST443 and the ETEC lineages L5 and L6. Interestingly, they found that ST production levels fluctuate by lineage, but not by type of CF.

2.5 The Microbiome and ETEC The composition of the intestinal microbiota can influence the establishment and progression of an enteric infection mediated by gut pathogens (Stevens, Bates, and King 2021). However, infection with a gut pathogen can induce changes and cause dysbiosis of the intestinal microbiota, which in turn can favor the infectious agent (Stevens, Bates, and King 2021). A study that evaluated the composition of the intestinal microbiota in the feces of Chilean children infected with a DEC pathogen (including ETEC), or a viral pathogen, and uninfected individuals found that children infected with DEC bacteria had a higher proportion of Proteobacteria (Escherichia albertii, Citrobacter werkmanii, and Yersinia enterocolitica subsp. paleartica) and a lower proportion of Firmicutes compared to the uninfected individuals and the viral group. Additionally, Haemophilus sputorum species were more abundant in the DEC group in comparison with the other groups (Gallardo et al. 2017). A comparison of the microbiota composition among symptomatic and asymptomatic carriers of ETEC-infected individuals in Bangladesh revealed that symptomatic individuals had a higher proportion of Proteobacteria, while most asymptomatic patients showed a higher proportion of Firmicutes like in the case of uninfected individuals. Symptomatic patients were also more prone to co-infection with other Enterobacteriaceae members, including enteroaggregative E. coli, Salmonella enterica, Citrobacter spp., and Klebsiella spp. The measurement of Shannon’s diversity index indicated a higher α-diversity in the microbiota of asymptomatic and uninfected individuals compared to symptomatic adult patients. Interestingly, in the symptomatic group, most of the samples were positive for the presence of genes for both LT and ST toxins, while the asymptomatic group showed the presence of the ST gene. Furthermore, the identification of antimicrobial resistance genes exhibited the presence of genes encoding enzymes for classes of beta-­ lactamase, trimethoprim sulfonamide, and fluoroquinolone in a higher proportion in both symptomatic and asymptomatic carriers compared to uninfected individuals (Higginson et al. 2022). An animal model of infection showed that piglets that developed diarrhea after inoculation with ETEC had a higher relative abundance of Lactobacillus, Citrobacter, Klebsiella, Salmonella, Enterobacter, Lactococcus, and Leuconostoc compared to control animals. The microbiota in the jejunum of diarrheal piglets exhibited lower diversity compared to controls measured by the Shannon and

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Simpson indices. In that study, some of the piglets were classified as resistant to ETEC infection, as they did not develop diarrhea. Interestingly, there was a marked difference in the microbiota composition of piglets with diarrhea compared to those with resistant conditions, while only small differences were observed between resistant piglets and non-infected animals, suggesting a dysbiosis of the microbiota in piglets with diarrhea and that the microbiota can play a role in the course of an infection, making some animals resistant or susceptible to disease (Bin et al. 2018).

2.6 ETEC Vaccines ETEC is associated not only with high morbidity and mortality, but also with long-­ term detrimental effects such as physical stunting and/or environmental enteropathy (Kosek 2017; George et al. 2018). Therefore, the development of vaccines against ETEC would clearly contribute to alleviating the associated burden of ETEC in vulnerable pediatric and adult populations that reside in LMICSs. Unfortunately, to date, there are no licensed vaccines to which these countries can access, despite world leading organizations acknowledging them as a priority (WHO 2021). However, there have been important advances in this field, and some of the ETEC vaccines are currently in advanced trial stages. Khalil et al. (Khalil et al. 2021) have published a detailed review of the various aspects that emerge for ETEC vaccines. Here, we will focus on three cellular vaccines and one subunit-based vaccine that have passed the preclinical phase and show promising results. ShigETEC is a cellular vaccine designed to generate an immune response against Shigella and ETEC. This Shigella attenuated strain has been engineered to heterologously express the LTB subunit of LT plus a modified, non-toxic form of ST(STN12S) (Harutyunyan et al. 2020). The results of the placebo-­ controlled randomized phase 1 trial of ShigETEC showed that this oral vaccine was well tolerated with a 4-time dose that elicited a serum and mucosal IgA response against Shigella and the generation of functional IgG antibodies that were able to neutralize the LTB subunit of ETEC (Girardi et al. 2022). Similarly, CVD 1208S-122 is a live attenuated bivalent oral vaccine directed against Shigella and ETEC that expresses colonization factor I (CFA/I) in addition to the LTb subunit (Medeiros et al. 2020). Phase 1 studies for this vaccine are being conducted. Fimbrial tip adhesin (FTA) is a subunit intramuscular vaccine that contains Class 5 fimbria combined with other CF/CS and is currently in phase II trial (Khalil et al. 2021). ETVAX is the only vaccine that has passed and is now in phase IIb. This is an inactivated oral vaccine consisting of four engineered E. coli strains that express recombinant versions of a set of colonization factors (CFA/I, CS3, CS5, and CS6) and also include a toxoid LCTBA, which is a chimera between CTB (Cholera toxin binding subunits) and the LTB subunit of LT (Svennerholm and Lundgren 2012; Lebens et al. 1996). The latter showed that the addition of the double-mutant heat-labile toxin (dmLT) increased the immunogenicity of ETVAX (Lundgren et al. 2014). Large phase I/II

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trials in Bangladesh have demonstrated that this vaccine elicited a mucosal immune response against the CFs and LTB in adults and children older than 12  months (Qadri et al. 2020; Akhtar et al. 2019), and it was recently reported that ETVAX triggered a mucosal and systemic immune response against O78 LPS, one of the most frequent O-antigens found in ETEC (Svennerholm et al. 2022). All these vaccines are planned to continue with subsequent trials; however, to our knowledge, none of the studies are taking place in Latin America.

2.7 Updates on Antibiotic Resistance in Intestinal and Extraintestinal E. coli 2.7.1 Antibiotic Resistance in Diarrheagenic E. coli (DEC) The intestinal pathotypes of E. coli are an important source and reservoir of antimicrobial resistance genes (ARGs). The overuse and misuse of antibiotics in the treatment of bacterial diarrhea in humans and animals has led to an increased incidence of bacterial resistance to commonly used antimicrobial agents. Although the AMR is a global problem, the burden of AMR falls disproportionately on LMICs, where it threatens sustainable development (Waddington et al. 2022). Several studies in Brazil have reported high levels of MDR (63% (Pitondo-Silva et al. 2015) to 67.7% (Rodrigues et  al. 2019)) among EPEC isolates from children with diarrhea that show high prevalence of resistance to cefuroxime, ampicillin, trimethoprim-­ sulfamethoxazole, and tetracycline (Pitondo-Silva et  al. 2015; Rodrigues et  al. 2019). A survey of the antibiotic susceptibility of DEC isolates from Mexican children showed that more than 80% of DEC isolates were MDR, and EPEC displayed the highest rates of resistance to tetracycline, ampicillin, trimethoprim-­ sulfamethoxazole, nalidixic acid, and chloramphenicol (Canizalez-Roman et  al. 2016). Also, 36.5% of the EAEC isolates from Brazilian children showed MDR and were resistant to ampicillin, trimethoprim-sulfamethoxazole, tetracycline, and cefotaxime (Taborda et al. 2018). In a study in Bolivian EAEC isolates from diarrheal and non-diarrheal cases, more than 50% of the isolates were MDR in both groups (Joffré and Iñiguez Rojas 2020), with significantly higher levels of trimethoprim-­ sulfamethoxazole among clinical isolates. Resistance to ciprofloxacin, which is widely used in Bolivia to treat diarrhea, was the lowest (Joffré and Iñiguez Rojas 2020). Studies in Latin America have reported the presence of antibiotic-resistant DEC isolates in wild or farm animals, food, and/or environmental settings (Borges et al. 2017; Murphy et al. 2021; Bessone et al. 2017). For example, MDR EPEC isolates were also found in wild animals and urban pigeons in Brazil that harbor the ESBL

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gene blaCTX-M-8. Another study with isolates of EPEC, ETEC, and EAEC from wild animals and birds from Mexico and Venezuela showed low rates of MDR but important levels of resistance to ampicillin (25/27 isolates) (Murphy et  al. 2021). The occurrence of MDR ETEC associated with neonatal diarrhea in dairy calves and pigs was reported in Argentina (González Pasayo et al. 2019) with high levels of resistance to gentamicin and ceftiofur (Bessone et al. 2017). Due to the interaction of urban and wild birds and animals with humans and other animals, they can constitute a reservoir for pathogenic E. coli with zoonotic potential (Borges et al. 2017; Murphy et al. 2021; Bessone et al. 2017). Another public health problem is contaminated water and/or food with resistant bacteria that need to be monitored. A study in Mexico has reported that all isolated ETEC and EPEC found in raw coriander from local markets were MDR (Gómez-Aldapa et al. 2016). Another study investigated the quality of surface water that is often used to irrigate food products or for human consumption. The authors identified that 90% of the DEC isolates were resistant to at least one antibiotic commonly prescribed in Mexico, while 17% of them were MDR (Canizalez-Roman et al. 2019). Similarly, Poma, Mamani, and Iñiguez (2016) and Guzman-Otazo et al. (2019) screened surface water samples from the La Paz River in Bolivia for the presence of DEC isolates, and reported that 78% and 35% of the isolates were resistant to at least one or three antibiotics, respectively. They were mostly resistant to ampicillin, nalidixic acid, trimethoprim-sulfamethoxazole, and tetracycline. These studies highlight the high potential risk of transmission of diarrheal diseases by consumption of contaminated water and vegetables, as well as the potential for the transfer of antibiotic resistance from the environment to the community. Together, the patterns of antibiotic resistance between DEC isolates reflect similar trends of antibiotic resistance in other LMICs with important levels of resistance to older antibiotics such as ampicillin, trimethoprim-sulfamethoxazole, and tetracycline, despite the large variation in the selection of antibiotics evaluated. The prevalence of ESBL-producing DEC isolates remains low in Latin American countries, but much higher levels have been reported from extraintestinal E. coli infections in hospitals in Asia and Africa (Ingle et al. 2018).

2.7.2 Antibiotic Resistance in Extraintestinal E. coli (ExPEC) E. coli is an extraordinarily versatile bacterial species with the ability to colonize numerous animal hosts, the environment, and food (Blount 2015). E. coli strains are non-pathogenic commensal bacteria; however, E. coli variants could cause infections inside and outside the gastrointestinal system (Pitout 2012). Extraintestinal pathogenic E. coli (ExPEC) are facultative pathogens that are part of the normal human intestinal flora. The ExPEC group includes uropathogenic E. coli (UPEC), neonatal meningitis E. coli (NMEC), sepsis-associated E. coli (SEPEC), and avian

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pathogenic E. coli (APEC). The virulence factors related to ExPEC pathogenicity are numerous and have a wide range of activities, from those related to bacterial colonization to those related to virulence, including adhesins, toxins, iron acquisition factors, lipopolysaccharides, polysaccharide capsules, and invasins, which are usually encoded on pathogenicity islands, plasmids, and other mobile genetic elements (Dale and Woodford 2015). These can be horizontally exchanged in related bacteria or in bacteria from different families, allowing their settlement in different environments. E. coli can be classified into the following phylogenetic groups: A, B1, B2, C, D, E, F, and clade I (Baldy-Chudzik, Bok, and Mazurek 2015). Commensal E. coli, without pathogenic characteristics, is present, among others, in the mucosa of the gastrointestinal tract. These most often represent group A or B1. Pathogenic E. coli responsible for intestinal infections represent phylogenetic groups A, B1, or D. The E. coli strains responsible for extraintestinal infections belong to groups B2 and D. Group E is related to group D (including O157:H7), while group F is related to the main group B2. The clones of E. coli strains, which are genetically diverse but phenotypically indistinguishable, have been assigned to the cryptic clade I (Köhler and Dobrindt 2011). Antimicrobial resistance rates in E. coli are increasing rapidly, especially resistance to fluoroquinolones and third- and fourth-generation cephalosporins. Furthermore, extended-spectrum β-lactamases (ESBLs) are a major cause of antimicrobial resistance among these bacterial strains (Fair and Tor 2014). Infections caused by ESBL-producing E. coli (ESBL-Ec) have increased worldwide both in hospitals (Paterson and Bonomo 2005) and in community settings (Pitout et  al. 2005). This phenomenon is related to the emergence of hyperepidemic clones, primarily E. coli sequence type 131 (ST131) and phylogroup B2. These clones cause UTIs and are a main source of bacteremia in humans (Tourret and Denamur 2017). E. coli ST131 was initially described among clinical isolates producing the CTX-­ M-­15 ESBL enzyme and resistant to fluoroquinolones in several countries (Pitout and DeVinney 2017). Currently, E. coli ST131 has a global distribution that is associated with various determinants of antimicrobial resistance (Garrido et al. 2017). E. coli ST131 isolates harboring the blaCTX-M-15 gene have been reported in Argentina, Brazil, Colombia, Ecuador, Mexico, and Uruguay, corroborating the expansion of this clone in this region (Zurita et al. 2019). Resistance to carbapenems has also been reported (Reyes et al. 2020) and the presence of the mcr-1 gene that confers resistance to colistin (Ortega-Paredes, Barba, and Zurita 2016). ESBL-E. coli strains have also been identified in urban rivers, companion animals, poultry products, ready-to-eat food, and vegetables (Ortega-Paredes et  al. 2018; Vinueza-Burgos et al. 2019; Zurita et al. 2020; Albán et al. 2020). Food safety is an important challenge for public health, both in the production stage and during its processing and distribution. Undoubtedly, the spread of ESBL-E. coli from community reservoirs to clinical environments appears relevant, especially when linked

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to lineages that harbor blaCTX-M genes, such as ST131, ST44, ST10, or ST410. CTX-M β-lactamases are widespread enzymes, and early variants of CTX-M enzymes could hydrolyze ceftriaxone and cefotaxime, although later variants could also hydrolyze ceftazidime (e.g., CTX-M-15 and CTX-M-55) (Castanheira, Simner, and Bradford 2021). Although treatments are available for isolates carrying these enzymes, combination with other resistance mechanisms may limit the activity of new agents (Castanheira, Simner, and Bradford 2021). Furthermore, many of these new treatment alternatives are not currently available in Latin America (Terreni, Taccani, and Pregnolato 2021). The worldwide burden of these extraintestinal infections is staggering, with hundreds of millions of people affected annually and considerable morbidity and mortality in cases of complication with bacteremia or sepsis syndrome. In addition, E. coli pathogens, particularly those that cause extraintestinal infections, have developed resistance to every class of antibiotics introduced to treat human and animal infections. The prevalence of resistance to first-line oral antibiotics, such as trimethoprim-sulfamethoxazole, fosfomycin, amoxicillin, and amoxicillin plus clavulanic acid, which are widely used to treat community-acquired E. coli infections, has increased steadily over time (https://www3.paho.org/data/index.php/es/temas/ resistencia-­antimicrobiana.html). The release on the market of fluoroquinolones and extended-spectrum cephalosporins in the 1980s increased expectations of treatment efficacy, but due to the appearance of E. coli-specific clone sequence type 131 (ST131) and its remarkable ability to survive and persist after its appearance in 2008, has suggested that this increase in resistance is related to the worldwide spread of this clone (Cummins et  al. 2021). In many Latin American countries, resistance to quinolones exceeds 50%, and the presence of ESBL-E coli probably exceeds 60% in some regions (https://www3.paho.org/data/index.php/es/temas/ resistencia-­antimicrobiana.html). The prevalence of ST131 emphasizes the relationship between this strain and infections that circulate in the Latin America region, so more studies should focus on the surveillance of AMR-ExPEC not only in hospitals, but also in the community and the environment. In fact, invasive ExPEC disease is one of the most common causes of death due to AMR. We must explore and develop more effective ways of dealing with AMR infections, including through innovative vaccines and therapeutics, as well as antibiotic management efforts to limit the development of more AMR pathogens. There are currently no ExPEC vaccines available on the market, and only a few candidates are in development. Janssen Vaccines, in collaboration with LimmaTech Biologics, is developing a vaccine (ExPEC4V) based on O antigens that correspond to four prevalent serotypes (Huttner and Gambillara 2018) (Table 2.1).

21 13 66 1.2k 20 431 85 31

2 8 8 151 3 43 9 6

132 79 545 54.k 130 1.4k 525 142

5 3 17 227 5 35 22 28

Carbapenems 18 29 27 44 121 4 15 15 15 11 11 46 29 183 1.1k 33 241 148 16

Fluoroquinolones 88 118 221 233 1.3k 13 45 147 75 55 67 30 21 159 821 33 388 171 25

Third-generation cephalosporins 78 105 215 230 1.9k 17 35 96 97 71 74

Source: MICROBE (Measuring Infectious Causes and Resistance Outcomes for Burden Estimation) https://vizhub.healthdata.org/microbe

Country Bolivia Ecuador Peru Colombia Mexico Costa Rica El Salvador Guatemala Honduras Cuba Dominican Republic Nicaragua Panama Venezuela Brazil Paraguay Argentina Chile Uruguay

Beta-lactam/ beta-lactamase Aminoglycosides inhibitor 31 60 31 63 106 116 11 132 142 274 4 27 4 30 6 18 7 33 7 40 18 40

Resistance to one or more antibiotic 370 433 841 933 4.3k 91 171 498 285 244 244

Table 2.1  Deaths attributable to bacterial antimicrobial resistance in E. coli, all ages, both sexes (2019)

9 5 23 590 11 147 32 14

Aminopenicillins 27 4 9 74 105 8 16 47 14 18 5

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Garrido D, Garrido S, Gutiérrez M, Calvopiña L, Harrison A, Fuseau M, Salazar Irigoyen R (2017) Clinical characterization and antimicrobial resistance of Escherichia coli in pediatric patients with urinary tract infection at a third level hospital of Quito, Ecuador. Bol Med Hosp Infant Mex 74:265–271 George CM, Burrowes V, Perin J, Oldja L, Biswas S, Sack D, Ahmed S, Haque R, Bhuiyan NA, Parvin T, Bhuyian SI, Akter M, Li S, Natarajan G, Shahnaij M, Faruque AG, Stine OC (2018) Enteric infections in young children are associated with environmental enteropathy and impaired growth. Tropical Med Int Health 23:26–33 Gill DM, Richardson SH (1980) Adenosine diphosphate-ribosylation of adenylate cyclase catalyzed by heat-labile enterotoxin of Escherichia coli: comparison with cholera toxin. J Infect Dis 141:64–70 Girardi P, Harutyunyan S, Neuhauser I, Glaninger K, Korda O, Nagy G, Nagy E, Szijártó V, Pall D, Szarka K, Kardos G, Henics T, Malinoski FJ (2022) Evaluation of the safety, tolerability and immunogenicity of ShigETEC, an oral live attenuated Shigella-ETEC vaccine in placebo-­ controlled randomized phase 1 trial. Vaccines (Basel) 10:340 Gómez-Aldapa CA, Segovia-Cruz JA, Cerna-Cortes JF, Rangel-Vargas E, Salas-Rangel LP, Gutiérrez-Alcántara EJ, Castro-Rosas J (2016) Prevalence and behavior of multidrug-resistant shiga toxin-producing Escherichia coli, enteropathogenic E. coli and enterotoxigenic E. coli on coriander. Food Microbiol 59:97–103 Gonzales L, Ali ZB, Nygren E, Wang Z, Karlsson S, Zhu B, Quiding-Järbrink M, Sjöling Å (2013) Alkaline pH Is a signal for optimal production and secretion of the heat labile toxin, LT in enterotoxigenic Escherichia coli (ETEC). PLoS One 8:e74069 Gonzales-Siles L, Sjöling Å (2016) The different ecological niches of enterotoxigenic Escherichia coli. Environ Microbiol 18:741–751 González Pasayo RA, Sanz ME, Padola NL, Moreira AR (2019) Phenotypic and genotypic characterization of enterotoxigenic Escherichia coli isolated from diarrheic calves in Argentina. Open Vet J 9:65–73 Gorbach SL, Banwell JG, Chatterjee BD, Jacobs B, Sack RB (1971) Acute undifferentiated human diarrhea in the tropics: I. Alterations in intestinal microflora. J Clin Invest 50:881–889 Guzman-Otazo J, Gonzales-Siles L, Poma V, Bengtsson-Palme J, Thorell K, Flach CF, Iñiguez V, Sjöling Å (2019) Diarrheal bacterial pathogens and multi-resistant enterobacteria in the Choqueyapu River in La Paz, Bolivia. PLoS One 14:e0210735 Harutyunyan S, Neuhauser I, Mayer A, Aichinger M, Szijártó V, Nagy G, Nagy E, Girardi P, Malinoski FJ, Henics T (2020) Characterization of ShigETEC, a novel live attenuated combined vaccine against Shigellae and ETEC. Vaccines (Basel) 8:689 Haycocks JR, Sharma P, Stringer AM, Wade JT, Grainger DC (2015) The molecular basis for control of ETEC enterotoxin expression in response to environment and host. PLoS Pathog 11:e1004605 Hazen TH, Nagaraj S, Sen S, Permala-Booth J, Del Canto F, Vidal R, Barry EM, Bitoun JP, Chen WH, Tennant SM, Rasko DA (2019) Genome and functional characterization of colonization factor antigen I- and CS6-encoding heat-stable enterotoxin-only enterotoxigenic Escherichia coli reveals lineage and geographic variation. mSystems 4:e00329-18 Higginson E, Abu Sayeed M, Pereira Dias J, Shetty V, Ballal M, Srivastava S, Willis I, Qadri F, Dougan G, Mutreja A (2022) Microbiome profiling of enterotoxigenic Escherichia coli (ETEC) carriers highlights signature differences between symptomatic and asymptomatic individuals. MBio 13:e00157–e00122 Huttner A, Gambillara V (2018) The development and early clinical testing of the ExPEC4V conjugate vaccine against uropathogenic Escherichia coli. Clin Microbiol Infect 24:1046–1050 Ingle DJ, Levine MM, Kotloff KL, Holt KE, Robins-Browne RM (2018) Dynamics of antimicrobial resistance in intestinal Escherichia coli from children in community settings in South Asia and sub-Saharan Africa. Nat Microbiol 3:1063–1073 Jennings MC, Tilley DH, Ballard SB, Villanueva M, Costa FM, Lopez M, Steinberg HE, Luna CG, Meza R, Silva ME, Gilman RH, Simons MP, Maves RC, Cabada MM (2017) Case-case

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Chapter 3

New Concepts on Domestic and Wild Reservoirs and Transmission of E. coli and Its Environment Adriana Bentancor, Ximena Blanco Crivelli, Claudia Piccini, and Gabriel Trueba Chapter Summary  Escherichia coli is a bacterium that has gone from a description of just being an intestinal habitat or environmental contaminant to its dispersion in various environments, in many of which it survives for long periods, showing great adaptation, including dryness. Virulence and antimicrobial resistance genes are functional on various genomic platforms, generating health risks. New concepts of domestic and wild reservoirs of E. coli strains as health risk reveal alternatives in the transmission of E. coli and its environment.

3.1 Introduction Many bacterial species are represented by a pan-genome, whose genetic repertoire far outstrips any single bacterial genome (Rosconi et al. 2022). The Escherichia coli genome was sequenced and published in 1997 (Blattner et al. 1997), describing the total coding capacity of this organism. In E. coli, the set of genes shared by all strains, the core genome, represents half the number of genes present in any strain. According to that, the remaining genes are related with a high diversity that results in potential adaptations (Rousset et al. 2021). The presence of mobile genetic elements facilitates the mobility of the so-called accessory genome which is responsible for the variability between strains. This variability is highly related to the A. Bentancor (*) · X. Blanco Crivelli Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Microbiología, CABA, Argentina e-mail: [email protected] C. Piccini Departamento de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay G. Trueba Instituto de Microbiología, Colegio de Ciencias Biológicas y Ambientales, Universidad San Francisco de Quito, Vía Interoceánica y Diego de Robles, Cumbayá, Quito, Ecuador © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 A. G. Torres (ed.), Trending Topics in Escherichia coli Research, https://doi.org/10.1007/978-3-031-29882-0_3

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environmental conditions selecting the accessory genes. Essential genes encode the processes that are necessary for the microorganism survival. Until recently, commonly applied binary classifications left no space between essential and non-­ essential genes. Hogan and Cardona (2022) explored how the quantitative properties of gene essentiality are influenced by the nature of the encoded process, environmental conditions, and genetic background, including the strain’s distinct evolutionary history. A bacterial pan-genome might influence gene essentiality, and some essential genes are initially critical for survival and can become non-essential in different environments (Rosconi et al. 2022). The study of genome dynamics takes a central spot in potentially pathogenic organisms (Duarte-Velázquez et al. 2022). In bacterial evolution, a central topic is acquiring genomic island capability, especially the transfer of pathogenicity-­ associated and antimicrobial resistance genes (Desvaux et  al. 2020). One of the best-studied horizontal gene transfers is the one mediated by phages (Duarte-­ Velázquez et al. 2022). The wide variety of pathogenic traits of E. coli is based on the ability to accept new genetic material such as plasmids, integrons, transposons, phages, and other mobile elements, resulting in the transfer of phenotypic traits linked to recombination events. E. coli is the most researched microorganism in the world. In addition to its harmless existence as a gut commensal, E. coli is a major cause of animal and human disease. Its impact varies on animal and human health, consisting of commensalism, gastrointestinal disease, or extraintestinal pathologies and has generated a classification of the isolates into pathotypes or pathovars. These are broadly split into two pathogroups, intestinal or diarrheagenic pathogenic E. coli (DEC) and extraintestinal pathogenic E. coli (ExPEC). At least 12 different E. coli pathotypes have been defined (Geurtsen et al. 2022). Although no specifically pathogenic virulence factors are associated with ExPEC, several pathotypes have been described: uropathogenic E. coli [UPEC], neonatal meningitis E. coli [NMEC], avian pathogenic E. coli [APEC], sepsis-associated E. coli [SEPEC], necrotoxic E. coli [NTEC], and others. On the other hand, DEC are characterized by their virulence factors. The pathotypes include enteropathogenic E. coli [EPEC], Shiga toxin-producing E. coli [STEC] which includes enterohemorrhagic E. coli [EHEC], enterotoxigenic E. coli [ETEC], enteroinvasive E. coli [EIEC], enteroaggregative E. coli [EAEC], as well as diffuse adhering E. coli [DAEC] and adherent-invasive E. coli [AIEC] (strains found in Crohn’s disease patients, but with a lack of consistent virulence factors) (Mirsepasi-Lauridsen et al. 2019). The presence of E. coli in feces and its ability to persist in a variety of environments outside of a (warm-blooded) host has resulted in its long-standing use as a fecal indicator bacterium for water quality (Gruber et  al. 2014; World Health Organization 2017). It is known that a switch on or switch off pathogenicity exists by exchanging mobile genetic elements as well as spontaneous mutations that will be efficient to the extent that they give an adaptive advantage to their environment, biotic or abiotic (World Health Organization 2022). Moreover, several strains adapt to the environment outside the hosts (De la Cuesta et al. 2022).

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It is estimated that 60% of human infectious diseases, and 75% of emerging diseases, are of animal origin. The “One Health” concept proposes a broad approach to minimize the impact of diseases on human and animal health which cause large economic losses and are linked to the ecosystems in which they coexist (World Organization for Animal Health 2014). Since the Germany outbreak by an STEC-EAEC hybrid strain, new combinations of virulence factors among the classic E. coli pathotypes have increased description of hetero-pathogenic E. coli or hybrid strains (Santos et al. 2020). E. coli could be detected everywhere, and there are no big differences among host species or among geographical zones. However, the distribution of virulence genes seems not to be random and may be related to a closer contact with humans/livestock settlements. Descriptive epidemiology and analytic epidemiology provide additional insights into the ecology of potentially pathogenic E. coli in multi-host settings, and the role of animals as intermediate reservoirs (Cabal et al. 2017). Also, whole genome sequencing (WGS) analysis allowed verification of the relevance of geographic variation of clones as well as indistinguishable profile of phage insertion sites, diversity, and genotypes between host reservoirs, e.g., STEC with cattle and sheep (Strachan et al. 2015). Besides, WGS of ExPEC belonging to the genetically diverse ST95 that includes avian pathogenic (APEC) causing colibacillosis in poultry, and human UPEC, demonstrated multiple lineages and overlapping suggested zoonotic source of infection (Jørgensen et al. 2019). However, Murphy et  al. (2021), while analyzing WGS, provided evidence for the clinical importance of wild animals as reservoirs for pathogenic strains and highlighted the need to include non-human hosts in the surveillance programs for E. coli infections.

3.2 Animal Reservoirs of Several Pathotypes Described in the Last Years The pathotype STEC, a relevant zoonotic E. coli, is a foodborne pathogen associated with contamination of water and food by feces of wild or domestic carrier animals. Food is a common source of infection, but many other potential sources are associated with outbreaks. STEC-associated diseases in animals are infrequent, which represents an additional difficulty in the epidemiological studies that showed the importance of the human-animal-environmental interface that is critical for disease development. Ruminants, especially cattle, constitute a vast reservoir of STEC, and human infection can frequently be traced to contamination of food or water with cattle manure. Besides ruminants, other small ruminants, such as sheep, goats, deer, and South American Camelids (SAC), are considered STEC reservoirs.

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3.2.1 Small Ruminants Among studies in sheep published in recent years, McCarthy et al. (2021) detected Shiga toxin positive in 83.8% of recto-anal swab samples from Irish sheep and confirmed 51.6% by positive culture. They found five animals shedding STEC O157, and three of these as super shedders. Young animals were the most frequent carriers, and the carriage during summer season was prevalent. Some of the STEC non-O157 isolated in the study harbored stx1c, besides dispersed virulence markers, but the relevant results were the identification of variants reported from clinical cases. The information highlighted in the results of WGS showed the same SNPs among animals coexisting in a geographical region (Strachan et al. 2015). In Southern Brazil STEC strains were isolated in 50% of the sheep tested, from different flocks (Martins et al. 2015). From the isolates, 87.1% harbored ehxA and eae genes, and putative adhesins and toxins were also detected. STEC from Brazil and Argentina sheep showed serotypes that are unusually reported in other regions (Martins et al. 2015; Blanco Crivelli et al. 2021). Moreover, in Argentina and Brazil, other E. coli pathotypes were identified, where aEPEC was isolated (Martins et  al. 2016; Blanco Crivelli et al. 2021) and EAEC was detected in sheep using PCR. In conclusion, sheep contribute to the geographical dispersion of the pathogenic E. coli and could contaminate their environment, as well as carcasses and sheep meat. Besides, the analysis of diarrheagenic lambs shows animals carrying ETEC, EIEC, and NTEC (Ghanbarpour et al. 2017). Goats are a potential source of diverse pathogenic STEC and aEPEC serotypes for humans. In South Africa, STEC was found in 80% of goat fecal samples. The serotyping revealed a carriage of O157:H7 and among non-O157 serotypes, O103:H8, O26:H2, O111:H8, and other serotypes (Malahlela et al. 2022). Moreover, strains isolated from goat milk were potentially pathogenic for humans, among them STEC O157:H7 and aEPEC O26:H11 (Álvarez-Suárez et  al. 2016). In Australia, STEC from goats, not belonging to the most prominent serogroups, harbored stx genes (Jajarmi et al. 2018). A study of 448 STEC strains from 2896 goat fecal samples in China revealed that 38% produced Stx2k. The emergent stx2k subtype was previously reported in isolates from human clinical samples, animals, and raw meat, only in that country. More than a half of the Stx2k-STEC are from the hybrid pathovar ETEC-STEC, carrying the heat-labile toxin (Yang et al. 2022). The domestic SAC were found carrying STEC O157, besides the wild SAC in captivity which were considered STEC reservoirs (Pritchard et al. 2009). Dias et al. (2019) reported wild roe deer and red deer carrying STEC O146[H21, H28], O27:H30, and ONT:H28 encoding stx2b or stx2g subtypes, intimin, enterohemolysin, and protease effector genes, found in Portugal. Moreover, Szczerba-Turek et al. (2020) identified 21.65% of red deer carrying E. coli O157:H7, and 24.63% of roe deer carrying STEC/AE-STEC in Poland; and these isolates showed stx2a/eae virulence profiles in both species. Previously, in the USA, Singh et al. (2015) provided evidence of STEC interspecies transmission between cattle and white-tailed deer in a shared agroecosystem, which highlighted the importance of the study of wildlife

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species in pathogen shedding dynamics and persistence in the environment. In this study, STEC and EPEC were detected in 22% of the fecal samples of white-tailed deer. The epidemiological study of reportable enteric diseases in Oregon showed O157:H7 in patients who hunt and harvest deer meat (Ladd-Wilson et al. 2022).

3.2.2 Other Ungulates The order Artiodactyla includes diverse ungulates, such as pigs, wild boars, peccaries, and hippopotamuses. Domestic swine harbor and shed several serogroups of E. coli involved in piglet diarrhea, post weaning diarrhea, and edema disease. A main stx-subtype was detected in 81–85% of swine strains carrying stx2e, which is rarely involved in human infections. The strains producing Stx1c and Stx2d toxins, associated with human infections, were detected in a low proportion in strains from pigs (Wang et al. 2017; Bok et al. 2020; Remfry et al. 2021). Other pathotypes isolated from domestic swine include ExPEC, ETEC, and EIEC. Contrary to what was reported by Baranzoni et  al. (2016), EPEC was prevalent in pigs from Mexico (Tamayo-Legorreta et al. 2020). STEC, EAEC, and aEPEC pathotypes were detected among wild boars, and the isolation of STEC O157:H7 and tEPEC O49:H10 carrying the eae-k and bfpA genes, a virulence profile previously reported in human disease, is noteworthy (Navarro-Gonzalez et al. 2015; Alonso et al. 2017; Bertelloni et al. 2020). In addition, the hybrid STEC/ETEC O139:H1 was detected in the wildlife, and was different from STEC O139:H1 strains obtained from domestic swine (Perrat et al. 2022). Additionally, swine in Chile was considered a source of a wide variety of STEC, which could be a potential cause of severe illness in humans (Galarce et al. 2019).

3.2.3 Pets Direct contact between humans and companion animals and the environmental reservoirs may be important routes for cross-species sharing of E. coli pathotypes. Among ExPEC, many sequence types (STs) were persistent for several years in dogs and humans. Due to the fact that most STs from canine infections are the dominant human ExPEC STs, which are similar in genomic information, plasmid carriage, and virulence gene, the hypothetical route of transmission via human dog is robust. Contrarily, a dominant canine STs 372 causes sporadic infections in humans; therefore, their zoonotic role is supported (Elankumaran et  al. 2022). Additionally, human STs shared 30% of identity with the STs prevalent in cats (Bourne et al. 2019). Other DEC pathotypes isolated from healthy dogs were ETEC, EPEC, STEC, and EIEC (Cui et al. 2022). Vasco et al. (2016) reported identical STs between children and domestic animals in a semirural community in Ecuador, supporting the

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role of zoonotic origin for aEPEC. Besides, isolated aEPEC obtained from diarrheal and asymptomatic kittens was genetically indistinguishable from each other and from humans. Therefore, kittens may also serve as a reservoir for aEPEC (Watson et al. 2021).

3.2.4 Birds Free-living birds in the wild, peri-urban, and urban contexts may host and spread pathogens. Their relevant role as potential reservoirs could cause the dissemination of pathogens across different environments. STEC O157 and O26 have been detected in Larus spp., yellow-legged gulls, and EPEC O26 was isolated from hunting birds in Italy (Gargiulo et  al. 2018; Russo et  al. 2021). Reports included the isolation of STEC and EPEC from different orders of wild birds in the last years (Caballero et  al. 2015; Konicek et  al. 2016; Borges et  al. 2017; de Oliveira et al. 2018). Isolates from Fregata magnificens from the oceanic coast of Brazil showed virulent genotypes, and serotypes of classical human ExPEC or APEC (Saviolli et al. 2016). Additionally, APEC (causing colibacillosis in chickens) has been reported to be genetically related to human ExPEC (Solà-Ginés et  al. 2015). Unexpectedly, hybrid tEPEC/STEC, which harbor eae, bfpA, and stx2f genes, were detected in birds (Gioia-Di Chiacchio et al. 2018). However, bfp operon types differed between human and bird hybrid tEPEC/STEC (van Hoek et al. 2019).

3.2.5 Rodents There is limited information about rats, mice, and bats which can also transmit E. coli. Mice and rats (Mus musculus, Rattus rattus, and Rattus norvegicus) have been found to carry DEC, including EAEC, EPEC, and STEC, that can potentially infect both humans and animals. Himsworth et al. (2015) detected STEC serotypes O26, O45, O103, and O145, which are relevant to public health. Additionally, Ramatla et al. (2022) and Azimi et al. (2021) identified Rattus spp. harboring aEPEC strains in a chicken farm. Moreover, Blanco Crivelli et al. (2018) reported the serotype O108 associated to STEC and aEPEC in Rattus rattus from Buenos Aires. The role of wildlife carrying a virulent profile in the epidemiology of E. coli is still unclear (Orden et al. 2021). The detection of wild big rodent capybaras carrying STEC living in an urban area of Brazil highlights the importance of including wild animals in the epidemiology of infectious diarrhea (Merker Breyer et al. 2022). On the other hand, the wild Marmota himalayana, which has little human interaction, carries virulence genes from different pathotypes, suggesting that in this reservoir new hybrids are developed and are potentially virulent to humans (Lu et al. 2016).

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3.2.6 Other Reports from Wild Animals Virulence genes eaeA, stx1, and stx2 were reported in samples from wild red foxes, which are proposed as potential carriers of DEC (Bertelloni et al. 2022). The study of reptiles in Brazil (free, in captivity, or companion animals) reported the carrying of NTEC and ETEC (Ramos et al. 2019). One of the missing pieces of information has been related to AIEC, which was described in 1999. The identification of this pathotype in the ECOR collection that was obtained from the intestine of healthy animals and humans showed several animals (elephants, pigs, monkeys, goats, and pumas) as carriers of strains from this pathogroup (Rahmouni et al. 2018).

3.3  E. coli Outside the Host E. coli is a normal inhabitant of the intestinal microbiota of humans and other warm-­ blooded animals. It is believed that the animal/human intestine constitutes its main habitat and its secondary habitat is the environment, or not associated with the host (Van Elsas et al. 2011). In this framework, it is considered that E. coli has a biphasic lifestyle alternating between both environments. The bacterium is eliminated by fecal matter; it proliferates massively in fresh fecal matter in the presence of oxygen (Russell and Jarvis 2001; Guerrero et al. 2020), and is able to die or persist for a variable time in the soil, water, or sediments, and then it can access to a new host. Since the intestine is its main habitat, its presence in water and soil has been considered an indicator of fecal contamination. However, numerous studies have reported that some E. coli strains can survive and potentially reproduce in extraintestinal environments. These “naturalized” E. coli populations can integrate into indigenous microbial communities (Jang et  al. 2017) sharing certain characteristics among them and differing genotypically from those of animal-host origin (Jang et al. 2017). E. coli strains have an extensive genetic substructure. They can be classified into 13 phylogenetic groups based on phenotypic and genotypic characteristic traits, A, B1, B2, C, D, E, F, and G, belonging sensu stricto to E. coli and cryptic clades (I to V) (Clermont et al. 2000, 2013, 2019). The comparative genomic analysis between environmentally adapted E. coli strains and other enteric E. coli strains showed that environmental strains belong to clades distantly divergent from enteric E. coli and other Escherichia strains, with no primary association with mammal hosts (Luo et al. 2011). Moreover, it has been found that environmental E. coli strains did not contain some genes of the pan-genome, but uidA, gadAB, and fimH genes were frequently present (Walk et al. 2009). Genetic exchange of core genes was detected, but only within the environmental clade or within enteric E. coli strains and not between environmental clades and enteric E. coli strains (Luo et al. 2011), indicating that there could be possible ecological barriers to gene transfer between both groups of strains.

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The survival of E. coli in natural environments can be influenced by abiotic and biotic factors (Rochelle-Newall et  al. 2015). Abiotic factors include temperature, which is considered the main factor for the survival and growth of the bacteria, as well as water, nutrient availability, pH, and solar radiation. Biotic factors include the presence of other microorganisms, the ability to acquire nutrients, the competition with other microorganisms, and the formation of biofilms in natural environments. Owing to the fluctuating conditions of the environment, E. coli needs to obtain diverse available nutrients and energy sources for survival. This is why most forms of E. coli have conserved particular key evolutionary factors, helping with adaptations, in their core genomes (Van Elsas et al. 2011). Moreover, the availability of carbon and nitrogen, the ability to regulate stress, and the presence of algal mats (e.g., Cladophora) affect the persistence of E. coli in freshwater beach sand (Rumball et al. 2021; Meyers and McLellan 2022). The importance of biotic factors in bacterial survival has been investigated. Several studies have shown that some Indigenous (natural) microbiota can cause E. coli population decline (Korajkic et al. 2013; Baker et al. 2020a, b). Moreover, Baker et al. (2020a) identified Paenibacillus alvei present in the soil microbiota that may reduce STEC O157:H7 in the soil environment while Ma et al. (2013) found that the survival of E. coli O157:H7 was positively correlated with the abundance of Actinobacteria and Acidobacteria and that negatively correlates with those of Proteobacteria and Bacteroides. The ability to adhere becomes important in the interaction between E. coli and plants and algae. In the environment E. coli could be associated with plant tissues, persisting as a biofilm, and being able to access different hosts when the plant products are ingested (Serra and Hegge 2021). Regarding the factors that participate in the formation of biofilm in plants, these include many adhesins, amyloid curli fibers, and exopolysaccharides; however, their role is not clear.

3.3.1  E. coli in the Aquatic Environment: Fecal Source or Adaptation? Entry of E. coli cells into aquatic systems occurs primarily through runoff, sewage discharge, and direct fecal deposition (Korajkic et al. 2019). In several countries, communities often suffer from defective sanitary infrastructure which many times exposes the communities to fecal matter contaminants. Open sewers are frequently visited by domestic and peri-domestic animals (including insects) which become transporters of fecal bacteria to human households. The number of microorganisms and the frequency at which they enter the ecosystem depends directly on human activities in the water system basin, whether they are agricultural activities, densely populated urban centers, or locations with low population density but without a sanitation system or sewage treatment (World Health Organization 2017). Once in the water, E. coli cells find environmental

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conditions that drastically differ from the gut in many different ways, from exposure to sunlight (ultraviolet radiation), low temperature, changing organic carbon and nutrients concentration or higher salinity (in the case of reaching coastal ocean) to the presence of other biotic components, such as bacteriophages, protists predators, or the native microbiota that control its chances of survival and decay (González et  al. 1992; Fenchel 2008; Wanjugi and Harwood 2013; Di Cesare et  al. 2022). Many of these variables have been proposed as inducers of a viable but non-­ culturable (VBNC) phase of E. coli in the environment (Xu et al. 1982; Ding et al. 2017). Under the VBNC state, bacterial cells are characterized by having physiological and molecular differences with culturable cells, including the composition of the cell membrane and the cell wall, the genes they express, cellular morphology, physical and chemical resistance, and virulence (Li et al. 2014). The VBNC cells can be reverted to their culturable condition in the appropriate laboratory conditions or after ingestion by the host (Oliver 2005), which represents a challenge for their use as indicators of fecal contamination because their abundance can be underestimated when using standard culture techniques, increasing their potential health risks. For example, a work performed in shallow groundwater wells in the touristic town of Cabo Polonio (Uruguay) (Soumastre 2016) revealed that, while the abundance of culturable fecal coliforms increased during summer, which was associated with the increased number of people, their total abundance assessed by in situ hybridization remained constant through the year. Furthermore, the pathogenic potential of the groundwater samples, assessed by qPCR, allowed to detect stx1, stx2, and eae genes, suggesting the presence of Shiga toxin-producing bacteria corresponding to the enteropathogenic and enterohemorrhagic pathotypes (Soumastre et al. 2022). Interestingly, Berthe et al. (2013) found that isolates from water lost their culturability more frequently than those of fecal origin. These findings suggest that under oligotrophic conditions, the presence of non-culturable E. coli in water could increase the outbreak risk because they cannot be detected by conventional culture-dependent techniques. In addition to the VBNC state, E. coli possess other mechanisms to survive and persist in the aquatic environment. For example, E. coli isolated from freshwater systems exhibit a smaller genome size than isolates from other sources, suggesting a streamlining-related evolutionary adaptation to the aquatic environment (Touchon et al. 2020). The genome streamlining theory proposes the reduction of genome size (reduction of non-coding DNA and fewer non-essential genes) as a strategy to allow a reproductive benefit to prokaryotes (Sela et al. 2016). It has been described as a common feature in the marine habitat among free-living bacterioplankton (Swan et al. 2013) and in pathogens (Moran 2002). Interestingly, it has been shown that in E. coli, the variations in genome size is associated with the source of isolation and with the phylogenetic group, being the smaller genomes associated with freshwater habitats. These freshwater small-genome E. coli isolates present few but highly diverse mobile genetic elements, implying the existence of a relationship between the size of the genome and the ability to exchange information, which can be especially helpful for adaptation to the aquatic environment (Touchon et  al. 2020). Moreover, several reports show that (a) E. coli can survive and grow outside the

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intestinal tract; (b) extraintestinal populations are in fact different from the host-­ associated ones (Jang et al. 2017); (c) freshwater E. coli genomes show the smallest number of antibiotic resistance genes, bacteriocins, and virulence factors, while harboring a higher number of capsule-producing systems; and (d) outbreak strains usually emerge or belong to a specific phylogenetic group (Touchon et al. 2020). In conclusion, all these results have challenged the idea that isolates from water are usually from fecal origin and strongly suggest that these strains have changed to adapt to water environments. Moreover, Power et  al. (2005) isolated free-living encapsulated E. coli strains from lakes that do not have fecal input and belong to different geographical regions. In a study that refers to the ecological conditions impacting mechanisms structuring E. coli in urban riverine communities in Argentina, Saraceno et al. (2021) found that the phylogroups A and B1 were dominant. Through experimental approaches, they demonstrated that B1 phylogroup persisted longer at low temperatures and exhibited phenotypic traits associated with a higher ability for plant colonization (Saraceno et al. 2021). This phylogroup was able to survive and persist in the water of different salinities (Walk et al. 2007; Ratajczak et al. 2010; Touchon et al. 2020). Furthermore, Berthe et al. (2013) found that the strains able to survive longer in estuarine water (up to 14 days) were from the B1 phylogroup, and they were able to grow at 7 °C. They also propose that several E. coli populations with different survival strategies coexist in the aquatic ecosystem, and those having a phenotype best fitted for survival outside the host will be positively selected (Berthe et al. 2013). Therefore, the evidence about the VBNC states, where oligotrophy-adapted cells cannot easily grow under nutrient-replete conditions, and the small genome which is characteristic of freshwater E. coli populations could be combined to hypothesize that the existence of small, encapsulated cells of E. coli that are indigenous to the aquatic environment and of non-fecal in origin are not necessarily associated with pathogenic processes. This should be considered when using E. coli as an indicator of water quality and when surveying environmental sources of outbreaks, which are unknown. Thus, research including the phenotypic, genotypic, and genomic comparison of environmental strains from a wide range of aquatic habitats is necessary to improve our knowledge about which the ecological mechanisms impacting E. coli communities in different water ecosystems are. This will help to elucidate the key environmental clues in selecting pathogenic strains, how long the different variants or phylotypes can survive in the environment, and how relevant their presence in water is for the generation of outbreaks.

3.3.2  E. coli in Soil Soils are a complex matrix with various characteristics, such as pH, presence of compounds with antimicrobial properties, presence of organic matter, and variable native populations of microorganisms. When in the soil sample, E. coli can enter a

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“dormant” state (viable but non-culturable), where cells cannot be easily recovered on standard laboratory media but are still present as viable cells (Van Elsas et al. 2011). Viable but non-culturable E. coli can recover culturability and pathogenicity, being a potential health risk (Ding et al. 2017). E. coli can be present in agricultural soil because animal manure is used frequently as a fertilizer. The behavior of different pathogens after biofertilization of clay and sandy soils with swine digestate was evaluated by Fongaro et al. (2017), suggesting that E. coli O157:H7 could serve as a useful microbial biomarker of depth contamination and leaching in clay and sandy soil. The analysis of soils from surrounding areas of the Buffalo River of Minnesota showed similar high prevalence of E. coli in soils from forests and pasture lands (Dusek et al. 2018). In sand terms, microcosm assays with pathogenic E. coli showed variable persistence of STEC strains according to serotype and in some cases viable but non-culturable states (De la Cuesta et al. 2022). Phylogroup B1 is usually more abundant in soil followed by the phylogroup A (NandaKafle et  al. 2017; Dusek et  al. 2018; Rumball et  al. 2021). Nevertheless, microcosm studies showed these phylogroups would be seasonally maintained in the environment while strains from phylogroups within B2 group, cryptic clade, and D/E could survive at least a year (Rumball et al. 2021).

3.3.3  E. coli in the Soil of Urban Areas In the urban environment, the presence of pathogenic E. coli was evaluated in sand from parks in the central region of the state of Sao Paulo, Brazil (Fernandes et al. 2013). E. coli strains detected represented 31.5%, and the virulence genes identified were eae, bfp, saa, iucD, papGI, sfa, and hlyA. Phylogenetic analysis showed 52.4% of E. coli strains belonging to the B1 phylogroup, 25.4% to the A phylogroup, and 22.2% to B2.

3.3.4 STEC in the Environment of Farm In the farming environment, the presence of E. coli pathovars, particularly STEC, has been studied. It has been observed that the persistence of STEC O157:H7 in this environment is due to its ability to colonize the bovine intestine, as well as biotic and abiotic surfaces outside the reservoir (Segura et al. 2018). The survival of the pathogen in drinking water from farmyards has been studied, observing that it can last between 70 and 240  days (LeJeune and Wetzel 2007; Polifroni et  al. 2014). Some STEC O157 strains can remain stably viable in the field for several years (Cobbaut et  al. 2008; Carlson et  al. 2009). The persistence could be due to the strains which are found in carrier ruminants, in environmental reservoirs, or in

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another species other than cattle that acts as a reservoir and that has not yet been identified (García et al. 2010). There is evidence that E. coli strains that have pastures as a niche differ phylogenetically from isolates that come from the bovine intestine (NandaKafle et al. 2017). The natural transmission of STEC among cattle can occur by direct contact (fecal-­ oral route) or indirectly from the contaminated rural environment (Segura et  al. 2018). Its persistence and excretion of the pathogen in these animals can be affected by different factors such as interaction with other microorganisms (antagonism relationships, competition for nutrients), physicochemical parameters (pH, temperature, solar radiation, rainfall), physiological parameters (the ability of the bacteria to form a biofilm, other factors that collaborate with persistence), or agricultural practices (use of manure as fertilizer) (Fremaux et al. 2006; Soon et al. 2011; Jeong et al. 2013; Vogeleer et  al. 2015). It is estimated that STEC strains can survive up to approximately 60 days in feces, their behavior varying between the serotypes studied (Polifroni et al. 2014). There are reports that confirm that serogroup O157 can persist in soil for 105 days (Ogden et al. 2002). Transmission between spatially close rural establishments should be considered, although the routes by which such transmission is established have not been clarified yet. However, it could include transport of fecal matter or soil from farms by personnel or machinery, the movement of food between farms, and wild animals, including birds, moving from one place to another (Chase-Topping et  al. 2008). Finally, fresh vegetables, green leafy vegetables, were detected as a source of EAEC, EPEC, EIEC, and STEC contamination in rural and urban areas (Priyanka et  al. 2021), and were also related to extended spectrum beta-lactamase (ESBL) production (Freitag et  al. 2018). Additionally, romaine lettuce was the source of outbreaks in the USA; the leaf contamination with STEC O157:H7 could be related to contaminated water for agriculture or cattle grazing on adjacent land, addressing the environmental contamination relevance (Waltenburg et al. 2021).

3.4  E. coli as a Source of Antimicrobial Resistance The diversity of antimicrobials and antimicrobial resistance genes (AMR) is the result of antagonistic co-evolution among different species of environmental bacteria through thousands of millions of years (Forsberg et al. 2014). Human society has massified the production and the use of these antimicrobials, and as a result, it has created a selective force promoting the proliferation and transference of these genes among bacteria associated with humans and other animals (Cantón 2009). A few years after the discovery of the antimicrobials, genes coding for mechanisms that either degrade, alter, or pump antimicrobials out of the cell appeared in E. coli (Tadesse et al. 2012). Throughout the years, AMR genes have disseminated among most E. coli lineages, and the presence of these genes has increased dramatically over the last 50  years (Tadesse et  al. 2012; Martínez and Baquero 2014). It is remarkable the speed of dissemination of some of these genes: the gene blaCTX-M

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(associated with resistance to third generation cephalosporins) started to disseminate in E. coli in the 1990s, from the environmental bacteria Kluyvera spp. (Cantón and Coque 2006). The gene mcr-1 (associated with colistin resistance) started to disseminate in E. coli in 2015 (Liu et al. 2016), and today E. coli carrying these genes could be found in rivers across the world. The acquisition of AMR genes causes fitness cost (Rajer and Sandegren 2022) which reduces the bacteria growth rate, the ability to colonize, or the ability to survive; however, it seems like these AMR genes and their bacterial hosts co-evolved; chromosomal mutations that ameliorate these fitness costs have been selected (Rajer and Sandegren 2022). Currently, some AMR genes no longer require selective pressure to thrive in E. coli without exposure to any antimicrobial (Hernando-Amado et al. 2017). Because of their wide dissemination and low fitness costs, many AMR genes (disseminated among E. coli more than 70 years ago) provided little information about their recent origin (whether these genes and their bacterial hosts come from domestic animals or humans). More recently, acquisition of AMR genes such as blaCTX-M or mcr-1 is still causing fitness costs (Rajer and Sandegren 2022), which may explain why they are present in a few E. coli lineages which are not numerically dominant in the intestinal E. coli population. These genes require antimicrobial pressure to disseminate and are overwhelmed by antimicrobial-sensitive bacteria when antibiotics are not present (Nobrega et  al. 2021). These genes are associated with environments where antimicrobials are used, and they decrease when environments with restricted antimicrobial use are present. The presence of these genes in a few E. coli lineages under antimicrobial pressure facilitates the evaluation of AMR gene transference from bacteria in animals to humans or vice versa (Fig.  3.1). This section is focusing on the sources and the transmission of blaCTX-M and mcr-1 genes.

3.4.1 Antimicrobial Resistance Crisis and Commensal E. coli The US CDC agency has nominated E. coli as one of the most dangerous bacteria when it comes to antimicrobial resistance (Kadri 2020). Although most E. coli strains are non-pathogenic commensal microorganisms, they can serve as reservoirs of antimicrobial resistance (AMR) genes, due to E. coli’s capacity of horizontal gene transfer, through conjugation (Porse et al. 2017). The presence of these AMR genes is mostly located in highly transferable plasmids (Porse et al. 2017), and the plasmids are populated with numerous transposable elements and integrons (associated with AMR genes); and transposable elements and integrons can mobilize the AMR genes from one plasmid to another (Loayza et al. 2020). Due to its abundance, commensal E. coli can easily be transmitted among animal hosts, and, within the host, AMR genes could be transferred to pathogenic or opportunistic Enterobacterales through horizontal gene transfer (Loayza et  al. 2020). These dynamics have been documented for the mcr-1 gene product, conferring resistance to colistin, which was first detected in 2015  in a commensal E. coli strain from

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Fig. 3.1  This cartoon aims to highlight the variety of alternatives in the transmission of E. coli, including strains harboring virulence and/or resistance genes that are a health risk. Contribution by Luciana Graciano PhD-student in Veterinary Science

chickens (Liu et al. 2016) and now is globally disseminated and present in many nosocomial Gram-negative opportunistic pathogens in Latin America and the world (Loayza-Villa et al. 2020).

3.4.2 AMR in the Environment E. coli is a facultative anaerobe, a member of the normal intestinal microbiota of warm-blooded animals, which not only tolerates but also uses oxygen in the environment outside the host (Guerrero et al. 2020); this feature may have contributed to its ability to be present in environments through fecal contamination. On the other hand, the number of antimicrobials used in livestock is greater than that used in humans, and it is expected to grow due to the increasing demand for animal protein for consumption (Tiseo et al. 2020). Consequently, E. coli from domestic animal waste is an important source of antimicrobial-resistant bacteria (Tiseo et al. 2020). Human waste is also a source of antimicrobial-resistant bacteria, and the volume of human waste concentrated in cities overshadows the amount of any other animal species (Wear et al. 2021). The water of these contaminated rivers in Latin America contains E. coli carrying blaCTX-M or mcr-1 and, in many cases, this water is used for

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irrigation (Dropa et al. 2016; Chavez et al. 2019; Guzman-Otazo et al. 2019; Ortega-­ Paredes et al. 2020; Díaz-Gavidia et al. 2021; Furlan et al. 2021).

3.4.3 AMR in the Food Animal products such as meat and dairy (including egg contents) may contain E. coli carrying blaCTX-M or mcr-1 from livestock (Fegan and Desmarchelier 2010; Adesiyun et  al. 2020), and many countries in Latin America have reported these genes in E. coli from food-producing animals (Faccone et al. 2019; Benavides et al. 2021; Cardozo et al. 2021; Salinas et al. 2021). Produce could also be a source of E. coli as soils used to grow vegetables in Latin America are fertilized with animal waste, which also contains substantial amounts of E. coli from livestock (Furlan and Stehling 2018; Prack McCormick et  al. 2022) or river water contaminated with human waste and used for irrigation (Montero et al. 2021). Several researches conducted in captive wildlife and free wildlife could have been biased towards a greater role of wildlife in the transmission of AMR (Milton et  al. 2019). Despite that, wild small mammals are a useful biomarker of spatial variation and the distribution of AMR, which was suggested as indicative risk of AMR transmission to mammalian hosts including humans (Furness et  al. 2017). Likewise, marine mammals and various fish species were proposed species sentinels of AMR from One Health purposes (Gross et al. 2022) Reports of wild birds carrying AMR in the Southern part of the American continent suggest a rapid adaptation and dissemination of relevant E. coli lineages at the human-animal-­ environment interface (Fuentes-Castillo et al. 2019).

3.4.4 Directionality of Domestic Animals and Humans It is difficult to determine whether antimicrobial-resistant E. coli from humans or livestock contribute more to the current antimicrobial-resistance crisis. It has been shown that the same clonal ESBL E. coli strains are present in domestic animals and humans in the same community in Latin America (Salinas et al. 2021); however, it is clear that many ESBL E. coli strains are generalists (they can colonize both human and livestock intestines) (Cardozo et al. 2021; Salinas et al. 2021). Isolation of carbapenem-resistant E. coli from food-producing animals strongly suggests the transmission of this antimicrobial resistance from humans to livestock, as carbapenems are not used in animal husbandry (Bonardi and Pitino 2019). Finally, the anthroposphere (defined as the part of the environment that is made or modified by humans) is tightly connected with the zoosphere which may suggest that these genes go back and forth between these two compartments.

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Chapter 4

New Molecular Mechanisms of Virulence and Pathogenesis in E. coli Fernando Navarro-García, Antonio Serapio-Palacios, Bertha González-­Pedrajo, Mariano Larzábal, Nora Molina, and Roberto Vidal

Chapter Summary  The rapid progress in diverse approaches and technologies such as genome sequencing, gene mutation, site-directed mutagenesis, proteomics, crystallography, high-throughput screening and cryo-electron microscopy has provided unprecedented knowledge regarding novel functions during E. coli pathogenesis. In this chapter, we discuss six novel mechanisms of virulence and pathogenesis into the different E. coli pathotypes. These mechanisms include two nanomachines (T3SS and T6SS) used to colonize and hijack the host cell functions (T3SS, T6SS) or for bacterial competition (T6SS), as well as the mechanisms of T3SS protein-­ protein interaction, which has allowed the development of blocking compounds or peptides as preventing strategies; likewise, mechanisms of some pathogenic E. coli strains, which have developed sophisticated strategies to overcome the colonization barriers imposed by the microbiota; and finally, mechanisms to regulate the expression of factors related with the lifestyle of these bacterial pathogens as those formF. Navarro-García (*) Department of Cell Biology, Centro de Investigación y de Estudios Avanzados, Mexico City, Mexico e-mail: [email protected] A. Serapio-Palacios Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada B. González-Pedrajo Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico M. Larzábal Instituto de Agrobiotecnología y Biología Molecular INTA-CONICET, Hurlingham, Buenos Aires, Argentina N. Molina Centro Universitario de Estudios Microbiológicos y Parasitológicos, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, Buenos Aires, Argentina R. Vidal Instituto de Ciencias Biomédicas, Universidad de Chile, Santiago, Chile © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 A. G. Torres (ed.), Trending Topics in Escherichia coli Research, https://doi.org/10.1007/978-3-031-29882-0_4

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ing bacterial communities or new mechanisms of remote interaction among bacteria and host cells, such as the delivery of vesicles containing cargo molecules.

4.1 Introduction Escherichia coli strains of different pathotypes share virulence factors and processes, not only for being the same species but also for their high plasticity to receive mobile genetic elements through horizontal transfer. Nowadays, the rapid progress in diverse approaches and technologies, such as genome sequencing, gene mutation, site-directed mutagenesis, proteomics, crystallography, high-throughput screening, and cryo-electron microscopy, has provided unprecedented knowledge regarding novel functions. These integrated set of approaches have allowed identification of nanomachines used to colonize and hijack the host cell functions. Likewise, mechanisms to regulate the expression of factors related with the lifestyle of these bacterial pathogens as those forming bacterial communities or new interaction mechanisms among bacteria and host cells, such as the delivery of vesicles containing cargo molecules. Here, we will discuss some of these new strategies that E. coli uses to colonize and persist in the host, including two secretion nanomachines (type 3 and type 6), interaction of E. coli with intestinal microbiota, biofilm formation, and E. coli outer membrane (OM) vesicles.

4.2 The Type III Secretion System (T3SS) in E. coli Important diarrheagenic human pathogens as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), which are major causes of infant morbidity and mortality in developing countries and of foodborne outbreaks in developed countries, respectively, depend on a T3SS (also called injectosome) for pathogenicity (Croxen et al. 2013). These extracellular enteric pathotypes use the T3SS for delivery of bacterial effector proteins into target intestinal cells (Gaytan et  al. 2016). More than twenty effector proteins are translocated to disrupt several key host cellular processes such as inflammatory signaling, host innate response, intracellular trafficking, tight junction integrity, cell death pathways, host cell survival, and cytoskeleton organization, thus allowing the establishment of infection and formation of a unique attaching and effacing (A/E) histopathological lesion (Pinaud et al. 2018). The A/E lesion is defined by intimate adherence of bacteria to the epithelial cell membrane, microvilli effacement, and formation of actin-rich pedestal-like structures beneath the bacterial attachment site (Croxen et al. 2013). All proteins needed for injectosome assembly, seven of the translocated effectors, chaperones, and regulators are encoded on a pathogenicity island termed the Locus of Enterocyte Effacement (LEE), which contains 41 genes organized into seven operons and four transcriptional units (Gaytan et al. 2016).

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4.2.1 Architecture of the Type III Secretion System Virulence-associated T3SS has evolved from the bacterial flagellum, which self-­ assembles by transport of its protein components through a T3SS, e.g., part of the flagellar structure was recruited to serve a new protein delivery function (Abby and Rocha 2012). Hence, the injectosome and the bacterial motility organelle share homologous proteins and a highly conserved architecture in the export apparatus. Injectosome is a syringe-like multiprotein complex (Fig.  4.1a) with a cylindrical basal body formed by ring structures embedded in the inner membrane (IM, including EscD, EscJ) and the outer membrane (OM, including EscC), which spans the periplasmic space and extends into an extracellular conduit constituted by a needle (a helical polymer of EscF subunits), a filament (a polymer of EspA subunits), and

Fig. 4.1  Two nanomachines of protein secretion. (a) The T3SS is assembled from more than 20 proteins into a nanosyringe-like structure that traverses three membranes. It is composed of three main elements: (i) the cytoplasmic components include the sorting platform (EscKQL) and the ATPase complex (EscNO); (ii) the basal body is a multiring structure spanning both bacterial membranes (EscJDC) and containing the export apparatus (EscRSTUV). The inner rod (EscI) connects the core export apparatus with (iii) the extracellular appendages, the needle (EscF), and the filament (EspA). The translocation pore (EspBD) is formed by hetero-oligomerization and insertion of these proteins into the host cell membrane. A continuous channel is formed throughout the whole structure for injection of effector proteins into eukaryotic host cells. (b) The T6SS is composed of a membrane complex and a tail complex. TssJ–TssL–TssM makes the membrane complex and is connected to the TssB–TssC tail sheath and the Hcp inner tube through the baseplate (composed of TssK, TssE, and VgrG). Effectors are recruited to the spike–tube complex through the extension domains of VgrG and/or PAAR-repeat proteins and through incorporation into the Hcp tube. An unknown extracellular signal triggers sheath contraction, which leads to the ejection of the spike–tube complex across the target membrane, thereby delivering effector proteins into the cell (the target cell could be a eukaryotic cell or another bacterium). The ATPase ClpV disassembles the contracted TssB–TssC sheath, which enables a new T6SS complex to be reassembled from the released subunits. (Both panels were created with BioRender.com)

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a translocon pore (formed by EspB and EspD subunits) that inserts into the host cell membrane. LEE and non-LEE encoded effector proteins are transported through an approx. 2.5 nm central channel that traverses this structure. The cytoplasmic components, the sorting platform, and the ATPase complex are assembled onto the cytoplasmic side of the EscD IM ring (Gaytan et al. 2016; Slater et al. 2018). Basal body houses the export apparatus, an essential highly conserved structure formed by the EscR, EscS, EscT, EscU, and EscV membrane-associated proteins (Fig. 4.1a). EscU and EscV are integral IM proteins with large cytoplasmic domains involved in recognition of type 3 secreted proteins and substrate specificity switching. The cytoplasmic C-terminal domain of EscV forms a nonameric export ring with a central pore through which substrates are funneled and subsequent secretion is enabled by the proton motive force. The core export apparatus in Salmonella SctR, SctS, and SctT (the unified nomenclature Sct will be used for non-E. coli proteins) is organized in a helical arrangement and is located in the periplasmic space (Wagner et  al. 2018). In EPEC, the EscRST complex possesses structural constrictions that are suggested to act as seals that prevent leakage of ions and metabolites (Tseytin et al. 2022). All these proteins form an entry gate for transport of secreted effectors through the IM. At the distal end of the export apparatus, the adaptor protein EscI forms an inner rod that connects the export core structure with the needle. Moreover, the injectosome export apparatus in E. coli is also used to form nanotubes that extract nutrients from host cells (Pal et al. 2019). Among the cytoplasmic components, the sorting platform is a cage-like structure also known as C-ring, composed by EscK, EscQ, and EscL (Fig. 4.1a), which plays a central role in the recruitment of different categories of type 3 substrates in a sequential manner (Lara-Tejero et al. 2011; Soto et al. 2017). Cryo-electron tomography studies in Shigella revealed it is formed by six pods of the SctK and SctQ proteins that are radially arranged at the base of the injectosome and six spokes at the cytoplasmic end, constituted by SctL (Hu et al. 2015). This structure has been proposed to have a dynamic behavior with free and injectosome-bound cytoplasmic complexes (Wagner et al. 2018). SctK attaches to the cytoplasmic part of the basal body through its interaction with the soluble domain of the IM protein SctD. ATPase complex is formed by the ATPase EscN and its negative (EscL) and positive (EscO) regulatory proteins, and it is anchored to the sorting platform through interactions between EscQ and EscL, and EscL and EscN (Fig. 4.1). ATPase EscN crystal structure shows structural similarity with the F0F1-ATPase α and β subunits. EscO is located at the center of an EscN homohexamer and interacts with the EscV cytoplasmic domain to enhance the proton motive force-induced secretion activity (Majewski et  al. 2019). ATPase complex plays a vital role as a docking site for chaperone-substrate complexes and in energizing the secretion process. ATP-­ hydrolysis has been proposed to serve for chaperone release from its cargo protein and effector unfolding for passage into the T3SS central channel.

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4.2.2 Hierarchical Substrate Secretion: Molecular Switches Proper biogenesis of the T3SS requires a strictly ordered secretion of its component proteins. This process is controlled by two molecular substrate specificity switches. After the Sec-dependent assembly of the export apparatus and membrane rings plus integration of the sorting platform and ATPase complex, formation of the inner rod and needle structure (early substrates) precedes that of the filament and translocon pore (translocators or intermediate substrates), which in turn occurs before secretion of effector proteins (late substrates). Secretion switches are regulated by protein-­protein interactions with the cytoplasmic domains of the export gate components EscU and EscV (Gaytan et al. 2016). The first secretion switch from early to intermediate and late substrates is achieved by a productive interaction between the EscP protein and the C-terminal domain of EscU. EscP belongs to a family of proteins that are secreted through the T3SS, interacts with early substrates EscF and EscI, and participates in needle length control. EscU belongs to the SctU/FlhB family of proteins that undergo spontaneous autocleavage at a conserved NPTH motif. Although the precise mechanism has not been elucidated, once the needle reaches its final length, EscP can interact with EscUC inducing a conformational change in this export gate component that switches substrate specificity (Monjaras Feria et  al. 2012). This first switching event arrests the growing of the needle structure. In addition, binding of SctP to SctU enhances the interaction between SctW (SepL in E. coli) and the C-terminal domain of the other export gate component, SctV, which participates in the second specificity switching event that ensures a timely translocation of effectors into target cells (Yu et  al. 2018). SepL forms a heterotrimeric complex with CesL and SepD, which interacts with EscVC (Portaliou et al. 2017; Gaytan et al. 2018). The switching model proposes that the SepL-SepD-­ CesL complex binds EscV, allowing recognition of intermediate substrates with high affinity, while blocking recognition of late substrates. When the translocon pore is formed in the host cell membrane, this protein complex dissociates from EscV, which can then recognize late substrates with high affinity (17). These molecular switches act in a coordinated manner for a controlled hierarchical secretion. It is thought that conformational changes in EscU after EscP binding could be transmitted to EscV via CesL (Diaz-Guerrero et  al. 2021). Moreover, it was demonstrated that SepL interacts with EscP for regulation of late substrate secretion (Shaulov et al. 2017). The success of the correct regulation of these switches allows the correct secretion of each effector inside the eukaryotic host cells.

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4.3 Type III Secretion System as Targets of Anti-virulence Agents for Pathogenic E. coli Unlike other secretion systems, presence of the T3SS is associated with the ability of the bacterium to cause disease and not with basic bacterial metabolism. This would indicate that specific agents that are directed against this virulence mechanism are unlikely to generate resistance by selective pressure (Tseng et al. 2009). Remarkably, mutation or deletion of T3SS structural proteins significantly reduces virulence without affecting bacterial viability (Lee and Kessler 2009). This suggests that the T3SS could be a target structure in an emerging strategy focused on the design and development of new anti-virulence agents. Therefore, one could assume that new ad hoc inhibitors could interfere with the formation and/or functionality of T3SS, which would prevent the presence of lesions, intestinal epithelium colonization, and pathogenic E. coli disease. A methodology widely used in the pharmaceutical industry as a starting point for agent development is high-throughput screening of large collections of compounds (Jj and Nh 1997). This complex method allows for the identification of virulence inhibitors against the target structure from a library of small molecules. Mühlen and colleagues proposed this approach by performing a high-throughput screening assay for T3SS inhibitory compounds (Mühlen 2021). The screening was based on six different libraries including more than 13,000 small molecular compounds. Thus, three substances were identified, which showed a significant dose-dependent effect on translocation of effectors into mammalian cells. These substances also affected effector-dependent cell detachment and in vitro A/E lesion formation by EPEC/EHEC. Two of these components only inhibited erythrocytes hemolysis, by interfering with the translocon pore-formation properties or translocation of effectors. The inhibitory effect of the third component was not involved in T3SS assembly, but at the level of translocation of effectors. Considering that the incorporation of the compounds is done with the T3SS already assembled in solution, these compounds would have no effect on the expression of genes or injectosome formation. Moreover, presence of substances, at least at the concentrations used, was harmless to eukaryotic cells or bacterial growth, making these drugs promising candidates for the therapeutic treatment of E. coli infection. Pan and colleagues using a similar methodology performed, whereby a high-­ throughput screening of 70,966 small molecules, identified 421 inhibitors of Yersinia pestis T3SS (Pan et al. 2009). They also screened inhibitory compounds against EPEC and demonstrated that two of them had inhibitory capacity against translocation of T3SS effectors, without causing cytotoxic activity against eukaryotic cells. These two compounds are based on a picolinic acid derivative and a symmetric dipropionate. Although the target on which they act remains unidentified, these findings open the possibility for the design of universal compounds against T3SS from a wide range of Gram-negative pathogens. Additionally, high-throughput screening methodology can focus on the search for compounds that affect regulatory mechanisms of virulence. A halogenated

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salicylaldehyde derivative compound, identified from a library of 20,000 small molecules, reduced the EPEC T3SS activity (Gauthier et al. 2005). This compound did not directly affect the secretion or assembly of T3SS, but its transcriptional expression. Since the compound inhibited the transcription of the master regulator Ler, affecting the T3SS structural protein and subsequent transcription regulated under this promoter. New research has returned to the first specific compound tested as an inhibitor of EPEC T3SS in 1972, e.g., aurodox (Kimura et al., 2011). This linear polyketide also exhibited antibacterial activity, which indicated that the use of this compound could generate bacterial resistance. However, molecules derived from aurodox (Kimishima et  al. 2022), the benzoyl derivative molecule, had the highest specific inhibitory capacity on EPEC T3SS, up to the levels of aurodox but with lower antibacterial activity. This suggests that the target site(s) for T3SS inhibition differ from the site(s) implicated for antibacterial activity. These results are promising since the absence of antibacterial activity would reduce the possibility of bacterial resistance. Besides, this compound could be evaluated against EHEC T3SS, without the risk of generating bacterial lysis and release of Shiga toxin. Other studies have focused on agents directed against a specific region of the T3SS to generate its inhibitory effect. Although the 20 components of T3SS can be diverse, they exist in a high prevalence of coiled-coil domains among their structural, translocator, and effector proteins (Pallen et al. 1997). These domains consist of amphipathic alpha-helical structures that are essential for the assembly of the translocation complex and in protein-protein interactions. Actually, point mutation of certain amino acids of these coiled-coil regions significantly decreases the virulence of these pathogens because of the inability of a proper T3SS assembly (Lee and Kessler 2009). Consequently, Larzábal and colleagues designed and synthesized peptides specifically selected from the coiled-coil regions of the structural and translocator proteins of the T3SS of EHEC O157:H7, EPEC and Citrobacter rodentium and demonstrated inhibition of secretion and translocation of secretory and effector proteins in vitro, generating a non-functional T3SS (Larzábal et al. 2010). They also detected inhibition of erythrocyte hemolysis and T3SS-dependent A/E lesion in vitro. The next step was to orally treat mice with these coiled-coil peptides before C. rodentium infection and during the infection procedure. Murine colonic infection was prevented as was C. rodentium T3SS-dependent colonic hyperplasia in mice (Larzábal et al. 2013), and no signs of mice toxicity were observed by these peptides. Neither did the cell monolayers used in the infection assays show alterations. Recently, Larzábal and colleagues have performed comprehensive molecular modeling studies to optimize T3SS-peptide interactions, thus generating new coiled-coil peptides with higher solubility and affinity to target proteins (Larzábal et al. 2019). The search for non-conventional therapies against the emergence and spread of antimicrobial multidrug-resistant bacteria has driven the development of new agents targeting their virulence mechanisms (Krell and Matilla 2022). Therefore, there is an urgent demand for the discovery, design, and development of anti-virulence agents that can range from small synthetic molecules to peptides. These agents

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should target the action of a virulence-specific system to generate little or no selective pressure for viability, to reduce the development of resistance. Furthermore, they should not harm commensal microbiota that do not possess such virulence mechanisms. Thus, the new ad hoc agents should have greater specificity than current antibiotics, which tend to act non-selectively against a wide range of bacteria, including non-pathogenic bacteria. This type of therapy would represent an even more important advancement for the treatment of patients infected with EHEC, since the use of antibiotics is not recommended, because of the increased release of Shiga toxin during treatment. Therefore, specific agents against the T3SS EHEC could reduce intestinal colonization and intestinal adherence and prevent the disease without aggravating the patient’s condition. In other pathogens, this type of alternative therapies could be an adjunct approach during antibiotic treatment to achieve an increased efficacy and to decrease the cytotoxic effects of antibiotics (Hotinger et al. 2021). Besides, T3SS is widespread in enteric pathogenic Gram-negative bacteria, which are responsible for severe intestinal diseases (Büttner 2012). While a specific inhibitor could be developed for each virulence mechanism, it should be considered that several of the T3SS components contain conserved sequences and structures. This suggests that an inhibitor directed against one component of the T3SS could be effective against a wide range of Gram-negative pathogens. In conclusion, research in this area has provided a wealth of knowledge about T3SS as a potential target of pathogenic E. coli and has elucidated much of its mechanism of action. Regarding the future development of agents against T3SS, research should focus on the design of inhibitors based on the knowledge of structures modeling with a pharmacophoric pattern of inhibitory activity already established. This progress could lead to new therapeutic or prophylactic strategies around infectiology or treatment-prevention.

4.4 Role of the Type VI Secretion System (T6SS) in E. coli Just after T6SS discovery in Vibrio cholerae (Pukatzki et al. 2006), two similar gene clusters of the pheU pathogenicity island were also found in enteroaggregative E. coli (EAEC) strain 17–2, named Sci-1 and Sci-2, encoding two distinct T6SSs (Dudley et al. 2006). T6SS gene clusters are widely distributed in proteobacteria and may exist in several chromosomal copies (Journet and Cascales 2016). T6SS was initially associated to bacterial virulence towards eukaryotic cells, but a scarce number are directly implicated in host cell disruption (Pukatzki et  al. 2007). However, the main T6SS role is related to bacterial competition through killing neighboring bacteria without cognate immunity protein. This is achieved by secretion of proteins with antibacterial activity directly into the periplasm of the target bacteria after cell-cell contact (Navarro-Garcia et al. 2019).

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4.4.1 Structure of the T6SS Three major complexes (13-core components) form the T6SS nanomachinery: the membrane complex (MC), the tail complex (TC), and the baseplate complex (BPC) (Fig. 4.1b). The MC is located in the IM resembling T4SS components (Pukatzki et al. 2006), the TC formed by components related to phage contractile tails (Leiman et al. 2009), and the BPC used for the tail assembles serving as a platform during tube and sheath assembly but also triggers the sheath contraction (Cherrak et  al. 2018). The structure of T6SS has extensively been studied in EAEC. The MC is specific to the T6SS unlike TC and BPC. Besides the anchor of EAEC baseplate to the IM, MC also functions as a channel to permit the passage of the tail tube-spike and for maintaining the attacking cell integrity throughout the inner tube translocation. The minimal MC is composed of the TssJ, TssL, and TssM proteins (Fig. 4.1b). In EAEC TssJLM complex, ten TssJ lipoproteins are bound to ten TssM proteins constructing a double concentric ring of arches and pillars throughout the periplasm. TssM is a key MC component as a connector of the IM and OM attaching the TC to the IM and OM (Navarro-Garcia et al. 2019). The TssB-TssC complex forms the tail sheath, which is both perpendicular to the membrane and a long tubular structure, and it is deeply extended into the cytoplasm (Fig. 4.1b). An inner tube is inside the tail sheath which is formed by stacked Hcp protein hexamers. In E. coli context, a direct interaction between the Hcp tube and the VgrG spike starts the correct Hcp tube polymerization and promotes sheath dynamics and Hcp release. VgrG crystal structure of the uropathogenic E. coli strain CFT073 showed that the VgrG trimer is located centrally in the PBC forming a spike. The ClpV chaperone acts during T6SS assembly and functions by causing the cytosolic TssB-TssC tubules’ depolymerization, leading to the TssB-TssC polymerization for forming the sheath structure again. ClpV also provides energy required for its contraction through depolymerizing the sheath (Navarro-Garcia et al. 2019). TssAFGK are essential core components of BPC (Fig. 4.1b), but an interaction network among several components of BPC and tail are required (TssE, TssG, TssF, TssA, TssK, and VgrG). EAEC TssK is a cytoplasmic trimeric protein associated with the IM forming oligomers. EAEC TssEFGK-VgrG phage-like baseplate is recruited to TssJLM MC via multiple contacts serving as assembly platform for tail tube/sheath polymerization. A cryo-electron microscopy of EAEC TssKFG subcomplex showed that TssKFGE wedges polymerize around the VgrG hub to form a hexagonal baseplate. Hexamerization of (TssK)6-(TssF)2-(TssG)1-(TssE)1 wedge leads to BPC formation around a VgrG trimer bound to a PAAR-repeat protein at its distal extremity (Navarro-Garcia et al. 2019).

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4.4.2  E. coli T6SS Effectors The effector must be associated with Hcp-VgrG-PAAR, which are components of the expelled structure; this puncturing protein delivers effectors at once in one lethal shot inside the target cell (Fig. 4.1b). Several T6SS effectors have been characterized including eukaryotic cell-targeting and antibacterial effectors, or for both targets (Navarro-Garcia et  al. 2019). For eukaryotic cell-targeting, a gene cluster deletion mutant (evfB to hcp1) in meningitis-causing E. coli K1 was impaired in binding/invasion of HBMEC (human brain microvascular endothelial) cells. Hcp1 and Hcp2 differentially interacted with HBMEC cells. An hcp2 mutant was deficient in the bacterial binding/invasion of HBMEC cells, while Hcp1 induced actin cytoskeleton rearrangement, apoptosis, and IL-6 and IL-8 release (Zhou et al. 2012). Avian pathogenic E. coli (APEC) SEPT362 mutants of clpV and hcp showed decreased adherence and actin rearrangement (de Pace et al. 2011). Additionally, hcp or clpV mutants APEC SEPT362 induce markedly decreased levels of filopodia formation and ruffle-like structures. Interestingly, hcp or clpV mutants were not involved in intra-macrophage replication (de Pace et al. 2010), but a tssM mutant reduced intra-macrophage survival (de Pace et al. 2011). Two putative T6SS loci were found in APEC TW-XM. T6SS1 mutants were deficient in adherence/invasion of several cells and displayed decreased pathogenicity in animal infection models. In contrast, T6SS2 mutants were impaired only in the adherence/invasion of mouse BMEC and duck brain tissue (Ma et al. 2014). In EHEC, a T6SS secreted effector Mn-containing catalase (KatN) helps bacterial growth during oxidative stress and is delivered in the host cytosol during intramacrophage engulfment of EHEC, reducing levels of intracellular reactive oxygen species and greater survival of phagocyted EHEC (Wan et al. 2017). E. coli T6SS gene clusters are classified in three distinct phylogenetic groups, T6SS-1 to T6SS-3, based on gene organization and homologies/similarities. Although most of the E. coli T6SSs studied participate in surface (biotic/abiotic) adherence, and in virulence to several infection models or in bacterial competition, most data on E. coli T6SSs suggests a role of T6SS-1 and T6SS-3 for antibacterial activity and of T6SS-2 for pathogenesis. The firsts T6SS-dependent antagonistic behaviors against neighboring bacteria were shown for T6SS1 and T6SS3 of EAEC 17–2 and T6SS1 of APEC TW-XM (Navarro-Garcia et al. 2019). The antibacterial T6SS function comprises a fascinating offensive and defensive mechanism of effector-immunity pairs, organized in bicistronic units. Antibacterial T6SS effectors are divided into effectors for targeting the cell wall, the membrane, and the nucleic acid, or other functions (Navarro-Garcia et al. 2019). These effectors include amidases, muramidases, and phospholipases that hydrolyze the cell wall (peptidoglycan bonds within the peptidic stem, Tae1–4 families, or glycosidic chains, Tge1–3 families), membrane lipids (phospholipids ester bonds, Tle1–5 families), and nucleases (DNase activity, Tde) (Journet and Cascales 2016). The cell wall targeting effectors include an amidase (VT1) in enterotoxigenic E. coli (ETEC) encoded within the vgrG island (Ma et al. 2018). By retrieving ­VT1/

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VTI1 effector/immunity pair in vgrG island from pathogenic E. coli, several putative effectors with diverse toxin domains were found (VT modules). Among them VT5, an effector widely encoded in ETEC, was found to act as a lysozyme-like effector and effectively kills adjacent cells. In silico, VT5s share a highly conserved catalytic motif GLxQ with known peptidoglycan glycoside hydrolase of T6SS effector Tge1 (Tse3) (Ma et al. 2018). While the membrane targeting effectors include Tde or Tle toxins bearing phospholipase A1, A2, or D activities, E. coli T6SS-1 gene clusters encode putative phospholipases upstream the vgrG genes, which might be transported by VgrG.  These phospholipases belong to different families; T6SS-1 clusters of adherent-­invasive E. coli (AIEC) LF82 or UPEC CFT073 carry putative phospholipases of the Tle3 family, while those on genomes of EAEC 042 and APEC TW-XM are closely related to phospholipases of the Tle1 and Tle4 families, respectively (Journet and Cascales 2016). EAEC Tle1 possesses phospholipase A1 and A2 activities. Auto-protection of the attacker cell is covered through the OM-lipoprotein; Tli1 and the Tle1-Tli1 binding inhibits the phospholipase activity (Flaugnatti et al. 2016). The in silico analyses identified E. coli T6SS phospholipase effectors by the existence of a conserved motif, GXSXG, such as tle1 to tle4, but not tle5 harboring HXKXXXXD motifs (Russell et al. 2013). Regarding nucleic acid targeting effectors, Rearrangement hot spot (Rhs) proteins are filamentous toxins displaying Rhs repeats at N-terminal regions, and highly variable toxin domains at C-terminal regions (Alcoforado Diniz and Coulthurst 2015). Rhs-CT modules are widely distributed as T6SS effector-immunity pairs in E. coli. Rhs-CT1 with an MPTase4 (metallopeptidase-4) domain showed an antibacterial effect. Ten different toxin domains were found in the Rhs-CTs, among them Rhs-CT3 and Rhs-CT4 which are putative DNases and Rhs-CT5 to Rhs-CT-8, which are putative RNases (Ma et al. 2017b). A template Rhs-CT1 was used for searching analogical targets in E. coli and detected more than 400 Rhs-CT proteins with an N-terminal PAAR motif (Ma et al. 2017b). The in silico analyses detected Hcps with a C-terminal extension carrying diverse toxin domains, designated as Hcp-ET, which are widespread in Enterobacteriaceae. Thus, an extended Hcp with a C-terminal HNH-DNase toxin domain was identified in E. coli O104 and piglet diarrhea isolate STEC004. This Hcp-ET1 and the T6SS2 cluster are widely prevalent in O104:H4 strains (Ma et al. 2017a). Further studies of Hcp-ETs, together with their immunity proteins, showed that Hcp-ET1 degrades DNA of the target cell via antibacterial predicted HNH-DNase activity. Hcp-ET3 with a C-terminal pyocin S3 toxin (DNase activity) in ETEC is severely impaired in its capacity to kill eti3/4− cells in a hcp-et3 mutant. Hcp-ET4 is also functional for interbacterial antagonism via growth inhibition by its colicin-DNase toxin (Ma et al. 2017a).

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4.5 Virulence Response of Pathogenic E. coli to the Microbiota During intestinal infection, invading pathogens encounter and compete with a dense and complex resident microbial community, or “microbiota,” to establish a colonization niche in the gut. Microbiota is a microbial community composed of hundreds of different bacterial species, each with its own biochemical repertoire. To establish a successful infection, some pathogenic E. coli strains have evolved sophisticated strategies to overcome the colonization barriers imposed by the microbiota (Buffie and Pamer 2013). While the microbiota can protect the host from infection, some members of this community have the potential to facilitate infection and/or regulate the severity of the disease. Pathogenic E. coli adaptively modulate the expression of critical virulence factors important for bacterial colonization by sensing microbiota-­ derived signaling molecules and metabolites. Additionally, microbiota members can influence E. coli ability to induce cell damage (Stevens et  al. 2021). Hence, understanding the intricate interactions between the microbiota and E. coli is critical for controlling infectious disease progression. Here, some of these new strategies that E. coli uses to thrive in the crowded gastrointestinal environment will be discussed.

4.5.1 Regulation of E. coli Virulence Factors by the Microbiota Pathogenic E. coli harbor several virulence factors to enhance proliferation in the gut, avoid clearance by the host immune system, and promote successful disease and transmission (Croxen et  al. 2013). Many intestinal environmental clues are known to regulate the expression of pathogen virulence genes; therefore, it is reasonable to consider that the microbiota also play an essential role in modulating E. coli pathogenic mechanisms. This occurs due to the need of E. coli to co-inhabit with a vast microbial community and the need to dynamically regulate energetically costly virulence behaviors. EHEC expresses two major virulence factors, Shiga toxin and a T3SS; the latter is used to inject effector proteins into host cells to manipulate cellular processes (Kaper et  al. 2004). These mechanisms are crucial for EHEC pathogenicity, and members of the microbiota can modulate these virulence factors (Fig. 4.2b). Indeed, Enterococcus faecalis increases the expression of T3SS-related genes in EHEC, leading to increased effector translocation and lesion formation (Cameron et  al. 2019; Martins et al. 2022). Because E. faecalis is mainly found associated with the intestinal epithelium, regulating gene expression could be a mechanism by which EHEC fine-tunes gene expression upon contact with the intestinal mucosa, allowing the pathogen to intimately associate with the epithelial cells (Barnes et al. 2017; Cameron et al. 2019). Bacteroides strains, abundant in human gut microbiota, can also regulate E. coli virulence and colonization (Fig.  4.2b). Bacteroides thetaiotaomicron increases

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Fig. 4.2 Pathogenic E. coli virulence is mediated by the microbiota. (a) Type VI secretion system (T6SS) is activated in the presence of the gut microbiota. E. coli senses the direct interaction with commensal bacteria and/or their metabolites. (b) Microbiota members enhance the virulence response of Pathogenic E. coli via microbiota-derived metabolites and/or signaling molecules. Bacteroides thetaiotaomicron; Escherichia albertii; Citrobacter werkmanii; Enterococcus faecalis. (Created with BioRender.com)

EHEC T3SS gene expression (ler, escC, escV, tir, and espA) and attachment to colonic epithelium (Martins et al. 2022). Interestingly, C. werkmanii and E. albertii (microbiota members) were found associated with diarrheagenic E. coli (DEC) infection in children under 5 years of age (Gallardo et al. 2017). Similar to B. thetaiotaomicron, these two commensal bacteria induce upregulation of virulence genes of STEC and EAEC (Izquierdo et al. 2022). It is remarkable that despite the phylogenetic distinctiveness of these commensal bacteria, they can all significantly regulate E. coli virulence gene expression and infection outcomes. This suggests that the E. coli pathogen might have evolved in response to the host microbiota to gain advantage by modulating its virulence gene expression.

4.5.2 Modulation of E. coli Pathogenesis by Bacterial Proteases New exciting research is taking place trying to uncover the contribution of bacterial proteases, proteolytic enzymes, in controlling pathogenic processes via proteolytic cleavage of virulence determinants. As mentioned above, enteric pathogens such as

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EHEC, EPEC, and C. rodentium deploy a T3SS to hijack cell functions through the secretion of protein effectors (Croxen et al. 2013). To translocate bacterial effectors into the host, these pathogens produce EspA, EspB, and EspD, which form the T3SS translocon in the host cell membrane (Garmendia et al. 2005). Surprisingly, host cell pore formation is a dynamic process that can be regulated by bacterial proteases. EHEC encodes the serine protease EspP, which cleaves EspB in a region essential for its function, limiting effector translocation to the host. Deletion of the espP gene increases the formation of actin-rich pedestals and cell damage characteristic of EHEC pathology, indicating that EHEC can self-inhibit its virulence (Cameron et  al. 2018). Like EspP, the serine protease EspC from EPEC also degrades the T3SS translocon components following cell contact, regulating T3SS-­ pore formation. In contrast, the commensal B. thetaiotaomicron, which has been found to induce the expression of espABD genes (for T3SS translocon components), enhances EHEC virulence by targeting these T3SS pore-forming proteins. Proteases produced by B. thetaiotaomicron cleave EspB to modulate T3SS function. Paradoxically, although B. thetaiotaomicron protease activity reduces the abundance of EspB, EspA, and EspD, this commensal bacterium promotes the higher translocation of bacterial effectors required for lesion formation (Cameron et al. 2018). This is a new role for microbiota members in potentiating disease caused by E. coli that could be widely distributed across other commensal bacteria. Indeed, the E. faecalis-secreted protease GelE also cleaves EspB, enhancing the translocation of the effector Tir by EHEC (Guignot et al. 2015). Therefore, influencing the pore formed in the host cells could be a new mechanism for microbiota-derived proteases to promote disease progression by pathogens.

4.5.3 Interbacterial Competition Using the T6SS Several pathogens use a T6SS to antagonize symbiotic gut bacteria (Fig. 4.2a). This mechanism for interbacterial competition allows pathogens to directly target microbial competitors to facilitate colonization and ultimately disease progression (Allsopp et  al. 2020). This has been clearly demonstrated during C. rodentium infection (Crepin et al. 2016), by leveraging this natural mouse pathogen, researchers were able to uncover evidence of the arms race between pathogens and the microbiota. C. rodentium deploys its T6SS to target members of the resident microbiota belonging to the Enterobacteriaceae family. Therefore C. rodentium activates the expression of its T6SS in the presence of commensal Enterobacteriaceae in the gut (Serapio-Palacios et al. 2022). Conversely, the removal of bacterial competitors, through antibiotic treatment, eliminates the need for the T6SS. These findings suggest that C. rodentium can sense bacterial competitors or their metabolites and respond by activating the expression of its T6SS nanomachinery. This helps C. rodentium to remove the protective colonization resistance imposed by competing gut microbes to establish its niche and allow for successful infection.

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Surprisingly, while pathogens are best characterized to use one or more T6SSs during host invasion, this sophisticated weapon is also used by commensal E. coli to counteract invading pathogens. Commensal E. coli strain Mt1B1 employs a T6SS to counteract C. rodentium intestinal colonization (Serapio-Palacios et  al. 2022), suggesting that it may be a widely shared strategy of gut microbes. Thus, the T6SS of Enterobacter isolates from healthy humans is also effective against EAEC strain 17–2 (Flaugnatti et al. 2021). T6SS loci are also widely distributed in Bacteroidales, a human gut microbiota major components (Coyne et al. 2016). This new mechanism for bacterial competition furthers our understanding that not only pathogens use T6SS to kill competitors to the microbiota, but also that the microbiota activates its T6SS to fight for a niche in the hostile gut environment. Furthermore, given the T6SS role in commensal bacteria, one might consider that the use of T6SSs could be capable of having a profound effect in shaping the gut microbiota composition. As gut microbiota composition has been linked to a wide variety of diseases, the effective modulation of the microbiota could have a significant impact on human health. However, scientists have just started to grasp the potential of the T6SS in pathogenic and commensal E. coli for modulating the microbiome.

4.6 Role of Bacterial Cell Surface Structures in E. coli Biofilm Formation Biofilms are surface-attached, structured microbial communities, containing sessile cells embedded in a self-produced extracellular matrix. About 80% of all bacterial infections are associated with biofilms, including recurrent urinary infections, endocarditis, prostatitis, persistent diarrhea, osteomyelitis, chronic wound infections, and biliary tract infections (Ballen et al. 2022). Biofilm formation by E. coli represents a change in thinking in infection pathogenesis and is a trait associated with bacterial persistence and virulence. E. coli biofilm formation is an intricate and highly coordinated process. The most accepted model to describe the formation of a mature biofilm comprises four stages: initial adhesion, early development of biofilm structure, biofilm maturation, and dispersion (Donlan 2002; Beloin et al. 2008). The initial event is the reversible attachment of planktonic bacteria to a surface (Ballen et al. 2022). This stage mostly depends on the presence of bacterial surface factors and physicochemical interactions between bacteria and surface. The main factors include bacterial adhesins and flagella, bacterial hydrophobicity and electrostatic charge, surface chemical structure, presence of conditioning film, hydrodynamic flow rate, pH, temperature, ionic strength, or nutrient availability (Donlan 2002; Beloin et al. 2008; Ballen et al. 2022). Adherence is a crucial step in the development of biofilm. E. coli adhesins include proteins from type 5 secretion system (T5SS) (classical autotransporter, two-pair system, trimeric autotransporter), usher chaperone fimbriae (CU secretion pathway), curli extracellular precipitation pathway, pili T4SS, and surface proteins

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assembled by T1SS (Korea et al. 2011; Ballen et al. 2022). In E. coli, type 1 fimbriae (T1F), curli fibers (thin aggregative fimbriae) and conjugative pili (F-pili) participate in this stage anchoring the bacteria to the surface and promoting the formation of biofilm on surfaces (Beloin et al. 2008; Ballen et al. 2022). Among clinical E. coli isolates, curli fimbriae are expressed in most EHEC and ETEC, but not in enteroinvasive E. coli (EIEC) or EPEC strains, suggesting a specific role in pathogenicity (Van Houdt and Michiels 2005). In UPEC, flagella, pyelonephritis-associated pilus (Pap), and other adhesins like Afa/Dr family can contribute to colonization bladder epithelium and dissemination to the kidney (Servin 2014). In EAEC, the main adhesins involved are adherence aggregative fimbria II (AAF/II), T1F, Fis, and YafK (Navarro-Garcia and Elias 2011). Unlike the other pathotypes, EHEC strains express a multitude of adhesins, but Long polar fimbriae (Lpf), E. coli common pilus (ECP), hemorrhagic pilus (HCP), and mucin-­ binding flagella are mainly involved in intestinal colonization (Rossi et al. 2018). The formation of mature biofilm includes the growth of the bacterial mass within a complex three-dimensional architecture. E. coli strains express combinations of adhesins that contribute to consolidate the biofilm structure. The autotransporter superfamily is a group of proteins often associated with the bacterial aggregation and biofilm formation (Vo et al. 2017). These proteins belong to the largest group of secreted OM proteins by T5SS. One of the best characterized autotransporters is Antigen 43 (Ag43), the major phase-variable OM protein of E. coli. Ag43 mediates the formation of biofilm-like intracellular communities and multispecies biofilms and enhances colonization and persistence in the bladder. Ag43 is associated to UPEC, EHEC, ETEC, EPEC, and commensal strains (Vo et al. 2017, 2022; Ballen et al. 2022). Adhesin Involved in Diffuse Adherence (AIDA-1) is another autotransporter that mediates bacterial attachment to mammalian cells and promotes autoaggregation via intercellular self-recognition. This molecule is a potent adhesin and is a highly efficient initiator of biofilm formation. AIDA-1 has been detected in diffusely adherent E. coli (DAEC), EPEC, ETEC, and EHEC (Sherlock et al. 2004; Charbonneau et al. 2006; Servin 2014; Vo et al. 2017). The autotransporter UpaB promotes adhesion to extracellular glycoproteins. UpaB was detected in UPEC and APEC. Instead, UpaC and UpaH promote strong biofilm formation and have been detected in UPEC and commensal strains (Rossi et al. 2018; Ballen et al. 2022). Furthermore, diverse extrachromosomal superficial colonization factors have been characterized in ETEC or EAEC, suggesting that the transfer of genes (encoded in mobile genetic elements) can constitute a relevant source of adhesion factors (Beloin et  al. 2008; Ageorges et al. 2020). Biofilm development is related to quorum sensing (QS), a cell-to-cell communication mechanism that regulates a variety of physiological functions, including the synthesis of biofilm matrix compounds (Beloin et  al. 2008; Saxena et  al. 2019; Ballen et al. 2022), playing an important role generating a microenvironment for bacterial growth. Biofilm protects the bacteria from host defenses, provides a favorable environment for horizontal gene transfer, and acts as a diffusion barrier for antimicrobials. The matrix contains extracellular polymeric substances, proteins,

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nucleic acids, lipids, nutrients, and metabolites that provide stability and cohesion to the biofilm. In E. coli, three principal exopolysaccharides have been detected in the biofilm matrix: β-1,6-N-acetyl-D-glucosamine polymer (PNAG), colanic acid, and cellulose. Other compounds like lipopolysaccharides and capsular polysaccharides may not accumulate significantly in the matrix, but still play an important indirect role in biofilm formation (Saxena et al. 2019; Ageorges et al. 2020; Ballen et al. 2022). The biofilm last stage includes bacteria dispersion by shedding fragments or the individual cells release. The dispersion is produced by decrease of cyclic diguanylate (c-di-GMP) in cells, which results in enzymes synthesis for breaking the biofilm matrix or by external factors such as physical triggers or enzymatic degradation (Ageorges et al. 2020; Ballen et al. 2022). The four-step model of biofilm formation is widely recognized. However, Sauer and colleagues (Sauer et al. 2022) have postulated that this model has some limitations making it difficult to extrapolate the results. The in vitro models do not consider differences in diversity, composition, and biofilm structure complexity outside the laboratory-controlled environment (e.g., patients, industry, or ambient). Also, this model does not incorporate the aggregation diversity and bacteria separation mechanisms, mixed biofilm formation, nor does it consider the succession of events in biofilms in open systems with a continuous influx of new colonizers.

4.6.1 Levels of Regulation in the Expression of Colonization Factors E. coli has a wide variety of adhesion factors that participate in surface colonization process. The expression of these factors is an extremely complex process involving the integration and coordination of signals and effectors of all the regulatory components. 4.6.1.1 Regulation at the Pre-transcriptional Level: Phase Variation Several mechanisms involved in phase variation have been described: DNA inversion, slipped strand mismatch, DNA methylation, and DNA deletion. Such mechanisms occur at the DNA replication stage, and a large majority of genes regulated by phase variation are molecular determinants of the bacterial cell surface. Adhesins like T1P, Ag43, and Pap are examples of regulation through this mechanism (Charbonneau et al. 2006; Navarro-Garcia and Elias 2011; Servin 2014). 4.6.1.2 Regulation at the Transcriptional Level: Regulators and Effectors Four regulatory mechanisms can be distinguished: positive or negative control of an inducible gene, and positive or negative control of a repressible gene. Bacteria can recognize environmental signals such as cell density (QS), carbon and energy

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sources, pH, osmolarity, temperature, or the accompanying microbiota and respond to stimuli by a wide range of signal transduction systems. The sensor detects an abrupt change in signal and activates a transcriptional regulator, which, in turn, represses or activates gene expression. PNAG synthesis, colanic acid, and cellulose are under the control of transcriptional regulators (Charbonneau et  al. 2006; Navarro-Garcia and Elias 2011; Ballen et al. 2022). 4.6.1.3 Regulation at the Post-transcriptional Level Three regulatory mechanisms are recognized: mRNA stability, riboswitch by the formation of hairpin termination of the ongoing process, and transcription attenuation (Navarro-Garcia and Elias 2011; Charbonneau et al. 2006). Post-transcriptional regulation mechanisms participate in biofilm formation, associated with small RNAs (sRNA) or riboswitches. These non-coding RNA molecules adopt a secondary structure that allows their binding to other molecules (including di-cGMP), and they act post-transcriptionally in cis. Ag43 expression level and PNAG production are examples of regulation through this mechanism (Ageorges et al. 2020; Ballen et al. 2022). 4.6.1.4 Regulation at the Translational Level Three main mechanisms have been described: production of antisense RNA and small RNA, riboregulation associated with conformational changes in mRNA, and translation efficiency depending on the codon present. PNAG production, colanic acid, and OmpA protein can be regulated by these mechanisms (Charbonneau et al. 2006; Sauer et al. 2022). 4.6.1.5 Regulation at the Post-translational Level A wide variety of molecular mechanisms regulate bacterial protein synthesis. Modulation of enzyme activity is a key mechanism in post-translational regulation. For example, enzymes of colonization factor synthesis can be modified by physical and chemical factors or through inducers and inhibitors. The second messenger C-di-GMP controls the motility and virulence of planktonic cells, as well as cell adhesion and the persistence of multicellular communities (Charbonneau et  al. 2006; Beloin et al. 2008; Ballen et al. 2022). This regulation can occur in a transcriptional, post-transcriptional, or post-translational way, with the participation of transcriptional regulators, ribonucleases, glycosyltransferases, and small RNAs (Charbonneau et al. 2006; Ballen et al. 2022; Sauer et al. 2022).

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4.7 Cross-Communication Mechanisms Between Pathogenic E. coli and Cell-Host It has been proposed that bidirectional cross-communication occurs between bacteria and cell-host, and a recent evidence indicates that bacteria using RNA-based signaling influence their hosts, as it was demonstrated with C. elegans-E. coli interactions, which are mediated by two sRNAs (Liu et al. 2012). In fact, bacteria secrete RNA involved in signaling (Ghosal 2017), and it is thought that the Hfq chaperone, an RNA-binding protein, may be playing a role in RNA export. Another interesting role for RNA export is the recent association with OM Vesicles (OMVs) described for various bacterial pathogens (Dauros-Singorenko et al. 2018). Bacterial OMVs are spherical structures with an approximate size of 100–300 nm, produced naturally by commensal and pathogenic Gram-negative bacteria, during bacterial growth. OMVs are comprised of lipid bilayers containing various biopolymers inside, such as lipopolysaccharides (LPS), phospholipids, peptidoglycans, OM proteins (OMPs), cell wall components, enzymes (autolysins), periplasmic proteins, cytoplasmic proteins, and membrane-binding proteins (Fig. 4.3). They also harbor nucleic acids (plasmid DNA, chromosomal DNA, and RNA), metabolites, signaling molecules, virulence factors, quorum-sensing-related molecules, and others (Jan 2017). OMVs production has also been described in Gram-positive bacteria of the phyla Actinobacteria and Firmicutes (Kesty et al. 2004). This structure biogenesis has not been fully clarified; however, they might be regulated by membrane proteins, and various biogenesis mechanisms have been suggested (Schwechheimer and Kuehn 2015). The entry mechanism shows OMVs interacting with host cells on lipid rafts in a temperature-dependent manner that is possibly facilitated by caveolin, and the enterotoxin receptor inside the lipid rafts (Kesty et al. 2004). Likewise, it exists the possibility that the OMVs enter the host cells through processes independent of lipid rafts. Besides, OMVs harbor various pathogen-associated molecular patterns (PAMPs) on their surface, which can initiate PRR signaling, facilitating their entry into the host cells (Kaparakis-Liaskos and Ferrero 2015).

4.7.1 Functionality and Effect of Bacterial OMVs The global epidemiological cargo attributed to the different E. coli pathotypes is associated with an extensive list of virulence factors that enable the development of different pathogenicity mechanisms (Gomes et al. 2016). Additionally, OMVs functionality must be added, which is related to a wide number of physiological/physio-­ pathological processes, including bacterial communication, pathogen-host communication, molecule transport, cell waste elimination, immunomodulation, cell survival, biofilm formation, release of virulence factors, and the DNA horizontal transfer mechanism (Jan 2017). Thus, the role of OMVs in bacterial

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Fig. 4.3  Biogenesis, structure, and composition of bacterial outer membrane vesicles (OMVs). Formation of bacterial vesicles from the outer membrane. Composition of OMV, cargo selection, and loading as part of OMVs are shown. This bud must be pinching-off to form a vesicle, which is showed at right of the drawing. This latter part shows a typical composition of bacterial OMVs. (OM) outer membrane; (IM) inner membrane; (OM protein) outer membrane protein; (OM-PG) outer membrane-peptidoglycan (LPS) Lipopolysaccharide; (DNA and RNA) nucleic acids. (Created with BioRender.com)

pathogenesis is of great importance, recognizing among their functions the release of toxins, modulation of the immune system, signaling molecule transport between bacterial cells, and the formation of biofilms (Jan 2017; Cecil et al. 2019). OMVs from commensal bacteria have the ability to induce maturation of the immune system (Shen et al. 2012), and the presence of OMVs in various tissues and/or fluids affected by infections suggests they have a role in the onset, development, and progression of inflammatory phenomena and a relevant role in bacterial pathogenicity (Kaparakis-­Liaskos and Ferrero 2015). Therefore, OMVs are used for delivery of virulence factors contained in them, as was observed for the cytolysin ClyA produced by E. coli and other enteric bacteria. A greater cytotoxic activity against mammalian cells was observed when OMVs were administered as compared to the protein purified from the bacterial periplasm. Simultaneously, the disruption of the epithelial barrier has been reported as a property attributable to OMVs as well as their ability to alter the integrity of the epithelial mucosa, allowing the bacterial components to enter the submucosa, promoting the progression of inflammation and infection (Wai et al. 2003). Regarding E. coli OMVs and their relation to the immune system; OMVs alter inflammation, thrombomodulin and the adhesion molecules P-selectin and E-selectin for human

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endothelial cells (Soult et  al. 2014), leading to recruitment of pro-inflammatory leukocytes, platelet aggregation and coagulation. Similarly, STEC/AIEC OMVs increase human epithelial cell proliferation, and induce reactive oxygen species, DNA damage, aneuploidy, and chromosomal instability (Tyrer et al. 2014).

4.7.2 STEC and AIEC as Pathogenicity Models of Cross-­Communication Mediated by OMVs The only zoonotic pathotype among the DEC strains, STEC, contains a sub-group called EHEC, in which besides the expression of Shiga toxins (Stx1 and/or Stx2), it has LEE, initially establishing it as the only STEC group associated to infections in humans (Gomes et al. 2016). Today, it is known that LEE-negative STEC can also produce clinical cases indistinguishable from EHEC caused illness (Montero et al. 2019). Regarding cross-communication between STEC and other bacteria or eukaryotic cells and its role in pathogenicity, it has been demonstrated that naturally secreted OMVs by STEC O157:H7 take and deliver to the cells a cocktail of biologically active virulence factors such as Stx2a, cytolethal distending toxin (CdtV-ABC), hemolysin, and flagellin, which are internalized via dynamin-dependent endocytosis. This delivery into the cells causes apoptosis induced by Stx2a and CdtV through the activation of caspase 9 (Bielaszewska et al. 2017). Additionally, the antigenic protease OmpT (confer resistance to antimicrobial peptide LL-37) is important in the biogenesis, composition, and size of STEC OMVs; ompT mutants clearly reduce their OMV production levels (Torres et al. 2020). An elongation factor associated with protein synthesis (EF-Tu) is transported in OMVs, but when secreted by O157:H7 and localized on the surface of this bacterium, favors adherence to eukaryotic cells, suggesting it as a possible export mechanism for this protein (Torres et al. 2020). Regarding RNAs mobilized in OMVs, this mechanism as only been described for E. coli K12 MG1655 and UPEC strain 536 (Blenkiron et al. 2016). AIEC (first isolates obtained from patients with Crohn’s disease [CD]) can adhere and invade intestinal epithelial cells. Although AIEC phenotypes of adhesion and invasion are undistinguished from DEC pathotypes, the AIEC invasive capacity is not associated with classic genetic determinants of invasiveness, such as the ipaC invasin of EIEC, or the tia gene product involved in the invasion process of ETEC strains (Darfeuille-­ Michaud et  al. 2004). Additionally, AIEC can modulate microRNA levels in the epithelial cells, reducing the protein expression related to the autophagy process, which would favor bacterial replication. In intestinal cells, the AIEC infection causes an increase in the expression levels of microRNAs MIR30C and MIR130A, and their increase reduces the levels of cell proteins ATG5 and ATG16L, suppressing the autophagy process. Interestingly, AIEC infection induces exosomes release by T84 epithelial cells and THP-1 macrophages, and the exosomes treatment of

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cells causes an increase in bacterial replication in comparison with cells stimulated with exosomes secreted from non-infected cells in both cell types (Larabi et al. 2020). Recent findings linking AIEC OMVs with the pathogenesis process emphasize that AIEC is associated with the ileal segment in CD, taking advantage of the overexpression of Gp96 (usually localized in the endoplasmic reticulum), which acts as a host cell receptor for the invasion of AIEC mediated through OmpA-enriched OMVs. Patients at high risk of developing severe ileal CD are those who, while expressing CEACAM6, also overexpress Gp96 in the ileal mucosa (Rolhion et al. 2010), and although it is known that the involvement of actin and microtubules is required for AIEC invasion (Darfeuille-Michaud et al. 2004), the associated molecular mechanisms are unknown.

4.8 Conclusions T3SS EHEC/EPEC structures have been elegantly elucidated (the injectosome), as has the secretion and function of most of the effectors delivered to host cells. New ongoing research is trying to understand how the secretion of so many effectors is regulated and what the hierarchy of secretion is, including the role of molecular switches and their specificity in regulating this hierarchy. It is also interesting to understand structural details at the level of each protein to determine protein-protein interaction processes, which allow us to use blocking compounds or peptides. These strategies will be relevant in preventing also several other T3SS-carrying Gram-­ negative bacteria. T6SS is novel in its ability to be used in microbial competition or pathogenesis. Previous advances in the structure of other secretion nanomachines allowed rapidly elucidation of the structure of the T6SS (the crossbow). Due to the nature of the effectors and molecular targets, both immunity proteins are needed for self-­ protection or sibling bacteria protection, as well as common targets for various bacteria and even for eukaryotic cells (e.g., phospholipases and nucleases). Bacterial competition is itself a critical issue in pathogenesis. To establish a successful infection, some pathogenic E. coli strains have developed sophisticated strategies to overcome the colonization barriers imposed by the microbiota. These include the regulation of E. coli virulence factors by the microbiota, the modulation of E. coli pathogenesis by bacterial proteases, and interbacterial competition using the T6SS, among others. In E. coli, biofilms are traits associated with bacterial persistence and virulence. For biofilm formation in E. coli, four stages are relevant for a mature biofilm: initial adhesion, early development of biofilm structure, biofilm maturation, and dispersal. The regulation of the expression of the factors involved is an extremely complex process that involves the integration and coordination of signals and effectors of all the regulatory components. Another mechanism is the cross-communication between pathogenic E. coli and host cells through OMVs. The OMVs deliver a wide variety of cargo molecules,

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including classical and new virulence factors such as cytosolic enzymes and nucleic acids. OMV functionality includes bacterial communication, pathogen-host communication, molecule transport, cellular debris removal, immunomodulation, cell survival, biofilm formation, virulence factor release, and DNA horizontal transfer. Acknowledgments  We thank Aylin Larrañaga and Marvin Paz for the drawings in Figs.  4.1 and 4.3.

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Chapter 5

Bovine Reservoir of STEC and EPEC: Advances and New Contributions Nora Lía Padola, Vinicius Castro, Analía Etcheverría, Eduardo Figueiredo, Rosa Guillén, and Ana Umpiérrez

Chapter Summary  Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) are diarrheagenic E. coli. While EPEC produce only diarrhea, STEC are a causal agent of Hemolytic Uremic Syndrome (HUS) in humans. Those pathotypes are zoonotic pathogens, transferred to humans through contaminated food and water, direct animal or environmental contact, and the bovine is the main reservoir, shedding in their feces those bacteria that could contaminate the environment and the derived foods. The One Health concept is applicable to those bacteria because the health of human is related to health of animals and their environments. This chapter discusses the finding of the last 5 years regarding virulence factors, serotypes, pathogenicity islands, and prevalence in cattle. In addition, the resistance mechanisms and survival strategies of STEC are also included. Prevention and control of the bovine with several strategies including animal handling, probiotics, and vaccines are also explained.

N. L. Padola (*) · A. Etcheverría Universidad Nacional del Centro de la Provincia de Buenos Aires, Facultad Ciencias Veterinarias, Departamento Sanidad Animal y Medicina Preventiva- CIVETAN, Tandil, Buenos Aires, Argentina e-mail: [email protected] V. Castro Department of Biological Science, University of Lethbridge (ULETH), Lethbridge, AB, Canada E. Figueiredo Departamento de Alimentos e Nutrição, Universidade Federal de Mato Grosso, Cuiabá, Mato Grosso, Brazil R. Guillén Instituto de Investigaciones en Ciencias de la Salud, Universidad Nacional de Asunción, San Lorenzo, Paraguay A. Umpiérrez Departamento de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 A. G. Torres (ed.), Trending Topics in Escherichia coli Research, https://doi.org/10.1007/978-3-031-29882-0_5

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Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) are E. coli pathotypes that produce histopathological lesions of “attaching and effacing” (A/E) in enterocytes of the gastrointestinal tract. This diarrheagenic E. coli are known as A/E E. coli (AEEC) (Moxley and Smith 2010) because they harbor a pathogenicity island named the locus of enterocyte effacement (LEE), carrying the eae gene encoding the intimin protein (Habets et al. 2022). However, there are STEC strains lacking LEE (LEE-negative) isolated from cases of severe illness, such as those suffering from hemolytic uremic syndrome (HUS). Cattle are the natural reservoir of STEC and EPEC, and different serotypes of those bacterial pathotypes are shed in their feces and are able to contaminate the environment and bovine hides with implications for public health and food safety (Moxley and Smith 2010; Arimizu et al. 2019). STEC and EPEC are zoonotic pathogens, transferred to humans via contaminated food and water, and direct animal or environmental contact. Human-to-human transfer via the fecal-oral route has been documented (EFSA 2020; Cáceres et al. 2020). STEC produces Stx that is the main virulence factor, responsible for serious diseases in humans such as HUS. EPEC strains do not produce Shiga toxins, but they cause diarrhea in children (Cáceres et al. 2020).

5.1 Shiga Toxin-Producing Escherichia coli (STEC) STEC O157:H7 strains are the most important cause of HUS worldwide. However, those infections have been decreasing significantly, in contrast to STEC non-O157 infections that have increased worldwide (Torres 2017). According to this, STEC is classified in O157 and non-O157 serogroups, such as O91, O104, O113, O26, O103, and O145. The serotypes or serogroups cannot predict virulence because serotypes should not be associated with a disease without considering the virulence factors that each strain carries (Colello et al. 2018; EFSA 2020). Another classification is according to the presence or absence of LEE-­ pathogenicity islands. LEE is present in some STEC strains isolated from severe human cases, but it does not appear to be essential for pathogenesis, since a large number of STEC strains isolated from sporadic outbreaks of hemorrhagic colitis (HC) and HUS do not possess the LEE (STEC LEE-negative strains) (Colello et al. 2018). While the marker for the presence of the LEE is the eae gene (encoding intimin), the LEE-negative strains are not easily identified. The importance of considering the virulence factors is because most of them are codified in mobile genetic elements such as plasmids, bacteriophages, integrons, transposons, and pathogenicity islands, and through the process of horizontal transfer, those virulence factors can be acquired for other E. coli or even get lost and define the virulence profile of a strain (Colello et al. 2018b).

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5.2 Enteropathogenic Escherichia coli (EPEC) EPEC is a diarrheagenic E. coli that has been defined through serogroups epidemiologically related to the disease. Currently, this bacterial group is defined as those capable of causing diarrhea by producing the A/E histopathology on the intestinal epithelium, mediated by intimin encoded by the eae gene (Scaletsky 2019). Some EPEC strains harbor the bfpA gene, encoding a structural subunit of the type IV pilus (BFP), present in the EPEC adherence plasmid (EAF). According to the presence or absence of BFP, EPEC strains can be classified as typical and atypical, respectively. The most common serotypes of typical EPEC (tEPEC) are O55:H6, O55:H-, O86:H34, O111:H2, O111:H-, O119:H6, O127:H6, O127:H40, O142:H6, and O142:H34, while O26:H11, O55:H7, O55:H34, O86:H8, O111ac:H9, O111:H25, O119:H2, O125ac:H6, and O128ac:H2 are the most frequent serotypes of atypical EPEC (Alonso et al. 2016).

5.3 Bovine Reservoir Zoonosis refers to an infectious disease that is transmissible under natural conditions by direct or indirect contact from vertebrate animals to humans. Indirect contact refers to vehicles, such as food or water that can transmit the bacteria. https:// www.cdc.gov/csels/dsepd/ss1978/lesson1/section10.html. E. coli represents a broad-based bacterium that is present in all environments, and it is also present in the most diverse means of agricultural production. The wide dissemination throughout the world is due to the ease of this microorganism to establish itself in a community and its rapid cell multiplication, factors that led E. coli to be present in the gastrointestinal tracts of several animals, including humans. Although the presence of E. coli does not necessarily pose a risk to production, the presence of certain strains can lead to a significant risk of causing disease to humans due to their pathogenic potential. Cattle are the main asymptomatic reservoir of STEC O157 and non-O157 strains that colonize the recto-anal junction (RAJ). These bacteria are shed in the feces and when more than 104 cfu/g of STEC are excreted, the bovine is considered “super-­ shedders.” This condition has been epidemiologically associated with certain Stx subtypes such as Stx2a. Although the definition for this event does not yet have a consensus in the literature, some authors classify as only the high presence of E. coli, other authors point out, in addition to the high microbial load, the frequent isolation of samples shedding more than 104 cells/g. These data reinforce the fact that the super-shedding event can represent one of the greatest challenges in food production and the need to investigate its causes. Several studies have investigated aspects related to the host, the pathogen, and the environment in which the animals are living in. Among the aspects of greater consensus in the animal host, we can mention an increase in cholesterol absorption in super-shedding animals when

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compared to non-shedders animals (Castro et al. 2022). The data referring to the pathogen indicate that most of the super-shedding isolates (approximately 99% of the isolates evaluated) presented mutations in the tir gene (translocated intimin receptor). This gene encodes a protein that is required for efficient pedestal formation in epithelial host cells during infection. Furthermore, the ability to form a biofilm has been classified as one of the key factors for the appearance of the super-shedding phenotype, since the formation of an intestinal biofilm represents the most likely explanation for this event. When STEC is repeatedly isolated from cattle feces over several months, these strains are defined as persistent (STECper), or sporadic (STECspo) colonizers of the bovine intestine if the strains have been isolated only a single or a few times. Virulence-associated genes could be correlated with these classifications (Barth et al. 2020; Pan et al. 2021). STEC and EPEC strains can grow in the intestinal tract of animals where they can adapt to low pH and compete with the microbiota, before reaching the colonization site (Jang et al. 2017). STEC use nutrients released from the mucus layer found in the bovine intestinal tract as a carbon source, and when mucus-derived carbohydrates are exhausted, they use gluconeogenic substrates such as glycerol, lactate, and amino acids (Jang et al. 2017). STEC strains possess several adhesins that participate in the adhesion to rectal epithelial cells: flagella, pili, and monomeric adhesins such as Eha, Saa, Ag43, Efa-1, and Iha, among others. Moreover, the interaction of STEC with native microbiota is essential to survive and persist in the gastrointestinal tract, and this condition can be affected by diet, acidic conditions, and use of antimicrobials added to cattle feed to improve animal weight gain (Cáceres et  al. 2017; Sapountzis et al. 2020). Several studies have evaluated the influence of animal diet on the dynamics of elimination of E. coli in feces. Grain-finishing diets were related to super shedding in the studies by Munns et al. (2015). Berg et al. (2004) determined that barley had a greater participation in E. coli shedding than corn-based diets. In the study performed by Berard et al. (2009), the authors concluded that the use of a forage-based diet could be a viable alternative in reducing E. coli shedding and that it would not affect animal production. The farm environment is a source of transmission and dissemination of STEC due to repeated fecal contamination produced by the animals. The level of environmental contamination depends on the strain’s ability to survive and the magnitude of shedding by animals. The survival of STEC strains (O157 and non-O157) in water troughs and bovine feces has been demonstrated. In water, the STEC strains have been detected up to 80 to 100 days, while in bovine feces, they survive until day 60. Several factors have been involved in the persistence of STEC in the animals and their environments, such as the capacity to produce biofilms, the activation of stress fitness genes in bovine feces, and the presence of competitive microbiota. The environment can play a key role in the transmission of STEC to newborn calves. They are exposed to this bacterium quickly after birth since it has been detected in newborn calves 8 months), which came from different regional production systems (dairy farm, feedlot, and grazing). Cáceres et al. (2017) detected a higher prevalence of espP among all STEC strains regardless of the category of cattle, and the production system, and stcE was the less prevalent. While espP was widely detected among LEE-positives and LEE-negative serotypes, stcE was detected in a few LEE-positive serotypes, such as O157:H7, O64:NM, and NT. A higher prevalence of katP in STEC isolated from young and rearing calves was found. LEE-negative STEC serotypes O113:H21, O116:H21, O130:H11, O178:H19, and ONT:H7 carried subA and were isolated mostly from rearing calves and adults from dairy and grazing systems. SubA-positive STEC harbored neither

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stcE nor katP. Efa1 was detected in LEE positive-STEC strains isolated from dairy young calves and feedlot rearing calves, declining with increasing age. Iha was detected in all STEC strains regarding the category of cattle, although it was more prevalent in those isolated from adults, and in LEE-negative STEC serotypes. This diversity of strains has been detected in cattle in Chile (Díaz et al. 2021). Iha was described as an adhesin in a LEE-positive STEC O157:H7 strain. Iha is an adherence-conferring protein and a siderophore receptor that is distributed among STEC strains of a variety of serotypes and was reported in Pathogenicity Islands (PAIs) and plasmids of LEE-negative STEC strains. Colello et al. (2019) described iha subtypes located in a PAI named Locus of Adhesion and Autoaggregation (LAA) and in a pO113 of LEE-negative STEC strains. Alignment and phylogenetic analysis revealed that iha LAA and iha pO113 subtypes were highly similar, whereas they have lower sequence similarity regarding iha gene with STEC EDL933, suggesting that iha genes from LEE-negative and LEE-positive STEC strains may have different origins. LAA was described by Montero et al. (2017), as a PAI associated with severe disease. LAA possesses four modules, and may be present as a “complete” structure with the four modules, module I (hes and other genes), module II (iha, lesP, and other genes), module III (pagC, tpsA, tpsB, and other genes), and module IV (agn43 and other genes), or as an “incomplete” structure when one of the modules is missing (< 4 modules). In Argentina, LAA was detected more frequently in LEE-negative STEC strains from adult cattle (grazing cattle, pre-slaughter cattle, feedlot cattle, adult dairy cattle) than from calves (milk-fed calves (2 months old), growing calves (2–8 months old), and newborn calves (< 24 h from birth)) (Colello et al. 2018c). LEE-negative STEC strains isolated from Chile and Paraguay harbored the complete LAA in 41.6% and 41.0%, respectively (Fig. 5.2) (Vélez et al. 2020). In Argentina, Colello et al. (2022) sequenced two O157:H7 strains isolated in the same period, one from a bovine carcass and the other one from a HUS case, and Fig. 5.2  Dynamic of LEE-­positive and negative STEC strains according to the age of animals

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each genome was compared. The O157:H7 isolated from bovine carcasses carries stx2a, astA, eae, ehxA, espAB, DFJP, gad, iha, iss, katP, nleABC, ompT, tccP, terC, tir, and toxB, while the genome of O157:H7 isolated from a human case possesses stx2a, stx2d, stx2c, astA, chuA, eae, ehxA, espAB DFJP, gad, iha, iss, katP, nle ABC, ompT, terC, tir, and toxB. Both STEC strains belonged to phylogroup E and to hypervirulent clade 8. Although both strains were assigned as ST11, the two genomes were grouped into different clusters, and they were not closely related to 31 STEC O157:H7 strains available in the database. Amadio et al. (2021) analyzed STEC O157:H7 isolated from cattle, and the isolates were classified as clade 8 (four strains) and clade 6 (four strains). The prophages encoding for stx2a were highly diverse, while those encoding for stx2c were conserved. Despite the differences found in the stx subtypes, the O157:H7 strains isolated from bovines are hypervirulent, and it indicates that the genomic content of STEC is influenced by the site of isolation, and E. coli genomic plasticity would allow for the evolution of a STEC population in a defined region. Regarding LEE negative-STEC O91 isolated from cattle and meat food, more than five serotypes (O91:H21, O91:H8, O91:H14, O91:H28, O91:H40) and eight virulence factors have been detected. Genes involved in adhesion as ehaA, elfA, espP, ecpA, and hcpA and a gene encoding the cytolethal distending toxin type-V (CDT-V) were also found (Hernandez et al. 2018). The O113:H21 strains studied harbor several toxins and adhesion genes (saa, espP, fimCD, ehaA, iha, hcpA, elfA, lpfO113, ecpA, subA, cdt-V) and stx subtypes associated with human disease (Sanso et al. 2018). STEC O22:H8 was isolated from recto-anal mucosal of a healthy Holstein-­ Friesian male calf in a dairy farm in Buenos Aires, Argentina. By whole-genome sequencing analysis and genomic comparative, Marques Da Silva et al. (2022) demonstrated that stx1a and stx2a were the most frequently found variants among the STEC strains while O22:H8 strain was predicted to have the stx1c and stx2d variants. The hes and tia genes were detected in this O22:H8. The tia gene is described as an important virulence factor of ETEC that contributes to both adhesion and invasion to this pathogenic group.

5.6 Antibiotic Resistance A consequence of inappropriate use of antimicrobials in veterinary medicine is the spreading of antibiotic-resistant strains, due to the potential risk of resistance transference through the food chain. Galarce et al. (2020) detected the distribution of 12 antimicrobial resistance genes in 54 STEC strains. All strains were phenotypically resistant to at least one antimicrobial drug (100% of resistance to cefalexin, followed by colistin (81.5%), chloramphenicol (14.8%), ampicillin and enrofloxacin (5.6% each), doxycycline (3.7%), and cefovecin (1.9%). Most detected antibiotic resistance genes were dfrA1 and tetA (100%), followed by tetB (94.4%), blaTEM-1 (90.7%), aac (6)-Ib (88.9%), blaAmpC (81.5%), cat1 (61.1%), and aac (3)-IIa

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(11.1%). Fluoroquinolone resistance was detected in 36.4% (96/264) of STEC isolates from different animal facilities throughout Uruguay. Concerning oxyimino-­ cephalosporin resistance, only 3 out of 96 isolates showed an ESBL phenotype, corresponding to ESBL CTX-M-14. The presence of a class 1 integron was detected in 5 of the E. coli isolates analyzed. Four different variable regions were identified (dfrA7, dfrA17-aadA5, dfrA1-aadA1, and dfrA12-orfF-aadA2), which may confer resistance to aminoglycosides and trimethoprim (Umpiérrez et  al. 2017). Also, fosfomycin-­trometamol resistance and fosA7 gene in STEC strains from cattle (Umpiérrez et al. 2022) in Uruguay were detected. Resistance to FOS is a major concern in human health since it is within the scarce therapeutic resources available for infections of multi-resistant microorganisms. Integrons are mobile genetic elements able to capture gene cassettes encoding antibiotic resistance from the environment and incorporate them by site-specific recombination. Class 1 and class 2 integrons are more frequently found in E. coli isolates that contribute to the spread of antimicrobial resistance genes. They carry an intI gene encoding an integrase, a recombination site (attI), and a promoter. Pérez Gaudio et  al. (2018) demonstrated the transference of integrons from an E. coli-­ resistant strain isolated from a swine farm to a negative STEC O157:H7 isolated from bovine in a period of only 4 hours. In fresh retail beef samples from Brazil, the integrons were detected in two isolates that showed intermediate resistance to streptomycin (Castro et al. 2019). In Argentina, seven out of 649 STEC strains, some from serogroups O26, O103, and O130, and isolated from cattle, chicken burgers, farm environments, and pigs were int1 positive. Different arrangements of gene cassettes were detected in the variable region of class 1 integron: dfrA16, aadA23, and dfrA1-aadA1. Phenotypic resistance to streptomycin, tetracycline, trimethoprim/sulfamethoxazole, and sulfisoxazole was detected. Microarray analyses showed that most of the isolates carried four or more antimicrobial resistance markers, and STEC strains were categorized as multidrug-­resistant (Colello et al. 2018b).

5.7 Biofilm Formation Biofilm formation is a spontaneous mechanism whereby STEC strains can resist a hostile environment and are able to survive and consequently infect the host through contaminated food and food-contaminated surfaces. Biofilm formation shows intra-­ and inter-serotype variability, and its formation does not depend only on the microorganisms involved. Other factors related to the environment (such as pH and temperature) and the surface (stainless steel and polystyrene) influence the biofilm expression. Most of the STEC and aEPEC strains found were curli-negative at 37 °C, while they showed curli-positive phenotypes at 20 °C. Both curli expression and biofilm formation were significantly influenced by temperature and incubation time. The ability to form biofilms on different environments can contaminate food

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and generate infections while protecting themselves against adverse conditions (Cáceres et al. 2020; Velez et al. 2022). All O157:H7 strains isolated from cattle were classified as strong-biofilm formers. The association between curli production and biofilm formation could not be determined, but the highest proportion of curli-positive strains at room temperature were strong biofilm-formers (Cáceres et al. 2022). Forty-eight STEC strains belonging to serogroups O113, O130, O171, O174, and O178 were assayed for their ability to form biofilm. Although all STEC strains were able to form biofilm, high variability was found between them. In this study, 64% of LAA-positive strains were classified as strong biofilm formers (Velez et al. 2021). In Paraguay, the biofilm formation capacity is very frequent in STEC isolates from cattle and butcher shops, being many cases categorized as SBF (Guillén 2022). STEC and aEPEC strains demonstrated to be able to form biofilm and produce curli fimbria under different conditions of medium and temperature, which is important due to the risk of survival and transmission of these pathogens from reservoirs and food to humans (Cáceres et al. 2020).

5.8 Prevention and Control 5.8.1 Animal Diet One strategy to control the contamination caused by super-shedding animals is the administration of different diets that can influence the reduction of contamination. One of the main alternatives is the use of forage instead of grain-based diets. In a study performed by Jacob et al. (2010), the authors verified that diets with the inclusion of up to 20% of distiller grains did not change the dynamics of shedding; however, the inclusion of 40% of distiller grain increased the prevalence of the pathogen in the feces. A key point was that diets based on distiller grains showed no relationship with pH values (Wells et al. 2009), which represents a positive aspect because a decrease in pH values with diets based on grains has already been associated to higher levels of E. coli shedding, especially when related to hay-fed diets (Lowe et al. 2010). Another point is that the evaluation of only the intestinal pH parameter can be a limiting factor because several other variables can be related to the elimination of the pathogen. In this sense, a study performed by Williams et  al. (2014) identified that pasture growth influenced shedding in dairy heifers; in addition, several other factors influenced increasing shedding in animals, such as body score, higher temperature, rainfall, and relative humidity. Additionally, in a study performed by Van Baale et al. (2004), the authors verified a result that differed from the others when the data from animals fed on forage were related to cattle with higher microbial counts and for a longer time than animals fed on a grain diet.

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5.8.2 Feed Additive The use of feed additives to combat contamination by STEC shedding represents a strategy that has been widely assessed by several research groups. The use of monensin, for example, was proposed by Paddock et al. (2011) and showed good potential for reducing O157:H7 shedding in feedlot cattle. In the same study, the authors concluded that the use of urea and ractopamine was not effective in reducing STEC in the feces. In addition, the use of natural compounds with antimicrobial capacity may represent a viable alternative in combating the elimination of STEC by animal feces. In a study performed by Braden et al. (2004), the authors reported that the use of Ascophyllum nodosum (Tasco-14) decreased the prevalence of STEC O157:H7 in animals when the additive was included in the diet. An interesting point is that in recent years, the presence of plant compounds with antimicrobial action has been extensively reported in the literature in studies performed in South America. For example, in a study performed by Moreira et al. (2022), the authors reported the antimicrobial effect of a waste extract of Pequi (Caryocar brasiliense) on the reduction of E. coli and other Enterobacteriaceae members. These and several other extracts could be explored in animal nutrition as potential inhibitors of pathogenic microorganisms. Additionally, the use of prebiotics (nondigestible carbohydrates fermented in the large intestine) showed promising results when administered in pre-ruminant calves (Roodposhti and Dabiri 2012). The use of a commercial prebiotic called Celmanax™ has been proposed to prevent the colonization of STEC strains in cattle. Although the product was not developed to reduce E. coli contamination in animal feces, the main objective was the prevention of cases of jejunal hemorrhage syndrome (JHS); however, it showed promising results in reducing STEC shedding (Baines et al. 2013).

5.8.3 Immunization and Bacteriophage Therapy Other STEC control strategies were developed and applied in cattle herds to prevent the colonization of this pathogen. Two commercial vaccines were developed as a strategy to reduce STEC O157:H7 in cattle. Although some studies have shown a high ability to inhibit the colonization of E. coli in animals, the use of vaccination as a means of controlling the STEC population has not been successful due to the low incentive for producers to vaccinate (Matthews et  al. 2013). However, some research aimed at the use of vaccines continues to be developed with the main objective of inhibiting the colonization of bacteria in animals. New vaccination strategies involve recombinant proteins (Martorelli et al. 2018) and live attenuated bacteria (Oliveira et al. 2012). The use of vaccines to inhibit colonization by STEC may influence elimination rates and represents a suitable alternative for controlling the spread of bacteria in environments. Another alternative is the use of bacteriophages (phages) as a strategy to reduce bacteria in animal feces. Phages that infect and kill bacteria hosts by lysis are termed

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lytic or virulent phages (Torres-Barceló 2018). The lytic potential of phages is being exploited in different areas in the agro-food industry against foodborne pathogens (Schmelcher and Loessner 2014; Denyes et al. 2017) or biocontrol agents (Havelaar et  al. 2015; Fernández et  al. 2018). Phage therapy has been used following the increasing emergence of antimicrobial-resistant bacteria (Kortright et al. 2019), but this strategy may contribute to the control of STEC non-O157 with biofilm-forming ability and antimicrobial resistance (Bumunang et al. 2019) and E. coli O157:H7 in ruminant rearing environments and cattle hides (Niu et al. 2009; Coffey et al. 2011). The potential of phage therapy against E. coli O157:H7 in ruminants was further supported in vitro (O’Flynn et al. 2004; Niu et al. 2009; Coffey et al. 2011; Carter et al. 2012) and in vivo when orally inoculating cattle (Rivas et al. 2010; Stanford et al. 2010; Niu et al. 2009a). The few reports available demonstrate the potential of phage therapy to reduce E. coli O157:H7 carriage in cattle, and the preparation of commercial phage products is being recently launched into commercial markets.

5.8.4 Inhibition of STEC Biofilm The use of Lactobacillus plantarum LP5 with probiotic potential may be an alternativeto reduce the formation of biofilms by STEC. Ruiz et al. (2022) demonstrated that L. plantarum LP5 reduced the biofilm formation of STEC by competition and exclusion tests, (p  93–100%) at the DNA level were found between some Stx2a prophages from O145 strains from cattle, or between the Stx2a prophage of a strain from a human patient and another from the environment of a dairy farm. In other cases, a high similarity was found except for the presence of insertion sequences. More regional and worldwide studies on Stx prophages are necessary to identify genomic features associated with higher virulence and to help identify STEC found in cattle or food that are most likely to cause serious human disease.

6.3.3 Stx Phage and HUS Development: The Forgotten Piece As mentioned before, infections caused by STEC are associated with the development of HUS, but too many aspects are not glimpsed yet. For example, it is still unknown which is the scientific explanation about the low percent (10–15%) of infected children with STEC that develop HUS and why only 10–100 colonies are enough to produce the syndrome. Experimental treatments and vaccine development are focused on STEC and Shiga toxin. However, there is a poor knowledge about the role of phage expressing Stx in the context of HUS being that the gene encoding Stx is carried and regulated by a Stx phage. Free Stx phages can infect other susceptible bacteria in the gut, exacerbating phage replication and Stx expression (Schmidt 2001). Indeed, phages are capable of infecting and lysogenizing laboratory strains of E. coli, as well as E. coli strains derived from the intestines of ruminants (Gamage et  al. 2003; Cornick et  al. 2006) and humans. The resulting lysogenic strains can produce toxins and infectious bacteriophage particles, facilitating the spread of toxin genes between E. coli and other Enterobacteriaceae. Interestingly, Tyler et al. (2013) have shown that mutant STEC strains in the phage excision mechanism do not induce kidney disease in an animal model. If bacteria and viruses in general can translocate across the intestinal barrier, it might be expected that phages can also translocate the intestinal wall. If translocation of phages does take place, this should lead to the presence of phages in peripheral blood known as “phagemia”. This phenomenon could have some functional consequences, especially in view of data suggesting that phages can exert immunobiological activities (Gorski et al. 2012) and are described as possible pathogen and immune modulators (Lengeling et  al. 2013). Indeed, there are several reports

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describing the presence of bacteriophages in animal sera used for in  vitro tissue culture (Merril et al. 1972) and within regions of the body that have been conventionally considered sterile, such as the blood, lymph, and organs (Nguyen et  al. 2017). Srivastava et al. (2004) showed that phages are translocated from blood to fetal tissue in a murine model. However, the main mechanism that phages use to bypass epithelial cell layers and gain access to the body remains unknown. Nguyen et al. (2017) used in vitro studies to demonstrate the rapid and directional transcytosis of various phages across confluent cell layers originating from the intestine, lung, liver, kidney, and brain. Recent reports have shown plenty of phages while analyzing human microbiomes, even more than eukaryotic viruses. The reports are based on skin, gut microbiota, lung, and ascitic fluid samples (Breitbart et al. 2003; Brown-Jaque et al. 2016; Oh et al. 2014; Virgin 2014). According to Górski et al. (2006), phages might be able to cross the intestine and pass to the peritoneal cavity. In addition, different lines of evidence have shown that phage lambda is capable of being internalized by mammalian cells, and that a mammalian gene expression cassette carried by phage lambda-based vectors can be expressed in target cells in vitro and in vivo (Lankes et al. 2007). The mechanisms through which phage lambda particles are taken up by mammalian cells are still unknown. However, different hypotheses for phage internalization have been  reported, including pathways such as phagocytosis, which could be increased by an antibody-dependent mechanism mediated by the Fc receptor, or macropinocytosis (Shimada et al. 1999). Recent studies demonstrated the ability of several phages to enter different eukaryotic cells (Timo et al. 2017; Nguyen et al. 2017). Timo et al. (2017) reported binding and penetration of E. coli bacteriophage PK1A2 into eukaryotic neuroblastoma cells in  vitro. This phage interacts with cell surface polysialic acid, which shares structural similarity with the phage receptor. The preceding data correspond to different phages, but what about Stx phages and mammalian cells? The ability of eukaryotic cells to recognize genetic sequences such as Stx2 promoters has been demonstrated (Bentancor et al. 2013a), as well as the ability to translate and express Stx2 toxin by mammalian cells. For this, cells were transfected with a plasmid construct containing the stx2 gene under its own promoter (pStx2) and cytotoxicity was observed, equivalent to incubating these cells with purified Stx2. Furthermore, in vivo studies were followed to expand these results. In the study carried out by Bentancor et al. (2013b), Balb/c mice were inoculated with the plasmid construct using the hydrodynamic inoculation procedure. Mice that received the plasmid by hydrodynamic inoculation died with typical signs of Stx2 intoxication (kidney damage, neutrophilia, and brain damage), and Stx2 toxin was detected in the brain by immunofluorescence, whereas mice immunized with a BLS-Stx2B chimera survived pStx2 inoculation. These results demonstrate the ability of host cells to express Stx in vivo, maintaining a toxic activity equivalent to the protein produced in the bacteria during infection. More studies were performed to assess the relevance of phage 933W in the development of HUS. Del Cogliano et al. (2018) showed that phage 933W in the absence of pathogenic bacterial factors produced intestinal, renal, and brain damage

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Bacterial environment

Eukaryotic environment

Lysogenic phase

Lytic phase

Fig. 6.1  Schematic representation of the role of the bacteriophage in HUS development, both in the bacterial and the eukaryotic environment. In the bacterial environment, phages are inside lysogenic bacteria, and there is a basal expression of Stx. Under stress conditions, for example, the intestinal environment, lytic phase is induced. At this moment, Stx and phage are released. Free bacteriophages can infect other susceptible bacteria in the gut. If the patient has susceptible bacteria, the expression of Stx and the phage replication are exacerbated, complicating the clinical picture. In the eukaryotic environment, cells can introduce free bacteriophage, or, in the macrophage case, they are able to internalize bacteria also. Once inside the cell, stx promoters are recognized and Stx is expressed keeping its biological activity. The mechanisms through which the bacteriophage DNA reached the core are still unknown. Anti-phage agents could prevent the infection of susceptible bacteria in the gut (reducing phage replication and stx expression) and prevent the internalization in eukaryotic cells (reducing stx expression). The schematic representation showing the capacity of free bacteriophage to infect susceptible bacteria in the gut could explain why only 10–100 CFU are enough to develop the syndrome

associated with HUS development. The absence of bacterial pathogenic factors in the E. coli C600 carrying Φ933W used in that work led to hypothesize that the damage observed could be associated with free phages infecting gut bacteria, which drove phage replication and Stx production. Moreover, those results evidence that it represents an excellent animal model useful to study phages as a therapeutic target. Figure 6.1 resumes the hypothesis about the role of the phage in HUS development. Studies focused on the phage as a new therapeutic target to prevent HUS are an alternative, innovative, and possible approach. If we search for compounds with antiphage activity, we can prevent the infection of commensal bacteria and inhibit stx expression by bacteria and host cells. Chitosan and cationic antimicrobial peptides have shown to have anti-phage activity (Amorim et  al. 2014; Del Cogliano et  al. 2018). Chitosan is a linear polysaccharide composed of randomly distributed chains

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of β-(1–4) D-glucosamine (deacetylated units) and N-acetyl-D-glucosamine (acetylated unit). It has many commercial and biomedical applications and is approved by the FDA (Food and Drug Administration) for use in humans. Strong antimicrobial activity of this compound against various microorganisms has been demonstrated (Kong et al. 2010). More recently, the anti-phage activity of chitosan has been demonstrated against phage c2, which infects strains of Lactococcus, and against phage MS2 with the capacity to infect strains of E. coli (Ly-Chatain et al. 2013). Therefore, chitosan was evaluated as an agent against phage 933W showing strong anti-phage effects, both under in vitro and in vivo conditions (Amorim et al. 2014). Because chitosan is a cationic compound and the mechanism by which it is capable of inactivating phage is unknown, other cationic agents were searched in the literature. Faccone et al. (2014) designed a group of novel cationic antimicrobial peptides (cAMPs) and tested them against a large panel of multiple-resistant clinical bacterial isolates. Five of these peptides were previously analyzed and showed antimicrobial activity on different bacterial strains and structure as an alpha helix in contact with lipid membranes (P5, P8, P8.1, P2 and P6.2). The other two peptides tested were Omiganan, a cAMP with a linear β-sheet structure derived from indolicidin (a heterocyclic chemical compound that forms the central nucleus of indolizidine alkaloids), which underwent clinical trials with activity against S. aureus; and a random sequence peptide (P. random) with cationic charge but no antimicrobial activity (Faccone et al. 2014; Hollmann et al. 2016). Due to its cationic property, a characteristic shared with chitosan, antimicrobial peptides were analyzed as possible anti-phage agents. Del Cogliano et  al. (2017) showed that cAMPs have strong anti-phage effects in vitro. However, more studies are necessary to advance in therapeutics approach with phage expressing Stx as a target.

6.4 Bacteriophages as Therapeutic Agents: Advantages and Disadvantages Phage therapy has existed since the description of the first phages. Phages were discovered by Frederick Twort in 1915 while he was trying to propagate vaccinia virus on agar plates and noted that contaminating bacteria grew but with the presence of glassy and transparent spots, where there was no bacterial growth. These cleared zones were transmissible to other cultures, as in the new cultures no growth was observed (Keen 2015; Salmond and Fineran 2015). Independent studies by Félix d’Hérelle described phages as “anti”-microbes that possess the ability to eliminate specifically one bacterial species without harming other types of cells. This description was derived from experiments, in which the recovery of patients with bacillary dysentery was achieved by administering orally a filtrate of the patient’s feces, with a recovery time of 48  h (Keen 2015). In the 1920s and 1930s phage research was focused on the development of phage therapy for the treatment of

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bacterial infections mainly in the extinct Soviet Union and other Eastern European countries (Salmond and Fineran 2015). However, in recent times, broad interest in the therapeutic use of phages has grown due to the increase in the rates of multi-drug-resistant bacterial infections (Kortright et al. 2019). There are some characteristics for phages to be chosen as biocontrol agents; among them, they must strictly follow a lytic cycle avoiding gene transduction and must not carry any virulence gene. The success of phage treatment will depend on choosing the phages with the best characteristics for the desired application (Hyman 2019). Phage therapy has several advantages when compared with the conventional antibiotic treatment and has become a promising alternative to eliminate multi-resistant bacteria. Some features that make phage useful over antibiotics are commented in the following paragraphs. Bactericidal agents:  Phages that follow the lytic cycle are capable of infecting and lysing bacteria; in this way the result of the infection will end in the death of bacteria, while some of the antibiotics only have a bacteriostatic effect, allowing the growth of the bacteria when antibiotic is withdrawn from treatment (Hatfull et al. 2022). Low doses:  When carrying out a successful infection, the consequence will be the destruction of the bacteria with the respective release of phages, which will grow exponentially in number; therefore, low doses would be enough to multiply the number of phages, allowing an increase in the effect of phage therapy (Luong et al. 2020b). In comparison, when using antibiotics, the doses must be high because some molecules lose effectiveness due to destruction or modification. Fast isolation and low cost:  Phages are widely distributed on earth, so their isolation is simple, not requiring the use of sophisticated methodologies. In addition, the cost to obtain a phage is not high compared to the research necessary to develop a new antibiotic which becomes more expensive due to the multiple stages that must be subjected to be finally marketed (Luong et al. 2020a). Despite showing many advantages over antibiotics, phage therapy has some disadvantages, for instance, narrow host range, the lysogenic phenomenon, the lack of relevant policies, and the lack of pharmacokinetic data (Lin et al. 2022). Some of the possible challenges and possibilities to overcome them are described in Table 6.1 (González-Villalobos et al. 2022). Table 6.1  Phage therapy: challenges and possibilities to overcome them Challenges Emergence of bacterial resistance to phage infection Biofilm establishment Side effects in microbiota

Possibilities Use of phage cocktails to ensure the lysis of all pathogenic bacteria Some depolymerases are encoded in a few phages Narrow host range of some phages

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6.5 Phage Biocontrol/Therapy Against Escherichia coli Pathotypes in Latin America 6.5.1 Phages Against Enteropathogenic E. coli Tomat et al. (2013) isolated phages from diarrheic samples of patients treated at the Centenary Hospital, Rosario, Argentina, with the goal of determining its host range and analyze their potential as biocontrol agents for EPEC strains in beef products. They evaluated the reductions of viable cells after exposure to two phages (DT1 and DT6) at different temperatures (5 and 24 °C) for 3, 6, and 24 h against EPEC strains. They found significant viable cell reductions, compared to controls without phages, at both temperatures, and mostly after 3 and 6 h of treatment. They showed that reductions were also influenced by phage concentration, being generally the higher concentration, 1.7 × 1010 (PFU/mL) for DT1 and 1.4 × 1010 PFU/mL for DT6, the most effective when tested against EPEC strains. Bourdin et al. (2014) studied 89 T4-like phages from their collection and tested them against four collections of childhood diarrhea-associated E. coli isolates, representing different geographical origins (Mexico versus Bangladesh) and various pathotypes (ETEC, EPEC, EIEC, EAEC, STEC). They also assayed different cocktails consisting of 3 to 14 T4-like phages and achieved 54% to 69% coverage against EPEC isolates from Mexico. They observed that the coverage of a given T4-like phage cocktail differed with the geographical area and epidemiological setting, and therefore, the composition of the phage cocktail must be adapted to the local situation for phage therapy approaches against E. coli pathogens.

6.5.2 Phages Against Shiga Toxin-Producing E. coli Phages DT1 and DT6 were also tested against two STEC strains by Tomat et al. (2013). They observed greater reductions at the higher temperature (24 °C), fact that could be explained by the active growth of bacteria which could allow an efficient bacteriophage replication. The biocontrol effect was found to be dose-dependent, with the highest phage concentration being the most effective. Although bacterial reductions obtained on meat were statistically significant, they were lower than expected with individual phages and with the two-phages cocktail. Consequently, in another study, these authors evaluated a cocktail of six lytic phages (including DT1 and DT6 phages) (Tomat et al. 2018). This phage cocktail proved to be effective, reducing levels of two STEC strains on food matrices (meat and milk) at refrigerated, room, and unsafe (37 °C) temperatures. Tomat et al. (2018) suggested that it may be used during cold storage of meat products, because STEC reductions increased as incubation was extended up to 6 days under refrigeration. As the six-­ phage cocktail was highly effective in both food matrices at 24 °C and 37 °C, they considered it could be also useful to prevent foodborne diseases especially when the cold chain is lost.

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Amarillas et  al. (2016) reported the complete genome sequence of phage phiE142, which can lyse Salmonella and multidrug-resistant (MDR) E. coli O157:H7 strains. The phage phiE142 belongs to the Myoviridae family and was isolated from animal feces collected on a farm in Northwestern Mexico, using an E. coli strain (EC-48) that was also isolated before from the same geographical region. The authors concluded that phiE142 is a member of T4-like virus genus of the Myoviridae family and the Tevenvirinae subfamily. The analysis of the genome revealed that this phage does not carry any genes coding for food-borne allergens, antibiotics resistance, virulence factors, nor genes associated with lysogenic conversion. These results and the broad host range of phiE142 make it a potential candidate as a biocontrol agent. Another phage that showed lytic activity against several MDR E. coli O157:H7 and Salmonella strains was isolated from horse feces in Mexico and characterized by Amarillas et al. (2021). This phage, named phiC120, is a member of Myoviridae family. Genes associated with lysogeny, virulence, toxins, and resistance to antibiotics were absent in the phiC120 genome. As one ORF was predicted to encode a long tail fiber assembly chaperone that showed potential allergenic and proinflammatory properties, toxicological testing is needed to guarantee the safety of this phage for human use. Interestingly, an ORF encoding a putative depolymerase was also detected, suggesting potential biotechnological applications as this type of enzyme can be useful to disrupt biofilms. Lopez et al. (2016) reported one phage, UFV-AREG1, isolated from cowshed wastewater, which belongs to Myoviridae family and showed lytic effect against EHEC O157:H7 (ATCC 43895), E. coli O111 (CDC O11ab), and E. coli ATCC 23229.  In another study, six phages belonging to the Myoviridae and 10 to the Podoviridae family were isolated from bovine minced meat and stool samples, and their host range was evaluated against STEC (Dini and De Urraza 2010). These authors concluded that phages CA91, CA933P, MFA933P, and MFA45D, which showed the highest resistance to acidic pH, were the best candidates for oral administration as therapeutic agents against STEC in cattle. On the other hand, Dini et al. (2016) assayed one phage and probiotics against STEC attachment to HEp-2 cells and cytotoxic effect in vitro. The treatment with phage alone increased cell detachment caused by STEC infection; the treatments with probiotics alone or in combination with phage proved to effectively diminish cell damage caused by STEC infection. Combined treatment showed a decrease in apoptotic cell count of 57.3% and a reduction in STEC adhesion to cell monolayer of 12 log CFU. This combination of phage and probiotics may be of great potential for reducing the number of pathogens adhered to epithelial cells during STEC O157:H7 infection and attenuating the cytotoxic effect derived from it. Four phages from cattle and poultry feces were isolated in Mexico and were analyzed for their ability to lyse Salmonella serotypes and STEC O157:H7 (López-­ Cuevas et al. 2011). All phages showed a lytic effect on STEC and lysed at least 70% of the 234 strains tested. Phages morphologies were consistent with members of the Myoviridae family. The wide host ranges of these phages suggested they

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could be used for simultaneous biocontrol of some Salmonella serotypes and STEC strains. In a study also performed in Mexico, Ramirez et al. (2018) isolated and characterized lytic phages against STEC O157:H7 strains with potential to be used as biocontrol agents in food. Their goal was to determine the physical stability and allergenic potential of free and microencapsulated (ME) phage cocktails against STEC. In vitro and in  vivo studies were performed to determine phage survival under different pH, temperatures, and UV light ranges. They found that the stability of ME phages was significantly higher than free phages after UV irradiation, a pH range from 3 to 7, and temperatures between −80 °C and 70 °C. The authors concluded that ME induced stability and assured a more suitable commercial formula for the biological control of E. coli O157:H7.

6.5.3 Phages Against Uropathogenic E. coli González-Villalobos et  al. (2021) described the isolation and characterization of three novel phages from wastewater samples, as well as their lytic activity against biofilm and adherence of uropathogenic E. coli (UPEC) to HEp-2 cells. The authors reported characteristics of three phages isolated from wastewater samples in Mexico City, considering the ability to eliminate bacteria adhered to HEp-2 cells and decrease biofilm formation, and their lytic activity against UPEC strains causing acute and persistent urinary tract infections (UTIs). Their results demonstrated that phage vB_EcoM-phiEc1 (ϕEc1) belongs to Myoviridae family, whereas vB_EcoS-­ phiEc3 (ϕEc3) and vB_EcoS-phiEc4 (ϕEc4) belong to Siphoviridae family. The phages showed lytic activity against UPEC and gut commensal strains. Phage ϕEc1 lysed UPEC serogroups, whereas phages ϕEc3 and ϕEc4 lysed only UTI strains with higher prevalence belonging to O25 serogroup. Moreover, phages ϕEc1 and ϕEc3 decreased both biofilm formation and adherence, whereas ϕEc4 was able to decrease adherence but not biofilm formation. In conclusion, these novel phages showed the ability to decrease bacterial adherence and biofilms, making them promising candidates for effective adjuvant treatment against UTIs caused by MDR UPEC strains. Another study, performed by Rodea et al. (2022), reported the genomic sequence and characterized the phages vB_EcoS-phiEc3 and vB_EcoS-phiEc4 that were shown to belong to the Kagunavirus genus, Guernseyvirinae subfamily and Siphoviridae family, respectively. Their lytic activity against MDR UPEC strains, as well as the absence of antibiotic resistance genes, lysogenic and virulence genes, enables vB_EcoS-phiEc3 and vB_EcoS-phiEc4 as a safe therapy option against UPEC infections. Vera-Mansilla et al. (2022) isolated and characterized three novel phages (MLP1, MLP2, and MLP3) from water samples from the Valdivia River in Chile. These phages belong to the Chaseviridae, Myoviridae, and Podoviridae families, respectively. They were able to infect and kill laboratory reference strains and MDR E. coli

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isolates from patients with UTIs, and were capable to infect intestinal pathogenic strains, like EAEC and DAEC. Their results suggest that these phages may represent an interesting alternative for the treatment of MDR E. coli UTI associated.

6.5.4 Phages Tested Against Biofilms Formed by E. coli Ribeiro et al. (2018) isolated the phage EcoM017 from sewage in Brazil and tested it against an E. coli strain isolated from a cow with mastitis. The highest phage titer used (109 PFU/ml) reduced bacterial growth and the quantity of biofilm formed by 90.0% and 87.5%, respectively. The minimum dose of EcoM017 capable of reducing biofilm formation of this bacterium was 101 PFU/mL after 24 h, but this low dose did not show an effect on mature biofilm. The authors indicated that EcoM017 phage was able to destroy biofilm architecture and penetrate to deeper layers. They proposed the use of this phage for the control of bovine mastitis. González-Gómez et  al. (2021) studied three phages, isolated from samples of ground beef and poultry liver in Mexico, to eliminate biofilms of E. coli strains isolated from the meat industry production environment. Phages were assayed individually and as a cocktail, at different developmental stages (2, 24, and 48 h) of E. coli biofilms developed on stainless steel surfaces. Biofilm reductions ranged from 0.95 log CFU/mL to 6.70 log CFU/mL after 1  h of treatment. The highest efficacy was obtained with 109 PFU/mL at 24 and 48  h of biofilm development. Noticeably, the individual phages showed reductions equal or greater than the cocktail in most treatments.

6.6 Conclusions Since the discovery of phages, their high potential to combat infections was shown; however, the discovery of antibiotics led to displacing phages from medical use. On the other hand, the emergence of MDR bacteria, which currently represents a major public health problem, renewed the interest in phage therapy which offers many advantages over antibiotics. However, the field of phages is not yet widely explored, so further studies are required to guarantee effective therapies with minimal risks.

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Oh J, Byrd AL, Deming C, Conlan S, Kong HH, Segre JA (2014) Biogeography and individuality shape function in the human skin metagenome. Nature 514:59–64 Pascal SB, Lopez JRL, Lucchesi PMA, Krüger A (2020) Subtypes of NanS-p Sialate O-Acetylesterase encoded by Stx2a bacteriophages. Proceedings 66(1):15 Ramirez K, Cazarez-Montoya C, Lopez-Moreno HS, Castro-del CN (2018) Bacteriophage cocktail for biocontrol of Escherichia coli O157:H7: stability and potential allergenicity study. PLoS One 13:e0195023 Ribeiro KVG, Ribeiro C, Sousa Dias R, Cardoso SA, Oliveira de Paula S, Cola Zanuncio J, Licursi de Oliveira L (2018) Bacteriophage isolated from sewage eliminates and prevents the establishment of Escherichia coli biofilm. Adv Pharm Bull 8:85–95 Rodea GE, Gonzalez-Villalobos E, Medina-Contreras O, Castelan-Sanchez H, Aguilar-Rodea P, Velazquez-Guadarrama N, Hernandez-Chiñas U, Eslava-Campos CA, Balcazar JL, Molina-­ Lopez J (2022) Genomic characterization of two bacteriophages (vB_EcoS-phiEc3 and vB_ EcoS-­phiEc4) belonging to the genus Kagunavirus with lytic activity against uropathogenic Escherichia coli. Microb Pathog 165:105494 Salmond GP, Fineran PC (2015) A century of the phage: past, present and future. Nat Rev Microbiol 13:777–786 Scheutz F, Teel LD, Beutin L et al (2012) Multicenter evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature. J Clin Microbiol 50(9):2951–2963 Schmidt H (2001) Shiga-toxin-converting bacteriophages. Res Microbiol 152:687–695 Serra-Moreno R, Jofre J, Muniesa M (2008) The CI repressors of Shiga toxin-converting prophages are involved in coinfection of Escherichia coli strains, which causes a down regulation in the production of Shiga toxin 2. J Bacteriol 190(13):4722–4735 Sharma S, Chatterjee S, Datta S, Prasad R, Dubey D, Prasad RK, Vairale MG (2017) Bacteriophages and its applications: an overview. Folia Microbiol 62:17–55 Shimada O, Ishikawa H, Tosaka-Shimada H, Atsumi S (1999) Exocytotic secretion of toxins from macrophages infected with Escherichia coli O157. Cell Struct Funct 24:247–253 Srivastava AS, Chauhan DP, Carrier E (2004) In utero detection of T7 phage after systemic administration to pregnant mice. BioTechniques 37:81–83 Tarr PI, Freedman SB (2022) Why antibiotics should not be used to treat Shiga toxin-producing Escherichia coli infections. Curr Opin Gastroenterol 38(1):30–38 Timo L, Pajunen M, Skog M, Finne J (2017) Internalization of a polysialic acid-binding Escherichia coli bacteriophage into eukaryotic neuroblastoma cells. Nat Commun 8(1):1915 Tomat D, Migliore L, Aquili V, Quiberoni A, Balagué C (2013) Phage biocontrol of enteropathogenic and Shiga toxin-producing Escherichia coli in meat products. Front Cell Infect Microbiol 3:1–10 Tomat D, Casabonne C, Aquili V, Balagué A, Quiberoni A (2018) Evaluation of a novel cocktail of six lytic bacteriophages against Shiga toxin-producing Escherichia coli in broth, milk, and meat. Food Microbiol 76:434–442 Torres AG, Amaral MM, Bentancor L et  al (2018) Recent advances in Shiga toxin-producing Escherichia coli research in Latin America. Microorganisms 6(4):100 Tyler JS, Mills MJ, Friedman DI (2004) The operator and early promoter region of the Shiga toxin type 2-encoding bacteriophage 933W and control of toxin expression. J Bacteriol 186(22):7670–7679 Tyler JS, Beeri K, Reynolds JL, Alteri CJ, Skinner KG, Friedman JH, Eaton KA, Friedman DI (2013) Prophage induction is enhanced and required for renal disease and lethality in an EHEC mouse model. PLoS Pathog 9:e1003236 Vera-Mansilla J, Sánchez P, Silva-Valenzuela CA, Molina-Quiroz RC (2022) Isolation and characterization of novel lytic phages infecting multidrug-resistant Escherichia coli. Microbial Spectr 10:e01678–e01621 Virgin HW (2014) The virome in mammalian physiology and disease. Cell 157:142–150 Wagner PL, Livny J, Neely MN, Acheson DW, Friedman DI, Waldor MK (2002) Bacteriophage control of Shiga toxin 1 production and release by Escherichia coli. Mol Microbiol 44:957–970

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Chapter 7

Insights into Animal Carriage and Pathogen Surveillance in Latin America: The Case of STEC and APEC Nicolás Galarce, Fernando Sánchez, Indira Kudva, Erika N. Biernbaum, Terezinha Knöbl, and André B. S. Saidenberg Chapter Summary  Shiga toxin-producing Escherichia coli (STEC) is a zoonotic diarrheagenic pathogen that can cause illness in humans and animals, and its circulating strains in the animal-human-environment interface exhibit great variability, where diverse animal species, mainly ruminants, play a fundamental role as reservoirs. Despite this impact, little is known in Latin America about the characteristics of the STEC strains circulating in animals. On the other hand, avian pathogenic E. coli (APEC) is an extraintestinal E. coli pathotype causative of avian colibacillosis. APEC has been traditionally considered as a secondary and opportunistic pathogen; however, some strains can act as primary disease agents and even with zoonotic potential. Considering the relevance of STEC and APEC for animal production and public health, we present here updated information on prevalence, genomic characteristics, and surveillance strategies in Latin American countries to provide state-ofthe-art information to improve the understanding of these p­ athogens under the One Health concept and thus contribute to their prevention and control. N. Galarce (*) Escuela de Medicina Veterinaria, Facultad de Ciencias de la Vida, Universidad Andrés Bello, Santiago, Chile e-mail: [email protected] F. Sánchez Departamento de Medicina Preventiva Animal, Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Santiago, Chile I. Kudva Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, USA E. N. Biernbaum Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, USA Oak Ridge Institute for Science and Education, Oak Ridge, TN, USA T. Knöbl · A. B. S. Saidenberg Department of Pathology, School of Veterinary Medicine and Animal Science – University of São Paulo (FMVZ-USP), São Paulo, SP, Brazil © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 A. G. Torres (ed.), Trending Topics in Escherichia coli Research, https://doi.org/10.1007/978-3-031-29882-0_7

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7.1 General Concepts Escherichia coli is the main facultative anaerobic bacterium in the intestinal tract of several species of animals, mainly mammals (Rivas et al. 2016). Most of its strains are commensal, but a small proportion are pathogenic, capable of producing intestinal and extraintestinal diseases. These pathogenic strains are classified into pathotypes according to the production of a wide variety of virulence factors, among others. The main pathotypes currently recognized with an impact on animal health are enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), avian pathogenic E. coli (APEC), and Shiga toxin-producing E. coli (STEC) (Kaper et al. 2004; Gyles and Fairbrother 2010).

7.2 General Concepts of Shiga Toxin-Producing E. coli (STEC) In Latin America, STEC infections remain endemic and have contributed to the burden of acute diarrheal syndrome in this region, with a considerable number of hemolytic uremic syndrome (HUS) cases mainly in Argentina, Chile, Uruguay, and Brazil (Torres et al. 2018; Castro et al. 2019; Cavalcanti et al. 2020). This could be explained by high beef consumption in those countries (OECD 2022), by the high cattle biomass in South America (FAO 2022), and by the presence of hypervirulent clones circulating in the cattle-beef-human interface (Pianciola and Rivas 2018). The pathogenicity of STEC is associated with the production of Shiga toxins (Stx), which are classified into Stx1 (of which there are four subtypes, Stx1a, Stx1c, Stx1d, and Stx1e) and Stx2 (including Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, Stx2g, Stx2h, Stx2i, Stx2j, Stx2k, and Stx2l) (Scheutz et al. 2012; EFSA BIOHAZ Panel et al. 2020; Biernbaum and Kudva 2022; Gill et al. 2022; Yang et al. 2022). Additionally, STEC may harbor other virulence factors that contribute to the development of severe illness in people. Among these is the eae gene, located in the locus of enterocyte effacement (LEE) pathogenicity island (PAI), which encodes intimin, a protein that mediates adhesion to enterocytes (Rivas et al. 2016). Although the presence of LEE increases STEC virulence, it is not essential for its pathogenicity, as a large number of LEE-negative strains have been associated with sporadic cases and outbreaks (Rivas et al. 2016; Smith et al. 2014). In these strains, other virulence factors have been described that allow them to generate illness in humans, such as the auto-agglutinating adhesin of STEC (Saa; Paton et al. 2001) and the PAI locus of adhesion and autoaggregation (LAA; Montero et al. 2017). In addition, STEC strains may possess several other virulence factors, such as long polar fimbriae (Lpf) associated with colonization of the intestine (Rivas et al. 2016) and the enterohemolysin A (EhxA; Amézquita-López et al. 2018).

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7.3 STEC in Animals Diarrhea and dysentery in neonatal calves, naturally or experimentally infected with STEC, have been demonstrated previously (Dean-Nystrom et al. 1997, 1999, 2008), with lesions in the terminal ileum, colon, and rectum, edema and neutrophil infiltration of the lamina propria, and exudate of neutrophils, mucus, and exfoliated epithelial cells in the lumen. Additionally, STEC strains of the Stx1c virotype have been described in diarrhea in sheep, while edema disease in swine has been associated almost exclusively with the Stx2e virotype (Gyles and Fairbrother 2010). Alongside its involvement in calf diarrhea, extraintestinal effects of STEC infection in cattle have been also described. For instance, several studies have demonstrated the presence of Gb3 (or CD77), the cellular receptor for Stx, on the surface of many B cells in mesenteric lymph nodes and on the surface of CD4+ and CD8α + T cells from these tissues and mucosal sites (Menge et al. 2001, 2006). In this sense, Menge (2020) points out that although Stx1 fails to induce apoptosis in bovine lymphocytes, it still may damage 28S rRNA, triggering a ribotoxic stress response, exerting a suppressive effect on bovine lymphocytes. Additionally, Fitzgerald et al. (2019) reported that Stx-induced damage of epithelial cells results both in alteration of local immune responses and in the renewal of intestinal epithelium. Moreover, the involvement of STEC in extraintestinal disease, such as bovine mastitis, has been described, with reported prevalence ranging from 10% to 66.7% (Galal et al. 2013; Turkyilmaz et al. 2017; Murinda et al. 2019). International reported prevalence of STEC in both diarrheic and non-diarrheic calves is highly variable ranging from 8% to 31% (Badouei et al. 2010; Ryu et al. 2020; Awad et al. 2020; Khawaskar et al. 2022). This high variability may be due to differences in ruminal development according to age, immune response, diet, cattle management, and even climate conditions (Cobbold and Desmarchelier 2000; Fernández et al. 2009).

7.4 STEC in Animals in Latin American Countries 7.4.1 Chile In Chile, there are few studies indicating the incidence of HUS, the last reported incidence rate being 3.4 cases per 100,000 children with a case fatality of 2.7% (Prado and Cavagnaro 2008). Likewise, few studies have been published in the last 10 years describing the carriage of STEC in animal species including cattle, its most common reservoir. These studies include Vidal et  al. (2012), where they investigated the frequency of intestinal carriage of STEC in 385 pigs and 759 steers, characterizing the virotype of the isolates (stx1/stx2, eae, ent, efa1, hlyA, lpf, iha, and saa) and the serogroup (O157/non-O157). STEC strains were isolated in 6% of pigs and 13.5% of cattle. STEC strains isolated from pigs did not present the virulence

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genes described in human clinical isolates; however, they were detected in strains isolated from cattle. In addition, by pulsed-field gel electrophoresis (PFGE), it was detected that no single circulating clones existed in both groups of animals. In the same context, Galarce et al. (2019) detected the presence of STEC in 17% of 300 stool samples collected from cattle at abattoirs and in 1% of 300 pig stool samples during 2017–2018. The detection of stx subtypes and other virulence genes showed 20 different virulotype profiles, with stx1a/ehxA/saa (29.6%, n = 16) as the most frequent, and only 1 strain corresponded to a clinically relevant serogroup (O111). Additionally, ​​PFGE analysis showed a high clonal diversity among isolated strains, describing 12 clades. This could suggest the presence of clones with the same virotype distributed in a low proportion in different farms, which could be due to husbandry practices or the fact that these animals may have had a common origin. On the other hand, strains isolated from pigs corresponded to samples obtained at different sampling times but from the same farm. These strains showed a high homology, which is not surprising because these strains harbored the same genes detected here, including the stx2e subtype. It should be mentioned that in Chile, despite E. coli being recognized as the main pathogen related to calf diarrhea (Paredes-­ Herbach 2011; Quiroga Ibarra 2012; Valenzuela Held 2014), most animal health laboratories do not conduct pathotype identification of the isolated E. coli strains; therefore, the true prevalence of STEC as etiological agent of calf diarrhea in this county remains unclear and probably underreported. More recently, Díaz et al. (2021) described the isolation rates and genomic features of STEC strains isolated from cattle in Central and Southern Chile. Thus, from 446 stool samples, they registered the presence of 56 STEC strains (12.6%) of which 30 non-O157 isolates were subjected to whole-genome sequencing. Serotypes O116:H21 ST58 and O168:H8 ST718 were the most frequently detected (13.3% each) and lpfA, gad, and stx2 the most detected virulence genes (100%, 90%, and 90%, respectively), while stx1 and eae were detected in much lower rates (26.7% and 13%, respectively). Additionally, the isolates were clustered based on serotypes rather than geographical origin, indicating the wide diversity of STEC strains colonizing Chilean cattle. Similarly, Galarce et al. (2021) analyzed the whole-genome sequences of 130 STEC strains isolated from the cattle-beef-human interface in Latin America, including 51 Chilean strains of bovine origin, to address the presence of 14 different adhesin-encoding genes. Most of Chilean strains harbored lpfA (100%), followed by ehaA (96.1%), iha (86.3%), saa (52.9%), and tia (45.1%), with 43.1% harboring LAA. Thus, 37 virotypes were found, with stx1a/stx2a/ehaA/ ehxA/hlyA/iha/lpfA/saa, stx2a/ehaA/ehxA/hlyA/hra/iha/lpfA/saa/subA/tia/hes/ pagC-like, stx2a/ehaA/ehxA/hlyA/iha/lpfA/saa/sab/subA, and stx2d/ehaA/iha/lpfA as the most frequent (5.9% each). Sequence types (STs) more reported corresponded to 443 and 297 (9.8% each) and 332 and 58 (7.8% each) while O178:H19 11.8%, O130:H11 9.8%, and O116:H21, O171:H2, and O183:H18 7.8% each. These results showed the wide variety of STs, serotypes, and adhesin-encoding genes harbored by STEC strains and highlighted the need for continuous monitoring and surveillance of STEC strains focusing on their genomic characteristics, including their virulence profiles.

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In the context of STEC in companion animals, the only study available is by Galarce et al. (2019), where 300 stool samples from dogs and 300 stool samples from cats were analyzed without detecting STEC in any of them. In relation to the detection of STEC in wild animals, González (2013) detected a 4% prevalence in fecal samples collected from pinnipeds belonging to a rescue center, while Marchant et al. (2016) evaluated the presence of STEC in 316 zoo animals and determined a prevalence of 4.4% among species of the order Artiodactyla. Despite the detection rates indicated above and that STEC strains circulating in animals in Chile harbor virulence and determinants of animal and human health concern current official surveillance for this pathogen is only conducted in beef and beef-derived products for export (ACHIPIA 2018).

7.4.2 Argentina Argentina is the country with the highest rates of HUS, with approximately 400 cases a year from 2002 to 2011, with an incidence ranging from 10 to 17 cases per 100,000 children under 5 years of age and a case fatality rate of 1–4% (Rivas et al. 2015). In this country, the incidence is ten times higher than in any other industrialized country, which may be due to beef being a traditional component of the diet in Argentina; ground beef, present in the diet of Argentinean children, was mainly associated with about 10% of the median number of HUS cases per year in children under 15 years of age (Rivas et al. 2015; Brusa et al. 2020). For these reasons, extensive research has been published on the characterization of STEC strains in the animals from this country. In this context, Fernández et al. (2012) characterized the virotype and serotype of STEC strains isolated from newborn, milk-fed, and growing calves. For this purpose, the researchers obtained 808 fecal samples, 378 from newborn calves, 252 from milk-fed animals, and 178 from growing calves. Of these samples, 38% were STEC-positive, from 25%, 43%, and 58% in newborn, milk-fed, and growing calves, respectively. The researchers explained these differences by certain husbandry practices and the presence of other domestic animals and insects that could influence the excretion of STEC. The detection rate in newborn calves also suggests that these animals are exposed to this bacterium quickly after birth and play a key role in vertical transmission of STEC. From the stx-positive samples, 148 STEC isolates were obtained: 26 (17.6%) from newborn, 46 (31.1%) from milk-fed, and 76 (51.4%) from growing dairy calves. Seventy-seven percent of STEC isolates from newborn calves carried stx2, 25% carried stx1, 81% carried ehxA, 65% carried eae, and 11% carried saa. In milk-fed calves, 46% of STEC isolates carried stx2, 37% carried stx1, and 17% carried stx1/stx2. In growing calves, 81% of the STEC isolates carried stx2, 8% carried stx1, and 10% carried stx1/stx2. Additionally, in newborns, stx2/eae/ehxA (58%) was the main virotype, whereas stx1/eae/ehxA (37%) and stx2 alone were the most frequently detected virotypes in milk-fed and growing calves, respectively. Regarding serotypes, a wide variety was detected from

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the three groups of animals, including O157 and non-O157. Noteworthy was that only two O157:H7 strains were isolated (one from milk-fed calves and one from growing animals) despite this serotype being responsible for most human clinical cases in Argentina. More recently, Okuno et al. (2021) investigated the prevalence of STEC among beef cattle from rural areas and analyzed STEC isolates for serogroup and virulence genes. STEC prevalence of 15.9% was reported following evaluation of rectal swabs from 283 beef cattle, selected from 31 different farms. Serogroups most detected corresponded to O145, O8, O171, and O185 (8.8% each), while stx2 (75.5%) was the most common stx subtype, followed by stx1/stx2 (17.8%) and stx1 (6.7%). Additionally, the detected virulence genes included iha (82%), eha and lpfA (78% each), efa (53%), saa (22%), eae (16%), and hlyA (2.2%). Regarding the surveillance of emerging non-O157 STEC serotypes, Miko et al. (2014) conducted the molecular characterization of stx-genotypes and 43 virulence genes in a collection of 39 STEC O178:H19 strains isolated from cattle in Argentina, an emerging serotype involved in human illness. Most of these strains harbored stx2c subtype (51.3%), followed by stx1a/stx2a (41%) and virulence genes espP (100%), iha (100%), lpfA (100%), pagC (61.5%), exhA (48.7%), and saa (46.2%). Strains were almost equally distributed in two clusters according to PFGE, where cluster A contained strains with virulence associated with disease in humans (48.7%), while cluster B contained strains with low virulence attributable to their reduced repertoire of virulence genes (51.3%). The authors concluded that these strains present risk to public health and hence should also be considered in STEC detection panels. In the same way, Krüger et al. (2015) characterized the stx subtypes, virulence genes, and MLVA of 21 STEC O26:H11 strains isolated from cows and calves. Most strains harbored stx2 alone (61.9%), followed by stx1 (33.3%) and stx1/stx2 (4.8%). All strains contained the virulence genes eae-b, tir, efa, iha, espB, cif, espA, espF, espJ, nleA, nleB, nleC, and iss. On the other hand, toxB and espI genes were exclusively observed in stx2-positive isolates, whereas katP was only found in stx1a-positive isolates. MVLA revealed the presence of three clusters, differentiated by the type of stx and accessory virulence genes present. More recently, Colello et al. (2019) characterized the subtypes of iha (encoding for the IrgA homologue adhesin) in nine LEE-negative STEC strains isolated from cattle. Most strains belonged to O91:H21 serotype (55.6%), and 88.9% harbored an iha subtype, of which 77.8% were associated with the presence of LAA and pO113, 11.1% were only associated with LAA, and 11.1% did not harbor any subtype. These results show that LEE-negative STEC strains frequently possess one or two iha genes located in mobile elements and that further studies are needed to reveal the actual role of Iha in STEC pathogenesis. Studies on STEC have also been conducted in Argentina on other animal species, used both for food production and as pets. In this context, Rumi et al. (2012) conducted a microbiological study of a dog and a cat living in the same household as an 11-year-old boy diagnosed with HUS resulting from STEC O157:H7 infection. Rectal swabs from both animals were first collected during the same week of HUS diagnosis and then weekly for 4 weeks. Thus, from the first sample obtained from the cat, a STEC O145:NM strain was isolated, with a stx2/eae/ehxA virotype. No

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STEC strains were isolated from subsequent samples obtained from the cat. In the case of the dog, at the third sampling, a STEC O178:H19 was isolated, stx2-­positive, and eae-, ehxA-, and saa-negative. No STEC isolates could be isolated in successive samplings. Although these strains differed antigenically from that from the child, both strains (the one from the child and the one from the cat) harbored stx2, eae, and ehxA virulence genes. These authors concluded that virulent STEC strains may be harbored by asymptomatic household dogs and cats and that more studies are needed to establish companion animals as a source of infection or accidental carriers of STEC in the epidemiological cycle of infections. On the other hand, Colello et  al. (2016) characterized the stx types (including stx2e subtype), serotype and virotype of STEC strains isolated from the pork production chain. In this study, STEC was isolated from 5.8% of the 277 rectal swabs that were collected from pigs. Among these strains, 37.5% carried stx1/stx2, 37.5% harbored stx2e, and 25% carried stx2. Serotypes more frequently detected corresponded to O8:H9 (25%) and O2:H32 (18.8%), while stx2e/agn43 (31.3%) and stx2/agn43 (25%) were the most detected virotypes. These results show that the presence of virulent STEC strains in food-producing animals pose not only a public health problem but also a threat for pork production with the presence of additional strains carrying stx2e. However, like the Chilean situation, official surveillance is only conducted in meat products for domestic consumption and export, but not on food-producing animals (Conicet 2016).

7.4.3 Brazil In Brazil, there are very few studies that indicate the incidence of HUS, and only the cases reported in the Sistema de Informação de Agravos de Notificação (SINAN) between the years 2009 and 2018 are available, where 112 cases of HUS were reported and, of these, 59 cases (52%) were confirmed (SVS 2021; MS 2022). Despite the existence of this reporting system and local laws in each state on the detection of STEC in food, it is possible that there is an underreporting of human cases due to the absence of a centralized and mandatory reporting system and the lack of a surveillance system for diarrheal E. coli strains (Castro et  al. 2019). Although low incidence rates of HUS are described, in the last decade, several studies have described a high carriage of STEC in cattle, as well as in new possible wildlife reservoirs. In this context, Andrade et al. (2012) identified virulence factors in E. coli strains isolated from 54 calves up to 60 days old in 12 dairy farms located in three regions of Minas Gerais. Of these, six presented liquid feces and were considered to have diarrhea. The prevalence of STEC was 57.1%. Different percentages of STEC-­ positive animals were found between diarrheic (66.7%) and non-diarrheic (37.5%) calves. Of the 33 STEC isolates from healthy calves, 32 were positive for stx1 and 1 for stx2. Of the ten isolates from five diarrheic calves, seven (70%) were positive for the stx1 gene. Also, an atypical association of stx1 and enterotoxin STa was

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detected in one isolate from a non-diarrheic calf, probably due to easy transmission through a plasmid in the case of enterotoxin or by a phage in the case of stx1. LEE-­ positive STEC were also detected in 20% of the isolates from healthy calves, and the eae gene was found to be associated only with stx1. No serotyping or further characterization of these strains was performed, but early carriage in young animals of strains that can cause disease in humans was demonstrated. In the same way, Freitas et al. (2014) collected rectal swabs from 31 calves (younger than 11 months old) and 21 cows (over 24 months old) from 21 farms located in different cities in the Southwestern State of Brazil, to detect and molecularly characterize O157:H7 isolates. A total of 260 colonies were isolated from these healthy animals, and 126 were STEC-positive. The prevalence of O157:H7  in cows was 4.8% (1/21), 0% (0/31) in calves and 1.9% (1/52) in all animals studied. The on-farm prevalence was 4.76% (1/21). One of the O157:H7 isolates was considered highly virulent, since it tested positive for the presence of stx2, eae, ehxA, saa, cnf1, and chuA, representing a potential risk to humans. In another study, Gonzalez et al. (2016) isolated STEC strains from fecal samples obtained from healthy cattle from 47 dairy farms in the State of Rio de Janeiro. From 1562 stx-positive fecal samples, 105 STEC strains were isolated, of which 50 (47.6%) strains belonged to 9 serotypes (O8:H19, O22:H8, O22:H16, O74:H42, O113:H21, O141:H21, O157:H7, O171:H2, and ONT:H21). The most frequent serotypes were O157:H7 (12.4%), O113:H21 (6.7%), and O8:H19 (5.7%). Regarding virulence genes, ehlyA (77.1%) was the most prevalent, followed by espP (64.8%), saa (39%), eae (24.8%), and astA (21.9%). All O157:H7 strains carried the gamma variant of the eae gene and the stx2c gene, while stx1/stx2 was prevalent among LEE-negative strains. A high number of serotypes described in human disease were detected. More recently, Arrais et al. (2021) analyzed 106 STEC isolates from the collection of the “Laboratório de Microbiologia Veterinária” of the “Universidade Federal de Jataí.” These were isolated from cattle (n = 59) and from healthy sheep (n = 47). In cattle, the stx1a/stx1c subtypes (63.2%) predominated over the stx1a subtype alone (36.8%) and for stx2, stx2a/stx2c (57.6%). In sheep, the stx1a/stx1c subtypes (56.1%), and the stx2b subtype (73.9%) were mostly detected. In the context of other animal reservoirs, Martins et al. (2015) isolated 70 STEC strains from 65 (50%) of 130 sheep sampled without diarrhea from southern Brazil. The most frequent serotype was O76:H19, followed by O65:H- and O75:H-. None of the STEC isolates belonged to serogroup O157. The majority of the STEC isolates (52.8%) possessed only the stx1 gene, while 32.9% carried both stx1 and stx2, and 14.3% harbored only stx2. Four of the STEC isolates (5.7%) were LEE-positive. In the case of companion animals, Puño-Sarmiento et al. (2013) did not detect any STEC strains from 50 dog and 50 cat stool samples. Conversely, Coura et al. (2018) detected two STEC strains from 154 dog stool samples. Both strains were positive for stx2, and one of these was subtyped as stx2e (isolated from a dog with diarrhea). Due to the proximity between wildlife animals and the community, as well as the substantial number of ecological parks located near or within urban centers, the investigation of these animals as carriers of zoonotic microorganisms is essential for surveillance and the development of strategies to mitigate human infection. In this

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context, Borges et al. (2017) sampled 123 free-ranging wild birds that were treated at the Wildlife Veterinary Hospital of UNESP/FCAV. Two STEC strains (0.8%) were isolated, and one of these was isolated from a specimen of Cariama cristata, with the virotype stx2/eae/saa/lpfAO113/nleE, belonging to serogroup O6 and ST2101. The other STEC strain was isolated from Rupornis magnirostris, with the virotype stx2/eae/exhA/iha/saa/lpfAO113/nleE, belonging to serogroup O48 and ST1423. In another study by Sanches et al. (2017), examining a total of 516 fecal samples isolated from captive birds belonging to 10 orders (including 70 species), only 3 STEC strains were isolated, carrying the stx2 gene. Subsequently, de Oliveira et al. (2018) registered the presence of three STEC strains from Columba livia domestica, which carried the stx2f/eae genes. More recently, Merker Breyer et  al. (2022) collected fecal samples from 21 capybaras (Hydrochoerus hydrochaeris) in an urban area of the City of Rio Branco, within the Brazilian Amazon region. Of these samples, 14 contained the stx1 gene (66.7%), 8 had stx2 (38.1%) and 5 had both genes (23.8%). Only one LEE-positive STEC strain was detected with the tir gene. These studies demonstrate the carriage of STEC outside its main reservoir, with the ability to cause serious illness in people, hence emphasizing the importance of monitoring it. Table 7.1 summarizes the principal STEC studies included in this review.

7.5 Foodborne Infection Surveillance in Latin America In the United States, all foodborne human infections became nationally notifiable since 1995 and 2000, respectively (Rivas et  al. 2014). In 1995, “FoodNet” was established by the Centers for Disease Control and Prevention (CDC) in collaboration with the USDA-FSIS, the Food and Drug Administration (FDA), and ten state health departments, as a surveillance network for cases of STEC and other foodborne diseases. FoodNet works in coordination with “PulseNet,” coordinated by the CDC and a network of North American laboratories, which uses strain-typing (PFGE) to identify case clusters and common source for outbreaks. Similar networks exist around the world besides networks of national reference laboratories that work in concert to detect and monitor foodborne infections (Rivas et al. 2014; Caprioli et al. 2014). In Latin America, outbreaks are typically first reported to the country’s Ministry of Health (MOH) (Fig. 7.1). The MOH is responsible for outbreak data collection, which are compiled under the Regional Information Surveillance System (SIRVETA) (Pires et al. 2012). Since the 1990s, the Pan American Health Organization (PAHO; www.paho.org) and the World Health Organization (WHO; www.who.int) have partnered with countries in Latin America to build disease surveillance. PulseNet Latin America and Caribbean (PNLAC), hosted by PAHO, was established around 2003–2004 to strengthen regional and national foodborne disease surveillance and communication between countries in the network (Fig. 7.2) (Chinen et al. 2019). PNLAC characterizes foodborne outbreak strains via PFGE analysis and is currently working toward incorporating whole-genome sequencing as a means of

Country Chile

stx1, stx2, stx1/stx2

lpfA, gad, and stx2

No data

O116:H21, O168:H8

stx2e/saa/lpfA

No data

12.6%

Cattle

O111

stx1a/ehxA/saa

Pinnipeds (Mirounga 4% leonina) Wild species of 4.4% Artiodactyla order

1%

Pigs

Non-O157

stx2

stx1a/stx2a/ehaA/ehxA/hlyA/iha/lpfA/saa, stx2a/ehaA/ehxA/ hlyA/hra/iha/lpfA/saa/subA/tia/hes/pagC-like, stx2a/ehaA/ ehxA/hlyA/iha/lpfA/saa/sab/subA and stx2d/ehaA/iha/lpfA sxt1

17%

Cattle

No data

Most detected virulence genes or virulotypes stx1/stx2, stx2 lpf, efa1, iha, and saa

O178:H19, O130:H11, O116:H21, O171:H2, O183:H18

6%

Pigs

More frequently detected STEC serotypes or of relevance No data

Cattle

Isolation rate 13.5%

Source of isolation Cattle

Table 7.1  Summary of STEC isolation by country and its source, isolation rates, serotypes, and virulence properties reported

González (2013) Marchant et al. (2016)

Reference Vidal et al. (2012) Vidal et al. (2012) Galarce et al. (2019) Galarce et al. (2019) Díaz et al. (2021) Galarce et al. (2021)

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–

Cat

O145:NM

O91:H21

O26:H11

O8:H9 and O2:H32

–

Cattle

5.8%

–

Cattle

O178:H19

Pigs

–

Cattle

O145, O8, O171, and O185

O178:H19

15.9%

Cattle

More frequently detected STEC serotypes or of relevance O157 and non-O157

Dog

Isolation rate 38%

Country Source of isolation Argentina Calves

stx2e/agn43 and stx2/agn43

stx2

stx2/eae/ehxA

stx2, stx1, stx1/stx2, eae-b, tir, efa, iha, espB, cif, espA, espF, espJ, nleA, nleB, nleC, and iss stx1, stx2, iha

stx2c, stx1a/stx2a, espP, iha, lpfA, pagC, exhA, and saa

stx2, stx1/stx2, stx1, iha, eha, lpfA, efa, saa, eae, and hlyA

Most detected virulence genes or virulotypes stx2/eae/ehxA, stx1/eae/ehxA, and stx2

(continued)

Reference Fernández et al. (2012) Okuno et al. (2021) Miko et al. (2014) Krüger et al. (2015) Colello et al. (2019) Rumi et al. (2012) Rumi et al. (2012) Colello et al. (2016)

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Country Brazil

O6 and O48

No data No data

No data

55.7% 44.3% 50%

1.3%

0.8%

0.6%

1.9%

81%

Cattle Sheep Sheep

Dog

Wildlife birds (Cariama cristata and Rupornis magnirostris) Captive birds

Free-ranging synanthropic birds (Columba livia domestica) Capybaras (Hydrochoerus hydrochaeris)

No data

6.7%

Cattle

O8:H19, O22:H8, O22:H16, O74:H42, O113:H21, O141:H21, O157:H7, O171:H2, and ONT:H21 No data No data O76:H19, O65:H- and O75:H-

O157:H7

48.5%

Cattle

More frequently detected STEC serotypes or of relevance No data

Isolation rate 57.1%

Source of isolation Cattle

Table 7.1 (continued)

Arrais et al. (2021)

Reference Andrade et al. (2012) Freitas et al. (2014) Gonzalez et al. (2016)

stx1, stx2, stx1/stx2, and stx1/tir

stx2f/eae

stx2

Merker Breyer et al. (2022)

Sanches et al. (2017) de Oliveira et al. (2018)

Martins et al. (2015) stx2 and stx2e Coura et al. (2018) stx2/eae/saa/lpfA O113/nleE, and stx2/eae/ehxA/iha/saa/lpfA Borges O113/nleE et al. (2017)

stx1a/stx1c, stx1a, and stx2a/stx2c stx1a/stx1c and stx2b stx1, stx2, and eae

stx1, stx2, stx2c, E-hlyA, espP, saa, eae, and astA

stx2/eae/ehxA/saa/cnf1/chuA

Most detected virulence genes or virulotypes stx1, stx2, stx1/STa, and stx1/eae

160 N. Galarce et al.

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Ministries of Health for each country responsible for outbreak data collection

Outbreak occurs in Latin America

Pan-American Health and World Health Organizations collaborate with member states to build foodborne disease surveillance

Partners with the CDC to establish Field Epidemiology Training Programs

161

Data compiled under the Regional Information System on foodborne diseases surveillance (SIRVETA) PulseNet Latin America and Caribbean (PNLAC) performs PFGE and analysis

Communicates between countries within the network

Improves disease surveillance and outbreak detection

Fig. 7.1  General surveillance in Latin America

further supporting outbreak investigations. Certain countries like Brazil have also partnered with the CDC to establish field epidemiology training programs to improve outbreak detection and disease surveillance (Fig. 7.1) (CDC 2014). In Chile, outbreak surveillance first occurs at the local level. Regional health authorities report cases to the Regional Health Ministerial Secretariats (SEREMI) and conduct interviews (Fig. 7.2) (Ministerio de Salud de Chile 2016). The regional delegate of epidemiology then conducts the initial investigation and notifies SEREMI of all outbreak cases. The SEREMI Epidemiology Unit reports data to official notification systems like MINSAL (Ministerio de Salud) and RAKIN (Sistema de Información y de Apoyo a la Gestión de las SEREMIS de Salud) and coordinates outbreak investigations. If this is a foodborne outbreak, the Health SEREMI Food Unit is responsible for collecting samples for testing, implements control measures, and disseminates information to regional and central authorities (Ministerio de Salud de Chile 2016). At the central level, the Department of Epidemiology-Department of Nutrition and Food monitors outbreak surveillance, prepares bulletins and reports, and coordinates with the Public Health Institute (ISP, Instituto de Salud Pública) Biomedical Laboratory and ISP Environmental Health Department. The ISP Biomedical Laboratory is responsible for confirming, characterizing, and reporting clinical outbreak samples, while the ISP Department of Environmental Health is responsible for confirming and reporting food and water samples (Fig. 7.2) (Ministerio de Salud de Chile 2016). Outbreak cases in Argentina are reported to the Ministry of Health MOH  of Argentina by regional health authorities (Fig. 7.2) (Ministerio de Salud de Argentina 2007). The MOH of Argentina conducts outbreak investigations and contact tracing, in partnership with the PAHO/WHO and CDC if needed. Testing is oftentimes first performed at local health laboratories and additional testing at the National Reference Laboratory (ANLIS; Administración Nacional de Laboratorios e Institutos de Salud). Public health measures are put into place, and the MOH reports information to the PAHO/WHO. Local, jurisdictional, and national levels all play a role in outbreak response and reporting (Ministerio de Salud de Argentina 2007).

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Outbreak occurs in Chile

N. Galarce et al.

Doctors report cases to the SEREMI and conduct interviews

Delegate of epidemiology conducts initial investigation and notifies SEREMI of all cases

SEREMI Epidemiology Unit reports outbreak data to RAKIN and official notification systems (MINSAL)

Health SEREMI Food Unit supervises associated facilities if food-related

Reports information in RAKIN and sends samples to environmental lab or ISP

Applies control measures and disseminates information to regional and central authorities

Department of EpidemiologyDepartment of Nutrition and Food (Central level)

Monitors surveillance and recommends control measures

Prepares bulletins and periodic reports

Coordinates with the ISP Biomedical Lab and Environmental Health Dept. and summons national Rapid Response Team if needed

ISP Biomedical Laboratory confirms clinical samples and characterizes outbreak strains

Report results of circulating strains and trains labs within the network

ISP Department of Environmental Health analyzes food and water samples

Reports results and trains Public Health labs

Report cases to the Ministry of Health (MOH) of Argentina

MOH conducts traceback and outbreak investigations with the help of PAHO/WHO/CDC, if needed

Lab tests conducted by local public health labs and the National Reference Laboratory (ANLIS)

Implements public health measures and report to PAHO/WHO

Local level

Reports cases to authorities, monitors and analyzes primary data, and alerts public

Initiates control actions, requests support at jurisdictional level if needed

Participates in surveillance training programs

Jurisdictional level

Coordinates surveillance, conducts investigations, and disseminates information

Promotes training involved in SINAVE, coordinates intervention strategies, and submits reports to superior levels

National level

Promotes training with Reference Institutions

Analyzes and disseminates provincial investigation data

Informs international organizations of outbreak information

Monitored by the Health Surveillance Secretariat

Participates with the Network of Strategic Information and Response Centers in Health Surveillance (Rede-Cievs)

Regional health facilities report cases to the Notifiable Diseases Information System (SINAN) or the VE-DTA of the MOH of Brazil

Regional epidemiology surveillance agencies investigate outbreaks

Municipal Secretariat notifies the State Health Secretariat

MOH partners with CDC for training and digital disease surveillance

Health Emergency Operations Center (COE) coordinates response

Disseminates information among SUS

Outbreak occurs in Argentina

Outbreak occurs in Brazil

Coordinates investigations at the regional level and disseminates information at the local, regional, and central levels

Fig. 7.2  Outbreak surveillance in Chile, Argentina, and Brazil

Local authorities are primarily responsible for notifying authorities of outbreak cases, analyzing primary data, initiating control measures, and partaking in surveillance training programs. At the jurisdictional level, authorities coordinate

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surveillance activities, conduct investigations, disseminate information and reports, and promote training in the National Epidemiological Surveillance System (SINAVE; Sistema Nacional de Vigilancia Epidemiológica). The national level updates the epidemiological surveillance standards as needed, promotes training with reference institutions, analyzes outbreak data reported from provincial health authorities, advises the public, and reports data to international organizations (Fig. 7.2) (Ministerio de Salud de Argentina 2007). Public health in Brazil is monitored by the Health Surveillance Secretariat in partnership with the Network of Strategic Information and Response Centers in Health Surveillance (Rede Cievs) (Fig.  7.2) (Ministério da Saúde Brasil 2022b). The Health Emergency Operations Center (COE) is responsible for coordinating actions in response to public health emergencies and disseminating information via the Unified Health System (SUS) (Ministério da Saúde Brasil 2022a). Foodborne outbreaks in Brazil increased in the early 2000s, and the National Epidemiological Surveillance System for Foodborne Diseases (VE-DTA; Sistema Nacional de Vigilância Epidemiológica das Doenças Transmitidas por Alimentos) was since developed to do reporting (Draeger et al. 2018). The VE-DTA is under the Health Surveillance Office of the MOH of Brazil. Each regional surveillance agency is responsible for conducting outbreak investigations and reporting data via VE-DTA, as well as notifying the State Health Secretariat. Cases can also be reported to the Notifiable Diseases Information System (SINAN; Sistema de Informação de Agravos de Notificação) (Fig. 7.2) (Rocha et al. 2020).

7.6 Avian Pathogenic Escherichia coli (APEC) APEC is a phylogenetically distinct pathotype from nonpathogenic commensal E. coli and diarrheagenic E. coli, and is part of the extraintestinal pathogenic E. coli (ExPEC) group, together with the uropathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC) pathotypes (Kaper et  al. 2004; Riley 2020). ExPEC possesses many virulence factors associated with biofilm formation, adherence and colonization of host tissues, iron uptake, serum survival, toxin production, and those facilitating persistent infection. Some strains also carry genetic determinants that encode antimicrobial resistance, often multidrug resistance (MDR, Nakazato et al. 2009; Cordoni et al. 2016; Lestrangea et al. 2017; Kim et al. 2020). APEC strains can be identified through serotyping and/or genotyping. However, proving that an isolate is pathogenic requires embryo mortality testing which is now rarely performed for ethical reasons. Serogroups O2, O78, and O1 represent 80% of the global frequency of APEC (Ferreira and Knöbl 2020). This prevalence may show regional variations (Wang et al. 2022). Currently, epidemiological studies based on the presence of the somatic antigen have become scarce, due to the difficulty to produce and standardize sera for specific diagnosis. Rapid sero-agglutination methods were replaced by molecular techniques, with the amplification of virulence

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genes in the late 1990s and more recently by bacterial genome sequencing techniques. The ability of APEC to cause disease is correlated with the presence of several virulence factors (Ewers et al. 2007; Johnson et al. 2008; Sarowska et al. 2019). A wide range of virulence genes have been associated with virulence in APEC, particularly the gene markers hlyF, iucD, iutA, ompT, papC, tsh, iroN, cva/cvi, and iss (Ewers et  al. 2005; Johnson et  al. 2008; Knöbl et  al. 2012; Cunha et  al. 2014). Currently, one of the most widely used protocols to classify APEC isolates is to assess for the minimal factors predicting virulence (hlyF, iss, iroN, iutA, and ompT) through a pentaplex PCR reaction described by Johnson et al. (2008). APEC generally carries large hybrid conjugative IncFII-/IncFIB-type plasmids (Johnson et al. 2006), which encode colicin V. However, the presence of the ColV plasmid itself is not unique to APEC and may be present in other ExPEC pathotypes. A new diagnostic study combining the identification of virulence factors, serogroups, and sequence types has been proposed by Johnson et al. (2022). The variety of APEC virulence factors and the overlapping of ExPEC markers represent difficulties in the study of this pathotype. Another challenge for the diagnosis is that, in addition to the primary cases, there are also outbreaks of opportunistic colibacillosis caused by non-virulent strains and linked to immunosuppression, stress, poor hygiene, husbandry, and biosecurity failures (Christensen et al. 2021). For several years, avian colibacillosis was considered a secondary and opportunistic disease, related to the concomitant infection by Mycoplasma spp. and infectious bronchitis virus, being aggravated by the addition of environmental factors, such as dust, ammonia, and sudden temperature changes (Ferreira and Knöbl 2009). There are no doubts about these factors contributing to the development of the disease. However, current whole-genome sequencing technologies reveal that some strains of APEC can act as primary disease agents, causing outbreaks with greater risk potential to the avian health (Cunha et al. 2017; Mehat et al. 2021; Saidenberg et al. 2022). Avian colibacillosis is one of the diseases with greatest economic impact on broiler breeding (Ferreira and Knöbl 2020). Disease control can be difficult due to the ease of horizontal transmission as well as the increasing rates of antimicrobial resistance posing therapeutic challenges (Cunha et al. 2017). APECs may harbor resistance genes to several antibiotics, disinfectants, and heavy metal compounds in addition to virulence determinants (Cunha et al. 2014, 2017; Kathayat et al. 2021). Some APEC can also be transmitted by food products, representing greater public health risk (Mehat et  al. 2021). The genetic similarity between APEC and other ExPEC pathotypes, in particular UPEC, suggests zoonotic risks (Ewers et al. 2007; Cunha et al. 2013), and since 2010, avian colibacillosis has been recognized as a foodborne disease, like foodborne urinary tract infection (FUTI). Several potential pathogenic strains have been detected worldwide (Vincent et  al. 2010; Bergeron et al. 2012). New in silico analysis techniques utilizing partial-genome sequencing (Multilocus Sequence Typing - MLST) or whole-genome sequencing allow monitoring of high-risk clonal groups commonly detected in avian species, such as ST117, ST95, and ST23 among others, besides tracing of isolates sharing zoonotic

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potential, such as ST131, ST73, and ST95 (Cunha et  al. 2017; Saidenberg et  al. 2020, 2022; Mehat et al. 2021; Cummins et al. 2022). Special attention has been given to some pandemic strains belonging to phylogenetic groups G and B2, such as ST117 and ST95. These strains cause outbreaks of primary disease, and unlike opportunistic strains (which are usually controlled with improved husbandry), controlling these APECs on farms requires the adoption of vaccination strategies (Christensen et al. 2021; Kathayat et al. 2020, 2021).

7.7 APEC in Latin American Countries A wide range of infections in avian species is associated with infection in extraintestinal sites by strains with virulent characteristics, particularly salpingitis, omphalitis, arthritis, cellulitis, sinusitis, airsacculitis, and sepsis (Ferreira and Knöbl 2009). The prevalence of colibacillosis can vary depending on the predisposing factors, such as quality of facilities, inefficient sizing and arrangement of the ventilation system, density above the maximum adequate numbers of birds, poor cleaning and disinfection of sheds and equipment, differences in temperature, litter humidity, heat stress, and high ammonia concentrations among other factors. Immunosuppressive diseases such as Marek, infectious bursal disease (IBD), and the presence of mycotoxins may also contribute to the occurrence of colibacillosis (Ferreira and Knöbl 2009). As a rule, any environmental, nutritional, or infectious factors damaging the respiratory tract epithelium or those that interfere with the immune system can make birds more susceptible to APEC infection. There is a general agreement that colibacillosis is a common disease found in poultry production flocks and that its various manifestations result in large economic losses in the poultry production chain. The losses occur in three main stages: in the hatchery with an increase in embryo mortality; in the field, negatively affecting zootechnical development of flocks; and at the slaughterhouse, causing an increase in carcass condemnation. After the onset of the disease in the flocks, treatment with antibiotics is inevitable, and this practice is extremely expensive, and often these treatments are palliative as the resulting damage such as mortality, increased feed conversion, and carcass condemnations at slaughter will be present in the affected flocks (Chansiripornchai 2019). It is estimated that economic losses in the United States exceed $40 million per year in slaughterhouses (Kathayat et  al. 2021). There is no consolidated data in Latin countries on economic losses, but the magnitude of the costs is significant. Most works on APEC are published in Brazil, given that the country is the second largest producer of broiler chickens in the world, with production exceeding the figure of 14 million tons of meat per year. In general, the total condemnations in slaughterhouses vary between 1.0% and 2.5%, of which about 40% are due to cases of colibacillosis (airsacculitis, ascites, gross meat appearance, and sepsis). The numbers increase when separate evaluations are calculated for dermatitis, cellulitis, airsacculitis, and contamination. At certain times of the year, these rates can increase

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significantly, exceeding the margin of 5–7%. Between the years 2016 and 2019, a total of 19,705,296,600 birds were slaughtered. Cellulitis represented 6.79% of condemnations, while airsacculitis represented 2.3% of the total number of condemned carcasses (EMBRAPA 2021). For instance, in the state of Santa Catarina in 2018, colibacillosis represented 16% of the total condemnation of chicken carcasses (De Quadros et al. 2019). The agent’s economic importance reinforces the need for studies on the epidemiology and spread of high-risk strains for birds. These studies can be performed using several techniques involving amplification and/or digestion of DNA, such as RFLP, AFLP, ERIC-PCR, or PFGE, the latter being considered the gold standard for regional comparisons. Usually, these techniques allow tracking of localized disease outbreaks with great efficacy in animal production chains when attempting to determine the presence of a specific bacterial lineage in different sites in the same facilities (e.g., breeder, hatchery, chickens, slaughterhouse, food, environment, etc.) or between different companies in the same region. However, this technique is limited when the objective is to establish global comparisons. Epidemiological studies to determine the global distribution of APEC strains use techniques amplifying or sequencing conserved parts of the genomic core. In this context, the use of the Clermont phylogenetic classification, MLST technique, and whole-genome sequencing (WGS)  are common (Clermont et  al. 2019; Mathers et al. 2015; Manges et al. 2019). By September of 2022, the EnteroBase possesses a total of 397 sequences of APEC from Latin America, with 53% from Ecuador, 23.4% from Brazil, 21% from Peru, and 3.2% from Paraguay. Clermont et al. (2013) described a phylogroup analysis technique which is based on the amplification of four bacterial DNA markers that allows rapid identification of 7 different E. coli phylogroups: A, B1, B2, C, D, E, and F (Clermont et al. 2013). Virulent strains isolated from extraintestinal infection sites (ExPEC) are usually grouped into phylogroups B2, F, or D. Diarrheagenic strains, commensal isolates, and those from environmental reservoirs are usually allocated to divergent groups E, C, A, and B1, respectively. The increase in the frequency of WGS and the use of in silico analysis showed the existence of an intermediate group between group B2 and F, which the authors began to call group G. This new classification resulted in an update of the previous PCR technique, with inclusion of the ybgD and cfaB genes (Clermont et al. 2019). Group G tends to cluster avian pathogenic samples (APEC), and although epidemiological studies in Latin America are still scarce, it is likely that this technique can improve the traceability of APEC and distinguish virulent from commensal and opportunistic isolates. Some of the strains of group G exhibit multiple resistance to antimicrobials, with high rates of resistance to tetracycline, sulfonamides, and phenicols, in addition to encoding enzymes of the CTX-M-1 group and other ESBL genes. They are strong producers of bacteriocins and can also be isolated from human microbiota and cases of sepsis, although at low prevalence. Group G consists of a large number of serogroups which includes five clonal complexes: ST117, ST657, ST454, ST738, and ST174. ST117 is the most widespread clonal group among commercial poultry, as well as the one with the highest levels of

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antimicrobial resistance and greater amount of virulence genes and showing more pathogenic effects after experimental inoculation in an animal model. ST117 strains were previously sporadic in Europe but have emerged in several countries since 2017, with predominance of O78 and O53 (Ronco et al. 2017). This strain is highly adapted to the avian organism and has a close relationship with virulence and extraintestinal infections due to virulence factors coding for high iron uptake capacity and carrying resistance to various antimicrobials (Ronco et al. 2017; Mehat et al. 2021). In addition to ST117, APEC-related MLST data also highlights the importance of the clonal groups ST23, ST95, ST140, and ST428/9 (Mehat et al. 2021).

7.8 Current Situation in Brazil In Brazil, there are few studies using the MLST technique, but the following sequence types have been reported in isolates of avian origin: ST10, ST73, ST88, ST93, ST95, ST117, ST131, ST155, ST359, ST648, and ST1011 (Maluta et  al. 2014; Cunha et al. 2017; Saidenberg et al. 2020, 2022). Monitoring of the ST117 strain showed the clonal group’s presence in broiler feces in the Paraná region in 2018. In 2020 and 2021, several companies reported increased losses due to colibacillosis outbreaks, reinforcing the importance of monitoring APEC pandemic strains. In the period from 2020 to 2022, a survey recorded the presence of ST117 in the states of Paraná, Santa Catarina, Rio Grande do Sul, Minas Gerais, and São Paulo. This clonal group was identified in commercial laying birds and broilers, with positivity in breeders, hatching eggs, weak chicks during the first week of development, and birds presenting colibacillosis lesions at slaughter. These strains were also present in swabs of the poultry environment and in insects (Alphitobius diaperinus beetles). These data warn about a possible spread of these strains in Brazil, belonging to phylogroup G, ST117, and with a profile of multiple resistance to antimicrobials (Knöbl 2022). Two additional studies highlighted the circulation of less common pandemic isolates in Brazilian poultry farms, but which are nevertheless of concern due to their zoonotic potential. In 2021, Saidenberg and colleagues reported the circulation of ST131:H22 in Brazilian poultry (Saidenberg et al. 2020). These isolates are part of the prevalent clonal group of MDR ExPEC affecting human patients around the world and were characterized by high antimicrobial resistance, including quinolones, beta-lactams, and colistin (Mathers et al. 2015; Manges et al. 2019). In 2022, these authors analyzed the whole-genome sequences of 11 APEC and 14 UPEC belonging to ST73/CC73, isolated from poultry on farms and from humans in hospitals in the same region of Brazil and compared it with 558 publicly available sequences. The poultry isolates presented a close evolutionary relationship with human isolates while also sharing potential virulence factors that facilitate the development of urinary tract infections and sepsis. The authors also reported the absence of some markers related to antimicrobial resistance as well as those usually described as classical APEC markers (Saidenberg et al. 2022).

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7.9 Perspectives and Control APEC infections are notoriously difficult to control in poultry due to its genetic variability and adaptability in the avian environment. Sometimes, the use of antimicrobials to control APEC has been ineffective due to the ongoing selection and emergence of resistant strains (Jahantigh et al. 2020; Kathayat et al. 2021). In situations of greater economic impact, the losses can be reduced with immunization using attenuated vaccines such as APEC O78:K80 ST23 mutant aroA (Poulvac) and with inactivated vaccines (bacterin and autogenous). In European outbreaks, the combined use of live attenuated vaccines, subunit vaccines, and autogenous vaccines was effective in controlling the colibacillosis outbreaks, with transfer of maternal antibodies (Christensen et al. 2021). The usage of epidemiological surveillance tools, based on constitutive gene sequencing, can reveal which sequence types are most prevalent on farms in Latin America. This epidemiological approach creates perspectives for specific control of important APEC isolates, such as ST117, developing new vaccines in a scenario of the restricted use of antimicrobials in poultry (El Jakee et al. 2016). It should be observed that in these cases the vaccine protection is homologous, being dependent on the passage of maternal antibodies, and it does not substitute good breeding practices and the adoption of improvement regarding management and biosecurity measures (Kathayat et al. 2021). The integrated control of the disease is dependent on the existence of noncarrier breeders to avoid vertical transmission, paying particular attention to flocks that are over 40 weeks old. Good incubation and breeding techniques must be used. Farming in all-in-all-out systems must maintain 15 days as the minimum interval between each flock and emptying facilities. It is also necessary to prepare the poultry environment, reducing dust, ammonia, and other substances that can irritate the mucous membranes of the respiratory tract. It is recommended to control vectors and promote adequate cleaning and disinfection of the facilities. Water quality monitoring is another important subject, giving preference to provide water with a drinking nipple. It is essential that the farm has a monitoring and control program for other respiratory and immunosuppressive diseases, in conjunction with the use of live virus vaccines. Nevertheless, even farms with elevated levels of biosecurity may have difficulties controlling colibacillosis (Ferreira and Knöbl 2020). As future perspectives, additional research is needed in relation to new vaccines, competitive exclusion products, bacteriophages, enzymes, probiotics, and essential oils as alternatives to using antimicrobials for the control of colibacillosis in poultry (Wang et al. 2019; Christensen et al. 2021). Acknowledgments  This work was supported in part (ITK, ENB) by the US Department of Agriculture-Agricultural Research Service (USDA-ARS) CRIS project 5030-32000-225-00D. ENB was supported by an appointment to the ARS Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the US Department of Energy (DOE) and the USDA. ORISE is managed by ORAU under DOE contract number DE- SC0014664.

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All opinions expressed in this paper are the authors’ and do not necessarily reflect the policies and views of the USDA, ARS, DOE, or ORAU/ORISE. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA, ARS, DOE, or ORAU/ORISE. The USDA is an equal opportunity provider and employer.

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Saidenberg ABS, Stegger M, Price LB, Johannsen TB, Aziz M, Cunha MPV, Moreno AM, Knöbl T (2020) mcr-positive Escherichia coli ST131-H22 from poultry in Brazil. Emerg Infect Dis 26:1951–1954 Saidenberg ABS, Vliet AV, Stegger M, Johannesen TB, Semmler T, Cunha MPV, Silveira A, Scaletsky ICA, Dalgaard A, La Ragione RM, Knöbl T (2022) Genomic analysis of the zoonotic ST73 lineage containing avian and human extraintestinal pathogenic Escherichia coli (ExPEC). Vet Microbiol 267:109372–109380 Sanches LA, Gomes MDS, Teixeira RHF, Cunha MPV, de Oliveira MGX, Vieira MAM, Gomes TAT, Knobl T (2017) Captive wild birds as reservoirs of enteropathogenic E. coli (EPEC) and Shiga-toxin producing E. coli (STEC). Braz J Microbiol 48(4):760–763 Sarowska J, Futoma-Koloch B, Jama-Kmiecik A, Frej-Madrzak M, Ksiazczyk M, Bugla-­ Ploskonska G, Choroszy-Król I (2019) Virulence factors, prevalence and potential transmission of extraintestinal pathogenic Escherichia coli isolated from different sources: recent reports. Gut Pathogens 11(1):1–16 Scheutz F, Teel LD, Beutin L, Piérard D, Buvens G, Karch H et al (2012) Multicenter evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature. J Clin Microbiol 50(9):2951–2963 Secretaria de Vigilância em Saúde (SVS) (2021) Doenças tropicais negligenciadas. Ministério da Saúde. Available online: www.gov.br/saude/pt-­br/centrais-­de-­conteudo/publicacoes/boletins/ epidemiologicos/especiais/2021/boletim_especial_doencas_negligenciadas.pdf (Accessed on 25 Sept 2022) Smith JL, Fratamico PM, Gunther NW IV (2014) Shiga toxin-producing Escherichia coli. Adv Appl Microbiol 86:145–197 Torres AG, Amaral MM, Bentancor L, Galli L, Goldstein J, Krüger A, Rojas-Lopez M (2018) Recent advances in Shiga toxin-producing Escherichia coli research in Latin America. Microorganisms 6(4):100 Turkyilmaz S, Parin U, Oryasin E, Bozdogan B (2017) Virulence genes, antimicrobial resistance and clonality of Escherichia coli O157:H7 isolated from mastitic bovine milk. Israel J Vet Med 72(1):17 Valenzuela Held BM (2014) Causas de muerte y resistencia antibiótica en terneros hasta 30 días de edad entre los años 2002 a 2012 examinados en el instituto de patología animal de la Universidad Austral de Chile. Bacherlor’s Thesis. Universidad Austral de Chile, Valdivia, Chile Vidal R, Corvalán L, Vivanco S (2012) Caracterización de cepas de Escherichia coli productor de Shigatoxina (STEC) aisladas desde cerdos y bovinos sanos, faenados en la Región Metropolitana. Av Cienc Vet 27(2). https://doi.org/10.5354/0716-­260X.2012.25987 Vincent C, Boerlin P, Daihnault D, Dozois CM, Dutil L, Galanakis C, Reid-Smith R, Tellier PP, Tellis PA, Ziebel K, Manges AR (2010) Food reservoir for Escherichia coli causing urinary tract infections. Emerg Infect Dis 16:88 Wang H, Liang K, Kong Q, Liu Q (2019) Immunization with outer membrane vesicles of avian pathogenic Escherichia coli O78 induces protective immunity in chickens. Vet Microbiol 236:108367 Wang Z, Zheng X, Guo G, Hu Z, Miao J, Dong Y, Zhengjun X, Qingan Z, Xiankai W, Xiangan H, Yuqing L, Zhang W (2022) O145 may be emerging as a predominant serogroup of avian pathogenic Escherichia coli (APEC) in China. Vet Microbiol 266:109358 Yang X, Liu Q, Sun H, Xiong Y, Matussek A, Bai X (2022) Genomic characterization of Escherichia coli O8 strains producing Shiga toxin 2l subtype. Microorganisms 10(6):1245

Chapter 8

Shiga Toxin and Its Effect on the Central Nervous System Alipio Pinto, Ana Beatriz Celi, and Jorge Goldstein

Chapter Summary Shiga toxin (Stx)-producing Escherichia coli (STEC) is responsible for hemorrhagic colitis, hemolytic uremic syndrome (HUS), and acute encephalopathy, which are triggered by the presence of STEC in food and water or cross contamination. When the central nervous system (CNS) is compromised, the mortality rate increases significantly. Like other Gram-negative bacteria, STEC also releases lipopolysaccharide (LPS). Although Stx causes brain damage directly through its canonical receptor Gb3 as well as indirectly through multisystemic factors, LPS, a proinflammatory element, amplifies the deleterious effects of STEC. This chapter aims to cover the latest clinical studies on neurological deficits resulting from HUS-associated encephalopathy as predictors of risk factors in typical HUS. Also, an update of experimental data from the behavioral to the cellular level on the pathophysiological processes produced by Stx in the CNS is provided. Finally, some pharmacological therapies are included that are considered experimentally and clinically relevant for future perspectives.

8.1 Introduction Shiga toxins (Stx) are also known as verocytotoxins due to their cytotoxic effect on Vero cells, e.g., kidney epithelial cells from the African green monkey (Chlorocebus sp.) (Konowalchuk et al. 1978; Petruzziello et al. 2009). Stx is a virulence factor released by both enterohemorrhagic E. coli (EHEC) and non-EHEC, which are generically called Shiga toxin-producing E. coli (STEC) (Menge 2020). This toxin belongs to an AB family of bacterial toxins which includes, among others, the A. Pinto (*) · A. B. Celi · J. Goldstein (*) Universidad de Buenos Aires, Facultad de Medicina, Departamento de Ciencias Fisiológicas, Buenos Aires, Argentina Universidad de Buenos Aires-CONICET, Instituto de Fisiología y Biofísica “Houssay” (IFIBIO), Laboratorio de Neurofisiopatología, Buenos Aires, Argentina e-mail: [email protected]; [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 A. G. Torres (ed.), Trending Topics in Escherichia coli Research, https://doi.org/10.1007/978-3-031-29882-0_8

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cholera toxin from Vibrio cholerae, anthrax toxin from Bacillus anthracis, diphtheria toxins from Corynebacterium diphtheriae, and tetanus toxin from Clostridium tetani (Fujinaga 2006; Zuverink and Barbieri 2018). AB toxins are named after their two basic protein components, which are noncovalently associated (Beddoe et al. 2010; Zuverink and Barbieri 2018). In the case of Stx, the B subunit (StxB) has a homopentamer structure that recognizes the cellular receptor globotriaosylceramide (Gb3) (Zuverink and Barbieri 2018), a glycosphingolipid present in the detergent-insoluble portion of lipid raft membranes rich in cholesterol (Fraser et al. 2004). On the other hand, the A subunit (StxA) is a catalytic component that exerts its action in intracellular molecules (Hall et al. 2017). Once the interaction of StxB-Gb3 occurs, a clathrin-dependent or clathrin-­ independent endocytic process takes place (Sandvig et al. 1993, 2002). This process culminates in a retrograde pathway in which early endosomes containing Stx escape the lysosomal pathway to the trans-Golgi network and the Golgi apparatus to finally reach the endoplasmic reticulum. StxA is then enzymatically cleaved by furin in two different chains: A1, which has a molecular mass of 27.5 kDa, and A2, with a molecular mass of 4.5 kDa (Fraser et al. 2004; Melton-Celsa 2014). The A1 chain, which contains the catalytic N-glycosidase activity, subsequently translocates to the cell cytosol and removes the adenine residue 2260 of 28S eukaryotic rRNA. Thus, protein synthesis is inhibited at the translational level as the elongation factor EIF2a no longer binds to ribosomes (Endo et  al. 1988; Furutani et  al. 1992; Hall et  al. 2017), which triggers a ribotoxic stress response consisting of proinflammatory and proapoptotic events (Iordanov et al. 2000; Tesh 2012). Stx has been classically known to produce its deleterious effect by interacting with its Gb3 receptor. This interaction is responsible for the hemolytic uremic syndrome (HUS), characterized by thrombocytopenia, microangiopathic hemolytic anemia, and variable degrees of renal compromise ranging from minor urine abnormalities to severe renal disease, mainly in children up to 5  years of age. Typical HUS is preceded by prodromal bloody diarrhea in STEC-infected patients (Karmali et al. 1983, 1985; Noris and Remuzzi 2005; Picard et al. 2015; Launders et al. 2016; Fakhouri et al. 2017). Acute encephalopathy is produced when the central nervous system (CNS) is affected, which entails a worse prognosis. The mortality rate derived from HUS adds up to 5% of total cases and up to 40% when the CNS is compromised (Alconcher et al. 2018). STEC causes more than 2.8 million annual acute illnesses worldwide, leading to 3890 cases of HUS and 230 deaths (Torres et al. 2018). Stx genes are encoded within a chromosomally integrated lambdoid prophage genome (Karch et al. 1999; Wagner et al. 2002; Wagner and Waldor 2002). There are two main groups of Stx: Stx1, which has a molecular mass of 70 kDa, and Stx2, with a molecular mass of 60 kDa. In turn, each group comprises various subtypes classified in letters (Scheutz et al. 2012; Baranzoni et al. 2016). Although the affinity of Stx1 to its receptor is ten-fold higher than that of Stx2, the latter has 400-fold higher toxicity than Stx1 in mice (Scheutz et al. 2012) and is primarily responsible for severe cases in human infections (Griffin and Tauxe 1991; Boerlin et al. 1999; Beutin et al. 2008).

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Although, as mentioned above, the risk of morbidity and mortality is known to increase dramatically with CNS compromise in STEC-HUS infections, experimental and clinical reports remain strikingly scarce. In this context, the latest relevant advances on predictors of neurological deficits by STEC-HUS used in the clinic are presented and draw a parallel with the latest experimental evidence from a behavioral to a molecular level with translational purposes. The topics include the loss of blood-brain barrier (BBB) functionality, neural cell alterations, influence of environmental factors, inflammatory role, and some pharmacological treatments of interest.

8.2 Predictors of Neurological Deficits as Worst Risk Factors in Typical HUS A multicenter study was conducted in Argentina in infants hospitalized with STEC-­ HUS between 2005 and 2016, as an attempt to relate the serotypes and genotypes detected with the severity of the disease in the acute period, understood as the need for dialysis, central neurological and severe intestinal compromise, and death risk. This study included neurological conditions as a significant risk factor, as 21.1% patients suffered severe neurological impairment and another 4.3% died with greater neurological damage. All these patients presented elevated levels of white blood cells, hematocrit and hemoglobin, and hyponatremia upon admission. STEC O157:H7 accounted for most of about 75% of the serotypes investigated, while the remaining 25% comprised O145 (16.8%), O121 (5.4%), and other serotypes (4.2%). In addition, 63.9% of O157:H7 harbored the stx2a/stx2c genotypes, which also included eae and ehxA, a predominance of virulence that did not vary over time. However, a statistically significant relationship could not be established between serotypes and genotypes identified and disease severity. Yet, this study demonstrated the importance of regarding neurological alterations as a severe risk factor for mortality, at least in the acute stage of the disease (Alconcher et al. 2021). Alterations in CNS vasculature can be diagnosed through retinal funduscopic examination (FE) on affected patients. Given that thrombotic microangiopathies in the CNS have been observed in patients with STEC-HUS, a prospective, longitudinal, observational study including FE was conducted to determine abnormalities at the level of the retinal vessels. In some cases, a Purtscher-like retinopathy was observed in those affected by STEC-HUS.  It was also found that the severity of STEC-HUS associated by colonic disease (hemorrhagic colitis) and neurological alterations (but not kidney disease) was statistically linked with abnormal FE findings. Of note, this is the first study in which FE has been used prospectively in patients with STEC-HUS and may pave the way for the use of this technique to determine the degree of neurological severity as a risk factor (Spizzirri et al. 2022). Moreover, it has been proposed that patients infected with STEC should undergo a set of neurological tests and CNS imaging at the time of hospital admission. In

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addition, the practice of biochemical analysis and supportive care, such as blood transfusion and/or renal replacement therapy, has to be performed, in the case STEC-HUS is diagnosed (Giordano et al. 2019). Another study in patients from the 2011 German E. coli O104:H4 outbreak found an interesting correlation between hemolytic anemia and neurological impairment (Lobel et al. 2017). In addition, venous contrast imaging revealed an increase in cerebral blood flow, which the authors hypothesized was meant to compensate for deficits in oxygen delivery to the brain. However, this compensation failed to prevent neurological damage. Although this hypothesis suggests an important clinical finding on the severity of anemia, it appears to leave out factors other than the cerebral vasculature which may produce neurological alterations, such as the direct effect of Stx on neurons or glial cells, as suggested in clinical (Meuth et al. 2013; Kramer et  al. 2015) and experimental studies (Pinto et  al. 2017; Berdasco et  al. 2019a), or the local or systemic production of cytokines (Goldstein et al. 2021). Studies have also been conducted on the relationship between plasma levels of C3 and neurological impairment (Netti et al. 2020). Patients with reduced plasma levels of C3 exhibited higher probability of neurological alterations and, consequently, a higher risk of developing clinical complications. The activation of the complement system could result from being a secondary factor in neurological damage. Therefore, the plasma level of C3 may serve as a biomarker of neurological involvement and, at the same time, determine the degree of STEC-HUS severity. Methylprednisolone treatment was introduced in Argentina as an experimental protocol including plasma infusions, methylprednisolone pulses (10 mg/k/day) for 3 consecutive days, and plasma exchange for 5 days in positive multisystemic diarrhea and HUS patients with neurologic involvement (Valles et al. 2005), with results showing positive outcomes in 9 out of 12 treated patients. Along the same line, encephalopathies caused by STEC-HUS in Japan were treated with anti-­ inflammatories such as methylprednisolone in addition to supportive care (Ishida et  al. 2018). This case has shown that anti-inflammatory treatment can reduce hypercytokinemia and brain inflammation. It has also shown an increase in brain glutamine in STEC-HUS patients which generates excitotoxicity and, together with hypercytokinemia, may play a key role in the pathogenesis of STEC-HUS-mediated encephalopathy. Moreover, the Argentinian group has determined in a murine translational model that the systemic administration of Stx2 induces the production of both proinflammatory and anti-inflammatory cytokines (Arenas-Mosquera et  al. 2022). Further, the administration of anti-inflammatory drugs such as dexamethasone or anti-TNF manages to significantly reduce the proinflammatory state (Pinto et al. 2018). The alterations found at the cellular level (Berdasco et al. 2022) could also be consistent with the excitotoxicity reported above. Indeed, the normalization of glutamine levels long after posttreatment with methylprednisolone (Ishida et al. 2018) may indicate a regulation which is independent from earlier cytokine reduction by methylprednisolone, although this point deserves further investigation. A previous experimental report in a murine model also showed that Stx2 induces glutamate release (Obata et  al. 2015), an event mediated by the neuronal Stx2 Gb3 receptor. These data support the notion that Stx2 acts directly on neurons and/or

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glial cells (Arenas-Mosquera et al. 2022; Berdasco et al. 2022), which differs from the widespread clinical perspective of endothelial brain cells being chiefly responsible for encephalopathy, in addition to electrolyte disorders, complement activation, and/or leukocyte disbalance.

8.3 Deleterious Action of Stx in Neurons In being embedded in the skull, surrounded by cerebrospinal fluid, and enveloped by the meninges, the human brain is remarkably well protected. Neurons are heterogeneously distributed throughout the brain, with gray matter mostly containing neuronal somas and white matter tracts mostly harboring axons (Herculano-Houzel 2014). The principal function of neurons is to process and communicate information (Azarfar et al. 2018). The average human brain consists of about 100 billion neurons (Herculano-­ Houzel 2009) living in a privileged organ from the microbiological/immunological point of view, as the barrier function of its microcirculation protects the brain against many insults (Varatharaj and Galea 2017). Alterations in neuron homeostasis, even the slightest ones, can lead to deleterious changes in the functional architecture of the brain. Therefore, the vulnerability of the brain lies in alterations in its metabolism, energy supply, anatomical structures, or the BBB (Kalia 2008; Grevesse et al. 2015). In the human CNS, the canonical Stx receptor Gb3 has been found both in neurons and endothelial cells (Obata et al. 2008). However, Gb3 has been only detected in neurons in the mouse CNS (Obata et al. 2008; Pinto et al. 2013, 2017; Arenas-­ Mosquera et al. 2022; Berdasco et al. 2022) and only found in endothelial cells in the rabbit CNS (Takahashi et al. 2008). On the other hand, the rat CNS shows Gb3 localization in neurons, reactive astrocytes (Tironi-Farinati et al. 2010), and microglia (Berdasco et al. 2019a). Stx2 seems to primarily target neurons and may alter neuronal physiological function, leading to an increase in glutamate release and the consequent increase in compensatory astrocytic Ca2+ transients (Obata et al. 2008). In a matter of fact, extensive evidence shows that Stx2 induces the enzyme glucosylceramide synthase (GCS) with the consequent upregulation of neuronal Gb3 (Celi et al. 2022). This upregulation may be regulated by the binding of Stx2 to Gb3. Alternatively, Gb3 upregulation may be activated by proinflammatory cytokines such as TNFα and IL-1B, triggering mitogen-activated protein kinases such as p38, JNK 1/2, and ERK 1/2 pathways, through NF-κB signaling, as it was observed in human HT29 colon epithelial cells (Moon et al. 2005). A rabbit model of systemic administration of Stx2 showed that the cerebrospinal fluid concentration of Stx2 increased for 6  h after administration and then declined to become undetectable after 24 h. In contrast, only 30% of the toxin was detected in plasma after 1 h, less than 10% was observed after 6  h, and undetectable levels were found after 12  h (Mizuguchi et al. 1996). These findings suggest that at least when Gb3 is present in rabbit and human BBB endothelial cells, Stx2 may cross the BBB, reaching the

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brain parenchyma. However, a mouse model of systemic administration of Stx2 rendered positive toxin immunodetection in the brain parenchyma (Pinto et  al. 2017), which indicates that Stx2 may cross the BBB in a Gb3-independent fashion. Interestingly, Stx2 was immunodetected in the brain parenchyma even in a mouse model of intragastric treatment with bacteriophage 933 (Bentancor et al. 2013; Del Cogliano et al. 2018). Studies in a rabbit model of systemic administration of Stx1 have revealed neuronal necrosis in the anterior brain, midbrain, cerebellum, medulla, and gray matter of the spinal cord, with areas of focal hemorrhage associated with edema. Ischemic damage and necrosis were also evident, with the appearance of neuronal karyorrhexis. The most severe lesions showed extensive areas of edema, hemorrhage, and infarction (Richardson et al. 1992). Given that Gb3 is expressed only in endothelial cells in the rabbit CNS, secondary damage to neural tissue due to vascular damage may be expected. However, results revealed selective neuronal damage 6  h after Stx2 administration, with neuronal atrophy and pyknotic nuclei (Mizuguchi et al. 1996). These findings suggest that even in rabbits, neurons may be selectively damaged by Stx in a Gb3-independent way. Furthermore, neuronal apoptotic changes have been diffusely observed in the granular layers of the cerebellum, basal ganglia, thalamus, cerebral cortex, and CA1 area of the rabbit hippocampus following systemic treatment with purified Stx2 (Takahashi et al. 2008). Mouse models of systemic Stx2 administration have shown deleterious effects both on neurons and the microvasculature (Pinto et al. 2013, 2017; Berdasco et al. 2019b; Arenas-Mosquera et al. 2022). In this case, mouse neurons showed an abnormal NeuN immune phenotype indicative of morphological alterations, although the reversibility of neuronal damage could not be established. Finally, Stx2 has been immunodetected in murine neurons after intracerebroventricular administration. Damaged Stx2-immunopositive neurons presented altered soma and fibers from the corpus striatum in different stages of degeneration (Goldstein et al. 2007). Bypassing the crossing of the BBB and cerebrospinal fluid-­ brain barrier, these studies provided compelling evidence that neuronal damage may be independent from vascular damage in encephalopathy produced by STEC infection.

8.4 BBB Functional Loss by Endothelial Cells The endothelium of brain capillary walls, unlike nonneural tissue, is highly selective in the passage of substances from blood to neural tissue. Thus, the CNS is a “privileged” system in which this unique characteristic of brain capillaries protects it from abrupt fluctuations of blood components that may disrupt the normal neuronal functions (Rolfe and Brown 1997). The BBB is made up of a non-fenestrated, continuous layer of specialized endothelial cells connected by tight junction complexes, which creates a high-resistance paracellular barrier to ions and small hydrophilic molecules (Varatharaj and Galea 2017). These highly specialized endothelial

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cells also have the following characteristics: Firstly, they are highly polarized cells (Betz and Goldstein 1978), which contributes to BBB properties. Secondly, they exhibit minimal vesicular trafficking, thus limiting the vesicle-mediated transcellular movement of cargo known as transcytosis (Tuma and Hubbard 2003). Thirdly, the combination of high polarization and restricting transcellular and paracellular transport allows endothelial cells of the CNS to employ highly polarized cellular transporters to dynamically control the influx of nutrients and efflux of toxins and metabolic wastes between blood and the CNS (Chow and Gu 2015). Finally, these cells lack leukocyte adhesion molecules, which prevents the entry of immune cells from the blood into the CNS (Feldt Muldoon et al. 2013). However, Stx2 has been reported to induce BBB permeability (Fig. 8.1) in the mice cerebellum, striatum, motor cortex, and spinal cord (Fujii et al. 1994; D’Alessio et al. 2016; Pinto et al. 2017) and in the rabbit hypothalamus (Fujii et al. 1996), which deprives the BBB of its barrier function and allows Stx2 crossing (Goldstein et  al. 2007; Bentancor et  al. 2013; Pinto et  al. 2017; Del Cogliano et  al. 2018). However, it is still unclear whether this is the only way Stx can reach the CNS or whether access through the circumventricular organs or the blood-cerebrospinal barrier may be at play. The BBB of mice systemically treated with Stx2 has shown endothelial glycocalyx alterations (Pinto et al. 2013, 2017; Berdasco et al. 2019b; Arenas-Mosquera et al. 2022) consistent with disruptions observed in other models of BBB damage (Varatharaj and Galea 2017). As the endothelial glycocalyx contributes to vascular protection (Nieuwdorp et al. 2005) and BBB function (Henry and Duling 1999; Vink and Duling 2000), its impairment may compromise BBB integrity and function (Ueno 2009). Microvessel damage has been associated with a decrease in the expression of the vascular endothelial growth factor (VEGF) (Pinto et al. 2013, 2017) and a decrease also observed in Stx-treated primary cultures of human podocytes (Psotka et  al. 2009). Recent reports indicate that alterations or loss of VEGF contributes to neuronal degeneration (Rosenstein et al. 2003). Conversely, VEGF treatment in response to stress situations (Silverman et al. 1999; Rosenstein et al. 2003; Khaibullina et al. 2004) enhances neuronal survival and neurite outgrowth in the explanted brain cortex or substantia nigra (Silverman et al. 1999; Rosenstein et al. 2003) and primary cortical neuron maturation (Khaibullina et  al. 2004). Therefore, the reduction in VEGF expression observed after Stx2 treatment in the mouse brain motor cortex and striatum (Pinto et al. 2013, 2017) may account for neuronal degeneration. BBB damage may occur before parenchymal impairment or be a consequence of it, the latter implying Stx access to the CNS through the circumventricular organs or the blood-cerebrospinal barrier. Indeed, astrocytes, important constituents of the BBB (Heithoff et al. 2021), are injured by Stx2 (Goldstein et al. 2007; Boccoli et al. 2008) and may hence induce or heighten ongoing BBB damage. However, convincing evidence hints at timeline in which endothelial cells are damaged before the brain parenchyma (Tironi-Farinati et al. 2013). Gb3 has been found in endothelial cells of the human and rabbit CNS (Obata et al. 2008; Takahashi et al. 2008). Furthermore, a rabbit model of systemic administration of Stx1 showed occlusive fibrin thrombi in capillaries and pyknotic

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Fig. 8.1  Mechanism of action proposed for Stx in the CNS. A conserved brain neurovascular unit is shown on the left side. On the right, a brain neurovascular unit is affected by the toxin. Disruptive damage to BBB endothelial cells: (1) Stx crossing the BBB reaching the brain parenchyma, perhaps by paracellular or transcellular means, and (2) changes in the expression of glycocalyx and tight junction proteins, with leucocyte transmigration. Once in the CNS, Stx produces astrogliosis (3) astrocytic edema, induction of proinflammatory cytokines, chemokines, and other components responsible for endothelial cell induction of procoagulant von Willebrand factor, adhesion molecules, and reduced expression of tight junction proteins are among the possible events that may occur. Stx activates microglia (4) phagocytic activity, and the expression and release of proinflammatory cytokines are increased. Neuronal Gb3 is upregulated by Stx and possible also by proinflammatory cytokines, and (5) neurodegeneration is an event caused by Stx. Stx-mediated glutamatergic excitotoxicity could be a deleterious event protected by an astrocytic stripping mechanism (6). Oligodendrocytes may also be affected by Stx-mediated proinflammatory cytokines, and (7) myelin sheath degeneration, oligodendrocyte death, and nerve fiber degeneration are events in this scenario (8). Stx also affects cerebrospinal fluid-brain barrier ependimocytes (9). Upregulation of proapoptotic protein Bax, downregulation of AQP4, intracellular edema, and the increase of apical cytoplasmic vacuoles are among the observed events. Red cells, erythrocytes; light violet cells, leukocytes. Cells on the left side: pink and plane, conserved endothelial cells; light blue, conserved astrocytes; brown, resting microglia; yellow, conserve neuron; green, conserved oligodendrocyte; pink and columnar, conserved ependimocytes. Cells on the right side: pink and plane, damaged endothelial cells; light blue, reactive astrocytes; brown, reactive microglia; beige, degenerative neuron; green, degenerative oligodendrocyte; pink and columnar, damaged ependimocytes. (The illustration is not at scale)

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vascular endothelial cell nuclei in thrombosed and non-thrombosed vessels in the anterior brain, midbrain, cerebellum, medulla, and gray matter of the spinal cord. These vascular changes were accompanied by focal hemorrhage associated with edema and damage in the neural parenchyma in those areas (Richardson et al. 1992). Systemically administered Stx2 has been immunodetected in the rabbit endothelium and tunica media of some arterioles from the cerebral parenchyma and subarachnoid space, which suggests Stx2-induced damage not only to the microvasculature but also to arterioles. Moreover, results showed arteriole thickening and subendothelial deposition of amorphous materials in the brain parenchyma and the arachnoid space 2 days after Stx2 administration. These changes in arterioles were accompanied by perivascular edema and erythrocyte extravasation. Likewise, intrathecal Stx administration in rabbits yielded comparable alterations in arterioles and produced thrombotic occlusion in both arterioles and capillaries, with multiple small infarcted areas (Mizuguchi et al. 1996).

8.5 Cerebrospinal Fluid-Brain Barrier Impairment: Involvement of AQP4 The ependyma is a simple ciliated epithelium. Ependymocytes are morphologically characterized by a cuboidal to columnar shape and a round nucleus with a fine stippled chromatin pattern and an inconspicuous nucleolus. These cells have a microvilli-covered surface, and most of them have a central cluster of long cilia. This epithelium lines the ventricular surface of the CNS, extending from the lateral ventricles to the filum terminale (Del Bigio 2010). Water channel AQP4 is abundantly expressed in the mammalian brain and can be found in the foot processes of astrocytes in direct contact with capillaries, in the glia limitans of the rat and human brain (Nielsen et  al. 1997), and in the basolateral membrane of ependymal cells (Nielsen et al. 1997). Two distinct isoforms of AQP4 have been identified so far, e.g., M1 and M23 (Lu et al. 1996). M23-AQP4 forms characteristic orthogonal arrays of intramembranous particles, while M1-AQP4 does not (Furman et al. 2003; Silberstein et al. 2004). These proteins are implicated in brain edema and are believed to participate in potassium clearance during neuronal activation (Amiry-Moghaddam et al. 2003; Verkman et al. 2006). Both isoforms of AQP4 are downregulated by Stx2 in the rat ependyma (Lucero et al. 2012), and increased expression of proapoptotic protein BAX by ependymal cells has been also observed in the rat third ventricle (Tironi-Farinati et al. 2010). Moreover, a rabbit model of systemic administration of Stx2 with concomitant intra cisterna magna treatment with horseradish peroxidase (HRP) has shown HRP throughout the cytoplasm of ependymal cells around the third ventricle, which suggests that Stx2 may also impair the cerebrospinal fluid-brain barrier. Stx2 was also detected by immunoelectron microscopy in these cells, accompanied by degenerative changes such as cellular edema and an increase in intracytoplasmic vacuoles of the apical portion of cytoplasm (Fujii et al. 1996).

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8.6 Involvement of Glial Cells in STEC-HUS Encephalopathy The human brain has two main types of cells, i.e., neurons and glial cells. Although glial cells were thought to outnumber neurons by ten to one for several decades, proportions are now known to be almost equal, with glial cells occupying half of the brain volume (Jessen 2004; von Bartheld et al. 2016; Allen and Lyons 2018). Glial cells present in the human and other vertebrates’ brain include radial glia, astrocytes, oligodendrocyte progenitor cells, oligodendrocytes, and microglia (Jessen 2004); however, strikingly little is known about the action of Stx on these cell types. This section will focus on the changes produced by STEC toxin release in astrocytes, microglia, and oligodendrocytes.

8.6.1 Reactive Astrocytes Triggering Neuroinflammation by STEC Toxins Astrocytes make up about 20% of all brain cells (Allen and Lyons 2018). Even though these cells were once only thought to fulfill support functions (Hamby and Sofroniew 2010), today, they are known to play important roles in neuronal survival and synaptogenesis, regulation of blood flow, blood-brain barrier maintenance, neurotransmitter uptake and recycling, and synapse development, among others (Hamby and Sofroniew 2010; Dossi et al. 2018). A widely accepted pathological hallmark of CNS insult (Hamby and Sofroniew 2010; Vasile et al. 2017; Dossi et al. 2018), astrogliosis is characterized by molecular changes that lead to progressive cell hypertrophy (cell body and processes), proliferation, and scar formation. These processes ultimately affect astrocyte function and may produce both positive and negative impacts on surrounding cells (Hamby and Sofroniew 2010). Astrogliosis may be caused by trauma, CNS infections, neoplasm, neurodegenerative diseases, and even systemic inflammation (Sofroniew 2009; Hamby and Sofroniew 2010). All these insults regulate the function of astrocytes through soluble mediators, such as interleukin (IL)-1, IL-6, TNF-α, LPS, and other pattern recognition receptor (PRR) ligands, glutamate, noradrenalin, adenosine triphosphate (ATP), reactive oxygen species (ROS), nitric oxide (NO), and many others, which may be produced by any cell type in the CNS, including endothelial cells and pericytes (Sofroniew 2009). Such molecular mediators trigger different intracellular pathways leading to glial fibrillary acidic protein (GFAP) upregulation, cell hypertrophy, proliferation, and anti-inflammatory or proinflammatory effects (Levison et al. 2000; Gadea et al. 2008; Neary and Zimmermann 2009; Hamby and Sofroniew 2010). Many authors have reported that reactive astrogliosis in animal models of STEC/ Stx-produced encephalopathy. Mice orally infected with E32511 STEC strain showed severe diffuse reactive astrogliosis in the spinal cord and medulla oblongata

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(Amran et  al. 2013). Furthermore, mice intravenously treated with Stx2 or Stx2 + LPS exhibited astrogliosis in the motor cortex (Pinto et al. 2013), striatum (Pinto et al. 2017), hippocampus (Berdasco et al. 2019b), and thalamus (Arenas-­ Mosquera et al. 2022), with the Stx2 + LPS group showing the strongest intensity. Transmission electron microscopy (TEM) studies have further shown degenerative changes in astrocytes up to 8 days after Stx2 intravenous treatment, together with cytoplasmic edema, disorganized endoplasmic reticulum, swollen mitochondria, and loss of nuclear electron dense elements (Tironi-Farinati et al. 2013). Astrocytic stripping, which consists of the interposition of astrocytic processes between the presynaptic and postsynaptic membranes, has also been observed in the mouse striatum (Tironi-Farinati et  al. 2013). In addition, intracerebroventricularly administered Stx2 produced reactive astrogliosis in the rat striatum and hippocampus and colocalized with GFAP (Boccoli et al. 2008). Moreover, Stx2 also produced cytoplasmic edema and glio-filament hypertrophy in perivascular astrocytes from the striatum and immunolocalized in astrocyte nuclei (Goldstein et al. 2007). Studies in vitro on rat cerebral cortex astrocytes showed that Stx1 cannot single-­ handedly produce significant astrocyte changes (Landoni et  al. 2010). However, pretreatment with 0.5 μg/ml LPS 18 h before Stx1 treatment sensitized astrocytes, which upregulated the expression of intermediate filament GFAP. Stx1 had a significant toxic effect, which was responsible for approximately 20% (10 ng/ml of Stx1) to 30% (25 and 100 ng/ml) of cell death. Additionally, the expression of Gb3, Stx1 internalization, and TNF-α and NO production were significantly higher in sensitized astrocytes treated with Stx1 (Landoni et  al. 2010). At the molecular level, these astrocyte changes were induced by TNF-α and the activation of NF-κB. By contrast, etanercept (a soluble receptor of TNF-α) and BAY 11–7082 (a NF-κB inhibitor) reduced the expression of GFAP and Gb3 and the internalization of Stx1 (Landoni et al. 2010). Furthermore, Stx2 (3 ng/ml) + TNF-α (50 ng/ml), but not Stx2 alone, produced the activation of NF-κB and upstream mitogen-activated protein kinase (MAPK) JNK and p38 pathways (Leu et al. 2016). Stx1-induced inflammatory mediator released by reactive astrocytes produced a chemotactic effect on polymorphonuclear cells (Landoni et  al. 2010, 2012; Leu et  al. 2016), which adhered to astrocytes and had a cytotoxic impact leading to almost 100% astrocyte death (Landoni et al. 2010). Comparable results have been observed in mice treated with intravenous Stx2, as an infiltrate of mast cells was found in the brain parenchyma (Tironi-Farinati et al. 2013), which suggests BBB compromise chemokine release (possibly by astrocytes and/or microglia). However, the pathophysiological implication of this finding remains unclear. Although Stx1 only produced astrocyte reactivity following cell sensitization with LPS (Landoni et  al. 2010), conditioned media from astrocytes treated with Stx1 was enough to produce a cytotoxic effect on human umbilical cord vein cells with properties of the cerebral endothelium (HUVECd) (Landoni et  al. 2012). Furthermore, conditioned media from astrocytes treated with Stx1 alone also reduced the expression of tight junction proteins ZO-1 and occludin. Moreover, a stronger effect was observed after astrocyte sensitization with LPS, including the upregulation of Gb3, the expression of adhesion molecules such as ICAM-1 and

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E-selectin, and the release of procoagulant von Willebrand factor by HUVECd. Furthermore, LPS pretreatment was also required to produce platelet activation, a key step for their aggregation. The factors present in conditioned media which induced HUVECd cell death still need to be elucidated; however, reactive astrocyte-­ produced TNF proved to be chiefly responsible for this effect preceded by NF-kB activation, as treatment of HUVECd with etanercept and BAY 11–7082 managed to significantly reduce damage (Landoni et al. 2012). One of the main clinical signs of encephalopathy produced by STEC infection is brain edema (Magnus et al. 2012; Kuroda et al. 2015; Yahata et al. 2015), which has also been reported in mice (Berdasco et al. 2019b). As described above, AQP4 has a critical role in brain edema formation (Yang et al. 2008; Ng et al. 2009; Donkin and Vink 2010; Ampawong et al. 2011) and is also expressed by astrocytes (Nagelhus and Ottersen 2013). Studies in vitro have shown that AQP4 upregulation is triggered by NF-kB in rat astrocyte primary cultures treated with Stx + LPS but not with Stx alone (Sugimoto et al. 2015). Edema is one of the four cardinal signs of inflammation, together with heat, redness, and pain, e.g., the Celsus tetrad (Talamonti et al. 2020). In addition to cellular edema produced by an increase in AQPs, vasoactive edema also contributes to brain swelling (Klatzo 1987; Donkin and Vink 2010). Abundant evidence has been reported of both cellular and vasoactive edemas (perivascular edema) in encephalopathy produced by Stx2 (Goldstein et  al. 2007). Furthermore, TEM studies of baboons treated with intravenous Stx1 have shown brain perivascular edema (Taylor et al. 1999). In terms of classical inflammation, prostaglandins are potent paracrine and autocrine lipid mediators implicated in many pathophysiological processes, including vasoactive brain edema (Sharma et al. 1994). The enzyme cyclooxygenase (COX) is key in the synthesis of prostaglandins and presents two isozymes, COX-1 and COX-2 (Smith et al. 1996). COX-2 is induced in many cell types in response to inflammatory mediators and is constitutively expressed in the normal brain (Yamagata et al. 1993). However, many authors have reported that astrocytes upregulate prostaglandin E2 in mouse brain injuries (Williams et al. 1994, 1997; Sandhya et al. 1998). In addition, NF-kB-dependent COX-2 upregulation has been observed in rat astrocyte primary cultures treated with Stx + LPS but not with Stx alone (Sugimoto et  al. 2015). Furthermore, COX-2 upregulation has been also implicated in seizures, neurotoxicity, ischemia, and inducing depression (Hirst et al. 1999). However, whether prostaglandin produced by COX-2 upregulation because of Stx + LPS treatment is at least partly responsible for these complications remains to be established. Worth highlighting, the in vitro sensitization with LPS or TNF-α required for the deleterious effects of Stx on astrocytes or HUVECd may not be essential in patients, as Stx alone produces significant changes in CNS astrocytes in animal models (Pinto et al. 2013, 2017; Tironi-Farinati et al. 2013; Berdasco et al. 2019b). However, it seems that practically all the changes induced by LPS and Stx in astrocytes are dependent of NF-kB activation (Landoni et  al. 2012; Sugimoto et  al. 2015; Leu et al. 2016), which may result for the interaction of Stx with other cell types in nervous tissue. Indeed, proinflammatory mediators and damage-associated molecular

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patterns released by injured cells acting on cytokines and PRRs, respectively, may produce the same sensitizing effect in astrocytes. Further evidence of the importance of astrocytic NF-kB activation in the development of encephalopathy produced by STEC is given by the protective effects of NF-kB-inhibitory drugs such as corticosteroids (Auphan et al. 1995) observed in patients (Goldstein et  al. 2021). Moreover, dexamethasone, betamethasone, and angiotensin-(1-7), which also inhibits NF-kB (El-Hashim et al. 2012), have managed to protect the brain of animals from the toxic effect of Stx2 (Fujii et al. 2009; Pinto et al. 2013, 2017, 2018; Goldstein et al. 2016). Therefore, the pivotal role of astrocytes in Stx-produced encephalopathy should be further explored.

8.6.2 Heat or LPS Modulate the Microglial Response to Stx Microglia represent 10 to 15% of all glial cells (Greter and Merad 2013). These cells are the tissue-resident macrophages of the CNS and maintain their CNS population by self-renewal, with little contribution from blood cells (Li and Barres 2018). Microglia work as resident innate immune sentinels capable of producing a strong inflammatory response to disturbances in homeostasis such as damage, infection, or any other insult detected by a wide range of receptors in their processes (Nayak et al. 2014). Microglial cells are exhaustively scanning the surrounding extracellular space and communicate directly with neurons, astrocytes, and blood vessels (Nayak et al. 2014; Li and Barres 2018). Once homeostatic imbalance is detected, microglia change their morphology from a highly ramified, small cell soma (traditionally called “resting”) to a variety of morphologies (traditionally called “activated”), becoming a poorly ramified, amoeboid cell soma. These cells are also highly specialized in cytokine secretion (Crews and Vetreno 2016; Pomilio et  al. 2016). Furthermore, microglia play an important role in synaptic organization, control of neuronal excitability, phagocytic removal of debris, and trophic support leading to brain protection and repair (Nimmerjahn et al. 2005; Denes et al. 2007). Studies in a rabbit model of systemic Stx2 administration have shown a marked increase in microglial activation as early as 6–12  h after Stx2 injection, that is, before the onset of neurological symptoms (28–42  h after Stx2 injection). Furthermore, 24 h after Stx2 treatment, TNF-α and IL-1β mRNA were significantly upregulated in the CNS parenchyma (Takahashi et al. 2008). These data support the notion that microglia might mediate secondary damage to neural cells after Stx2 treatment. However, rabbit neurons do not express Gb3 and may thus be an unsuitable model to evaluate direct damage to neurons by Stx2. In contrast, a mouse model, in which neurons do express Gb3, of systemic administration of Stx2 revealed ERK1/2-independent NF-kB activation 2  h after treatment. In addition, IL-6 and TNF were significantly upregulated in the CNS parenchyma after 24 h, and behavioral alterations occurred after 2 and 4 days (Pinto et al. 2017; Berdasco et al. 2022). This represents stronger evidence that neuronal damage produced by Stx2 may occur after microglial activation.

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In turn, in vitro studies on rat microglia primary cultures treated with Stx2 in different contexts (Berdasco et al. 2019a) have shown microglial capability to internalize Stx2 in a concentration-dependent manner, an ability increased by LPS or heat-shock stimuli. Of note, this effect may be mediated by Gb3, as immunofluorescence studies revealed Stx2 and Gb3 colocalization. Stx2 internalization by microglia was followed by microglial activation, increased metabolic and phagocytic activity, and IL-1β release, which suggests differentiation to a proinflammatory phenotype. However, no cytotoxic effects were found. Interestingly, a similar effect was observed following Stx2B subunit incubation, which indicates that the microglial response to Stx2 may be triggered by a noncanonical toxin mechanism of action.

8.6.3 Oligodendrocytes Are Oxidative and Proinflammatory Targets Oligodendrocytes are the cells responsible for myelin sheath production in the vertebrate CNS. Myelin provides axonal electric isolation, which allows saltatory propagation of action potentials at high velocity (Tognatta and Miller 2016). Oligodendrocytes can also sense neuronal activity by neurotransmitter receptors located on their membrane, which probably accounts for oligodendrocyte differentiation and ability to adequately myelinate axons (Bakiri et al. 2009). Only a few studies have documented the action of Stx on the myelin sheath (Fujii et  al. 1994, 1996; Taylor et  al. 1999; Goldstein et  al. 2007; Pinto et  al. 2018; Berdasco et al. 2019b), and even fewer have reported the action of Stx on oligodendrocytes themselves (Richardson et al. 1992). Some of these studies have shown myelin sheath degeneration in mouse and rabbit models of systemic administration of Stx2 (Fujii et al. 1994, 1996) and pyknotic nuclei in oligodendrocytes in a rabbit model of systemic administration of Stx1 (Richardson et  al. 1992). In addition, dose-dependent degeneration of myelinated nerve fibers was reported in a baboon model, in the cerebral cortex and cerebellum, including the unraveling of large myelin nerve fibers (Taylor et al. 1999). Likewise, rats treated with intracerebroventricular Stx2 have shown demyelinated sheaths of fibers in the corpus striatum (Goldstein et al. 2007). Furthermore, myelin basic protein (MBP), one of the main proteins of the myelin sheath, has been shown to decrease in the internal capsule and the fimbria of the murine brain hippocampus following systemic Stx2 administration (Pinto et al. 2018; Berdasco et al. 2019b). Worth pointing out, damage to myelin may be due to an indirect action of Stx through the production of a proinflammatory and/or oxidized state. Indeed, microglial cells express proinflammatory cytokines such as TNF-α and/or IL-1β (Takahashi et al. 2008; Arenas-Mosquera et al. 2022; Berdasco et al. 2022) and produce ROS (Berdasco et al. 2019b) following Stx2 treatment murine models and may thus damage oligodendrocytes and/or myelin (Beck et al. 1988; Sharief and Hentges 1991). This event occurs in demyelinating diseases such as multiple sclerosis, where

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myelin destruction is associated with activated microglia (Trapp et  al. 1998; Lassmann et al. 2007). In addition to ROS, microglial production of NO radicals is an important source of oxidative damage observed in the pathogenesis of demyelinating diseases (Torre-Fuentes et al. 2020).

8.7 Current Pharmacological Treatments Used for STEC-HUS Encephalopathy So far, treatments for acute encephalopathy caused by STEC infection  have included steroid pulse therapy (Koehler 1995; Kaal and Vecht 2004; Forster et al. 2006; Fujii et al. 2009; Pinto et al. 2013, 2017), immunoglobulin G (IgG) immuno­ adsorption (Flam et al. 2016), and complement factor binding antibody (Arvidsson et al. 2015; Licht et al. 2015; Greenbaum et al. 2016; Giordano et al. 2019; Mahat et al. 2019).

8.7.1 Steroid Pulse Therapy Steroids have proven effective in the treatment of a plethora of brain inflammation disorders which involve edema, including traumatic brain injury. Dexamethasone is one of the synthetic glucocorticoids most widely used in the clinic for disorders of the nervous system with BBB alterations such as edema, neuro-oncological diseases, and ischemia (Koehler 1995; Kaal and Vecht 2004; Forster et al. 2006). The mechanism of action of dexamethasone may be mediated by the glucocorticoid receptor, which can bind to DNA sequences in the 5′ flanking region of target genes and thus activate the transcription of genes such as occludin (Forster et al. 2006). Increased expression of occludins between endothelial cell tight junctions may be associated with a decrease in BBB permeability in inflammatory processes. In experimental studies with mice, treatment with dexamethasone increases survival by 50% in groups infected with a lethal dose of Stx2, as it reduces inflammation and BBB permeability in the CNS. In addition, dexamethasone induces the restoration of the endothelial glycocalyx and the basal expression of VEGF, a decrease in reactive astrocytes/microglia, and the recovery of around 60% of degenerating neurons (Pinto et al. 2013, 2017). Betamethasone sodium phosphate (BSP) is an adrenocortical hormone analog that has been evaluated in trials with rabbits inoculated with an intravenous injection of Stx2 (1.4  mg/kg: 1.6 LD50). Assays using pulse therapy with usual doses (4  mg/kg) of BSP rendered 80% mortality rates. In contrast, pulse therapy with high doses (36 mg/kg) of BSP administered for 2 days with a frequency of two doses every 24 h was effective in reducing death rates and brain injury (Fujii et al. 2009). As an example of this therapy and its clinical application, Rosazza et  al. (2021) reported on a child diagnosed with

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STEC-­HUS that presented late and severe CNS involvement characterized by subacute encephalitis which progressed to a coma. Of note, intravenous administration of high-dose steroid pulse therapy rendered full neurological recovery (Rosazza et al. 2021).

8.7.2 Immunoglobulin G (IgG) Immunoadsorption Patients with decreased neurological function and renal lesions caused by Stx intoxication have been treated with IgG depletion by immunoadsorption (Flam et  al. 2016). The treatment consisted of the use of a staphylococcal A column where 11 L of plasma were processed for 6 and a half h, 2 days in a row. After 5 days, this process was repeated, and a rapid improvement in neurological and renal function was demonstrated. Although the specificity of the antibodies eliminated toward CNS antigens remains to be proven, the elimination of autoantibodies or harmful complexes with Stx may be linked to the effect of IgG depletion.

8.7.3 Complement Factor Binding Antibody The complement is a proteolytic cascade made up of several subunits (C1-C9) that lead to cell lysis through the formation of a pore-shaped structure called the membrane attack complex (MAC) (Mathern and Heeger 2015). Complement activation products such as C3b, C3c, C3d, and factor B, C5 convertase, and soluble C5b–C9 have been seen in plasma from children with STEC-HUS (Arvidsson et al. 2015). Eculizumab is a humanized monoclonal IgG2/4 antibody approved for use in humans which has demonstrated efficacy in reducing kidney damage and fostering improvement in children with neurological damage associated with atypical uremic syndrome (aHUS) (Gulleroglu et al. 2013) and HUS-STEC (Giordano et al. 2019). Eculizumab acts by binding to the complement protein C5 and thus inhibits the action of the C5 convertase. In this way, the formation of C5a, C5b and MAC is prevented (Arvidsson et al. 2015; Licht et al. 2015; Greenbaum et al. 2016). The use of eculizumab has been reported in a retrospective analysis of thrombotic microangiopathy cases due to typical STEC-HUS in a pediatric cohort, with successful results in four out of five severe cases of neurological involvement. Although the number of treatments is still low, eculizumab may be regarded as effective, as it promoted remission in a wide range of neurological signs and symptoms, imaging (basal ganglia and cortex), and laboratory data (hemoglobin, platelet count, LDH, serum creatinine, C3 levels) (Giordano et al. 2019). However, eculizumab efficacy and safety are not entirely clear yet. Although evidence has shown improvements in clinical cases of neurological involvement associated with STEC-HUS, reports so far have been based on nonrandomized retrospective studies, case reports, or series, which may fail to yield objective,

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conclusive results. Therefore, prospective randomized controlled trials are expected to determine the efficacy of eculizumab in STEC-HUS (Mahat et al. 2019).

8.8 Perspectives on Stx-Produced Encephalopathy Treatment So far, the most effective treatment to block the deleterious action of Stx on its target cells has been passive immune therapy with anti-Stx antibodies (Goldstein et al. 2021; Henrique et al. 2022) which may neutralize the toxin before brain damage occurs. The strategy in this case is to produce neutralizing antibodies with the ability to block the binding of Stx to cellular receptors. Chimeric murine-human monoclonal antibodies have been developed since the late 1980s (Perera et  al. 1988). Monoclonal antibodies used in many in vitro and in vivo models have successfully demonstrated the ability to neutralize the toxic and lethal effect of Stx (Yamagami et al. 2001; Kimura et al. 2002, 2003; Sauter et al. 2008; Mejias et al. 2016; Hiriart et  al. 2018). Phase 1 safety and pharmacokinetics trials have been successful in healthy adult volunteers receiving anti-Stx antibodies (Dowling et al. 2005; Bitzan et al. 2009; Lopez et al. 2010; Hiriart et al. 2018). These phase 1 studies have proven that these antibodies are safe and well tolerated, which makes them excellent candidates to be employed as preventive therapeutic treatment in patients infected with STEC. In addition, multilineage-differentiating stress-enduring (Muse) cells are non-­ tumorigenic stem cells with reparative functions that exist naturally in humans and whose effectiveness has been recently evaluated in regenerative medicine (Kuroda et  al. 2010). Muse cells are pluripotent and can be isolated from bone marrow, peripheral blood, and connective tissue (Wakao et al. 2012) or else obtained from cultures of fibroblasts and mesenchymal stem cells (Kuroda et al. 2010). These cells could be used for the treatment of acute encephalopathy caused by HUS with multiple benefits. Muse cells injected intravenously are first directed to the site of damage through receptors that recognize sphingosine signals generated by damaged cells (Yamada et al. 2018). Additionally, these cells offer anti-inflammatory, anti-­ apoptotic, anti-fibrotic, anti-modulatory, and paracrine protective effects (Hosoyama and Saiki 2018; Yamada et al. 2018), and they replace damaged cells by spontaneous differentiation into constituent cells of the tissue (Hosoyama and Saiki 2018; Yamada et  al. 2018). Moreover, Muse cells express histocompatibility leukocyte antigen G (HLA-G), which mediates immune tolerance, preventing acute rejection reactions (Dezawa 2018). Other advantages of Muse cells are their ability to reside in damaged tissue (Uchida et al. 2017; Yamada et al. 2018). Muse cells have been assessed as treatment in cardiovascular diseases and spinal cord damage. These clinical trials were based on the intravenous injection of Muse cells derived from an HLA-unmatched donor to an immunocompetent receptor (Uchida et al. 2017). Muse cells also tolerate stress by secreting survival molecules that play a role in regulating cellular DNA damage (Alessio et al. 2018). Ozuru et al. (2020) reported the benefits of treatment with allogeneic human Muse cells. In

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NOD-SCID mice, the authors generated a model of acute encephalopathy similar to that of HUS patients (Fujii et al. 1994; Ozuru et al. 2020) and evaluated the inoculation of 5 × 104 Muse cells following an injection of a lethal dose of STEC O111. Results showed that the treatment effectively prolonged mouse survival up to 3 months, caused a decrease in reactive astrocytes, and suppressed the activation of caspase 3  in the brain. Histological analysis of the brain of treated mice showed Muse cell integration and neuron-like morphology (Ozuru et al. 2020). One of the possible mechanisms of action of Muse cells is through the expression of granulocyte colony-stimulating factor. This 19.6-KDa glycoprotein is associated with the integrity of AQP4 and the stimulation of endothelial growth factor production, which acts in the prevention of BBB damage and the reduction of neuronal death and apoptosis (Cosler et al. 2007; Chu et al. 2014). This glycoprotein is also thought to stimulate the proliferation of glial and neuronal progenitor cells, improving neurocognitive functions and repairing damaged white matter (Dietrich et al. 2018).

8.9 Conclusion One of the greatest challenges to be faced in the coming years is the application of a standardized and efficient pharmacological protocol to neutralize the acute neurological effects in STEC-HUS, as deaths are caused by neurological involvement. For this purpose, drugs are being evaluated in animal models and clinical scenarios, although prospective, controlled, and randomized studies are still necessary to determine drug efficacy. It is also essential to continue investigating the pathophysiological mechanisms underlying acute encephalopathy in experimental models. To date, no early biomarkers or clinical diagnosis tools with sufficient sensitivity and specificity are available to predict neurological damage in the context of STEC-­ HUS infection, which hinders the use of preventive treatment; e.g., retinal FE is a simple procedure that could be used to predict neurological damage. In this chapter, neurons are described as primary targets of Stx. However, the inflammatory state should also be mentioned as a determining factor in the severity of encephalopathy, associated with other events of cell degeneration and/or death, dependent or not on the Gb3 receptor in the brain. For example, molecules such as NF-kB, cells such as astrocytes and microglia, or inflammatory or excitotoxic mechanisms may be relevant brain targets to consider in future research. Although a final solution would be to eradicate pathogenic E. coli contamination in food and water or cross contamination, work must be done to find solutions to alleviate or neutralize the deleterious effects through pharmacological means as soon as the disease occurs.

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Chapter 9

Relevance of Escherichia coli in Fresh Produce Safety Juan J. Luna-Guevara, Magaly Toro, Christian Carchi-Carbo, Juan L. Silva, and M. Lorena Luna-Guevara

Chapter Summary  Fresh produce demand and consumption have increased due to their nutritional value in the human diet. However, produce has been linked to large and deadly outbreaks of foodborne disease caused by pathogenic Escherichia coli strains, demonstrating the risk of this food/bacteria combination for human health. Moreover, E. coli has traditionally been used as an indicator of fecal contamination, including food products such as produce. In this chapter, various aspects associated with the presence of E. coli in produce will be addressed, such as factors involved in contamination, mechanisms of attachment and survival of the pathogen in vegetables, contamination rates in produce in Latin America, and control strategies to reduce the risk of contamination. This information could contribute to developing effective control measurements that guarantee food safety.

J. J. Luna-Guevara · M. L. Luna-Guevara (*) College of Food Engineering, Faculty of Chemical Engineering, Meritorious Autonomous University of Puebla, Puebla, Mexico e-mail: [email protected] M. Toro (*) Joint Institute for Food Safety and Applied Nutrition, University of Maryland, College Park, MD, USA Institute of Nutrition and Food Technology (INTA), University of Chile, Santiago, Chile e-mail: [email protected] C. Carchi-Carbo Institute of Nutrition and Food Technology (INTA), University of Chile, Santiago, Chile J. L. Silva Department of Food Science and Technology, Mississippi State University, Starkville, MS, USA

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 A. G. Torres (ed.), Trending Topics in Escherichia coli Research, https://doi.org/10.1007/978-3-031-29882-0_9

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9.1 Introduction Fresh produce (FP) delivers valuable nutrients to the human diet. However, foodborne outbreaks associated with these products have risen significantly. E. coli is not an exception and has been increasingly linked to foodborne outbreaks involving produce (Luna-Guevara et al. 2019). Pathogenic E. coli strains pose a health risk and are harmful foodborne pathogens transmitted by drinking water, fruits, and vegetables (e.g., tomatoes, melons, parsley, cilantro, lettuce, spinach), raw milk, or fresh meat. These foods could be contaminated during the food manufacturing process or at the point of origin. Food industry contamination may occur during preharvest or harvest due to using a contaminated water supply, among other contamination sources. The pathogen can survive in the environment and foods using different mechanisms. In humans, pathogenic E. coli can cause diarrhea, hemorrhagic colitis, hemolytic uremic syndrome, and other implications. Diarrheagenic E. coli comprises distinct pathotypes, including enteropathogenic E. coli (EPEC), Shiga toxin-producing E. coli (STEC), and its more pathogenic version known as enterohemorrhagic E. coli EHEC, enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC), enterotoxigenic E. coli (ETEC), and adherent invasive E. coli (AIEC) (Croxen et al. 2013). Each pathotype is characterized by virulence factors associated with toxin formation and colonization factors (CFs) required for the pathogenesis. Virulence factors of ETEC are located mainly on plasmids. EPEC and some STEC share a 35-kb virulence gene cluster on a chromosomal pathogenicity island (PAI) known as the locus of enterocyte effacement (LEE), which is required for their characteristic attaching and effacing (A/E) phenotype linked to virulence (Qadri et al. 2005). Genes encoding virulence factors (e.g., LEE or Shiga toxins) may contribute to the bacteria’s survival in the environment or commensalism in other hosts and may provide an adaptive driving factor for the retention of specific traits (Croxen et al. 2013. Shiga toxin-producing E. coli (STEC), particularly serotype O157:H7, can synthesize one or more Shiga toxins (Holger et al. 2011). In ETEC, the heat-labile toxin (LT) and heat-stable toxin (ST) mediate the deregulation of ion channels in the epithelial cell membrane. Enteroaggregative Escherichia coli (EAEC) is the second most common cause of travelers’ diarrhea after ETEC (Vogeleer et al. 2014) and is associated with foodborne outbreaks of diarrhea in developing counties. Their main virulence factors are the heat-stable toxin (EAST1), Shigella enterotoxin (ShET1), and hemolysin E (Estrada-Garcia et  al. 2002). A Shiga toxin-producing EAEC strain was responsible for one of the largest foodborne outbreaks in Europe, and that was found contaminating sprouts: the E. coli strain O104:H4. This chimera strain was a combination of the chromosomal backbone of EAEC with the bacteriophage encoding Stx2 from STEC and caused numerous cases of disease and death (Scheutz 2015). In this context, this review aims to describe the behavior and importance of pathogenic E. coli in fresh produce and vegetables. This information could contribute to developing effective control measurements that guarantee food safety.

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9.2 Relevance of Total and Fecal Coliforms and E. coli Detection in Produce 9.2.1 Total and Fecal Coliforms The coliform group includes microorganisms of fecal and no fecal origin, and E. coli is one of the most used as an indicator of fecal contamination. These microorganisms may be established on equipment and utensils through the formation of biofilms and can contaminate processed foods (Matthews et al. 2017). Bacterial indicator groups such as coliforms are helpful in food safety, including for produce, when criteria are set to reduce or eliminate foodborne hazards. These microbiological criteria can help to verify that the food processing is adequate, eliminating contamination or reducing the risk. When a specific type of food is repeatedly linked as a vehicle related to food disease outbreaks, then the criteria also have a public health application. The value of testing for coliforms has recently been reduced in importance about their fecal origin, as other Klebsiella, Enterobacter, and Citrobacter bacteria have been considered false positives since they can grow in non-fecal environments, including water, food, and manure. However, it is used as a standard indicator to monitor fecal contamination in products, including fresh vegetables. According to Jay et al. (2008), coliform counts in fresh and frozen vegetables are an indicator that reflects the sanitary quality of the processing steps, and a reduction presented can be considered by stages, such as blanching prior to freezing processes. Some examples of the highest coliform counts in fresh vegetables are found at 7–8 log cfu/g in produce, such as red and green chicory and carrot salad, sprouts, and bean sprouts. It also depends on the method used for counting because coliforms can be quantified on a plate or Petrifilm (Fig. 9.1) and using the most probable number (MPN) method. In frozen vegetables, the MPN values were ≤ 20 in cauliflower, corn, and peas, while the blanched products had an absence of fecal coliforms. Finally, determining E. coli in foods is useful when fecal contamination may have occurred. This contamination of food also implies the risk that other enteric pathogens contaminate the food.

9.2.2 Isolation and Identification of E. coli (Generic and Pathogenic) in Fresh Produce Pathogenic E. coli has been frequently isolated from vegetables. Interestingly, isolation procedures vary depending on the E. coli pathotype and type of vegetable. In general, blend or stomach, a 1:10 sample dilution in brain-heart infusion (BHI) broth is used for pre-enrichment for all pathogenic E. coli except by O157:H7 and other STEC (Feng et al. 2020).

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Total coliforms count (CFU/g)

37°C

Homogenization

Isolation in selective media Enrichment stage

Biochemical test

DNA extraction

Serotype confirmation

Pathotype molecular confirmation

Strains storage -80°C

Fig. 9.1  Quantification, isolation, identification, and confirmation of E. coli in fresh vegetables. (Adapted from Leyva-Abascal (2022))

For STEC strains, sample preparation and enrichment are modified depending on the type of food. Leaf products, coriander, parsley, juice, milk, bottled water, and other beverages have specific protocols that differ from all other foods in dilution and homogenization methods. In the case of leafy produce and cilantro and parsley, it is essential to consider that blend or stomach must never be used since some vegetables’ components might inhibit bacterial growth (Feng et al. 2020). Selective and differential media are used to isolate generic E. coli after enrichment with a nutrient-rich culture media, such as Trypticase soy broth (TSB). Some of the most frequently used agars are MacConkey agar (MCA) and eosin methylene blue (EMB). In MCA, pink colonies (lactose positive) suggest E. coli growth as well as a metallic luster evidence of E. coli presence on EMB. Basic biochemical tests such as IMViC (indole, methyl red, Voges-Proskauer, and citrate) are used for biochemical identification. Traditional tests are still conducted to differentiate among different E. coli isolates, such as serological or genetic tests; the first ones are based on the three main surface antigens: the “O” (somatic), “H” (flagella), and the K (capsule). Pathogenic E. coli strains are classified into specific groups (pathotypes) (EPEC, ETEC, EAEC, EHEC, EIEC) based on the presence of gene products linked to virulence, such as fimbriae, flagella, toxin, capsule, and lipopolysaccharides. For the identification of

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virulence traits, molecular techniques such as PCR (polymerase chain reaction) are used to amplify the genes associated with virulence factors (Meng et al. 2013). Diverse techniques have been used for differentiating among E. coli of the same serotype or pathotype, especially in epidemiological investigations. These subtyping techniques rely on genetic and genomic differences driven by evolutionary changes across time. Pulsed-field gel electrophoresis (PFGE) was the gold standard for defining differences among E. coli isolates. However, a decade ago, whole-­ genome sequencing (WGS) demonstrated its value after the first E. coli O104:H4 isolates were sequenced and unveiled the characteristics of this new pathogenic E. coli causing a massive outbreak of foodborne disease in Europe (Rohde et al. 2011). WGS is becoming a gold standard for investigating relatedness among E. coli and other foodborne bacteria worldwide, and an increasing number of studies are using WGS to investigate E. coli in Latin America (Montero et al. 2017; Gutiérrez et al. 2021; Galarce et al. 2021).

9.3 Colonization and Internalization of E. coli Different mechanisms participate in E. coli retention, binding, or association with plants, including fresh produce. Researchers evaluated E. coli retention after vigorous washing in diverse produce varieties (e.g., cut leaves, fruits, and sprouts). Cut lettuce retained E. coli as soon as 5 min after inoculation, while more than 1 h was required in leaves with an intact epidermis or fruits and more than 1 day for sprouts (Mathews et al. 2014). Conditions such as an aerobic atmosphere, low temperature, and pH and important level of UV (ultraviolet) energy, aerial surfaces (phyllosphere), poor level of nutrients, and the presence of antimicrobial secondary metabolites offer a hostile environment for the growth of E. coli (Brandl 2008). Nevertheless, E. coli can contaminate vegetables. This is paramount since produce is often consumed raw, and low infective doses are required to cause intestinal disease. Indeed, some authors described that even 10 CFU of E. coli O157:H7 can cause illness (Ackers et al. 1998).

9.3.1 Adherence Enteric pathogens produce an array of adherence structures and proteins to colonize animal hosts (McWilliams and Torres 2015; Chhetri et  al. 2019). Such factors include curli, fimbriae, flagella, LPS, colanic acid, and several outer membrane proteins. These adhesion factors are also crucial for EHEC to colonize vegetables or produce biofilms on abiotic surfaces (Carter et al. 2016). EAEC shows adherence to arugula leaf (Eruca vesicaria) by two different phenotypes, including the aggregative adherence fimbriae (AAF) and the flagella. The flagellar mutant adhered to the epidermis but did not attach to the stomatal region.

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In contrast, the aaf mutant lost the ability to adhere to the epidermis but maintained stomatal adherence. In this way, it was demonstrated that the interaction of EAEC with raw vegetables involves multiple adherence factors (Berger et al. 2009a, b). According to Scallan et al. (2011), adherence is a survival mechanism, which allows enteropathogens such as E. coli to remain on tomatoes, lettuce, peach, spinach, broccoli, alfalfa, and apple. Ibarra-Cantún et al. (2022) determined that ETEC survived with populations of 7 log CFU g−1 at 7 °C and 9.2 log CFU g−1 at 22 °C until 120 h in tomatoes. Bacterial adherence and colonization on tomato surfaces under both storage conditions were confirmed by scanning electron microscopy.

9.3.2 Mechanisms of Colonization E. coli possess different mechanisms for human epithelial colonization, which have been confirmed to serve for adherence to raw vegetables (Teplitski et  al. 2009). STEC strains have different colonization factors such as adhesins, fimbriae, flagella, and type III secretion system (T3SS). T3SSs are complex multiprotein organelles that allow bacteria to transport proteins across the bacterial cell envelope directly into the cytosol of eukaryotic cells (Galan and Wolf-Watz 2006; Cornelis 2006). EHEC possesses specific plant colonization factors that are not present in nonpathogenic E. coli strains. For instance, E. coli O157 and non-O157 strains can adhere to E. vesicaria, spinach, and lettuce leaf epidermis via EspA filaments. EHEC exploits the same molecular mechanism to colonize mammalian intestinal cells as those used to bind to a plant phyllosphere; however, the adhesion of EHEC to leaves is independent of the effector protein translocation required for mammalian cell colonization. Another study showed that ETEC strain H10407 could adhere to vegetables such as lettuce, basil, and spinach through EtpA filaments, which form a flagellar tip structure, and colonization factor I, which is dispensable for fixing on leaves (Shaw et al. 2008). Temperature plays a significant role in E. coli plant colonization. Experiments were performed at 4, 26, 37, and 42 °C for 24 h to determine the temperature at which EHEC (strain EDL933) colonizes spinach and lettuce leaves most efficiently. The most favorable conditions for E. coli colonization in this experiment were between 26 and 42 °C. At 4 °C, no significant colonization was observed because of the bacteria’s inability to replicate at this temperature (Xicohtencatl Cortes et al. 2009).

9.3.3 Internalization Studies showed that EHEC could be introduced into the inter-mesophyll cellular space; however, these bacteria are only opportunistic epiphytes in plants (Shaw et al. 2008).

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E. coli O157:H7 can colonize and internalize living spinach and lettuce plants and can be retained in diverse parts of the plant, such as leaves, shoots, and fruits, even after vigorous washing or disinfection (Luna-Guevara et al. 2019) Furthermore, EHEC can adhere diffusely to the epidermis, then aggregate around the stomata, and later penetrate the stomata and junction zones (spongy mesophyll) of cut lettuce leaves at a depth of 20 to 100  μm. Additionally, it has been shown that E. coli O157:H7 can move inside the plant through the root system and reach the edible portion of lettuce (Wright et al. 2013).

9.3.4 Biofilms Biofilm is defined as a ubiquitous lifestyle of bacteria associated with surfaces. This form of life provides bacteria metabolic benefits and physical protection in harsh environments and and accelerate genetic exchanges (Yaron and Römling 2014). Biofilm-associated cells are more resistant to many toxic substances, including antibiotics, chlorine, and detergents; some are used in the produce industry (Ryu et al. 2004; Uhlich et al. 2014; Carter et al. 2016). In the colonization of vegetables, such as sprouts and tomato root, EHEC forms biofilm by producing poly-beta-1,5-n-acetyl-D-glucosaminecellulose (PGA), cellulose, and colonic acid (CA) (Matthysse et al. 2008). Crozier et al. (2016) demonstrated differential regulation of specific genes associated with metabolism, biofilm, and stress response when EHEC was exposed to spinach (S. olercera) or lettuce (Lactuca sativa) plant extracts during colonization. Some clinically relevant non-O157:H7 serogroups are commonly called “the big six” (O26, O45, O103, O111, O121, and O145). The ability to form biofilms on abiotic surfaces was demonstrated by strains from the big six group and three other serogroups: O91, O113, and O128. Furthermore, strains of these serotypes also exhibited resistance to multiple antimicrobials (Vogeleer et al. 2014). ETEC has been shown to form biofilms in different environments, using curli, the extracellular matrix of PGA, cellulose, and CA, on sprouts and tomato roots grown in water (Ageorges et al. 2020). ETEC also demonstrated flagella-mediated adherence to lettuce and leafy vegetables. Because of nutrient leakage, plant tissue with mechanical damage promotes the growth of enteric pathogens such as ETEC (Seo and Matthews 2012). Furthermore, ETEC regulates the expression of their genes differently when they come into contact with different parts of the plant or its extracts. ETEC responds differently with acidic pH in intact lettuce leaves (Gonzales-Siles and Sjöling 2016). EAEC is not the only E. coli that produces CA, but EHEC, EPEC, and EIEC can also synthesize this acid. EAEC is not the only E. coli that produces CA, but EHEC, EPEC, and EIEC can also synthesize this acid. Borgersen et al. (2018) found that the pathogenic Shiga toxin-producing EAEC O104:H4 makes threefold to sixfold higher levels of exopolysaccharide CA structures than the EAEC O42 strain. CA and biofilm structures are formed on sprout surfaces for plant colonization. Biofilm expression is

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downregulated by bile at 37 °C and turns on the expression of other important virulence factors at the intestinal level. All of this suggests that EAEC exists in a “biofilm competent state” in the environment, promoting its persistence on raw vegetables, and when the bacteria enter the human gastrointestinal tract, it is protected from the acid pH of the stomach by the biofilm that covers it.

9.3.5 Other Mechanisms According to research, different proteins or structures are required to colonize different plants. It has been reported, for example, that STEC requires the EspA fiber of T3SS to attach to and colonize arugula, lettuce, and spinach leaves (Shaw et al. 2008; Xicohtencatl Cortes et  al. 2009). The colonization of baby spinach leaves requires flagella, curli, E. coli common pilus (ECP), and hemorrhagic coli pilus (HCP). At the same time, the curli is also involved in the adhesion of EHEC to alfalfa sprouts (Saldaña et al. 2011).

9.4 Presence of Generic and Diarrheagenic E. coli in Fresh Produce in Latin America Since 2012, a few studies have focused on the presence of E. coli in fresh produce in Latin America. In most of these studies, E. coli has been used as an indicator of hygienic quality, and limited research has assessed the prevalence of pathogenic E. coli in this group of foods. Below, representative studies surveying E. coli in produce are described, primarily classified by the country where the study was conducted. Brazil has published the majority of the studies on the presence of E. coli in produce in Latin America. The microbiological quality of lettuce in diverse forms has been addressed for whole fresh (Scherer et al. 2014; Maior et al. 2021), minimally processed (Santos et al. 2020), and ready-to-eat lettuce, including lettuce sampled at restaurants (César et al. 2015; Lima et al. 2017). In these studies, sampling sizes varied from 9 to 100 units, and results showed that E. coli was found in 0 to 66.7% of samples. Interestingly, 5.6% (2/36) of ready-to-eat lettuce samples were contaminated with E. coli, although contamination levels were low (3.6 cfu/g) (César et al. 2015). Brazilian studies surveying other products included one that collected a diverse group of minimally processed leafy vegetables (Da Cruz et  al. 2019), in which 50% (16/32) of vegetables analyzed carried E. coli. Another study that considered a higher number of samples of diverse nature examined raw salads from restaurants; E. coli was present in 7.8% (7/90) of these samples. Moreover, two pathogenic E. coli were found among the isolates (Lima et al. 2017). Despite these results, it has been reported that eating raw vegetables was linked only to 1.2% of foodborne diseases in Brazil between 2000 and 2015 (Lima et al. 2017).

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The hygienic quality of fruits and vegetables has been reported in Ecuador. E. coli was present in 29% (16/54) of the cilantro and 44% (25/54) of the parsley samples evaluated (Cerón-Salgado and Grijalva-Vallejos 2015). Another study analyzed 128 samples of 10 types of vegetables, and 25% of the samples carried E. coli. The vegetable most frequently contaminated was ready-to-eat lettuce, followed by package romaine lettuce and American spinach, and E. coli levels reported for all contaminated samples were 6.2 cfu/g (Hualpa et al. 2018). A broader selection of ready-to-eat and packaged fruits and vegetables, representing 20 types, was later studied; 11% of vegetables (13/117) and 9% of fruits (11/119) carried E. coli. Notably, 11% and 30% of the isolates obtained were of extended-spectrum beta-­ lactamases (ESBL) antimicrobial-resistant E. coli phenotype (Montero et al. 2021). Furthermore, another research group reported that out of 90 vegetable samples, 3 carried ESBL resistance in a lettuce, alfalfa, and parsley/cilantro compound sample (Ortega Paredes et al. 2018). These two publications demonstrate that antimicrobial-­ resistant bacteria are a rising hazard present in produce. In Argentina, one study reported that out of 62 vegetable samples, including lettuce, parsley, cabbage, and tomato, 24% (15/62) carried generic E. coli (Pellegrini et al. 2022). Another study detected E. coli in 38.6% (144/373) of vegetable samples; 14 samples were presumptive to carry diarrheagenic E. coli (3.75%), including eight ETEC and two STEC isolates (González et al. 2017). Interestingly, STEC but not ETEC was included in the list of E. coli most frequently causing diseases in Argentina; however, other sources of infection, rather than vegetables, can be linked to E. coli disease (Torres 2017). Studies have detected DECs in multiple types of produce in Mexico. For example, 36% of Jalapeño and 14% of serrano peppers were contaminated with STEC and up to 12% with ETEC (Cerna-Cortes et al. 2012). In restaurants in Pachuca, 85% (110/130) of raw, ready-to-eat salads were contaminated with E. coli, and DECs were detected in 8 samples. Vegetables frequently contaminated were Spanish (STEC and ETEC) and mixed salads (STEC and EIEC) (Castro-Rosas et al. 2012). Nopalitos, a commonly consumed food in Mexico, revealed that 80% (80/100) of raw and 74% (74/100) of raw cut nopalitos were contaminated with E. coli. STEC, EPEC, and ETEC were isolated from 10% of the nopalitos analyzed, and finally, raw beetroot was investigated for DEC (Gómez-Aldapa et al. 2016). Generic E. coli was present in 53% of samples obtained in Pachuca, and 9% of beetroot juice had DECs, including STEC, EPEC, and ETEC (Gómez-Aldapa et al. 2014). Because this product is a more processed food than vegetables, other factors, such as cross-­ contamination, may have affected the E. coli results. In Chile, a few published studies have focused on E. coli. Specifically, studies have searched for STEC and multidrug-resistant E. coli in vegetables. After surveying 1200 samples of lettuce, alfalfa, tomatoes, and spinach, a single sample was identified as STEC-positive: a STEC serotype O22:H8 was detected in one lettuce sample (Sánchez et al. 2021). In 2021, multidrug-resistant E. coli were detected in parsley and lettuce obtained from vegetables in markets in southern Chile (Díaz-­ Gavidia et al. 2021).

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Studies from other Latin American countries have also addressed E. coli in produce. A survey in La Habana, Cuba, assessed 100 vegetables of diverse types and found E. coli in 18% of them (Puig-Peña et al. 2013). In Cundinamarca, Colombia, four STEC O157:H7 isolates were obtained from guava, tangerine, pea, and spinach samples among distinct types of produce (Patiño et al. 2020). Although not numerous in Latin America, these reports show that fresh produce and minimally processed and ready-to-eat fruits and vegetables were contaminated with E. coli. E. coli has been traditionally considered the fecal indicator by excellence. Its presence is connected to inappropriate food handling, poor hygiene, cross-­ contamination, and/or improper food storage. Depending on the legal regulations of each country, E. coli can be present in levels up to 103 in fresh fruits and vegetables and up to 102 cfu/g in ready-to-eat vegetables, but not every country has legislation considering the presence of E. coli or diarrheagenic pathotypes in produce (Código Alimentario Argentino. Capitulo XI. 2021; Regulamento Técnico Sobre Padrões Microbiológicos Para Alimentos. 2001; Chile and MINSAL 2015; Peru 2003). The evidence also demonstrates that pathogenic E. coli are present in these types of food; STEC, EPEC, ETEC, and EIEC have been detected in small percentages in produce, including fruits. Moreover, the international concern about antimicrobial-­ resistant bacteria has not been irrelevant to foods. A few recent studies in Latin America have centered on detecting resistant E. coli in produce. These studies found that they are vehicles for this type of organisms, including multidrug-resistant E. coli and the worldwide recognized threat of ESBL-positive E. coli strains (Fuga et al. 2022).

9.5 Sources of Contamination During the Preharvest and Postharvest of Fresh Vegetable Production Fresh produce has been heavily linked to outbreaks of foodborne illness. Pathogens identified as hazards on fresh vegetables include Shigella spp., Listeria monocytogenes, Staphylococcus aureus, Aeromonas hydrophila, and the spore-formers Bacillus cereus, Clostridium botulinum, and C. perfringens. However, the species implicated in most fresh fruits and vegetable outbreaks are Salmonella enterica and E. coli (Faour-Klingbeil et al. 2016). Produces are consumed raw or minimally processed; therefore, appropriate risk management strategies must be implemented at the field production stage to minimize food safety risks (Chhetri James 2006).

9.5.1 Preharvest Factors As illustrated in Fig.  9.2, numerous factors contribute to primary and cross-­ contamination of produce. In preharvest and postharvest handling, some factors include animal fecal contamination, insect transmission, use of untreated manure,

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Fig. 9.2  Main sources of contamination in fresh vegetables: (1) contaminated water, (2) hands, (3) insects or pests, (4) untreated manure, (5) irrigation water, (6) soil. (Adapted from Leyva-­ Abascal (2022))

application of contaminated irrigation water, floods with contaminated water, or direct contact with dirty hands (Tomás et  al. 2011). Among practices that may increase the risk of produce contamination include using irrigation systems such as aerial spraying and flood irrigation since they expose more edible parts of the plant to direct contact with contaminated water and/or soil particles (Miceli and Settanni 2019). Fresh produce can become contaminated at any stage of the production chain, from farm to fork. Such contamination can prevail during produce cleaning, packaging, and storage (Kinsinger et al. 2017). According to research, the risk of product contamination is high during three stages: in the field, during initial processing, and in the kitchen (Ailes et  al. 2008). Although washing and sanitizing vegetables at home are essential in preventing foodborne infections, different studies have shown that this procedure does not guarantee the complete inactivation of pathogenic bacteria in leafy products, especially considering internalization in vegetable tissue. Therefore, the absence or low load of pathogens in the harvest is essential for food safety (Lenzi et al. 2021). Moreover, additional research is needed to ensure that the same rinsing and sanitizing processes performed to clean, remove, and prevent contamination are not pathways for cross-contamination that could amplify foodborne illness outbreaks and health risks to public health (Kinsinger et al. 2017). Soil and non-composted animal manure are considered essential factors of contamination before harvest. A wide range of human pathogens, including pathogenic

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E. coli, can be found in soil, due to the presence of animal waste (Heaton and Jones 2008). Research shows that E. coli O157:H7 can survive in the soil for 7 to 25 weeks, depending on soil type, moisture level, and temperature. The application method used for organic waste may increase survival time: clumping of material applied above ground and injection application of liquid manure can protect bacteria from desiccation and elevated temperatures (Hutchison et al. 2004). Fertilization and irrigation management may impact produce contamination and proliferation of human pathogens. Avoiding contaminated fertilizers/water is one of the significant musts to ensure produce safety. First, organic fertilizers and irrigation water can be a means of dissemination for Enterobacteriaceae (Heaton and Jones 2008). In organic food production, animal manure fertilization is a frequent practice; several reports relate this farming system to fecal contamination, particularly during the harvest of leafy vegetables (Denis et al. 2016). Solomon et  al. (2003) showed that E. coli O157:H7 could enter the vascular system of lettuce and reach the edible parts of the plant via contaminated water. However, it has been pointed out that unrealistic inoculum concentrations were used in that study. Although hydroponics is a more controlled production system than cultivation in the soil, it is not exempt from the risk of pathogenic microorganisms: it is necessary to pay special attention to sources of contamination, such as substrates, water, pipes, and seeds (Lenzi et al. 2021). On the other hand, pathogenic bacteria can also come from domestic animals and wildlife, especially during the preharvest stages of green leafy plants (Stuart et al. 2006). Some studies have shown that feces from wildlife participate in plant contamination and can cause E. coli O157:H7 outbreaks. Some reports found that intensive farming practices have forced crop fields to locate too close to animal production areas. The ecological consequences of this proximity are the increased likelihood of E. coli O157:H7 contamination associated with wildlife. Potential reservoirs for pathogenic E. coli included coyotes, dogs, deer, and rabbits’ feces (Lynch et al. 2009). Some insects could also be a source of plant contamination; flies have been shown to transfer E. coli to plant leaves or fruit (Berger et al. 2010). Finally, the personal hygiene of field workers, including using portable toilets and handwashing stations, plays a vital role at the preharvest level (Lenzi et al. 2021).

9.5.2 Postharvest Factors During postharvest, major risks are associated with inadequate hygienic procedures. In some cases, the presence of E. coli on vegetables, such as alfalfa sprouts, fresh spinach, and raw clover sprouts, is significantly higher in the final postharvest stages compared to the early handling stages. Confirmation of E. coli in postharvest packing steps indicates possible fecal contamination and the potential presence of enteric pathogens from fecal origin (Luna-Guevara et al. 2019). In the journey that vegetables make from farm to table, fresh products are subject to various risks of microbial contamination due to an important variety of handling, processing, storage, and

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transport activities that, in case of unfavorable conditions, can result in microbiological risks being present (Gil et al. 2015). According to Faour-Klingbeil et  al. (2016), water is recognized as one of the most important vectors of human enteric pathogens in vegetable crops. Water is used in many steps, such as washing, tank cooling, spraying, and shipping ice during the postharvest process. The first function of washing is to remove dirt and debris, and the second process is designed to remove contamination acquired in the field (Barrera et al. 2012). However, as knowledge accumulated, it became apparent that postharvest washing under commercial conditions has limited decontamination efficacy and, if anything, can potentially lead to cross-contamination events (Murray et al. 2017). Furthermore, using of contaminated water in dry coolers where fresh products are stored can result in vegetable contamination (Gagliardi et al. 2003). Most of the internalization would occur via the hydrocooling process or through having a temperature difference between produce and water (Li et  al. 2008). Other potential E. coli contamination sources may include fresh produce processing operations involving multiple unit operations, which may provide opportunities for cross-­ contamination. If implemented, preventive sanitation programs such as good agricultural practices (GAP), good manufacturing practices (GMP), and Sanitation Standard Operating Procedures (SSOPs) will minimize the possibility of contamination by pathogenic bacteria (Gil et al. 2015). Lynch et al. (2009) has mentioned that the presence of pathogens in vegetables, such as E. coli, can occur through cross-contamination by the hands of the food handler due to a lack of hygiene when raw meat or poultry is also prepared. Finally, consumers could contaminate fresh produce when they touch fresh vegetables while deciding to purchase the product. If a person’s hands become contaminated by inadequate hygiene, this product could be affected by cross-contamination (James 2006). Other causes of fresh vegetable contamination include a lack of temperature control during storage and poor knowledge about the importance of washing and disinfecting vegetables, among others.

9.6 Preventive Measures as Safety Mitigation Strategies in Fresh Produce First, it is critical to recognize that it is impossible to eliminate the risk of contamination in raw produce, especially when grown in open fields (De Keuckelaere 2015). Produce contamination can occur at any point in the production chain and can be amplified from origin to consumption (Nüesch-Inderbinen and Stephan 2016). Therefore, mitigation strategies for produce safety have primarily focused on two approaches: pathogen contamination prevention in the field and interventions to reduce pathogens in fresh produce via washing and inactivation technologies (Oni et al. 2015).

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Some control strategies advocate focusing on seeds before sowing, which is one of the essential preharvest preventive measures (FAO 2011). On the other hand, irrigation water, manure, and soil are the main reservoirs and transmission routes for foodborne pathogens during preharvest; however, these are difficult to control (Gil et al. 2015; Jung et al. 2014). Interrupting activities such as irrigation 2 to 67 days before harvest is one suggested control measure to reduce the risk of contamination (Moynihan 2013). This method aids in reducing pathogen introduction spills caused by various irrigation systems, particularly in manure-treated soil. Furthermore, irrigation water can act as a reservoir and a vehicle for transmitting foodborne pathogens into fresh produce (Allende 2017; Bozkurt 2021). Preventive measures include using surface irrigation techniques such as dripping or furrow irrigation rather than spray irrigation, routine irrigation equipment maintenance and cleanliness, and water exchange (Iwu and Ifeanyi 2019; Bozkurt 2021). Because manure can carry harmful bacteria, improper composting has been linked to foodborne illnesses. Proper composting treatment needs the appropriate temperature and humidity levels to render foodborne bacteria inactive. Proper composting methods and allowing enough time between manure application and harvest can reduce food safety risks. The FAO recommends limiting manure use to specific time frames based on whether the edible part of the plant comes into contact with the soil (>120 days before harvest) or not (>90 days before harvest) (USDA 2023; GLOBAL GAP 2016; FDA 2017). Monitoring activities near crop-growing farms is another recommended measure to reduce the risk of produce contamination. Understanding what activities occur nearby is necessary to take precautions, such as adding physical barriers, building levees and water-diversion channels around farms, or reducing run-off from animal-­ producing operations and waste management. Using sanitizers and disinfectants after harvest may mitigate the introduction of microorganisms of human health importance to freshly harvested produce; this could help reduce the bacterial load from postharvest cross-contamination (FDA 2017). According to some experts, washing vegetables before consumption is one of the most crucial components of a multi-barrier approach to health risk reduction (Losio 2015). Water is widely used in fresh produce postharvest processing to cool, hydrate, clean, and transport products. However, if water becomes contaminated with pathogens, it can contaminate produce (Mensah et al. 2002), resulting in cross-­contamination, especially if the same water is used to wash other produce (Pushpakanth et al. 2019). As a result, water quality is critical in reducing contamination risk. Sanitizers added to wash water can help control microbial hazards; however, factors such as product concentration and type, contact time, and target microorganism type must be considered. Otherwise, sanitized water can serve as a vehicle for the spread of microbial contamination. Furthermore, sanitizers should be added in lesser amounts, as direct contact with the product may leave harmful residues (Gil et al. 2015; Gombas 2017; Meireles et al. 2016; Murray et al. 2017; Souza 2019). Several studies have established that washing, handling, and storage are the primary sources of numerous pathogen contaminations in the traditional fresh vegetable market, though a direct link between contamination of these products and

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foodborne disease outbreaks is challenging to establish (Denis et  al. 2016; Kim et al. 2017). To ensure the safety of fresh produce, it is crucial to prevent contamination in the field and minimize cross-contamination during postharvest processing (Murray et al. 2017). Hurdle technologies, or combinations of different preservation methods, have been proposed to have safe produce, extend shelf life, and improve food safety (Singh and Shalini 2016). Bactericidal preservation methods can reduce the number of bacteria or inhibit their proliferation. One of the leading technologies to use is the application of low temperatures at harvest and throughout the food chain (processing, packaging, storage, and distribution). A continuous cold chain at harvest and postharvest (0–4  °C) could be a helpful barrier because bacterial multiplication would be limited (Mogren et al. 2018). Several other preservation methods (e.g., heating, cooling, drying, curing, preserving, acidifying, oxygen removal, fermenting, and adding preservatives) have been used for centuries, and they are now part of the concept of hurdle technologies (García Carrillo et al. 2020; Singh and Shalini 2016). Currently, pulsed-field technologies, UV light treatments, high hydrostatic pressures, electron beam irradiations, and the use of active packaging that captures oxygen in the packaging material, in conjunction with hazard analysis and critical control point (HACCP) and good manufacturing practice (GMP) systems, are widely used to ensure food safety (Singh and Shalini 2016). Finally, it is crucial to recognize that improving food safety, prevention, and control requires a multidisciplinary approach that combines animal and plant production knowledge and practices and takes a risk-based approach throughout the food supply chain (FAO 2011). The USDA recommends following a Quality Assurance Program (QAP) (USDA 2018) which has a requirement that producers conduct risk assessments throughout the crop production cycle, e.g., water source assessment, animal manure management, and worker hygiene, producers’ education to improve their knowledge of food safety and good agricultural practices (GAP). Outreach programs that meet the needs of producers and motivate them to adhere to production standards and maintain the food safety cycle would be beneficial in preventing contamination (Gil et al. 2015; Balali et al. 2020). As a result, production standards should be developed from farm to table using the principles of Sanitation Standard Operating Procedures (SSOP), good agriculture practices (GAP), good manufacturing practices (GMP), good hygiene practices (GHP), and hazard analysis and critical control point (HACCP) (Maldonade et al. 2019).

9.7 Antimicrobial Alternatives for Postharvest Use in Fresh Vegetables Postharvest processes focus on food safety and conserving food properties such as nutritional value, taste, aroma, and good appearance. Several physical, chemical, and biological methods are being developed to eliminate foodborne

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microorganisms and ensure fresh vegetables’ safety (Palou et  al. 2016; Leyva et  al. 2017). Biological methods have received attention as a viable and safer alternative, and antagonistic microorganisms as biological control agents have been studied. Another option during the postharvest disinfection stage is using essential oils as antimicrobial agents against foodborne microorganisms, including E. coli. Table 9.1 summarizes some alternatives that can be used in raw vegetable postharvest stages. Furthermore, these agents can be used with other technologies, such as encapsulation or sonication, to improve or preserve antimicrobial effectiveness. According to Gul et al. (2015), the encapsulation process represents an interesting alternative to promote antimicrobial compounds’ stability since it consists of covering the active compound with polymeric agents like maltodextrin or Arabic gum, being the most used materials. These polymeric compounds form a three-­ dimensional matrix that reduces active compound interaction with the environment, thereby reducing degradation (Jiménez-González et al. 2022). The inhibitory effect of sonication on E. coli increases with intensity, independent of treatment duration. In this case, microbial viability is lost due to mechanical effects, specifically cavitation, which raises temperature and pressure, damaging the bacterial cell wall and membrane (Scherba et al. 1991). When selecting suitable agents or methods, it is necessary to consider vegetable systems, pathogen molecular mechanisms, and antimicrobial mechanisms. Other important factors to consider include cost-benefit, toxicity, and ecotoxicity evaluation to ensure the successful implementation of postharvest activities.

9.8 Conclusion There is a great demand for fresh vegetables for their nutritional value. However, the raw consumption of these products represents a significant challenge since their safety cannot be 100% guaranteed. E. coli can adhere to plant surfaces and subsequently transit to the edible parts of plants. Contamination of products by enteric pathogens represents a risk to consumers’ health and stands for an essential role in the occurrence of foodborne outbreaks. Total elimination of pathogenic E. coli from the preharvest environment is almost impossible. Therefore, emphasis should be placed on preharvest factors that affect food safety and, subsequently, on the identification of mitigation strategies since all enteropathogens survive on fresh produce, during commercially relevant periods, despite the use of multiple disinfection systems. Finally, it is necessary to continue with the study on the effectiveness of preventive measures, which contribute to guaranteeing the safety of fresh agricultural products, thus allowing to protect consumers’ health.

Table 9.1  Antimicrobial alternatives for postharvest use in fresh vegetables and their effect on inhibiting E. coli Agent type Citrus species such as mandarin (Citrus reticulata blanco), lemon (Citrus aurantifolia (Christm.) Swingle), and orange (Citrus sinensis L.) Thyme and oregano oils

Antimicrobial agent Monoterpenes, in particular limonene

Antimicrobial use 0.2 μl/ml, in combination with a mild heat treatment (54 °C/10 min), showed synergistic lethal effect on E. coli

References Aguilar-­ Veloz et al. (2020)

Thymol, carvacrol, α-pinene, γ-terpinene, and p-cymeno Phenolic extracts

10 ppm and sonication treatments for 10 min in tomato disinfection

Luna-­ Guevara et al. (2015)

237–1000 μg/ml

Conventional agents

Peracetic acid Sodium hypochlorite Chlorine dioxide Ozone

Resveratrol

Resveratrol

Methanolic extract of Berberis lycium Dilutions of apple cider vinegar (ACV)

Berberine

Ozonated water

Ozone

Ultraviolet irradiation

Ultraviolet-C irradiation

Organic acids

Citric and lactic acids

Peracetic acid (80 ppm), chlorine (100 and 200 ppm), chlorine dioxide (3 and 5 ppm), and ozone (3 ppm). Reduce populations >5 log of E. coli O157: H7 inoculated on apples, lettuce, strawberries, and cantaloupe Solutions of resveratrol at 400 μg/ mL; the inhibition percentage observed for E. coli was 81% Effective against E. coli (zone of inhibition 41 ± 1 mm) The minimum dilution of ACV required for growth inhibition for E. coli cultures; a 1/50 ACV dilution was required Ozone of 1 mg/L and a time of treatment of 15 min are recommended for the disinfection of tomatoes UV-C dose (7.5 kJ/m2) on inhibition of E. coli (2.4 log CFU/g) in “Rocha” fresh-cut pears Maximum reduction for E. coli about 2.0 log10 CFU g−1 with 0.5–1 × 104 ppm. Without significant efficacy enhancement from incrementing dipping Times from efficacy of organic acids dipping in inactivation of E. coli on fresh-cut iceberg lettuce 2 to 5 min The inclusion of nisin (IU mL−1) in a pectin coating applied on “Rojo Brillante” persimmon. E. coli populations were reduced by more than 1.0 log10 after 4 days of storage at 5 °C

Loizzo et al. (2010) Luna-­ Guevara et al. (2019)

Jackfruit tree (leaves)

Apple cider vinegar

Nisin in a pectin coating Nisin

Paulo et al. (2010) Malik et al. (2017) Yagnik et al. (2018)

Santa Cruz et al. (2017)

Graça et al. (2017) Akbas and Ölmez (2007)

Sanchís et al. (2016)

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Chapter 10

Quantitative Microbial Risk Assessment of Hemolytic Uremic Syndrome due to Beef Consumption: Impact of Interventions to Reduce the Presence of Shiga Toxin-­Producing Escherichia coli Victoria Brusa, Mariana Cap, Gerardo Leotta, Marcelo Signorini, and Sergio Vaudagna Chapter Summary  In the last decades, several quantitative microbial risk assessments (QMRA) of hemolytic uremic syndrome (HUS) from beef consumption have been conducted. In general, all models reported a similar probability of illness from consumption of a single serving of beef contaminated with Shiga toxin-producing Escherichia coli (STEC). However, differences in the probabilities estimated by the different models worldwide reflect the local conditions of food production, distribution, storage, and preparation. In general terms, most of the QMRA published to date agree on the importance of the main factors identified in the transmission of the disease. The sensitivity analysis on ground beef showed that the risk of infection and HUS probability were linked to the following variables: no application of hazard analysis and critical control point (HACCP) for STEC in abattoirs, no application of Sanitation Standard Operating Procedures (SSOP) in butcher shops, no application of good manufacturing practices (GMP) and good hygiene practices

V. Brusa IGEVET – Instituto de Genética Veterinaria “Ing. Fernando N. Dulout” (UNLP-CONICET LA PLATA), Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, Buenos Aires, Argentina M. Cap · S. Vaudagna Instituto Tecnología de Alimentos, INTA, Buenos Aires, Argentina Instituto de Ciencia y Tecnología de Sistemas Alimentarios Sustentables (UEDD INTA-­ CONICET), Buenos Aires, Argentina G. Leotta (*) Instituto de Ciencia y Tecnología de Sistemas Alimentarios Sustentables (UEDD INTA-­ CONICET), Buenos Aires, Argentina M. Signorini Instituto de Investigación de la Cadena Láctea (IdICaL) (CONICET-INTA), EEA Rafaela, INTA, Santa Fe, Argentina © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 A. G. Torres (ed.), Trending Topics in Escherichia coli Research, https://doi.org/10.1007/978-3-031-29882-0_10

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(GHP) in butcher shops, and application of GMP and GHP at home and cooking preference. Knowledge of the impact of the variables identified in the sensitivity analysis allows optimizing resources and time to direct actions with greater certainty, as well as to avoid taking measures that will not have an impact on the risk of HUS-STEC.  Among the interventions under controlled experimental conditions evaluated to control STEC in beef products, gamma irradiation was the most effective one. The second most effective intervention was high-pressure processing (HPP). With respect to interventions against STEC on beef carcasses in commercial abattoirs, the inclusion of intervention treatments in HACCP programs should help achieve significant reductions in pathogenic bacteria on carcasses. The following interventions applied on carcasses in abattoirs will be briefly described: organic acids, hot water, steam vacuum, electrolytically generated hypochlorous acid (EGHA), ozone (aqueous and gaseous), and combined interventions. However, a single intervention at one stage of the production chain is not enough to eliminate pathogens reliably and entirely from beef surfaces. Undoubtedly, it is necessary to consolidate the epidemiological surveillance of HUS and generate information on each of the STEC transmission routes. In this way, new QMRA could be carried out, risk variables identified, and new scenario analysis performed so that health authorities can take targeted measures and actions to mitigate all STEC-caused diseases.

10.1 Introduction Hemolytic uremic syndrome (HUS) is a group of diseases characterized by the triad of nonimmune microangiopathic hemolytic anemia, thrombocytopenia, and decreased renal function (Karmali et al. 2010). This syndromic disease can present two forms: (1) infection-induced HUS, mainly caused by Shiga toxin-producing Escherichia coli (STEC-HUS) and to a lesser degree by other microorganisms (Streptococcus pneumoniae-HUS, influenza-HUS), and (2) atypical HUS, caused by alternative complement pathway dysregulation-HUS, complement-independent HUS (cobalamin C, DGKE, or INF2 mutation), HUS with coexisting disease (transplantation, autoimmune disorders, drugs, malignant hypertension, malignancy/cancer chemotherapy), and idiopathic HUS (Rivas et al. 2006; Loirat et al. 2016; Feng et al. 2022). The information about HUS cases around the world is scarce and limited to notifiable disease data from different World Health Organization (WHO) regions and population estimates on exposure, age distribution, and clinical course of illness (Majowicz et  al. 2014). The available epidemiological data do not discriminate HUS cases from other STEC illnesses (diarrhea, bloody diarrhea, hemorrhagic colitis), making it difficult to interpret and compare information (Kirk et  al. 2015; JEMRA 2018). Additionally, epidemiological surveillance systems differ among countries, with the consequent diverse ways of presenting the information on STEC-­ HUS cases Table 10.1.

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Table 10.1  Incidence of HUS in different countries per each 100,000 total people and 100,000 children under 5 years old

Country New Zealand Argentina China Ireland France Belgium USA

Incidence 100,000 total people 0.8

100,000 children