Non-traditional Approaches to Combat Antimicrobial Drug Resistance [1 ed.] 9789811991677, 9789811991660

Table of contents :
Foreword
Preface
Contents
About the Editors
1: Recent Strategies to Combat Multidrug Resistance
1 Introduction and Background
1.1 Multidrug Resistance
1.2 Global Incidences
1.3 Economic Impact
1.4 Currently Available Therapies
1.5 Need for Alternative Therapies
2 Nanotechnology to Combat MDR
3 Quorum Sensing and Biofilms
3.1 QS Network
Cyclic di-GMP (c-di-GMP)
3.2 Biofilm-Associated Antimicrobial Resistance
4 Strategies to Inhibit the Biofilm Formation
4.1 Targeting Initial Stages of Biofilm
4.2 Surface Coating
4.3 Affecting Pili and Flagella
4.4 Inhibiting the Production of EPS
4.5 Reducing Polysaccharide Production
4.6 Reducing Matrix Protein Production
4.7 Reducing eDNA Production
4.8 Inhibiting QS System
4.9 Anti-Virulence Compounds
4.10 Phage Therapy
4.11 Antimicrobial Peptides
5 Conclusion
References
2: The Role of Advanced Therapeutic Techniques to Combat Multi-drug Resistance
1 Introduction
1.1 Non-traditional Approaches to Counter Multi-drug Resistance
Bacteriophage-Based Strategy
Quorum Sensing
Targeted Laser Therapy
CRISPR-Cas
2 Bacteriophage-Based Strategy
2.1 Bacteriophages Can Be Used to Combat Various Multi-drug-Resistant Bacterial Isolates
2.2 Bacteriophages Can Be Used to Combat Multi-drug-Resistant Acinetobacter baumannii
2.3 Bacteriophages Can Be Used to Inhibit Multi-drug-Resistant P. Aeruginosa
2.4 Bacteriophages Can Be Used to Treat Multi-drug-Resistant Salmonella serovars
3 Quorum Sensing
3.1 Application of Quorum Sensing to Inhibit Multi-drug-Resistant P. aeruginosa
3.2 Application of Quorum Sensing to Suppress Multi-drug-Resistant Vibrio cholerae
3.3 Utilisation of Quorum Sensing to Suppress Multi-drug-Resistant E. coli
3.4 Utilisation of Quorum Sensing to Counter Multi-drug-Resistant Chromobacterium violaceum
4 Laser Therapy
4.1 Efficiency of Laser Therapy to Inhibit Different Multi-drug-Resistant Bacterial Pathogens
4.2 Effectiveness of Laser Therapy to Attenuate Multi-drug-Resistant Mycobacterium tuberculosis
5 CRISPR-Cas
5.1 Activity of CRISPR-Cas to Suppress Diverse Multi-drug-Resistant K. pneumoniae
5.2 Activity of CRISPR-Cas to Counter Various Multi-drug-Resistant Osteosarcoma Malignancies
6 Discussion and Conclusion
References
3: Strategies to Combat Multidrug Resistance by Non-traditional Therapeutic Approaches
1 Introduction
2 Antibiotic Failure due to Multidrug Efflux Pumps
3 Anti-virulence
4 Toxins
5 Microbiome-Modifying Therapies
6 Nanomedicine: A Novel Approach
7 Non-traditional Approaches
7.1 Anti-virulence Approaches
Targeting Toxins
Adhesins and Biofilms
Inhibitors of Specialized Bacterial Secretory Systems
VF Secretion
Inhibitors of Organism-Specific Cell-to-Cell Signaling or Bacterial Communication
Counteracting Immune Evasion
8 Microbiome-Modifying Therapies
9 Phage Therapy
9.1 Phages as Carriers
9.2 Phage-Derived Products
10 Immunotherapy
11 Other Bio-antibacterial Approaches
11.1 Antimicrobial Peptides (AMPs)
11.2 Bacteriocins
11.3 Peptide Nucleic Acid (PNA)
11.4 Nanoparticles
11.5 Antisense RNA
11.6 Resistance Modulation and Removal of Drug-Resistant Plasmids
12 Utilizing Emerging Drug Targets in Drug Development
12.1 Targeting Iron Acquisition and Storage
12.2 Targeting Membrane Protein Large
12.3 Targeting ClpP Protease
12.4 Targeting Central Carbon Metabolism
12.5 Targeting Energy Generation Which Further Inhibits Respiratory Chain and ATP Synthesis
12.6 Targeting ROS and NOS
13 Drug Repurposing: Combination Therapies of Multiple-Drug Resistance (MDR)
13.1 Drug Repurposing
14 Difference Between Traditional Drug Discovery and Drug Repurposing
15 Drug Repurposing Strategies
15.1 Drug Repurposing Approaches
16 Mechanisms of Microbial Drug Resistance
17 The Use of Combination Therapy to Counter Multidrug Resistance
18 Future Perspective
References
4: Treatment Strategies to Combat Multidrug Resistance (MDR) in Bacteria
1 Background
2 MDR
3 Global Incidence
4 Economic Impact
5 Currently Available Therapies
5.1 Polymyxins
Mechanism of Action
5.2 Aminoglycosides
5.3 Tigecycline
5.4 Fosfomycin
5.5 Ceftolozane/Tazobactam
5.6 Ceftazidime/Avibactam
5.7 Eravacycline
5.8 Meropenem/Vaborbactam
5.9 Linezolid
5.10 Iclaprim
5.11 Daptomycin
6 Non-traditional Approaches
6.1 Anti-Virulence Factors
6.2 Targeting Biofilms
6.3 Targeting Quorum Sensing
6.4 Drugs Repurposed as Antibiotics
7 Conclusion
References
5: Alternative Therapy Options for Pathogenic Yeasts: Targeting Virulence Factors with Non-conventional Antifungals
1 Introduction
1.1 Global Burden of Yeast Infections and Associated Risk Factors
1.2 Antifungal Resistance and Need for Alternative Therapies
2 Virulence Factors as Alternative Therapeutic Targets in Pathogenic Yeasts
2.1 Virulence Factors: Overview and Therapeutic Concepts
2.2 Inhibition of Biofilm Formation
2.3 Inhibition of Morphogenesis: Yeast-to-Hyphae Transition
2.4 Inhibition of Secreted Aspartic Proteinases
2.5 Inhibition of Prostaglandin E2 Production
2.6 Inhibition of Polysaccharide Capsule and Other Virulence Factors of Cryptococcus Spp.
3 Alternative Therapeutics for Pathogenic Yeasts
3.1 Natural Antifungal Peptides
3.2 Probiotics
3.3 Nanoparticles and Antifungal Drug Delivery
3.4 Phytochemicals and Essential Oils
3.5 Antifungal Free Fatty Acids and Derivatives
3.6 Antifungal Photodynamic Therapy
3.7 Antifungal Vaccines and Immunotherapeutics
4 Conclusions and Future Considerations
References
6: Role of Bacteriophages as Non-traditional Approaches to Combat Multidrug Resistance
1 Introduction
1.1 Multidrug Resistance and Development of Multidrug Resistance
1.2 Classification of Bacteriophages
1.3 Genome Structure of Bacteriophages
2 Culture and Characterization of Bacteriophages
3 Phage Therapy against Multidrug Resistance Pathogens
4 Phage Engineering and Antimicrobial Activity
4.1 Phage Engineering andits Role in Widening the Antimicrobial Spectra
4.2 Phage Engineering and Reduced Host Immune Response
4.3 Phage Engineering and Enhanced Antimicrobial Efficacy
4.4 Bacteriophages and CRISPR-Cas System
5 Phage-Based Proteins as Antibacterial Agents
5.1 Polysaccharide Depolymerases (PSDs)
5.2 Virion-Associated Peptidoglycan Hydrolases (VAPGHs)
5.3 Endolysins
5.4 Holins
5.5 Pyocin
6 Phage-Based Pathogen Detection
6.1 Identification of Bacterial Pathogens Using Antibody-Based Method
6.2 Labelled Phages
6.3 Ice Nuclease Reporter Bacteriophages
6.4 Phage-Based Biosensors
7 Phage-Based Vaccine Development
8 Future Prospects
8.1 Bacteriophages as an Emerging Tool to Control Antimicrobial Resistance
8.2 Bacteriophages and their Role in One Health Approach
8.3 Possibility of Microbial Resistance against Bacteriophages
9 Conclusion
References
7: Drug Repurposing: An Approach for Reducing Multidrug Resistance
1 Background
2 Multidrug Resistance (MDR): The Problem
3 Drug Repurposing: A New Alternative Approach
3.1 Mechanism of Repurposing
Biological Methods
Computational Methods
4 Database Resources for Repurposing
5 Chemical Structure and Molecule Information Strategy
6 Challenges in Drug Repurposing
7 Rationale for Drug Repurposing
8 Advantages of Drug Repurposing
9 Conclusion
References
8: Quorum Sensing as an Alternative Approach to Combatting Multidrug Resistance
1 Introduction
2 Quorum-Sensing Regulation Mechanism
3 Quorum Sensing in Gram-Positive Bacteria
3.1 Streptococcus pneumoniae ComD/ComE Competence System
3.2 Bacillus subtilis ComP/ComA Competence/Sporulation System
Competence
Sporulation
3.3 Staphylococcus aureus AgrC/AgrA Virulence System
4 Quorum Sensing in Gram-Negative Bacteria
4.1 Vibrio fischeri LUXI/LUXR Bioluminescence System
4.2 Pseudomonas aeruginosa LasI/LasR-RhlI/RhIR Virulence System
5 Quorum Sensing in Fungi
6 The Role of Quorum Sensing in Fungal Adaptation Strategies
6.1 QS Regulation of Fungal Morphology
6.2 QS Mechanisms Associated with Inter- and Intraspecies Communication
6.3 Quorum Sensing and Fungal Infections
6.4 QS Molecules and Virulence Factor Modulation
6.5 QS Molecule Control of Cell Shape, Size, or Physiological Status
7 Regulation of the Microbial Resistance by Quorum Sensing and Biofilm Inhibition
8 Biofilms
8.1 Biofilms in Gram-Positive and Gram-Negative Bacteria
The Role of Cell Wall Components in Biofilm Formation
The Role of Motility in Biofilm Formation
The Role of Biofilm Matrix Components in Biofilm Formation
8.2 Biofilms in Fungi
Matrix Composition
Host Immunity and Candida Biofilm Interactions
Aspergillus
Matrix Production
Host Immunity and Aspergillus Interactions
Saccharomyces Cerevisiae
Molecular Basis for Yeast Biofilm Formation
Molecular Basis for Cell Surface Adhesion
Quorum Sensing
Extracellular Matrix and Biofilm Resistance
References
9: Nanoengineering Approaches to Fight Multidrug-Resistant Bacteria
1 Introduction
2 Advantage of Nanotechnology-Based Approach to Fight MDR Bacteria
3 The Mechanisms of Antimicrobial Resistance
4 Classification of Infections
5 Effect of Nanoparticles on the Bacterial Resistance
6 Can Nanoparticles Cause Bacterial Resistance?
7 Designing Nanoparticles as Antimicrobial Agents
7.1 Inorganic and Metallic Nanoparticles
7.2 Antibiotic-Conjugated Nanoparticles
7.3 Small Molecule-Conjugated Nanoparticles
7.4 Antimicrobial Peptide-Conjugated Nanoparticles
7.5 Antimicrobial Peptide-Conjugated Organic Nanoparticles
8 Mechanisms of Antimicrobial Activity of Nanoparticles
9 Conclusions and Prospect
References
10: Quorum Sensing-Mediated Targeted Delivery of Antibiotics
1 Introduction
2 Bacterial Resistance to Antibiotics
3 Bacterial QS Systems
3.1 QS in Gram-Positive Bacteria
3.2 QS in Gram-Negative Bacteria
4 QS Antagonists and Agonists
4.1 Natural Product-Based and Natural Product-Derived QS Inhibitors
4.2 Synthetic QS Inhibitors
5 QS Mediated Delivery: Design
6 Conclusions
References
11: Metal Chelation as a Promising Strategy to Combat Fungal Drug Resistance
1 Introduction
2 Chemistry and Biology of Magnesium
3 Significance of Magnesium in Yeast
4 Magnesium Channels
5 ALR1 and ALR2
6 MRS2
7 MNR2 and LPE10
8 Magnesium Exchange Mechanism
9 Targeting Mg Homeostasis Against Candida
10 Conclusion
References
12: Propolis: A Natural Antibiotic to Combat Multidrug-Resistant Bacteria
1 Introduction
2 Propolis: A Natural Antibiotic
3 Propolis: An Effective Alternative Medicine Against MDR Microbes
4 Synergistic Effects of Propolis with Different Antibiotics Against MDR Microbes
5 Role of Propolis as an Anti-Biofilm and Anti-Quorum Sensing Agent
6 Conclusions
References
13: Therapeutic Potential of Himalayan Ayurvedic Herbs Against Multidrug-Resistant Fungal Pathogens
1 Introduction
2 Fungal Pathogens
3 Current Antifungal Agents and Their Toxicity
4 Himalayan Medicinal Plants and Their Therapeutic Potential
5 Conclusion
References
14: Antimicrobial Stewardship Programme: Why Is It Needed?
1 Introduction
2 Antibiotic Resistance
3 Mechanism of Antibiotic Action
4 Emergence of Antibiotic Resistance
5 Drivers for Resistance
6 Clinical and Economic Consequences of Antibiotic Resistance
7 Stewardship a Key to Combat the Antibiotic Resistance
8 Antibiotic Stewardship Programmes and the COVID-19 Pandemic
References

Citation preview

Mohmmad Younus Wani Aijaz Ahmad   Editors

Non-traditional Approaches to Combat Antimicrobial Drug Resistance

Non-traditional Approaches to Combat Antimicrobial Drug Resistance

Mohmmad Younus Wani • Aijaz Ahmad Editors

Non-traditional Approaches to Combat Antimicrobial Drug Resistance

Editors Mohmmad Younus Wani Department of Chemistry University of Jeddah Jeddah, Saudi Arabia

Aijaz Ahmad Clinical Microbiology and Infectious Diseases University of the Witwatersrand Johannesburg, South Africa

ISBN 978-981-19-9167-7 ISBN 978-981-19-9166-0 https://doi.org/10.1007/978-981-19-9167-7

(eBook)

# The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 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 Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Foreword

The heralding of the modern antibiotic era is typically associated with the discovery of penicillin by the Scottish Microbiologist, Alexander Fleming, in September 1928. It was not until the 1940s, however, that purification, compound stability, and mass production of penicillin became possible. By 1944–45, after clinical studies established the effectiveness of penicillin for a wide variety of infections, production of this drug was substantially upscaled for it to be made more widely available. The optimism that penicillin, the so-called miracle drug, as well as other drugs developed during the golden age (1950–1960) of antibiotic discovery, would drive infectious diseases to extinction was unfortunately unfounded. At his Nobel Prize lecture in 1945, Alexander Fleming, who understood the rapid adaptability of microbes under selective pressure, forewarned that the injudicious use of penicillin by physicians would lead to the emergence of penicillin-resistant bacteria and that physicians had a moral obligation to use this drug correctly and only when necessary. In 2022, we find ourselves facing the real and global threat of a post-antibiotic era. The injudicious use of antimicrobial agents over the decades has created a situation where some microbes are resistant to many, if not all, antimicrobial agents. Injudicious use of antimicrobials is not just an issue for physicians and veterinarians to grapple with. It also needs to be addressed in the agricultural sector, which is

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responsible for more than two-thirds of global antimicrobial consumption. The haunting impact of global warming and the impact of climate change on infectious diseases and their distribution and redistribution is already evident. More than ever before, we need integrated and collaborative prevention and treatment strategies to deal with current, emerging, and novel pathogens. “One Health” provides an appropriate, inclusive, and integrative approach. Regrettably, the pipeline for the production of new antimicrobial agents is somewhat dry. The costs and time scales associated with the development of antimicrobial agents, the complexity of conducting clinical trials, and getting these agents through lengthy licensure processes are financial disincentives for private investors and for large, profit-driven pharmaceutical corporations. Against this backdrop, it is clear that in addition to the quest of finding and funding new antimicrobial agents and implementing strict antimicrobial stewardship programs to extend the life of the currently available antimicrobials, it is imperative for academics, research institutions, governments, and international funding agencies to come together in search of alternative strategies. Non-traditional approaches such as combination therapies, targeting virulence factors, disruption of biofilms, and interference with quorum sensing as well as developing new drugs with multiple drug targets have the potential to deal with drug-resistant pathogens. Understanding the close relationship between the host microbiota and infectious pathogens could unravel different factors for controlling these infections. There has been a renewed interest in bacteriophages, bacteriophagederived lytic enzymes, and bacteriophage-antibiotic combinations. Due to their high specificity, bacteriophages have the potential to eliminate infectious pathogens without disturbing the normal flora of the hosts. The role of biofilms in drug resistance is well established, and therefore agents controlling biofilm formation or drugs targeting pre-formed biofilms will be advantageous. All these strategies are plausible to inspire the formulation of newer generations of antimicrobial agents to control multidrug-resistant infectious pathogens. The book entitled Non-traditional Approaches to Combat Antimicrobial Drug Resistance is a laudable initiative by the Editors to address alternative antimicrobial strategies. Different authors from around the globe have been strategically chosen for this issue to address various possible ways to handle drug resistance. This book is a collection of invaluable chapters written by subject experts in their field. I have no doubt that this book will not only generate great interest for experts in the field but also form a wider readership. Drug resistance, due to its high morbidity and mortality, is among the various top global threats that are facing humankind. It behooves us to consider and develop alternative approaches to combat drug resistance. It is the least we can do to better this otherwise fragile world. Clinical Microbiology & Infectious Diseases, School of Pathology of the NHLS & University of the Witwatersrand Johannesburg, South Africa

Adriano G. Duse

Preface

On September 13, 2016, UN General Assembly has declared antimicrobial resistance (AMR) as one of the top ten worldwide public health issues. This threat has further been amplified by the climate change, which worsens the challenge by providing breeding ground for resistant bacteria and other pathogens. AMR poses a challenge to the efficient prevention and treatment of an expanding range of illnesses brought on by bacteria, parasites, viruses, fungi, and other microbes. It happens when microbes are excessively exposed to antimicrobial drugs and in response the microbes undergo mutations or develop different resistance mechanisms to combat the drug for its survival. AMR is becoming an increasingly serious problem in both developed and the underdeveloped nations by posing a challenge to fight against HIV, tuberculosis, cancer, and other life-threatening diseases. Without adequate and effective antimicrobials for the prevention and treatment of infections, medical procedures such as cancer chemotherapy, organ transplantation, diabetes management, and major surgeries become extremely risky. AMR is placing the benefits of the Millennium Development Goals at risk and threatens the achievement of the Sustainable Development Goals. It is therefore a significant problem that needs to be tackled by all realms of government and society as it offers a threat to public health globally. Furthermore, a lack of access to quality antimicrobials remains a major issue. Shortage of antimicrobial drugs is affecting countries of all levels of development and especially in healthcare systems. COVID-19 pandemic has unarguably made the entire world realize the importance of availability of adequate therapeutic agents and the preparedness of any microbial invasion. The clinical pipeline of new antimicrobials is dry. According to WHO annual analysis, in 2021, there were only 27 new antibiotics in clinical development against priority pathogens, of which only six were classified as innovative. More broadly the report describes that, of the 77 antibacterial agents in clinical development, 45 are traditional direct-acting small molecules and 32 are non-traditional agents. Examples of the latter are monoclonal antibodies and bacteriophages, which are viruses that can destroy bacteria. Currently, at least 700,000 people die each year due to drugresistant diseases, including 230,000 people who die from multidrug-resistant tuberculosis. This figure has been predicted to reach 10 million deaths a year by 2050, if

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no measures are taken to control drug resistance. In recent decades, bacterial resistance to antibiotics has developed faster than the production of new antibiotics, making bacterial infections increasingly difficult to treat; same is true for other antimicrobial drugs. In addition, a decreasing interest of pharmaceutical companies in developing new antimicrobials is also on rise. Scientists worry that a particularly virulent and deadly “superbug” could one day join the ranks of existing untreatable microbes, causing a public health catastrophe. Although AMR occurs naturally over time, usually through genetic changes, misuse and overuse of antibiotics further accelerates and aggravates this problem by applying an evolutionary stress on the population. Antimicrobials are also frequently administered to humans, and animals without a proper medical supervision in many countries. This also increases the cost of healthcare with longer hospital stays and need for more intensive treatment. Without effective tools for the prevention and adequate treatment of drug-resistant infections and improved access to existing and new quality-assured antimicrobials, the number of people for whom treatment is failing or who die of infections will increase. Therefore, in addition to intensifying efforts to find novel drugs, new strategies should also be employed to deal with drug resistance. Apart from the traditional or conventional approaches that are being used to tackle the drug-resistant pathogens, the use of non-traditional approaches is an attractive and fascinating option to curb the process of resistance. Non-traditional approaches include the development of therapies that target pathogen virulence, rather than growth, phage-based approaches, development of antimicrobial peptides, drug repurposing, and other combination regimens. Although a plethora of material is currently available in the form of research articles and reviews covering the multi-drug resistance problem, there is no comprehensive coverage of the use and development of non-traditional approaches to combat AMR in the form of a book. This book is intended to serve as a comprehensive literature guide for the beginner researchers and a reference material for the academic and research community involved with tackling multi-drug and AMR problem. This book is aimed to grab the attention of researchers in pharmaceutical industries to develop different non-traditional approaches to tackle drug-resistance problem. Furthermore, this book will also guide clinicians and infection control practitioners to understand the need for utilizing non-traditional approaches to combat drug resistance. Several non-traditional approaches have already been successfully used to combat drug resistance in many cases, but work is still needed to be done, both from the research community and the industry sectors to streamline the efforts and understand the need to develop different successful non-traditional approaches that can augment or replace the currently used therapies. This book serves two purposes, one, it covers the most relevant information about the prospects of using non-traditional approaches to combat multi-drug resistance and second it will help motivating the industrial sector and government agencies to invest more in the research and development of non-traditional approaches and strategies, besides the conventional therapies as a weapon to tackle the multi-drug

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resistance problem. Since antimicrobials now have a limited lifespan before drug resistance emerges, non-traditional approaches offer new opportunities to tackle infections from resistant microbes from different angles as they can be used complementarily and synergistically or as alternatives to established therapies. Jeddah, Saudi Arabia Johannesburg, South Africa

Mohmmad Younus Wani Aijaz Ahmad

Contents

1

Recent Strategies to Combat Multidrug Resistance . . . . . . . . . . . . . Nikky Goel, Zohra Hashmi, Nida Khan, Razi Ahmad, and Wajihul Hasan Khan

2

The Role of Advanced Therapeutic Techniques to Combat Multi-drug Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Musa Marimani, Aijaz Ahmad, and Adriano Duse

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Strategies to Combat Multidrug Resistance by Non-traditional Therapeutic Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Harsh Yadav, Anand Maurya, Alka Agarwal, Anurag Kumar Singh, Satish Dubey, Aditya Moktan Tamang, Reshu Agrawal, and Sushil Kumar Chaudhary Treatment Strategies to Combat Multidrug Resistance (MDR) in Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bisma Jan, Rafia Jan, Suhaib Afzal, Mehrose Ayoub, and Mubashir Hussain Masoodi

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Alternative Therapy Options for Pathogenic Yeasts: Targeting Virulence Factors with Non-conventional Antifungals . . . . . . . . . . . 101 Obinna T. Ezeokoli, Ntombikayise Nkomo, Onele Gcilitshana, and Carolina H. Pohl

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Role of Bacteriophages as Non-traditional Approaches to Combat Multidrug Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 Koushlesh Ranjan, R. A. Siddique, M. K. Tripathi, M. K. Bharti, and Akshay Garg

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Drug Repurposing: An Approach for Reducing Multidrug Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 Ruchi Khare, Sandeep Kumar Jhade, Manoj Kumar Tripathi, and Rahul Shrivastava

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Quorum Sensing as an Alternative Approach to Combatting Multidrug Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 Aimee Piketh, Hammad Alam, and Aijaz Ahmad

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Nanoengineering Approaches to Fight Multidrug-Resistant Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 Sahadevan Seena and Akhilesh Rai

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Quorum Sensing-Mediated Targeted Delivery of Antibiotics . . . . . . 249 Mohmmad Younus Wani, Manzoor Ahmad Malik, and Irfan A. Rather

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Metal Chelation as a Promising Strategy to Combat Fungal Drug Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 Sandeep Hans, Zeeshan Fatima, and Saif Hameed

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Propolis: A Natural Antibiotic to Combat Multidrug-Resistant Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 Deepak M. Kasote, Archana A. Sharbidre, Dayanand C. Kalyani, Vinod S. Nandre, Jisun H. J. Lee, Aijaz Ahmad, and Amar A. Telke

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Therapeutic Potential of Himalayan Ayurvedic Herbs Against Multidrug-Resistant Fungal Pathogens . . . . . . . . . . . . . . . . . . . . . . 297 Indresh Kumar Maurya, Rahul Jain, Ruchi Badoni Semwal, and Deepak Kumar Semwal

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Antimicrobial Stewardship Programme: Why Is It Needed? . . . . . . 309 Mohd Younis Rather, Ajaz Ahmad Waza, Yasmeena Hassan, Sabhiya Majid, Samina Farhat, and Mohammad Hayat Bhat

About the Editors

Mohmmad Younus Wani is an associate professor of Organic and Medicinal Chemistry in the College of Science, University of Jeddah, Jeddah, Saudi Arabia. He received his Ph.D. in Organic and Medicinal Chemistry from Jamia Millia Islamia, New Delhi, India, and postdoctoral training from the University of Coimbra, Portugal, and Texas Therapeutics Institute, UTHealth, Houston, Texas, USA, on the development of new treatment strategies and non-traditional approaches to combat antimicrobial drug resistance (AMR). With an extensive experience in the field of medicinal chemistry and drug discovery, he has authored/co-authored over 50 peerreviewed research articles and 04 books, and is the recipient of many prestigious honors, awards, and grants. He is a professional member of many scientific societies and is on the editorial board of many reputed scientific journals. Dr. Wani is working at the interface of chemistry and biology to advance the field with new questions and pertinent issues of the twenty-first century. Aijaz Ahmad is a Senior Medical Scientist in the Division of Infection Control of National Health Laboratory Service at Charlotte Maxeke Johannesburg Academic Hospital and a Lecturer in the Department of Clinical Microbiology and Infectious Diseases, School of Pathology, University of the Witwatersrand. His core research interest is understanding fungal pathogenesis and development of antifungal vaccines and novel antifungal drugs. He has obtained his Ph.D. in Medical Mycology. During his trainings as a Postdoctoral Scientist from different institutions, he gained lot of experience to be a medical microbiologist. He is an HPCSA registered Medical Scientist in Microbiology and is also National Research Foundation (NRF) rated researcher. With an extensive research experience in infectious diseases, microbial pathogenesis, and drug development, he has authored/co-authored over 90 peer-reviewed publications, supervised more than 25 postgraduate students, and is recipient of several research grants and awards.

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Recent Strategies to Combat Multidrug Resistance Nikky Goel, Zohra Hashmi, Nida Khan, Razi Ahmad, and Wajihul Hasan Khan

Abstract

Multidrug resistance (MDR) has emerged as one of the most important global health issues of the twenty-first century, posing serious threats to humans, animals, and the environment. Infections caused by antibiotic-resistant bacteria result in over 700,000 deaths worldwide every year. Most of the infecting agents, such as viruses, bacteria, fungi, and other parasites, have developed significant levels of drug resistance, which has resulted in increased morbidity and mortality, as well as a detrimental impact on antimicrobial drug development. The World Health Organization (WHO) reports a very high rate of resistance among bacterial strains like vancomycin-resistant Enterococci, methicillin-resistant Staphylococcus aureus, and rifampicin, isoniazid, and fluoroquinolone-resistant Mycobacterium tuberculosis in developed and underdeveloped countries. Concerns about the wide range of MDR infections that cause prolonged illness, increased medical expenses, and a greater risk of mortality demand an immediate action. This chapter emphasises on the use of non-traditional approaches to combat MDR, including the development of quorum sensing (QS)-targeted therapy, nanoparticle-based therapy, anti-virulence therapy, bacteriophage therapy, and

N. Goel · R. Ahmad (✉) Department of Chemistry, Indian Institute of Technology Delhi, New Delhi, India Z. Hashmi Mycobacterial Pathogenesis Laboratory, Infection and Immunology Department, Translational Health Science and Technology Institute, Faridabad, Haryana, India N. Khan Department of Chemical Engineering, Indian Institute of Technology Delhi, New Delhi, India W. H. Khan (✉) Department of Microbiology, All India Institute of Medical Sciences, Delhi, New Delhi, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. Y. Wani, A. Ahmad (eds.), Non-traditional Approaches to Combat Antimicrobial Drug Resistance, https://doi.org/10.1007/978-981-19-9167-7_1

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antimicrobial peptides. The mechanism of action of these novel approaches and potential issues restricting their transfer from laboratory to clinical use is also discussed. Keywords

Multidrug resistance · Antimicrobials drugs · Antiviral · Nanomedicine · Biofilms

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Introduction and Background

Antimicrobial resistance has become a significant global public health risk that demands coordinated regional, national, and international solutions. Overuse of antibiotics and the lack of control mechanisms to prevent the development of resistant bacteria in healthcare settings have resulted in an alarming rise in the incidence of diseases caused by resistant bacteria (Fernández et al. 2016). The World Health Organization (WHO) developed a priority list of antimicrobial drugresistant (AMR) pathogens in response to the global growth in drug-resistant strains of different bacterial pathogens (Singh and Chibale 2021). Antibiotic resistance in numerous microbial species necessitates the development of new therapeutics and approaches to tackle this challenge. A new line of defence against multidrugresistant (MDR) microbes has been recognised as a result of recent developments in nanotechnology that allow the fabrication of nanoparticles with desired physicochemical properties (Singh et al. 2014). In this book chapter, we summarised and discussed the recent development demonstrating the potential ways to evade the antimicrobial drug resistance.

1.1

Multidrug Resistance

Resistance to antimicrobial medications, often known as multidrug resistance (MDR), has developed naturally among pathogenic microorganisms to aid in their survival. Almost all the capable infecting agents (e.g. bacteria, fungi, virus, and parasite) have gained high levels of MDR with enhanced morbidity and mortality. Deaths from severe acute respiratory infections, diarrhoeal diseases, measles, AIDS, malaria, and tuberculosis account for more than 85% of the mortality from infection worldwide (https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resis tance). MDR is one of the major global health problems, conferring significant threat to humans, plants, and animals. Misuse and overuse of antimicrobials in hospitals, food packaging, etc. and faster rate of resistance than antimicrobial drug development as well as poor infection prevention and control practices have contributed to the spread of MDR infections. Antimicrobial resistance is a matter of concern in immunocompromised patients and is becoming an increasingly serious issue in both developed and developing nations. Studies have shown very high rates of resistance among bacterial strains like vancomycin-resistant Enterococci,

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methicillin-resistant Staphylococcus aureus, and rifampicin-, isoniazid-, and fluoroquinolone-resistant Mycobacterium tuberculosis (Fair and Tor 2014). Prolonged drug exposure and continuous viral replication develop varied resistant strains of virus and persistence of infections despite therapy. Antiviral drug resistance has been seen in oncology patients infected with either cytomegalovirus (CMV), herpes simplex virus (HSV), varicella-zoster virus (VZV) (Strasfeld and Chou 2010), human immunodeficiency virus (HIV), influenza A virus, hepatitis C (HCV), or hepatitis B virus (HBV) (Margeridon-Thermet and Shafer 2010). Malaria is one of the prime MDR protozoan diseases, caused by Plasmodium falciparum (Yang et al. 2011), other being Entamoeba spp. causing amoebiasis, a major public health issue in many tropical and subtropical countries (Bansal et al. 2006). Isolates of Candida spp., Aspergillus spp., Cryptococcus neoformans, etc. show resistance to antifungal drugs like amphotericin B, ketoconazole, fluconazole, flucytosine, and echinocandins (Loeffler and Stevens 2003). Antivirals are the first line of defence against a variety of illnesses that may be lethal for several pathogenic viruses, including HIV and HBV. Antiviral medications may become less effective or inefficient as flu viruses evolve. Antiviral medicines may help lower flu virus susceptibility but may not perform as effectively against viruses with reduced susceptibility, and virus replication might start early in the disease, as we witnessed with COVID-19. Multidrug regimens are the most often used antiviral therapy for HIV today, and they usually include drugs from multiple classes, particularly when cross-resistance mutations are unknown (Pau and George 2014). This regimen increases the genetic barrier by demanding many mutations to bestow resistance. Nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, and/or integrase inhibitors are the most prevalent combinations. Reverse transcriptase inhibitors, particularly lamivudine, are the most common HBV therapy. However, there have been reports of a number of lamivudine resistance mutations, many of which have minimal genetic barriers. In antiviral management, combating antiviral drug resistance in life-threatening infections should be prioritised.

1.2

Global Incidences

Estimating the global impact of antimicrobial resistance in terms of mortality and of public health cost is quite difficult, and there are few studies to address this issue. AMR is a major public health problem to progress achieved against infectious diseases, cancer therapy, organ transplant, and intensive care. Globally, about 700,000 deaths are caused by drug-resistant infections every year and without effective intervention are projected to cause ten million deaths and a worldwide financial loss of US$100 trillion by 2050. Furthermore, the direct and indirect consequences of antimicrobial resistance are predicted to disproportionately affect low- and middle-income countries (LMICs) in Asia and Africa (Manesh and Varghese 2021). The stark consequences of AMR were featured in a 2019 report of the UN Interagency Coordination Group on Antimicrobial Resistance, which

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assessed that drug-resistant infections could cause ten million deaths each year globally by 2050, without sustained efforts to contain antimicrobial infections. The document additionally reported that currently at least 700,000 people die annually from drug-resistant infections, including 230,000 deaths from MDR-TB. It also alarms the world of the need of urgent actions as the antimicrobial resistance can have disastrous impact within a generation. According to US CDC’s Antibiotic Resistance (AR) Threats report (2019), more than 2.8 million antibiotic-resistant infections occur in the United States each year, and more than 35,000 people die as a result. In addition, the estimated national cost to treat infections caused by six multidrug-resistant germs was more than $4.6 billion annually. It has also categorised drug-resistant bacteria and fungi as urgent threats, serious threats, concerning threats, and watch list (Table 1.1). Concerning such a vast variety of MDR infections leading to prolonged illness, higher expenditures for health care, and an immense risk of death, the World Health Organization (WHO) has acknowledged the utmost requirement of an improved and coordinated global effort to contain MDR which has provided a framework of interventions to slow the emergence and reduce the spread of MDR microorganism (World Health Organization 2001). In 2015, the WHO launched the Global Antimicrobial Resistance and Use Surveillance System (GLASS) to foster filling knowledge gaps and to inform strategies at all levels, and it proposed a combination of interventions that incorporate strengthening health systems and surveillance, improving the use of antimicrobials in hospitals and in community, infection prevention and control, empowering the development of suitable new drugs and vaccines, and political commitment. In 2020, the World Antimicrobial Awareness Week (WAAW) started, in November18 to 24, which was previously known as World Antibiotic Awareness Week, to raise awareness of antimicrobial resistance worldwide and encourage the best practices among the public, health workers, and policy makers to slow the development and spread of drug-resistant infections. According to the May 2021 GLASS report, 109 countries and territories worldwide have enrolled in GLASS, comprising 107 in the GLASS-AMR module and 19 in the GLASS-AMC (antimicrobial consumption) module. The report describes developments over the past years of GLASS and other AMR surveillance programmes led by the WHO, including resistance to anti-human immunodeficiency virus and anti-tuberculosis medicines, and antimalarial drug efficacy.

1.3

Economic Impact

The AMR expense to the economy is significant. In addition to death and disability, prolonged illness leads to longer hospital stays, the requirement for more costly medicines, and financial challenges for those affected. GLASS 2020 report estimated that only one-third of AMR burden is on high-income countries, while the other two-thirds in LMICs where access to antibiotics has risen with increases in gross domestic product per capita. Global per capita antibiotic consumption has been assessed to have an increment of 39% in 2000–2015, and that of watch antibiotics—

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Table 1.1 CDC category of bacteria and fungi threats S. no. 1 2 3

4 5

6 7

8

9 10

11

12

13

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15

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Microorganism (type) Acinetobacter (bacteria) Candida auris (fungus) Clostridioides difficile (bacteria) Enterobacterales (bacteria) Neisseria gonorrhoeae (bacteria) Campylobacter (bacteria) Enterococcus (bacteria) Mycobacterium tuberculosis (bacteria) Candida species (fungus) ESBL-producing Enterobacterales (bacteria) Staphylococcus aureus (bacteria) Salmonella Typhi (bacteria) Group A Streptococcus (bacteria) Group B Streptococcus (bacteria) Aspergillus fumigatus (fungus) Mycoplasma genitalium (bacteria)

Infection caused Pneumonia, UTI, Blood infection Bloodstream infections Diarrhoea and colitis Nosocomial infections STD (gonorrhoeae)

Drug resistance Carbapenem All antifungal drugs Multi-antibiotics, (fluoroquinolones, penicillin, lincomycin, tetracycline, etc.) Carbapenem All antibiotic drugs

Category Urgent threats Urgent threats Urgent threats Urgent threats Urgent threats

Diarrhoea, fever, abdominal pain Bloodstream and surgical site infections, UTI Tuberculosis

Fluoroquinolones, tetracycline

Rifampicin, isoniazid, and fluoroquinolone

Serious threats

Candidiasis

Amphotericin B, ketoconazole, fluconazole, azole Penicillin and cephalosporin

Serious threats Serious threats

Sinusitis, Food poisoning

Methicillin

Serious threats

Typhoid fever

All but antibiotics macrolides and carbapenems

Serious threats

Pneumonia, sepsis

Erythromycin

Concern threats

Pneumonia, UTI, Blood infection

Clindamycin

Concern threats

Azole

Watch list

Quinolone

Watch list

Pneumonia, UTI

Urethritis, cervicitis

Vancomycin

Serious threats Serious threats

ESBL extended-spectrum β-lactamase, UTI urinary tract infection, STD sexually transmitted disease

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antibiotics that the WHO suggests only for explicit indications due to their high AMR potential, under the AWaRe classification—has been accounted for to have increased by 91%, over the same period of time, with the increments being most prominent in LMICs (Devi 2020). A significant driver of resistance is antibiotic use in humans and in terrestrial and aquatic animals raised for human consumption. Global antibiotic consumption in humans increased by 65% between 2000 and 2015, whereas consumption in animals is expected to increase by 11.5% between 2017 and 2030. If nothing changes to modify these directions, antibiotic consumption is probably going to increase worldwide by 200% between 2015 and 2030 (Schar et al. 2020). Many nations lack laboratory capacity and antimicrobial stewardship programmes. Besides, unchecked and unregulated utilisation of antibiotics in many regions is likely to drive the development and spread of AMR and ought to be tracked. Studies report that BRICS (Brazil, Russia, India, China, and South Africa) countries consumed 76% of the overall increase in global antibiotic consumption between 2000 and 2010, of which 23% was attributed to India. Of all the medicine sales worth $12.6 billion in India between 2013 and 2014, antimicrobials added to around 16.8%. A critical extent of these sales was made through out-of-pocket (OoP) expenditures without universal health coverage and financial risk protection mechanisms (Saksena et al. 2014). India faces a complex situation. On the one hand, it has extreme utilisation of newer generation of antibiotics in urban areas and in the private sector, which is potentially prompting AMR. On the other hand, there is poor access to antibiotics in rural areas leading to avoidable morbidity and premature mortality. Alsan et al. have demonstrated that OoP health expenditures were strongly correlated with antimicrobial resistance in LMICs (Alsan et al. 2015). The economic damage of unlimited antimicrobial resistance could be comparable to the shocks experienced during the 2008–2009 global financial crisis because of drastically expanded medical expenditures; impact on food and feed production, trade, and livelihoods; and eventually increased poverty and disparity. In higherincome countries, a package of simple interventions to address antimicrobial resistance could pay for itself due to costs averted. In lower-income countries, additional but still relatively modest investments are urgently needed. Looking at this global urge to contain AMR, the United Nation has come up with the goal of strengthening One Health, and multisectoral actions to address antimicrobial resistance (AMR); learn from the COVID-19 pandemic (2020–2021) to tackle the growing threat of AMR, which has been referred to as a ͞‘silent tsunami’; and deliver on the 2030 Agenda for Sustainable Development and commitment of actions at the global, regional, and national levels have been implemented. This includes keeping AMR high on the political agenda, building awareness, and understanding and strengthening coordination, political leadership, and collaboration on AMR actions, addressing the growing global threat of AMR in all countries through a coordinated, multisectoral, inclusive One Health approach to contribute to the achievement, encouraging all member states to have a multisectoral AMR national action plan, working towards sufficient and sustainable funding for AMR-specific and AMR-sensitive actions, developing global and national economic studies, as appropriate, that account for addressing AMR across One Health, incentivising and

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prioritising investment needs. Also, development and sustainable strong partnerships with the relevant private sector, investors, central and development banks, academia, and research institutions are required to tackle AMR by incentivising AMR financing, research and development, innovation, and equitable access.

1.4

Currently Available Therapies

Antimicrobial drug discovery has always been considered as one of the most relevant discoveries of the twentieth century. Identifying and exploring new potential antimicrobial targets and exploring new chemical entities as antimicrobial drugs are in great demand. Although the spread of the antimicrobial drugs formally started with the antimicrobial era, in the pre-antimicrobial era, numerous remedies belonging to the traditional medicine like antimicrobial activity of herbs and plants have constantly put microorganisms under selective pressure and are still being used to fuel the pipeline of antimicrobials used by mainstream medicine. The origin of antibiotic era is associated with names of Paul Ehrlich, who discovered that a chemical called arsphenamine was an effective treatment for syphilis. He named it ‘magic bullet’ as it selectively targeted only disease-causing microbes and not the host, and Alexander Fleming, to be credited for the serendipitous finding of penicillin, later to be claimed as the miracle drug of the twentieth century (Aminov 2010). Since then, plenty of antibiotics have been discovered contributing to the control of infectious diseases that were the leading causes of human morbidity and mortality for most of human existence. However, over the last 30 years, antibacterial drug discovery has faced a discovery gap, and antibiotic resistance has accelerated its pace during the same time leading to more severe spread of MDR bacterial diseases. During World War II, pharmacologic agents such as weak acids, phenolic dyes, and undecylenic acid were the only available treatment for fungal infections. The status of antifungal therapy changed dramatically in the late 1950s with the introduction of newer broader-spectrum agents, such as the amphotericin B, iodinated trichlorophenols, and the imidazoles, that acted by disruption of the fungal cell membrane. Regardless of widespread use, however, these agents became subject to various clinically significant limitations identified with their suboptimal spectrum of activity, the emergence of resistance, the induction of dangerous drug-drug interactions, their less than optimal pharmacokinetic profile (itraconazole capsules), and toxicity (Maertens 2004). Immunotherapy is another potent therapy available for antimicrobial infections. The dawn of vaccination is linked with remark discovery of smallpox vaccine by Edward Jenner in 1796 which eventually culminated in its global eradication in 1979. Louis Pasteur’s research headed the improvement of live attenuated cholera vaccine and inactivated anthrax vaccine in 1897 and 1904, respectively. Also, viral tissue culture practices in the mid-late twentieth century (from 1950 to 1985) have led to the development of the Salk (inactivated) polio vaccine and the Sabin (live attenuated oral) polio vaccine. Mass polio vaccination has now eradicated the disease from numerous regions across the globe (Plotkin

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2014). Besides these successful events of vaccination, a bitter truth is that some vaccines like Bacillus Calmette-Guerin (BCG) against tuberculosis (TB), the only available vaccine, though proved to protect against childhood TB, have not performed up to the mark in case of adult TB and hence more potent therapy is required to tackle the deadly infection (Andersen and Doherty 2005). Nevertheless, the rapid emergence of mutant strains poses a major obstacle in development of vaccine.

1.5

Need for Alternative Therapies

The twentieth century is considered as the golden period for antimicrobial therapy. However, the dawn of the antimicrobial era has sadly corresponded to the rise of the phenomenon of antimicrobial resistance in hospitals, communities, and the environment concomitant with their use. Antimicrobial resistance (AMR) is a natural process whereby microbes evolve in such a way to withstand the action of drugs. The effective use of any therapeutic agent is limited by the potential emergence of tolerance or resistance in pathogen to that compound from the time of its first administration. This is true for drugs used in the treatment of bacterial, fungal, parasitic, and viral infections. Misuse, overuse, and incomplete course of antimicrobial therapy are the major factors accelerating the development and spread of AMR. Fleming had predicted that the insensitive utilisation of the discovery could prompt the determination and proliferation of selected mutants of bacteria resistant to antibiotics. Indeed, alarming signs of developing resistance were reported just after a few years of the golden age of antimicrobials in countries of all income levels (Spellberg and Gilbert 2014). AMR has emerged as one of the most important global threats of the twenty-first century, posing serious dangers to humans, animals, and the environment. Infections due to antimicrobial resistance are one of the top reasons of deaths worldwide. There is no time to wait. Unless the world takes no essential actions, antimicrobial resistance will have a terrible impact within a generation. Antimicrobial resistances are developing faster than antimicrobial production, with the result that common diseases are becoming untreatable and lifesaving medical procedures riskier to perform. Hence, promising alternative or non-conventional approaches with potential benefits such as a broad spectrum of efficacy and greater potency are required to tackle deadly MDR diseases.

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Nanotechnology to Combat MDR

Antimicrobials can be synthetic, semisynthetic, or natural in nature (i.e. from plants and animals). However, antimicrobial overuse has resulted in the development of multidrug-resistant bacteria, which is one of the most difficult situations for healthcare providers. The spread of resistant organisms is the main issue with the development of antibiotic resistance. Antimicrobial resistance is being combated by replacing traditional antimicrobials with new technology. Nanoscale materials

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Fig. 1.1 Different route for the synthesis of nanoparticles

marked a new hope for combating drug resistance (Rudramurthy et al. 2016). Nanomaterials hold an enormous promise in both the medical fields. To combat microbial pathogens, several nanostructures containing metallic particles have been developed. The development of effective nanomaterials necessitates a thorough understanding of the physicochemical properties of NPs as well as the biological aspects of microorganisms. The risks of using NPs in healthcare must be addressed. Nanoparticles (NPs) with a median size of 1 to 100 nm have unique qualities due to their nano-chemical and physical characteristics, notably in terms of chemical and physical composition (Sardar et al. 2014). Depending on their elemental structure, surface area, and coating agent composition, nanoparticles can exhibit a variety of physical and chemical properties (Ahmad et al. 2013; Sadaf et al. 2020). Nanoparticles exhibit a variety of unusual properties, including mechanical properties (Mishra et al. 2015; Gao et al. 2020), antimicrobial activity (Ahmad et al. 2014a; Ahmad et al. 2015; Abdulla et al. 2021; Goel et al. 2021b), antiviral activity (Goel et al. 2021a; Zeyaullah et al. 2021a; Zeyaullah et al. 2021b), drug delivery capacity (Ahmad et al. 2021; Srivastava et al. 2021), optical properties (Albrecht et al. 2018), catalytic activity (Mishra et al. 2016; Liu and Liu 2017; Perwez et al. 2017; Ahmad and Khare 2018; Ghosh et al. 2021a; Ghosh et al. 2021b), and refolding of protein (Ahmad et al. 2014b; Ghosh et al. 2019). Nanomaterials can be made in a variety of ways, including chemical, physical, and biological approaches, depicted in Fig. 1.1. Physical and chemical approaches are often costly, take more time and energy to complete, involve complicated operations, and also produce harmful by-products (Castro et al. 2014; Ngoepe et al. 2020). Biological synthesis, on the other hand, is a more sustainable, cost-effective, and gentle process than chemical or physical methods (Mishra et al. 2013; Khatoon et al. 2015; Mazumder et al. 2016; Jacob et al. 2020). Furthermore, biological synthesis provides

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the ability to manipulate the desired characteristics and limits the size and form of desired molecules (Castillo-Henriquez et al. 2020). This interest has focused on organisms such as bacteria, actinomyces, fungi, viruses, and algae which produced secondary metabolite act as reducing or stabilising agents for synthesis of nanoparticles (Ghosh et al. 2021b). These days, nanomaterials have been used in various fields like optical, chemical, and biological. Superconductors, catalysts, optical devices, gene and drug delivery, fuel cells, cell and tissue imaging, and biosensors are just a few examples of the nanoparticle potential applications (Adibkia et al. 2007; Tiwari et al. 2011; Zinjarde 2012; Bahrami et al. 2014; Maleki Dizaj et al. 2015). Nanoparticles have intrinsic properties to kill the microbes and have ability in diagnostic immunoassays (Nam et al. 2003; Chen et al. 2008; Osterfeld et al. 2008). Different research groups have developed and evaluated various types of NPs, including various metal and metal oxides, to demonstrate the antimicrobial effect examples which include silver, gold, magnesium oxide, zinc oxide, titanium dioxide, magnesium oxide, copper oxide, silicon dioxide nanoparticles, etc. (Ahmad et al. 2015; Khatoon et al. 2015; Maleki Dizaj et al. 2015; Mazumder et al. 2016; Ghosh et al. 2021a; Ghosh et al. 2021b; Goel et al. 2021b). NPs act on microbes in a variety of ways, and the mode of action of these NPs varies depending on the type (Table 1.2 and Fig. 1.2).

3

Quorum Sensing and Biofilms

Microorganisms have evolved to contain quite complex behavioural and developmental systems to ensure their progressive existence. These microbes need to compete and cooperate to meet different environmental challenges. To effectively comply, microbes can differentiate into alternate modes of life triggered by sensing various environmental/unfavourable conditions, such as the free-living form to surface-attached communities (Nadell et al. 2016). The attachment of microbes to the surface, either abiotic or biotic, leads to the formation of thick exopolysaccharide (EPS) layer, a process known as biofilm formation. The biofilm EPS layer is composed of mainly proteins, polysaccharides, lipids, and eDNA (Goel et al. 2021c). The microbes have evolutionarily developed the trait to sense the density of the other microorganisms residing in their environs via the production of chemical signals known as autoinducers. The attribute is called quorum sensing (QS), an interconnection with the formation of biofilm (Kindler et al. 2019). Once the autoinducers threshold value crossed that corresponds to a high population of local bacteria, a mutual response is triggered, leading to biofilm formation (Fancher and Mugler 2017). The autoinducers released help the bacteria regulate the expression of various genes involved in virulence, pathogenicity, competition, resistance, biofilm formation, and dispersal. QS also regulates the synthesis of surfactants such as rhamnolipids, which help maintain the channels for distributing nutrients and oxygen and help remove wastage. The extracellular DNA (eDNA) released upon autolysis of subpopulation of bacteria via these channels is an important component of EPS biofilm that helps later during the maturation of biofilm (Yan and Wu 2019).

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Table 1.2 Mode of action of different nanoparticles against pathogenic microbes (Reprint from Rudramurthy et al. (2016)) Type of nanoparticles Silver (Ag)

Magnesium oxide (MgO)

Titanium dioxide (TiO2)

Zinc oxide (ZnO)

Gold (Au)

Copper oxide (CuO)

Iron

Mode of action Interfere with the electron transport chain and transfer of energy through the membrane Inhibit DNA replication and respiratory chain in bacteria and fungi Formation of reactive oxygen species (ROS), lipid peroxidation, electrostatic interaction, alkaline effect Formation of superoxide radicals, ROS, and site-specific DNA damage Hydrogen peroxide generated on the surface of ZnO penetrates the bacterial cells and effectively inhibits growth Zn2+ ions released from the nanoparticles damage the cell membrane and interact with intracellular components Generate holes in the cell wall Bind to the DNA and inhibit the transcription process Reduce bacteria at the cell wall Disrupt the biochemical processes inside bacterial cells Through ROS-generated oxidative stress. ROS, superoxide radicals (O2-), singlet oxygen (1O2), hydroxyl radicals

Susceptible microbes Methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis Vancomycin-resistant Enterococcus faecium and Klebsiella pneumoniae S. aureus, E. coli, Bacillus megaterium, Bacillus subtilis

References Morones et al. 2005; Rai and Bai 2014; Franci et al. 2015; Khatoon et al. 2015

E. coli, S. aureus, and against fungi

Hirakawa et al. 2004; Wong et al. 2013; Ahmad et al. 2014a; Besinis et al. 2014; Ahmad et al. 2015 Yamamoto 2001; Brayner et al. 2006; Padmavathy and Vijayaraghavan 2008; Liu et al. 2009; Tayel et al. 2011

E. coli, Listeria monocytogenes, Salmonella, and S. aureus

Koper et al. 2002; Krishnamoorthy et al. 2012

Methicillin-resistant S. aureus

Gil-Tomás et al. 2007; Perni et al. 2009; Pissuwan et al. 2010; Rai et al. 2010

B. subtilis, S. aureus, and E. coli

Ruparelia et al. 2008; Raffi et al. 2010; Chatterjee et al. 2012; Sampath et al. 2014

S. aureus, S. epidermidis, and E. coli

Behera et al. 2012

(continued)

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Table 1.2 (continued) Type of nanoparticles

Aluminium (Al) Bismuth (Bi)

Carbonbased

Mode of action (OH-), and hydrogen peroxide (H2O2) Disrupt cell walls through ROS Alter the Krebs cycle and amino acid and nucleotide metabolism Severe damage to the bacterial membrane, physical interaction, inhibition of energy metabolism, and impairment of the respiratory chain

Susceptible microbes

References

E. coli

Ruparelia et al. 2008; Sadiq et al. 2009 Luo et al. 2013; Nazari et al. 2014

Multiple antibiotic -esistant Helicobacter pylori E. coli, Salmonella enteric, E. faecium, Streptococcus spp., Shewanella oneidensis, Acinetobacter baumannii, Burkholderia cepacia, Yersinia pestis, and K. pneumonia

Kang et al. 2008; Yang et al. 2010; Dong et al. 2012; Leid et al. 2012; Shvedova et al. 2012

Fig. 1.2 Nanoparticle effect on microbial cell mechanism

The mechanism of biofilm formation usually follows four steps: initial attachment to the surface, formation of discrete microcolonies, maturation of biofilm followed by dispersal after perceiving signals such as nutrient limitation, and poor oxygen availability depicted in Fig. 1.3 (Goel et al. 2021c). The two systems, QS and cyclic di-GMP (c-di-GMP), play a fundamental role in establishing the biofilm and are discussed in detail.

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Fig. 1.3 Four stages involved in the formation of biofilm

3.1

QS Network

QS is the intercellular communication system dependent on QS autoinducer concentration and cell density (Lin Chua et al. 2017). The QS bacteria generally produce three classes of autoinducers, acyl-homoserine lactones (AHLs) produced by Gramnegative bacteria, auto-inducing peptides by Gram-positive bacteria, and autoinducer-2 (AI-2), which usually acts as non-species-specific molecules produced irrespective of Gram-negative or Gram-positive bacteria to establish the intra- and inter-species communication. In addition to these common QS signalling molecules, several bacteria species produce distinct autoinducers (Suresh et al. 2021). The QS network is well studied in Pseudomonas aeruginosa which has three QS system: (i) LasI-LasR system which produces N-(3-oxo-dodecanoyl)-Lhomoserine lactone (3OC12-HL) which acts on LasR, (ii) RhlI-RhlR system which produces N-(butyryl)-L-homoserine lactone (BHL), and (iii) Pseudomonas quinolone signal (PQS)-based QS, PqsABCDH-PqsR system that is related to the synthesis and the use of 2-heptyl-3-hydroxy-4-quinolone (HHQ). The first two QS systems essentially are N-acylated homoserine lactone (AHL)-based QS systems and exist in many bacteria (Pena et al. 2019; Yan and Wu 2019). Many transcriptomic studies were conducted to infer the gene regulation at various conditions, of which 94 experimental data showed that 30 studies had no effect on the three QS systems with each

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other and 29 showed good correlation between LasI-LasR and RhlI-RhlR. In contrast, a weak correlation was observed in the case of the PQS system with both LasI-LasR and RhlI-RhlR. These transcriptomic studies are conducted to explore the interplay between these three systems and hypothesise whether the biofilm formation can be reversed by inhibiting or downregulating the synthesis of autoinducers and signalling molecules (Yan and Wu 2019).

Cyclic di-GMP (c-di-GMP) The switch between biofilm and planktonic state in P. aeruginosa, like many bacteria, is controlled by a secondary messenger molecule, c-di-GMP. C-di-GMP is synthesised by diguanylate cyclases (DGCs) containing the GGDEF domain and degraded by phosphodiesterases (PDEs) containing the EAL or HD-GYP domain (Fu et al. 2021). A high level of c-di-GMP molecule favours biofilm formation by inhibiting ATPase activity. Therefore, FleQ cannot regulate the downstream flagella genes responsible for synthesising flagella, thereby restricting mobility and hence biofilm formation, whereas a low level of intracellular c-di-GMP is accountable for biofilm dispersal and return to the planktonic state (Lin Chua et al. 2017; Fu et al. 2021). Lin Chua et al. (2017) studied the transcriptomic profiles at low and high intracellular levels of c-di-GMP. The authors concluded that to overcome the stress and prevent themselves from phagocytosis, an immune response, low levels of c-diGMP induce both QS systems: pqs and rhl. PqsR acts as a mediator to activate both the QS systems that eventually lead to increased production of pyocyanin and rhamnolipids correlated to higher virulence and mobility (Lin Chua et al. 2017).

3.2

Biofilm-Associated Antimicrobial Resistance

Bacteria in the biofilm are more resistant to antibiotics than bacteria growing planktonically. The thick layer of EPS renders protection to the bacteria residing in the biofilm against exposure to the antimicrobials. EPS acts as a physical barrier preventing antibiotic penetration, and also negative components of EPS can bind to the positive antibiotics, for instance, aminoglycosides, resulting in decreased diffusion of the antibiotics (Sherrard et al. 2014). The gradient of nutrients and oxygen available developed deep within the biofilm results in non-growing populations that are highly drug-resistant (Sánchez et al. 2019). In addition, the β-lactam class of antibiotics acts on actively dividing microbial cells, but the bacteria in biofilm are metabolically inactive (Sherrard et al. 2014), as depicted in Fig. 1.4. Also, heterogeneous communities of bacteria exist in the biofilms, so there are chances of uptake of eDNA by susceptible bacteria released from resistant bacteria carrying the resistant genes. This is known as a horizontal gene transfer mechanism that results in resistance in sensitive bacteria after acquiring resistant genes (Goel et al. 2021c). The QS in biofilm also contributes to increased virulence, biofilm formation, and restricted mobility. The efficacy of the antibiotics is affected by the concentration that reaches the target due to compromised penetration of antibiotics in the biofilm, subinhibitory concentration reaches, that has low or no effect on the bacteria

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Fig. 1.4 Biofilm and antimicrobial resistance (Sherrard et al. 2014)

growing inside the biofilm (Sherrard et al. 2014). In continuation with the efforts to combat antimicrobial resistance, researchers are trying to inhibit early-stage biofilms in addition to the discovery of antibiotics to target drug-resistant bacteria.

4

Strategies to Inhibit the Biofilm Formation

The biofilm inhibition can be targeted at various steps of its formation such as (1) early stage of biofilm targeting the pili and fimbriae, bacterial appendages, and modifying the surface of attachment; (2) inhibiting the production of EPS by reducing production of exopolysaccharides, proteins, and eDNA; and (3) inhibiting the QS system by blocking and degrading the secondary molecule, preventing binding of the secondary molecule to the corresponding receptors.

4.1

Targeting Initial Stages of Biofilm

As mentioned in the mechanism of biofilm formation, the first step is attachment to the surface which is reversible during the early stages of biofilm formation. The bacteria adherence to the surface is weak, and EPS secretion has not started yet. Bacteria instability at the surface makes it easy to return to the planktonic stage,

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preventing biofilm development. The surface properties and bacterial appendages such as pili and fimbriae affect the bacterial adhesion to the surface (Fu et al. 2021).

4.2

Surface Coating

Applying a hydrophilic coating to the surface reduces bacterial attachment due to the repulsive effect as the surface of the bacteria has hydrophobic properties. 2-Methacryloyloxy ethyl phosphorylcholine (MPC) has been explored and found that MPC-coated polymer has significantly inhibited the adhesion of Staphylococcus aureus and reduced surgical site infections. In addition, antibacterial agents and antimicrobial peptides (AMPs) are coated on the surface to kill attached microorganisms directly (Kaneko et al. 2020). Wang et al. (2016) reported silver nanoparticle-functionalised titanium surfaces to inhibit the biofilm of Staphylococcus epidermidis (S. epidermidis, RP62A) and Staphylococcus aureus (S. aureus, USA 300). Real-time PCR (RT-PCR) data suggested that Ag-0.01 surfaces downregulated the expression of icaA while upregulating the transcription level of icaR, and both genes help regulate the biofilm formation in S. epidermidis. Also, Ag-0.01 surfaces downregulated fnbA and fnbB expression in S. aureus biofilm, and fnbA and fnbB genes are required for FnBP-mediated biofilm development (Wang et al. 2016).

4.3

Affecting Pili and Flagella

Pili and flagella are cell surface appendages that help bacteria adhere to the surface by overcoming surface tension. Flagella act as a bacterial adhesin promoting bacterial adhesion. Three types of pili are well-studied: type I, type IV, and curli (AritaMorioka et al. 2018). Type I are functional amyloids that primarily help bacteria in the attachment to the surface; type IV are motility appendages (also, twitching motility) that plays a critical role in microcolony formation; and curli are major extracellular surface appendages that help in cell-to-cell contact and bacterial attachment (Fu et al. 2021). Recently, the compounds 1-amino-4-hydroxyanthraquinone (Disperse Red 15 or DR15) and 4-(4′-chloro-4-biphenylylsulfonylamino) benzoic acid (CB1) were reported inhibiting the biofilm formation at initial stages without affecting the planktonic growth of E. coli. The compounds were inhibiting the FimH adhesin of type I pili; the coating did not leach out when incubated with artificial urine samples (Miryala et al. 2020).

4.4

Inhibiting the Production of EPS

EPSs allow the irreversible attachment of bacteria to the surfaces by promoting cohesion with each other. The irreversible attachment is enhanced by producing

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various polysaccharides, proteins, eDNA, lipids, and other biomolecules. The maturation of the biofilm at this stage can be inhibited using anti-EPS molecules, as discussed further.

4.5

Reducing Polysaccharide Production

Exopolysaccharides are the significant components of the EPS layer of the biofilm that act as a molecular glue providing stability and help maintain the biofilm’s integrity (Goel et al. 2021c). Many commercial antibiotics, natural source-derived compounds, antimicrobial peptides, and probiotics are reported to inhibit the production of the polysaccharide. Chen et al. (2016) reported 5H6 (2-(4-chlorophenyl)4-[[(6-methyl-2-pyridinyl)amino]methylene]-1,3-oxazol-5(4H)-one) that reduced the biofilm maturation of Streptococcus mutans biofilm. Glucosyltransferase (Gtf) enzymes synthesise extracellular polysaccharides (EPSs), and 5H6 inhibited the Gtf activity, hence reducing biofilm maturation (Chen et al. 2016). Shang et al. (2021) in a recent study demonstrated the activity of Trp-containing peptides against biofilm formation of multidrug-resistant P. aeruginosa. The peptide has inhibited the biofilm formation by downregulating the pelA, algD, and pslA transcription associated with the exopolysaccharides production (Shang et al. 2021).

4.6

Reducing Matrix Protein Production

Matrix proteins are well known for their contribution in providing stability and biofilm formation. Mutational studies revealed that the absence of matrix proteins led to reduction of biofilm formation and altered biofilm structures. In Vibrio cholerae, three major proteins are studied, namely, RbmA, Bap1, and RbmC, that are important for biofilm formation which helps in cell-to-cell binding, scaffolding, and adhesion on abiotic surfaces. Some proteins secreted out have enzymatic activity such as proteases, DNases, and glycosyl hydrolase dispersin B that hydrolyses EPS matrix components facilitating biofilm matrix degradation and dispersal (Fong and Yildiz 2015). Parrino et al. (2021) reported 1,2,4-oxadiazole topsentin analogues that showed inhibition of sortase A (Srt A), a transpeptidase enzyme with 50% biofilm inhibitory concentrations (BIC50s) below 10 μM for the most active compounds. Srt A is a membrane enzyme that helps in adhesion of matrix molecules to the peptidoglycan cell wall in Gram-positive strains (Parrino et al. 2021).

4.7

Reducing eDNA Production

eDNA aids adhesion by reducing the repulsive force exerted by the surface molecule charges towards the bacterial cell membrane. It also provides structural stability and guides motility in biofilms via interactions with other components such as type IV pili. The role of eDNA was also studied during host immune response, and it was

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observed that eDNA chelates divalent cations, which trigger genetic response to increasing pathogenicity and resistance to antimicrobials (Okshevsky and Meyer 2015). Inhibiting eDNA could help in inhibiting biofilm formation at the initial stages. Karygianni et al. (2020) studied the combined effect of enzymes proteinase K and DNase I on Actinomyces oris, Fusobacterium nucleatum, Streptococcus mutans, Streptococcus oralis, and Candida albicans biofilm. The presence of Proteinase k and DNAse I alone or in combination had an effect on the overall bacterial count and the structural integrity of the biofilms. For antimicrobial effects, antibiotics with enzymes could be a promising approach to help combat antimicrobial resistance (Karygianni et al. 2020).

4.8

Inhibiting QS System

As discussed, the role of QS system in mediating biofilm maturation, inhibiting and degrading the signalling molecules such as c-di-GMP, and blocking interaction of the QS molecules with the corresponding receptors could provide better insights in inhibiting the biofilm formation and maturation. Malešević et al. (2019)) studied the effect of Delftia tsuruhatensis 11304 ethyl acetate extract on P. aeruginosa MMA83 biofilm, resulting in biofilm inhibition formation but no effect on preformed biofilms. QSI extract with meropenem and gentamycin reduced the production of virulence factors, elastase, rhamnolipid, and pyocyanin, and significant downregulation of lasI, lasR, rhlI, rhlR, pqs, and mvfR expression was also observed (Malešević et al. 2019). Recently, researchers are trying to exploit the already approved FDA against biofilm formation. Aminoglycosides, a commonly used class of antibiotics, exhibit biofilm inhibition by targeting the QS regulatory protein LasR in P. aeruginosa (Zhou et al. 2020). Recently, SM23, a boronic acid derivate designed as β-lactamase inhibitor, inhibited biofilm formation of P. aeruginosa and production of virulence factors, pyoverdine, elastase, and pyocyanin, without affecting bacterial growth. Also, it resulted in a reduction in levels of 3-oxo-C12-HSL and C4-HSL, two QS-related autoinducer molecules of lasR/lasI system (Peppoloni et al. 2020).

4.9

Anti-Virulence Compounds

Compounds that target the pathways necessary for pathogenesis but not for microbial growth are referred to as anti-virulence therapy. Because these chemicals only block virulence components without affecting pathogen survival, they impart weaker selection for resistance than traditional antibiotics, minimising the chance of resistance insurgence (Allen et al. 2014; Vale et al. 2014). Another significant benefit of anti-virulence therapy is that it has a less harmful effect on the host microbiome, avoiding the substantial side effects of antibiotic therapy (Buroni and Chiarelli 2020). However, the treatment causes inadequate pathogen clearance, which might be especially troublesome for immunocompromised patients (those

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with AIDS, cancer, cystic fibrosis, and those in critical care units), whose immune systems are presumably too weak to eliminate even disarming bacteria. Combining anti-virulence chemicals with antibiotics to benefit from both virulence inhibition and successful pathogen elimination might solve the problem (Vale et al. 2014; Brown 2015; Dickey et al. 2017). Furthermore, when regulatory mechanisms are involved, treatment timing must correspond with the length of the target factor’s activity. This necessitates a thorough understanding of the virulence factor’s mechanism throughout the pathogenesis phase. Furthermore, the expression of virulence factors might fluctuate depending on the infection location. Currently, there is a lack of complete knowledge on how different types of antibiotics and anti-virulence agents interact, whether interactions are primarily synergistic or antagonistic, and how combinatorial therapies impact antibiotic resistance strain development and spread. As a result, the translation of preclinical findings into clinical practise is hampered. Rezzoagli et al. revealed in a recent study that combining antibiotics with anti-virulence agents can have synergistic effects and reverse antibiotic resistance selection in Pseudomonas aeruginosa (Rezzoagli et al. 2020). Quorum sensing, a communication system widely used by bacteria to regulate the expression of many virulence factors (including the production of toxins, siderophores, proteases, and immune-evasion factors), as well as biofilm formation, which renders bacteria much more resistant to antibiotic action, is an important target in the development of antivirulence molecules (Papenfort and Bassler 2016). As a result, quorum quenching or quorum sensing blocking can decrease numerous virulence features at the same time, leaving bacteria harmless to their hosts. Most anti-virulence medications are still in the early stages of development, and clinical trials have yet to be completed (Dickey et al. 2017).

4.10

Phage Therapy

Phage therapy is a term used to describe the use of bacteriophage viruses to treat bacterial illnesses. It has been in use for more than a hundred years. Viruses that can infect both Gram-positive and Gram-negative bacteria are known as phages. From the first successful delivery of phage to treat infants with Shiga dysentery in 1921, to its widespread usage in people throughout the world, the usefulness of phage treatment has been hotly debated (Summers 2012). The golden age of antibiotics began in 1945, and phage products were taken off the market. The impending death of all existing antibiotics, on the other hand, has rekindled interest in phage therapy. Antibiotic resistance is usually unaffected, and unlike other antibiotics, they may target bacteria trapped in biofilms (Forti et al. 2018; Hansen et al. 2019). There are more than 1031 phages in the biosphere, which may be easily separated from soil, bodies of water, dung, and sewage (Suttle 2007). The majority of phages identified contain double-stranded DNA genomes and have non-enveloped icosahedral head and tail structures. Phages provide an endless supply of antibacterial drugs against all human bacterial infections, owing to their large abundance, great variety, and relative simplicity of separation. Phages precisely destroy bacteria while causing

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no damage to other creatures. Phage treatment has the benefit of self-regulating at the site of infection and decaying once the pathogenic bacteria have been destroyed. The disadvantage is bacterial and phage co-evolution, which is vital to consider when new harmful bacterial pathogens emerge and new demanding conditions occur. Despite its safety and success, phage therapy clinical functioning still requires precise criteria. The endeavour is made more difficult by information gaps in the areas of acceptable routes of administration, phage selection, frequency of administration, dose, phage resistance, and phage pharmacokinetic and pharmacodynamic features. Furthermore, phages necessitate a careful examination of the immune response they may elicit. Most current human data have been generated on a single-patient compassionate use basis due to a lack of well-controlled clinical trial data and complicated regulatory frameworks. These findings promote further investigation of the phage-antibiotic combination, which appears to be a potential option for clinical development in the near future. With the present antibiotic crisis, phage treatment has the potential to reduce the ever-increasing problem of infectious diseases, either as an alternative to antibiotics or in conjunction with medicines.

4.11

Antimicrobial Peptides

Antimicrobial treatments are facing a severe problem because of the increasing rise of drug-resistant infections. The inability of the most powerful antibiotics to destroy many pathogens including recent emergence of superbugs highlights the urgent need for new control agents to be developed. Antimicrobial peptides (AMPs) are a rising class of natural and synthetic peptides with a broad range of targets including viruses, bacteria, fungi, and parasites (Bahar and Ren 2013). AMPs derived from synthetic and natural sources have broad-spectrum antibacterial action with excellent specificity and minimal toxicity (Zhang et al. 2021). The AMP database [Data Repository of Antimicrobial Peptides (DRAMP) [http://dramp.cpu-bioinfor.org/] includes 22,259 entries as of March 1, 2022, which includes 5891 general AMPs (including natural and synthesised AMPs); 16,110 patent AMPs; and 77 AMPs in therapeutic development (preclinical or clinical stage). Four thousand one hundred two antibacterial, 1816 antifungal, 5485 antimicrobial, 5550 insecticidal, 5485 antiviral, 5485 antiprotozoal, and 5491 antiparasitic are among the entries in DRAMP. When it comes to the antimicrobial actions of AMPs, bacteria vary in their intrinsic sensitivity and resistance mechanisms to these peptides. In general, direct competition between bacterial species, as well as host-pathogen interactions, promotes the evolution of AMP resistance mechanisms. Several studies have revealed various mechanisms of bacterial resistance to AMPs, including the production of proteases and trapping proteins, the modification of cell surface charge, membrane fluidity, and efflux pump activation, and the use of biofilms and exopolymers, as well as the development of sensing systems through selective gene expression (Moravej et al. 2018). AMP research has been driven by the pressing demand for novel antimicrobials. More AMPs may reach clinical testing

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and therapy in the near future, thanks to significant advances in related understanding and lead compounds.

5

Conclusion

Only two antibiotics of the eight approved since 2017 represent a new chemical scaffold. The remaining antibiotics are derivatives of existing classes of compounds that bring benefits and advantages over traditional antibiotics. The eight new antibiotics all have activities against ESBL (extended spectrum β-lactamase) enzymes; most of them are effective against carbapenem-resistant Enterobacteria (KPC producers), while very few compounds are active against carbapenemresistant P. aeruginosa and multidrug-resistant A. baumannii. Unfortunately, there are still an extremely limited number of therapeutic alternatives for the latter. These antibiotics are mainly used in the treatment of cUTI and cIAI. Further scientific evidence is needed to assess their actual effectiveness in the treatment of other infections. The WHO Model List of Essential Medicines includes the combination of vaborbactam, meropenem, and plazomicin. There is a significant progress in research. The number of new effective antibiotics against Gram-negative bacteria has increased. Majority of the drugs authorised or under clinical development from 2017 to date, whose targets are pathogens on the WHO’s 2016 list (critical priority, high priority, and medium priority), are combinations of a-lactam and a-lactamase inhibitor. Cefiderocol is the only antibiotic that is active against all three pathogens of critical priority, along with the compound called SPR-206 phase I (an analogue of polymyxins with an excellent antibacterial spectrum). At the end of 2020, there were 43 antibiotics in clinical development, of which 15 were phase I, 13 in phase II, and 13 in phase III. As many as 19 antibiotics are shown to be effective in vitro in the treatment of infections caused by pathogens of the so-called ESKAPE group, an acronym that includes the Enterococcus faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter species, responsible for the six main nosocomial infections related to care. It is, of course, essential that the new antibiotics developed do not have cross-resistance with other existing compounds. In fact, the search for new antibacterial drugs that result from the modification of traditional antibiotics is also based on knowledge of cross-resistance mechanisms. However, finding innovative chemical structures with new targets and binding sites is very difficult and yields fewer results than other approaches. In addition to the small and large molecules that have been analysed in this review, there are other potentially effective non-traditional approaches such as faecal bacteriotherapy (also called faecal microbiome transplantation) in the treatment of recurrent C. difficile infections. Other non-traditional approaches (such as immunomodulators and phage products) have not yet entered clinical development due to considerable obstacles. Unfortunately, unfavourable market trends remain: Although public investments in the development of new antibiotics have increased slightly in recent years (mainly from Germany, the United Kingdom, and the United States, thanks to organisations such as BARDA, CARB-X, and GARDP), private investment has fallen further.

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Many pharmaceutical companies are abandoning research in this area, not least because of the high costs involved in the clinical development of a new antibiotic. In view of the rather long time required for clinical development, 11 new antibiotics are expected to be approved in the next 5 years, while many compounds are likely to remain stagnant in phases II and III due to the costs involved.

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The Role of Advanced Therapeutic Techniques to Combat Multi-drug Resistance Musa Marimani, Aijaz Ahmad, and Adriano Duse

Abstract

A wide variety of microbial pathogens use distinct mechanisms to evade selection pressure and subsequently develop drug resistance towards conventional drugs. Ultimately, this leads to survival and proliferation of drug-resistant microorganisms that cause diseases and remain untreatable even after applying recommended conventional therapies. Globally, this puts an enormous burden in public and private clinical institutions as a large number of patients succumb to diseases induced by drug-resistant mutant pathogens. Traditionally, antimicrobial drugs are designed to target and suppress key molecular factors that are critical during microbial entry, penetration, infection and replication. These factors mainly include DNA, RNA and virulence proteins, which are employed as therapeutic targets to treat human diseases. Unfortunately, the presence of various drug-resistant pathogens has rendered this method ineffective. Accordingly, different non-traditional mechanisms have been tested to combat diseases caused by drug-resistant strains. It is against this background that this book chapter will discuss the antimicrobial activity and benefits of applying some of these non-traditional therapeutic techniques to treat pernicious human diseases caused by drug-resistant microorganisms.

M. Marimani Anatomical Pathology, School of Pathology, Health Sciences, University of the Witwatersrand, Johannesburg, South Africa A. Ahmad · A. Duse (✉) Clinical Microbiology and Infectious Diseases, School of Pathology, Health Sciences, University of the Witwatersrand, Johannesburg, South Africa Infection Control, Charlotte Maxeke Johannesburg Academic Hospital, National Health Laboratory Service, Johannesburg, South Africa e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. Y. Wani, A. Ahmad (eds.), Non-traditional Approaches to Combat Antimicrobial Drug Resistance, https://doi.org/10.1007/978-981-19-9167-7_2

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Keywords

Drug resistance · Conventional drugs · Non-traditional approaches

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Introduction

1.1

Non-traditional Approaches to Counter Multi-drug Resistance

Bacteriophage-Based Strategy Bacteriophages or phages are viral agents capable of infecting bacteria. These obligate intracellular parasites are employed to control bacterial infections. Bacteriophages are the most abundant microbial agents and offer an alternative mechanism of treating bacterial diseases (Alvi et al. 2020). As a therapeutic strategy, phages are beneficial over antibiotics in that they confer improved specificity, they are self-replicating within bacterial cells, their activity depends on the presence of bacteria, they have low toxicity (Wright et al. 2009; Sarker et al. 2012), they often do not affect the human normal flora population and they are highly specific. Furthermore, phage therapy is cheaper compared to production of antibiotics as bacteriophages are abundant in nature and their preparation methods are costeffective. However, the major setback associated with phage therapy is that most bacteriophages are only effective against specific bacterial pathogens. Nevertheless, appreciable decreases in bacterial replication have been achieved after employing phage therapy. These promising results were obtained following application of normal or modified viral agents (Sabitova et al. 2018; Dedrick et al. 2019; Bao et al. 2020; Luong et al. 2020). Phages may display various lifestyles depending on their infectivity (Hobbs and Abedon 2016). The virulent or obligately lytic phages hijack the host cellular processes and initiate replication. This results in the production of novel virions capable of killing bacterial cells. By contrast, temperate or lysogenic bacteriophages integrate into the host cell genome to initiate replication. Generation of specific cellular signals prompts these phages to exit the infected host by cell lysis. The rise in the emergence of drug-resistant microorganisms and manifestation of diseases that are untreatable by conventional antibiotics has prompt the use of phage therapy. By virtue of their specificity, phages can be conveniently applied to target and disrupt the replication machinery of the infecting bacterial pathogen without inducing marked toxicity or immunological effects (Gogokhia and Round 2021). The major success of bacteriophage-mediated strategy depends on comprehensive knowledge of the infecting bacterial agent, nature of the bacteriophage and phagebacteria interactions (Yuan et al. 2019). Accordingly, adequate extraction of new phages and deciphering their activities against bacterial agents, particularly those capable of forming biofilms and conferring drug-resistant traits, are critical for longterm control of bacterial proliferation and subsequent spread. To this end, a number of studies have investigated various phages for their adaptability and utility to

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abrogate infections caused by susceptible and various drug-resistant bacterial strains (Mah et al. 2003; Debarbieux et al. 2010; Santajit and Indrawattana 2016; Das and Manefield 2012).

Quorum Sensing Quorum sensing (QS) describes a process whereby cell-to-cell communication and gene expression are regulated based on variations in cell population density. For this reason, repression of cellular pathways that regulate QS is used to avert communication among microbial cells, aberrant gene expression profile, transmission of drugresistant genes and subsequent emergence of drug-resistant microbes. This process is regulated by secretion of auto-inducers, small molecules involved in maintaining appropriate levels of virulence factors. These include adhesion, biofilm formation, toxin production and motility factors (Yin et al. 2017). Such a model exists in Vibrio cholerae which consists of three analogous QS systems that are responsible for the secretion of toxin-coregulated pilus, cholera toxin as well as biofilm and haemolysis (Hema et al. 2016). Signals generated by auto-inducers are transmitted within the microbial community and foster interaction with associated regulators leading to collective virulence gene expression. These include genes implicated in the production of adhesion and motility factors, biofilms, extracellular protease enzymes and efflux pumps (Wolloscheck et al. 2018; Rezaie et al. 2018; Navidifar et al. 2019). Type IV pili are cell surface structures that act as potent virulence factors associated with driving microbial adherence to living cells and non-biological surfaces (Rasko and Sperandio 2010). Pili are also implicated in the flagellum translocation process known as twitching motility. Consecutively, twitching motility is responsible for initiating the adhesion process and formation of biofilms in pathogens such as Pseudomonas aeruginosa (LaSarre and Federle 2013). With regard to Gram-negative bacterial species, the majority of cell-to-cell communications are mediated by auto-inducers called acyl-homoserine lactones (Muras et al. 2020). For example, the iqs, rhl, pqs and las genes are coordinated by four different QS systems in P. aeruginosa. Within these QS networks, transcriptional regulatory proteins IqsR, RhIR, PqsR and LasR control the expression of these genes. The transcriptional regulators bind to particular auto-inducers and trigger the expression of specific virulence genes (Hurley and Bassler 2017; McCready et al. 2019). This is accompanied by the synthesis of several virulence proteins, some of which are responsible for enhancing microbial pathogenicity, inducing biofilm formation and development of drug resistance (Ansari et al. 2019). To date, various compounds extracted from natural plants have undergone biological experimentations to decipher their utility to abolish cell-to-cell communication mediated by several microbial QS networks (Kessler et al. 1993; McLean et al. 2004; Adonizio et al. 2008; Husain et al. 2015; Ansari et al. 2014; Ansari et al. 2017). Impressively, other studies have focused on delivering anti-QS agents using nanomaterials to enhance and prolong their antibacterial activity, stability and biocompatibility, increase target specificity and decrease cellular toxicity (Ilk et al. 2017; Gómez-Gómez et al. 2019; Shah et al. 2019). In contrast to antibiotics, which are designed to target and inhibit certain cellular processes that are essential for

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pathogenicity, anti-QS compounds repress cell-to-cell interaction without applying selection pressure on the bacteria. Eventually, this therapeutic mechanism suppresses bacterial replication while averting development of drug-resistant mutant pathogenic strains that emerge due to long-term application of selection pressure associated with conventional antibiotic monotherapy (Wáng et al. 2018; Dixit et al. 2019a; DeNegre et al. 2019). Therefore, QS is advantageous in repressing bacterial pathogenesis, emergence of drug resistance and transmission of mobile genetic elements that facilitate the spread of drug-resistant genes, particularly within microbial communities existing in biofilms.

Targeted Laser Therapy Laser therapy is a clinical procedure whereby laser is applied to damaged or diseased cells or tissues to provide medical relief. Low-level laser therapy (LLLT) elicits tissue irradiation by applying low power lightning (Carroll et al. 2014). This type of laser has a narrow spectrum that ranges from 600 nm to 1000 nm. In particular, the use of LLLT involves application of near infrared (NIR) and red light to cells or tissues at low levels. This causes biostimulation of cells or tissues and bolsters photobiological and photochemical effects. These beneficial effects include cell and tissue regeneration as well as eradication of pain and inflammation (Hirschl et al. 2004; Mizutani et al. 2004). As opposed to other laser methods, exposure to LLLT does not generate thermal effects on irradiated cells or tissues courtesy of the low power density employed by this procedure. For this course, LLLT has been harnessed to support patient treatment by complementing surgery and conventional antimicrobial drugs. Indeed, several investigations have highlighted that LLLT is capable of treating inflammation caused by musculoskeletal pain, oral diseases and diabetic ulcers (Bjordal et al. 2011) as well as producing beneficial effects on cellular metabolism (Ratkay-Traub et al. 2001). The LLLT procedure unleashes its activity by providing luminous energy to the mitochondria, leading to excitation of chromophores (Huang et al. 2009). This inhibits oxidative stress and promotes the production of adenosine triphosphate (ATP) (Karu 1989; Huang et al. 2013). Such biostimulatory properties alter cellular mechanisms by activating adhesion and migration while decreasing the frequency of programmed cell death in treated cultured cells and animals (AlGhamdi et al. 2012). Of clinical importance is the demonstration that application of LLLT stimulates growth of endothelial cells (Li et al. 2020), macrophages (Young et al. 1989), keratinocytes (Grossman et al. 1998) and fibroblasts (Yu et al. 1994). Likewise, this therapeutic technique has been demonstrated to improve angiogenesis in various biological experimental systems with the aim of ameliorating the formation of blood vessels (Tuby et al. 2009; Oubina et al. 2021). Previously, appropriate modulation of progesterone and appreciable increases in oestrogen levels corresponding to upregulation of the 3-β-hydroxysteroid dehydrogenase gene were reported upon exposure of porcine granulosa cells to helium-neon laser (630 nm) (Gregoraszczuk et al. 1983). These cellular events have a significant impact on the female health and reproductive function (Gregoraszczuk et al. 1983). Recently, LLLT has been applied as a treatment strategy for male infertility by stimulating considerable increases in the

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number, viability and survival of spermatozoa (Oubina et al. 2021; Gabel et al. 2018). Unlike LLLT, photothermal therapy (PTT) uses photoabsorbers to produce heat to burn infected cells or tissues. This method allows spatiotemporal controllability, minimum light exposure and deep tissue penetration. Administration of PTT with NIR irradiation has been shown to facilitate blood circulation (Guo et al. 2016) and wound healing (Aller et al. 2009), display antibacterial activity (Wecksler et al. 2006; Wang et al. 2013; Fan et al. 2015) and obliterate cancerous cells (Guo et al. 2017). The widely reported drawback of using PTT is that the procedure has limited specificity and tends to target healthy non-infected cells and tissues (Xiao et al. 2017; Qian et al. 2018). Nevertheless, both LLLT (Thomé et al. 2018; Kouhkheil et al. 2018; Dixit et al. 2019b) and PTT (Qing et al. 2019; Yan et al. 2020; Wang et al. 2020) have been tested against a variety of drug-resistant bacteria that persist in the presence of conventional antibacterial drugs. Considerable decreases in bacterial growth, spread and biofilm formation were documented after administration of laser therapy, thus illustrating the advantage of LLLT and PTT over conventional drugs with regard to eliciting potent antimicrobial activity.

CRISPR-Cas The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPRassociated protein 9 (Cas9) is a gene editing technique which targets and cleaves double-stranded DNA. Cleavage of DNA leads to formation of double-stranded breaks at the cleavage sites and disrupts normal cellular processes such as DNA replication, transcription and protein synthesis. The CRISPR-Cas9 system was first detected in bacteria where it acts as a natural defence mechanism involved in adaptive immune response against bacteriophages (Makarova et al. 2006; Barrangou et al. 2007). In this system, the CRISPR RNAs (crRNAs) coupled with associated Cas proteins have been demonstrated to repel viral replication in Escherichia coli (Brouns et al. 2008). Moreover, the ability of CRISPR-Cas9 to cleave bacteriophage target DNA has been uncovered in Streptococcus thermophilus (Barrangou et al. 2007) and Staphylococcus epidermidis (Marraffini and Sontheimer 2008). Structurally, the CRISPR-Cas9 loci contain a range of similar CRISPR repeats embedded in DNA-targeting spacers that code for crRNA components and an operon composed of Cas genes that encodes specific proteins. In different environments, bacteriophages can be paired to archaea or bacteria based on the CRISPR spacer regions (Andersson and Banfield 2008; Sun et al. 2013). The CRSIPR-Cas9 exerts its adaptive immunity-mediated antiviral response as follows: (a) Introduction of bacteriophage DNA as spacer sequence within CRISPR (b) Precursor crRNA (pre-crRNA) transcription to produce crRNAs containing a virus targeting spacer domain and repeat portion (c) Cleavage of viral DNA by Cas proteins guided by crRNA at complementary crRNA spacer sequences

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Interestingly, three different CRISPR-Cas9 systems (I, II, III) with contrasting modes of action to cleave the invading foreign DNA have been identified (Makarova et al. 2011a; Makarova et al. 2011b). The protospacer adjacent motif (PAM) is a short sequence situated in close proximity with the sequence targeted by crRNA on the foreign DNA. In type I and II, this sequence motif is crucial for adaptation and allows Cas nuclease to cut invading foreign nucleic acids (Mojica et al. 2005; Deveau et al. 2008; Horvath et al. 2008; Shah et al. 2013). A large Cas nuclease complex is required for foreign DNA targeting in type I and III (Brouns et al. 2008; Hale et al. 2009; Haurwitz et al. 2010; Hatoum-Aslan et al. 2011; Nam et al. 2012; Rouillon et al. 2013). Oppositely, a single Cas nuclease protein is necessary for crRNA-directed DNA detection and cleavage in type II systems (Jinek et al. 2012; Gasiunas et al. 2012). Extensive bioinformatics data mining and analysis have revealed that Cas9 is a large protein with multiple cellular functions (Makarova et al. 2002). This protein consists of two domains, the RuvC (Makarova et al. 2006) and HNH domains (Makarova et al. 2006; Bolotin et al. 2005; Haft et al. 2005). In S. thermophilus, Cas9 was shown to block bacteriophage invasion (Barrangou et al. 2007; Deltcheva et al. 2011), cleave phage DNA (Garneau et al. 2010) and allow targeting of viral and plasmid DNA in vivo (Deltcheva et al. 2011; Sapranauskas et al. 2011). Strikingly, the Cas9 protein was demonstrated to have a dual RNA-directed DNA nuclease activity and employs the crRNA and trans-activating CRISPR RNA (tracrRNA) complex (Deltcheva et al. 2011) to introduce double-stranded breaks in Streptococcus pyogenes (Jinek et al. 2012). Mechanistically, the RuvC domain of Cas9 functions by cleaving the DNA strand opposite the complementary sequence, while the HNH region cuts the strand complementary to the crRNA sequence (Jinek et al. 2012; Gasiunas et al. 2012). However, mutation in either domain produces a protein variant that induces single-stranded DNA breaks, while genetic alterations in both regions generate a DNA-binding protein directed by RNA (Jinek et al. 2012; Gasiunas et al. 2012). Correspondingly, identification of foreign DNA necessitates the presence of the PAM sequence and crRNA binding to initiate cleavage by Cas9 protein (Jinek et al. 2012; Gasiunas et al. 2012). In general, functional Cas9 exerts efficient target DNA cleavage with high specificity (Sternberg et al. 2014; Kuscu et al. 2014) and minimal off-target effects (Doudna and Charpentier 2014). Overall, the CRISPR-Cas9 gene editing mechanism is well suited to combat the persistence of multi-drug-resistant microbes. Cleavage of target DNA significantly hampers microbial replication, gene expression and protein production stages. For this reason, repeated DNA cleavage cycles dramatically disrupt the expression of virulence genes and proteins and severely diminish the fitness of microorganisms. This inevitably suppresses the microbial load and compromises the pathogenicity and virulence of targeted microorganism. Appreciably, in biofilm communities CRISPR-Cas9 may be applied to counter and undermine microbial growth as well as to prevent the transfer and acquisition of drug-resistant genes. The impact of this versatile system has universal applications and creates drastic changes that are of immense global health importance pertaining to inhibition of multi-drug-resistant microorganisms and averting the emergence of drug-resistant mutant strains.

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Bacteriophage-Based Strategy

2.1

Bacteriophages Can Be Used to Combat Various Multi-drug-Resistant Bacterial Isolates

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In recent years, genetic mutations leading to proliferation of multi-drug-resistant bacterial pathogens coupled with extensive biofilm formation among pathogenic microorganisms have become major public health problems. Due to the general failure of conventional antibacterial agents to treat diseases caused by multi-drugresistant strains, combination therapy has been applied to disrupt microbial replication and alleviate disease symptoms. Unfortunately, long-term application of this strategy often results in development of unintended side effects. For example, although administration of combination therapy markedly reduces microbial load in patients infected with tuberculosis (TB) and human immunodeficiency virus (HIV), adverse effects such as hepatotoxicity, stomach cramps, hypersensitivity, heart complications and kidneys failure usually accompany their long-term use. Traditionally, antimicrobial compounds are extracted from natural plants. This is followed by characterisation of the extracted material, identification of chemical groups with antimicrobial activity and synthesis of active compounds. An alternative method is to artificially synthesise the therapeutic agents by chemical synthesis. Conventional drugs attained from natural products or by various chemical procedures usually have common modes of action. These compounds are designed to target and inhibit essential molecules that are essential in the replication cycle of the microorganism in question. These target molecules include DNA, RNA, virulent genes and proteins. This simple and straightforward drug design approach can combat disease-causing microorganisms. Nonetheless, the major challenge arises when the pathogens develop mutations that render these conventional drugs ineffective against a variety of drug-resistant mutant strains. This devastating outcome necessitates evaluation of alternative treatment strategies to treat medical conditions that are unresponsive to conventional therapies. Bacteriophages are viruses that infect and replicate within bacterial cells (Fig. 2.1). For clinical purposes, this strategy is attractive and advantageous as these viruses have a high infectivity rate for bacteria but are incapable of infecting humans, animals and plants. As such, the utility of applying bacteriophages to treat human diseases induced by multi-drugresistant bacteria has been explored. This method has been tested against multi-drugresistant bacteria retrieved from patients with septic wounds (Pallavali et al. 2017). The collected bacterial pathogens were assayed against several antibiotics and various bacteriophages. Detailed analysis revealed that the dominant species obtained from these septic wounds were P. aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae and E. coli, respectively (Pallavali et al. 2017). Other Gramnegative and positive microorganisms were also present in patient swab samples but in lower quantities. In addition, these microorganisms conferred resistance against penicillin, amoxicillin, ampicillin, tetracycline and vancomycin (Pallavali et al. 2017). In vitro administration of bacteriophages induced considerable lytic activity against all tested multi-drug-resistant bacteria. It was also reported that phage

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Fig. 2.1 Mechanism of action of bacteriophages against bacterial pathogens. Bacteriophages attach to replicating bacterial cells to initiate host penetration. Phage DNA is injected into bacterial cells leading to disruption of host replication cycle. Attenuated host cells are lysed resulting in eradication of pathogenic bacteria

therapy did not augment multi-drug resistance in bacteria. Thus, phages may be used as potential prophylactic agents to prevent wound sepsis and may also be applied as a mixture of different bacteriophages to combat emergence of drug-resistant pathogens (Pallavali et al. 2017).

2.2

Bacteriophages Can Be Used to Combat Multi-drug-Resistant Acinetobacter baumannii

A spike in multi-drug resistance has advanced the onset of diseases caused by the opportunistic microbes such as Acinetobacter baumannii (Jansen et al. 2018). The phage KARL-1 is a filamentous virus belonging to the Myoviridae family. The antibacterial activity of this virus was assayed alone and in conjunction with other

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antibiotics. These antibacterial agents included colistin, ciprofloxacin and meropenem. Considerable decreases in bacterial replication were noted after introducing KARL-1 phage at various multiplicities of infection (MOI), with the optimum being 10-1 (Jansen et al. 2018). On the other hand, the most effective antibacterial agent was meropenem at a drug concentration of less than 128 mg/l. Moreover, synergistic effects were observed after simultaneous application of KARL-1 with colistin, ciprofloxacin or meropenem (Jansen et al. 2018). Impressively, robust A. baumannii clearance was achieved by co-administration of KARL1 phage at an MOI of 10-7 and meropenem at dosage of 128 mg/l. This promising outcome illustrated that the novel KARL-1 bacteriophage is capable of countering bacterial proliferation and is clinically versatile as it can be applied with already existing antibiotics to enhance microbial clearance and prevent development of drug-resistant mutant strains (Jansen et al. 2018).

2.3

Bacteriophages Can Be Used to Inhibit Multi-drug-Resistant P. Aeruginosa

P. aeruginosa is a Gram-negative, facultative aerobic, rod-shaped bacterium that causes a wide spectrum of human diseases. These include among others bacteremia, dermatitis, soft tissue infections, bone and joint infections and urinary tract and respiratory system infections. Previously, the use of the phage vB_Paem_LS1 against multi-drug-resistant P. aeruginosa has been interrogated (Yuan et al. 2019). Genome characterisation disclosed that this phage had a high sequence similarity to viruses that belonged to the Pbunavirus genus, composed of a GC content of 55.7% and contained a double-stranded DNA of about 66,095 base pairs in size. The lytic activity of the phage vB_Paem_LS1 was evaluated in vitro against P. aeruginosa (Yuan et al. 2019). Thereafter, bacterial counts and scanning electron microscopy were employed to examine antibacterial inhibitory effects. A remarkable decline in bacterial load and replication coupled with a sharp decrease in biofilm formation accompanied application of the bacteriophage to replicating, multi-drugresistant P. aeruginosa. This positive result showed that the phage vB_Paem_LS1 is therapeutically useful owing to its adaptability to restrict bacterial growth and inhibiting microbial spread by abolishing biofilm formation (Yuan et al. 2019). In a more recent investigation, P. aeruginosa growth was neutralised by the bacteriophage RLP (Alvi et al. 2020). The lytic system of this phage consisted of four genes, namely, R/z, R/z1, endolysin and holin. The bacteriophage displayed improved stability under different temperatures and was capable of inhibiting microbial growth. The phage also displayed enhanced utility by infecting a broad range of different multi-drug-resistant bacteria. In infected mice, injection of RLP resulted in 92% survival relative to 7.4% survival rate in untreated animals (Alvi et al. 2020). Consequently, considerable decreases in bacterial load, proliferation and other critical makers of microbial replication in vitro and in vivo augur well for the use of RLP phage for provision of efficient antibacterial therapy (Alvi et al. 2020).

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Bacteriophages Can Be Used to Treat Multi-drug-Resistant Salmonella serovars

The vast diversity of Salmonella species to cause human infections and confer drug resistance is of global public health concern. Mahmoud et al. (2018) examined the impact of infecting Salmonella serovars collected from broilers in Egypt with three distinct bacteriophages (Mahmoud et al. 2018). The host Salmonella serotypes were Salmonella typhimurium, S. Kentucky and S. enteritidis, while the infecting phages were Salmacey1, Salmacey2 and Salmacey3. With the exception of S. Kentucky that was resistant to seven of the ten antimicrobial drugs, all other Salmonella serovars were resistant to more than two therapeutic agents (Mahmoud et al. 2018). The three phages were shown to belong to two distinct families: Salmacey1 and Salmacey2 were classified under the Siphoviridae family, while Salmacey3 belonged to the Myoviridae group. This classification was made following morphological introspection of phage particles by transmission electron microscopy (Mahmoud et al. 2018). In vitro bioassays highlighted that all phages had polyvalent properties and were capable of infecting all tested Salmonella serotypes. The bacteriophages also displayed efficient infectivity following delivery into replicating Citrobacter freundii cells. Additionally, Salmacey1 and Salmacey2 induced a dramatic increase in cell lysis after administration into Enterobacter cloacae, while Salmacey3 elicited a pronounced effect in E. coli (Mahmoud et al. 2018). Conversely, none of the phages were able to infect Salmonella Paratyphi, Bacillus cereus and Staphylococcus aureus. The results illustrated that the three phages evaluated in this study possess antibacterial efficacy against a broad range of multi-drug-resistant microorganisms and may potentially be utilised to repress infections caused by these mutant bacterial species (Mahmoud et al. 2018).

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Quorum Sensing

3.1

Application of Quorum Sensing to Inhibit Multi-drug-Resistant P. aeruginosa

Quorum sensing relies on preventing communication among cells to ameliorate stimulation of pathogenicity and disease development (Fig. 2.2). Biofilms are composed of a network of several microbial species contained within extracellular polysaccharides and proteins. This community of microorganisms offers protection and prolonged survival of pathogens that would otherwise be eliminated by antimicrobial molecules. In particular, horizontal gene transfer and activation of efflux pumps by some pathogenic species within the biofilm promote persistence due to emergence and transmission of drug-resistant genes. This allows bacterial growth, spread and survival even in the presence of antibiotics. Eventually, this has significant and overwhelming clinical outcomes as it results in the manifestation of persistent medical conditions that are untreatable by conventional medications. Therefore, this requires innovation of novel and effective therapeutic strategies to

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Fig. 2.2 Depiction of quorum sensing in bacteria. Bacteria communicate via cell-to-cell interactions in response to environmental changes. This triggers a change in cell growth and regulates bacterial population density. Signalling molecules called auto-inducers are produced in response to changes in bacterial growth. Secretion of auto-inducers causes a change in gene expression which subsequently regulates bacterial replication

treat and/or prevent diseases caused by multi-drug resistance organisms. Accordingly, the antiquorum sensing activities of propolis extracts, Humulus lupulus, Ruta graveolens, Olea europaea, Styrax benzoin, Camellia sinensis and Ocimum basilicum were evaluated against 204 P. aeruginosa clinical isolates that conferred drug resistance (El-Sayed et al. 2020). Furthermore, the effect of Olea europaea on

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the expression profile of biofilm-related genes was also investigated. The disc diffusion assay indicated that 49% of the bacterial isolates were multi-drug resistant and over 90% of these were capable of biofilm formation (El-Sayed et al. 2020). Olea europaea and Camellia sinensis caused the most pronounced decrease in biofilm formation at subinhibitory concentrations, with antibacterial activities of 82.2% and 84.8%, respectively. A sharp decline of about 97–99% in the expression of genes associated with biofilm formation such as lasR, lasI, rhlR and rhll was triggered following administration of these natural extracts. These observations highlighted the importance of utilising natural compounds to inhibit cell-to-cell communication to prevent expression of biofilm-related genes leading to a marked decrease in microbial replication and bacterial load (El-Sayed et al. 2020).

3.2

Application of Quorum Sensing to Suppress Multi-drug-Resistant Vibrio cholerae

Staggering elevation in the emergence of multi-drug-resistant Vibrio cholerae drives the onset of the cholera epidemic and overwhelms both the private and public health facilities. To counter this, a study focusing on employing the antiquorum sensing compound derived from 2,3-pyrazine dicarboxylic acid has been carried out (Hema et al. 2016). Thus, the antiquorum sensing agents targeted the LuxO regulator responsible for facilitating cell communication in V. cholerae strains. Delivery of these derivative compounds led to a significant decrease in cell communication and successively caused a remarkable decline in biofilm formation. This dramatic decrease in biofilm formation was achieved after applying the compounds at a low dosage (IC50 ranged between 1 mM and 70 mM). To extensively augment antibacterial efficacy, combination therapy involving simultaneous delivery of 2,3-pyrazine dicarboxylic acid derivatives and antibiotics was applied (Hema et al. 2016). The tested antibacterial agents were erythromycin, chloramphenicol, tetracycline and doxycycline. Remarkably, co-administration of derivative compounds with antibiotics was synergistic and led to improved inhibitory effects, thus proving the therapeutic benefits of utilising the 2,3-pyrazine dicarboxylic acid derivatives as anti-QS agents (Hema et al. 2016).

3.3

Utilisation of Quorum Sensing to Suppress Multi-drug-Resistant E. coli

Multi-drug resistance conferred by E. coli towards carbapenem antibiotics is attributed in part to the pathogen’s ability to form extensive biofilm networks (Thakur et al. 2016). This necessitates intricate biological and physiological processes that include quorum sensing, adhesion and complex biofilm development. Three herbal products, namely, Berberis aristata, Camellia sinensis and Holarrhena antidysenterica, were assessed against E. coli conferring multi-drug resistance (Thakur et al. 2016). Holarrhena antidysenterica exhibited antiquorum sensing

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activity that was comparable to that achieved by conventional antibiotics. Camellia sinensis induced both antiquorum sensing (90.0%) and antibiofilm formation (84.1%) activities. A dramatic increase in antibacterial effects was observed after application of Berberis aristata, which elicited antiquorum sensing (96.06%), anti-adhesion, (91.3%) and antibiofilm activities (51.3%). All these results validated molecular docking predictions of phytoligands analysed against Pilin and Lux S receptors in E. coli (Thakur et al. 2016). Moreover, pairwise correlation analysis of assayed antibacterial activities with bioactivity function, qualitative and quantitative descriptors, illustrated that decreased tannins content, average flavonoids levels and enhanced alkaloid quantities correspond to antiquorum sensing, anti-adhesion and antibiofilm production activities displayed by all three herbal extracts (Thakur et al. 2016). Specifically, superoxide and nitric oxide scavenging qualities were positively associated with antiquorum sensing activity. The findings showed that all three herbs, particularly Berberis aristata, displayed appreciable antimicrobial efficacy which may be harnessed to counter multi-drug-resistant E. coli conferring resistance to carbapenem antibiotics (Thakur et al. 2016).

3.4

Utilisation of Quorum Sensing to Counter Multi-drug-Resistant Chromobacterium violaceum

The antibacterial compound citral and its derivatives have been examined for their antiquorum sensing activities towards Chromobacterium violaceum (Batohi et al. 2021). Elucidation of antiquorum sensing efficacy by qualitative and quantitative methods indicated that the compound CD1 was the most effective. The other antibacterial molecules such as CD2, CD3 and citral also exerted potent antimicrobial activity, but their efficacy paled in comparison to that exerted by CD1 (Batohi et al. 2021). Notably, all tested compounds repressed C. violaceum growth and biofilm formation after being administered at subinhibitory concentrations. Successive gene expression profiling revealed that the test compounds triggered a marked decrease in the mRNA transcription levels of quorum sensing genes as compared to untreated controls (Batohi et al. 2021). These encouraging observations indicted that citral and its derivative compounds possess critical therapeutic properties that disrupt both cell-to-cell communication and biofilm formation mechanisms. These features may as such be utilised to regulate bacterial growth (Batohi et al. 2021). Since cell-to-cell communication is one of the crucial mechanisms employed by microorganisms in response to environmental changes, molecules that inhibit quorum sensing may be applied to avert cellular signalling. These antiquorum agents may be conveniently used to repress the growth of both drug-susceptible and drug-resistant microorganisms.

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4

Laser Therapy

4.1

Efficiency of Laser Therapy to Inhibit Different Multi-drug-Resistant Bacterial Pathogens

Laser therapy entails administration of laser to achieve cellular irradiation (Fig. 2.3). The effectiveness of laser therapy in disrupting biofilms has been tested. This approach was explored in S. aureus and P. aeruginosa strains that conferred resistance to methicillin (Kirui et al. 2019). Co-delivery of gold nanoparticles and pulsed laser therapy led to appreciable biofilm disruption levels of up to 99% as compared to untreated samples. Furthermore, a combination of gold nanoparticledirected pulsed laser therapy and amikacin or gentamicin exhibited synergistic effects and exerted 5-log and 4-log antibacterial activities, respectively (Kirui et al. 2019). In contrast, a 1-log decrease in biofilm formation was noted after application of either gold nanoparticle-directed pulsed laser or antibiotics, thus indicating the importance of combination therapy in effecting extensive antibacterial inhibition. Noticeably, a combination of gold nanoparticle and laser therapy efficiently reversed antibiotic susceptibility to the level comparable to that demonstrated by drug susceptible strains. This promising outcome indicated that co-administration of gold nanoparticles and laser therapy may be used to disrupt biofilm formation and eliminate drug resistance (Kirui et al. 2019). Production of nanomaterials with various functional characteristics has been exploited for regulating diseases caused by multi-drug-resistant bacteria (Zhao et al. 2020). Hence, multifunctional nanomaterials called boronic acid functionalised thiolated graphene-based nitric oxide nanogenerators have recently been investigated for antimicrobial activity against multi-drug-resistant Gram-negative bacteria. Delivery of these nanomaterials led to a considerable decrease in bacterial growth, spread and biofilm formation in cultured cells and infected mice (Zhao et al. 2020). Simultaneous application of a single near-infrared (NIR) laser therapy and multifunctional nanomaterials caused hyperthermia followed by release of nitric oxide (Zhao et al. 2020). This synergistic therapeutic approach resulted in enhanced degradation of bacterial membrane, disruption of intracellular components and apoptotic activity. With regard to functional properties, nitric oxide and photodynamic therapy markedly improved antibacterial activity. Oppositely, a combination of thiolated graphene, nitric acid and boronic acid is crucial for enhancing biocompatibility in vitro and in vivo. This strengthens and sustains antibacterial efficacy and inhibits production of biofilms (Zhao et al. 2020). This consolidated the prospect of utilising a combination of NIR laser and multifunctional nanomaterials to suppress proliferation of multi-drug-resistant Gram-negative bacteria, which included A. baumannii, P. aeruginosa and K. pneumoniae. Moreover, inhibition of biofilm production is essential as it prevents aggregation of microbial pathogens that would otherwise advance transfer of drug-resistant genes by horizontal gene transfer (Zhao et al. 2020).

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Fig. 2.3 Strategy of targeted laser therapy. Laser therapy is applied to irradiate diseased cells or tissues from a given population. Irradiation eradicates diseased cells or tissues, but the majority of healthy cells and tissues survive. Successive cell repopulation promotes symptom alleviation and patient recovery

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Effectiveness of Laser Therapy to Attenuate Multi-drug-Resistant Mycobacterium tuberculosis

Cases of multi-drug-resistant TB are challenging to treat and result in high morbidity and mortality rates globally. As an alternative strategy, the N2 laser has been exploited to suppress replication of multi-drug-resistant Mycobacterium tuberculosis (Mtb) strains that persisted even after provision of chemotherapy (Bhagwanani et al. 2015). To this end, the advantage of using N2 laser therapy was analysed in 567 drug-resistant Mtb isolates in vitro. Furthermore, the robustness of the laser therapy was successively evaluated in patients with pulmonary TB (Bhagwanani et al. 2015). Approximately 21.5% of the Mtb clinical isolates conferred resistance to rifampicin, while 12.8% were sensitive to all anti-TB agents. In contrast to control isolates, exposure of multi-drug-resistant pathogens to N2 laser had a negative effect on bacterial survival. Subsequently, this positive clinical manifestation was also replicated in patients who received N2 laser therapy, leading to considerable recovery in 90% of the individuals. Other markers of Mtb replication were also significantly impeded following exposure to N2 laser therapy. In 75% of the patients, the acid-fast bacilli remained undetectable by immunohistochemical assays for up to 15 days. Additionally, clinical improvements were also noted in 60% of patients following lung monitoring by chest X-rays. These findings uncovered that N2 laser therapy is potent against multi-drug-resistant TB when tested in vitro and in patients and sets a promising prospect for detailed characterisation and analysis for potential clinical application to combat drug-resistant Mtb strains (Bhagwanani et al. 2015).

5

CRISPR-Cas

5.1

Activity of CRISPR-Cas to Suppress Diverse Multi-drug-Resistant K. pneumoniae

The CRISPR-Cas9 is a gene editing method that disrupts the normal replication cycle by casing double-stranded breaks in the target DNA molecule (Fig. 2.4). To date, several key microbial regions and virulence-promoting markers have been attenuated using the CRISPR-Cas9 method (Li et al. 2017; Tashkandi et al. 2018; Ortigosa et al. 2019; Berlec et al. 2018; Fokum et al. 2019). More importantly, this gene editing approach has also been administered to diminish resistance to different clinically useful antimicrobial therapies (Lage 2016; Chen and Zhang 2018; Dong et al. 2019; Wei et al. 2019; Wang et al. 2019). The CRISPR-Cas9 mechanism targets and cleaves DNA to create double-stranded breaks in the sequence of interest. This process is accompanied by DNA repair, which enables continuous binding and cleavage of target DNA sequence. The DNA repair is accomplished by two distinct mechanisms, namely, homology-directed repair (HDR) and non-homologous end joining (NHEJ) (Zaboikin et al. 2017; Paulsen et al. 2017; Schiermeyer et al. 2019). The HDR process results in accurate repair of disrupted target DNA with the use of homologous DNA sequences. Conversely, NHEJ introduces errors in the cleaved

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Fig. 2.4 Therapeutic mechanism of CRISPR-Cas9 gene editing system. The guide RNA and Cas9 form a complex that recognises the target sequence. The Cas9 nuclease cleaves the target sequence to induce double-stranded breaks in DNA. DNA repair is achieved by homology directed repair (HDR) and non-homologous end joining. The HDR mechanism leads to accurate repair of target DNA by using homologous DNA sequences. The NHEJ process causes errors in the cleaved DNA sequence and introduces insertion and deletion mutations. Overall, application of CRISPR-Cas9 disrupts DNA replication and successive transcription and protein synthesis processes

DNA resulting in the development of insertion and deletion mutations. Therefore, this technology is utilised to attenuate microbial fitness after repeated cleavage events. This has many adaptable and useful medical qualities and applications that

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include eradication of pathology-causing genes, repression of virulence protein production and inactivation of drug-resistant genes. The blaKPC-2 and blacKPC-3 genes mainly mediate resistance of K. pneumoniae to carbapenem drugs. Indeed, the vast majority of K. pneumoniae strains conferring resistance harbour these drug-resistant genes following acquisition via horizontal gene transfer facilitated by bacterial plasmids (Mackow et al. 2019). Overwhelmingly, infection with these pathogens results in manifestation of diseases that are intricate to treat and are associated with high mortalities and clinical outbreaks. Consequently, an investigation to unravel the importance of the CRISPR-Cas in preventing plasmid-mediated transmission of drug-resistant genes was elucidated by Mackow et al. (2019). To assess this, the presence of the CRISPR-Cas system was screened in 304 K. pneumoniae clinical isolates. Data showed that isolates that contained CRISPR-Cas displayed decreased resistance to antimicrobial agents as compared to isolates lacking the CRISPR-Cas system. More significantly, CRISPRCas-positive isolates were capable of blocking transmission of drug-resistant blakPC genes by repressing plasmid-mediated horizontal gene transfer (Mackow et al. 2019). Additionally, it was also demonstrated that isolates that failed to acquire drug-resistant genes harboured CRISPR sequences that were very similar to those present in bacterial plasmids responsible for eliciting multi-drug resistance. These critical findings highlight that the presence or absence of the gene editing CRISPRCas system determines the drug susceptibility or resistance profile of K. pneumoniae strains (Mackow et al. 2019). Previously, 176 K. pneumoniae clinical isolates were collected from patients presenting with urinary tract infections or bloodstream complications in Taiwan (Li et al. 2018). Besides, 97 K. pneumoniae genomic DNA sequences were obtained from the Integrated Microbial Genomes & Microbes genome database. The Cas1 gene sequence alignment and CRISPRFinder tools detected CRISPR-Cas systems in 41.2% of the retrieved bacterial genomic DNA sequences. With regard to chromosomal location and Cas1 and Cas3 protein sequences, the identified CRISPR-Cas systems were categorized into type I-E and subtype I-E (Li et al. 2018). Interestingly, CRISPR-Cas systems were not identified in genome sequences of clonal complex isolates (n = 258) representing the majority of K. pneumoniae strains displaying multi-drug resistance. Subsequent genomic DNA amplification with primers specific for Cas revealed that 30.7% of clinical isolates possessed CRISPR-Cas. Within these isolates, type I-E CRISPR-Cas was prevalent in urinary tract infections (11.2%), whereas subtype I-E CRISPR-Cas was mainly associated with bloodstream complications (28.7%). Extensive analysis indicated that pathogens containing subtype I-E system displayed increases susceptibility to ampicillin-sulbactam, gentamicin and cefazolin as opposed to isolates lacking CRISPR-Cas system (Li et al. 2018). These observations suggest that the subtype I-E CRISPR-Cas system in K. pneumoniae disrupts acquisition of drug resistance genes and may be applied to safeguard the genetic integrity of the microorganism (Li et al. 2018).

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Activity of CRISPR-Cas to Counter Various Multi-drug-Resistant Osteosarcoma Malignancies

Multi-drug resistance remains a hurdle for successful recovery of patients with osteosarcoma (Liu et al. 2016). The primary source of drug resistance is dysregulation of P-glycoprotein (P-gp), a membrane-bound protein responsible for drug transportation. Notably, dysregulation of this protein is a result of overexpression of the ABCB1 gene. Various strategies have been exploited with the goal of reversing multi-drug resistance elicited by ABCB1 gene; however, none has proven effective (Liu et al. 2016). Consequently, this required exploring other treatment options capable of reversing drug resistance corresponding to gene dysregulation and P-gp dysfunction. CRISPR-Cas9 was applied to overturn the detrimental effects accompanying multi-drug resistance associated with aberrant regulation of ABCB1 and P-gp in cultured human cells (Liu et al. 2016). As opposed to untreated control cells, the application of CRISPR-Cas9 gene editing tool effectively nullified resistance to the anticancer drug doxorubicin in multi-drug-resistant cell lines U-2OSR2 and KHOSR2 (Liu et al. 2016). This meta-analysis investigation suggests that CRISPR-Cas9 may be conveniently used to target and modify the ABCB1 gene to circumvent multi-drug resistance associated with its protein product P-gp. This has crucial clinical benefits for patients with osteosarcoma as it bolsters the success of cancer therapy, mitigates disease severity and prolongs drug activity (Liu et al. 2016). In a related study, the cluster of differentiation 44 (CD44) was the target for CRISPR-Cas9-mediated gene editing (Xiao et al. 2018). In particular, CD44 is an important receptor for binding of hyaluronic acid. Hyaluronic acid-CD44 binding initiates a cascade of cellular events, which include tumour growth, metastasis and disease severity (Xiao et al. 2018). This has an enormous clinical impact for patients with osteosarcoma. The study examined 56 osteosarcoma patients and retrieved 96 tissue samples to assess and analyse CD44 expression by immunohistochemistry. The transwell technique and wound healing were used to determine cell invasion and migration patterns. The cellular proliferation rate was analysed by 3D cell culture, and drug resistance was interrogated by drug uptake method in conjunction with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay (Xiao et al. 2018). The immunohistochemistry data showed that enhanced CD44 levels were correlated with increased risk of metastasis, poor patient recovery and development of drug resistance. Nevertheless, a robust suppression of cell invasion and migration coupled with reversal of drug resistance were noted following CD44 gene silencing by CRISPR-Cas9. Therefore, CD44 knockdown by CRISPR-Cas9 represses the activity of markers associated with aggressive osteosarcoma and advances patient recovery and survival by increasing drug sensitivity (Xiao et al. 2018).

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Discussion and Conclusion

A dramatic surge in global antimicrobial resistance has necessitated the use of other therapeutic approaches to abolish drug resistance. These therapeutic techniques have been evaluated as conventional antibiotics are incapable of inhibiting most mutant strains including drug resistance and multi-drug-resistant pathogens. As conventional drugs mainly focus on repressing the activity of virulent genes, proteins or other markers associated with pathogenicity, novel treatment strategies use different mechanisms to treat clinical conditions caused by mutant strains. In addition, some of these strategies are versatile and adapted to suppress the growth of drug-resistant pathogens as well as to avert the emergence of successive mutant microbes by blocking the transmission of drug-resistant genes. This is crucial for controlling the replication and growth of microbial pathogens harbouring drug-resistant genes. This is also essential for alleviation of disease symptoms, enhancing positive treatment outcomes, facilitating clearance of persistent microbes in both the public and private clinical institutions and sustaining the efficacy of antimicrobial agents. Unconventional therapeutic methods intended to treat and prevent the development of drug-resistant microorganisms include the use of bacteriophages, QS, laser therapy and CRISPR-Cas9. Although these methods use different mechanisms to reverse multi-drug resistance, they have been successfully implemented to counter microbial growth and to avert subsequent development of mutant strains. The bacteriophages infect bacteria followed by active viral replication using the host cellular machinery. This significantly restricts bacterial growth and spread and lessens the bacterial load and virulence. Owing to their efficient lytic activity, specificity and limited toxicity, phages have been exploited to combat multi-drugresistant bacterial pathogens (Kesik-Szeloch et al. 2013; Jung et al. 2017; Tkhilaishvili et al. 2019). This application extends to microorganisms existing within biofilms, to counter drug resistance and transmission of genes conferring drug resistance. To be specific, this approach has been used to suppress proliferation of multi-drug-resistant P. aeruginosa (Alvi et al. 2020; Yuan et al. 2019; Pallavali et al. 2017), S. aureus, K. pneumoniae, E. coli (Pallavali et al. 2017), A. baumannii (Jansen et al. 2018), S. typhimurium, S. Kentucky and S. enteritidis ((Mahmoud et al. 2018). Natural plant products that block cell-to-cell communication have been harnessed to circumvent the pathogenicity of multi-drug-resistant bacteria. These extracts represent a wide variety of plants including Olea europaea, Ocimum basilicum, Styrax benzoin, Camellia sinensis, Humulus lupulus and Ruta graveolens, all of which caused appreciable decreases in virulence and biofilm formation in P. aeruginosa (El-Sayed et al. 2020). These markers of replication were markedly repressed after downregulating the expression of genes implicated in QS (El-Sayed et al. 2020). Similarly, citral and derivative compounds accomplished anti-QS activity in C. violaceum at low concentrations. Likewise, this impressive antibacterial activity accompanied suppression of QS genes responsible for initiating and facilitating cellular communication and advancing microbial pathogenesis (Batohi et al. 2021). Co-administration of the anti-QS agent 2,3-pyrazine

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dicarboxylic acid derivatives and conventional antibiotics that included erythromycin, chloramphenicol, tetracycline and doxycycline mitigated the growth of the water-borne multi-drug-resistant V. cholerae (Hema et al. 2016). Interestingly, other plant extracts exerted antibacterial efficacy by various modes of action. For example, Camellia sinensis caused a decrease in QS and biofilm production, while Berberis aristata triggered considerable depreciation in QS, adhesion and biofilm formation (Thakur et al. 2016). Laser therapy relies on exposing cells or tissues to targeted laser to achieve irradiation. This intervention has previously been utilised to attenuate the virulence of S. aureus (Kirui et al. 2019), P. aeruginosa (Kirui et al. 2019; Zhao et al. 2020), K. pneumoniae and A. baumannii (Zhao et al. 2020) conferring multi-drug resistance. Noticeably, this technique has also been employed to regulate the growth and spread of multi-drug-resistant cancer (Guo et al. 2017) and Mtb in vitro, in vivo and infected humans (Bhagwanani et al. 2015). These documented reports suggest that laser administration may potentially be employed as a viable and effective method of controlling various diseases. The CRISPR-Cas9 method executes its therapeutic activity by cleaving the target DNA molecule, thus creating double-stranded breaks. This alternative treatment mechanism has been exploited to abrogate the pathogenicity of K. pneumoniae (Mackow et al. 2019) containing multi-drug-resistant genes as well as multi-drug-resistant osteosarcoma (Liu et al. 2016; Xiao et al. 2018). These observations illustrated that the CRISPR-Cas9 strategy is ideally suited for disease control by weakening the cellular replication machinery following cleavage of target DNA. Going forward, the ideal therapeutic methods will be the ones capable of mitigating the replication of sensitive and various drug-resistant microorganisms. Preferably, these diverse and dynamic alternative treatment strategies must be able to achieve therapeutic efficacy at low concentration to minimise cytotoxic and other unintended effects. Equally important will also be the ability to avert the emergence of drug-resistant pathogens by blocking transmission of drug-resistant genes from mutant strains to susceptible microbes by horizontal gene transfer. This is of critical importance in persistent microorganisms sharing a common environment such as biofilms, where drug resistance is remarkably increased due to the high rate of transmission and acquisition of drug-resistant genes. Additionally, in biofilm communities there is activation of efflux pump systems that flush out or dilute the concentration of conventional drugs. Ultimately, this creates a high degree of selection pressure against the drug and allows adaptation and drug tolerance, thus rendering the drug ineffective. As a result, extensive research investigations, particularly directed at identifying new molecular markers for therapeutic targeting in vitro and in appropriate animal models, are essential for innovation of novel and potent antimicrobial agents. This is essential for decreasing the selection pressure currently being applied against licensed conventional therapeutic molecules. Practically, implementation of these ideal therapeutic techniques has to be matched with appropriate and responsible drug use by both patients and the general public to prolong their efficacy and prevent development of strains conferring drug resistance. Overall, a synergy among hospital staff, drug users and the public as a

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whole is necessary for efficient management and sustainability of novel treatment approaches. In conclusion, discovery of other alternative strategies for disrupting microbial growth and spread constitutes a global emergency. Alternative treatment methods offered by bacteriophages, laser therapy, QS and CRISPR-Cas9 have been demonstrated to attenuate the virulence of multi-drug-resistant pathogens that are not responsive to conventional drugs. In accordance with the positive clinical outcomes associated with application of these unconventional treatment approaches, invention of other alternative treatment techniques may provide much-needed drug efficacy and coverage as well as alleviating the clinical burden associated with multi-drugresistant diseases. Thus, comprehensive drug testing, appropriate and timely diagnosis and correct drug prescription in conjunction with responsible use of the antimicrobial agents are of critical importance for eradicating the scourge and onset of drug-resistant microorganisms. Acknowledgements The authors are grateful for research funding offered by the National Research Foundation Thuthuka grant and the Carnegie DTA research grant (grant code: RKSMM21). Author Contributions Musa Marimani compiled the book chapter with the help of Aijaz Ahmad and Adriano Duse. Competing Interests The authors have no competing interests to declare.

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Strategies to Combat Multidrug Resistance by Non-traditional Therapeutic Approaches Harsh Yadav, Anand Maurya , Alka Agarwal , Anurag Kumar Singh , Satish Dubey, Aditya Moktan Tamang, Reshu Agrawal, and Sushil Kumar Chaudhary

Abstract

Drug resistance is currently acknowledged as the most concerning healthcare issue worldwide. Antibiotic resistance in all clinically significant pathogens, a halt in the discovery and development of new antibiotics, and recurrent infections brought on by multidrug-resistant pathogens are some of the problematic aspects of the current antibiotic crisis that affect both developing and developed countries. All these issues are impeding the effectiveness of conventional antibiotics as a therapeutic option, which is why non-traditional approaches are becoming more popular. Despite several non-traditional approaches to fight drug

Harsh Yadav and Anand Maurya contributed equally with all other contributors. H. Yadav · A. Moktan Tamang · R. Agrawal Department of Pharmacy, Indira Gandhi National Tribal University, Lalpur, Amarkantak, Madhya Pradesh, India A. Maurya · A. Agarwal Department of Medicinal Chemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India A. K. Singh Cancer Biology Research and Training, Department of Biological Sciences, Alabama State University, Montgomery, AL, USA Centre of Experimental Medicine and Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India S. Dubey Laboratory of Molecular Taxonomy and Medicinal Plant Biology, Department of Botany, Guru GhasidasVishwavidyalaya, Bilaspur, Chhattisgarh, India S. K. Chaudhary (✉) Institute of Bioresources and Sustainable Development, Takyelpat, Imphal, Manipur, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. Y. Wani, A. Ahmad (eds.), Non-traditional Approaches to Combat Antimicrobial Drug Resistance, https://doi.org/10.1007/978-981-19-9167-7_3

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resistance have gained interest, their use is restricted since they call for advanced diagnostics that go beyond pathogen identification, and only a few therapies have reached late-stage clinical trials. Exotoxin-targeted therapies are one of the most advanced non-traditional therapies used to treat infections caused by Staphylococcus aureus and Clostridium difficile. Another important non-traditional approach to treat or prevent C. difficile infection is the microbiome therapy. It is more likely that after approval of any non-traditional therapy, it would be used concomitantly with antibiotics to fudge multidrug resistance. This chapter discusses the characteristics of such unconventional therapies and how they can be applied to treat multidrug-resistant diseases. Keywords

Antimicrobial · Antibiotics · Multidrug-resistant pathogens · Non-traditional approaches

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Introduction

Since the time of Hippocrates and Galen, medicine has advanced steadily, with each new discovery extending and bettering patient lives. By the eighteenth century, medical science had grasped the concept of vaccines and made significant strides in the prevention of infectious diseases. Despite this, a diagnosis of bacterial infection was typically viewed as a death sentence. Patients had the choice of living or dying because medication was mostly used for sympathetic care. The breakthrough of antimicrobials in the twentieth century, however, changed that. It is not an exaggeration to say that this transformed medical research forever, as it permitted for the improvement of many medical discoveries from small medical procedures to the complete replacement of organs, as well as the installation of various clinical gadgets, that greatly helped in improving the quality of life for countless individuals (Kirienko et al. 2019). The term “antibiotics” was first used in 1942 which means any substance that is produced by the microorganism and eventually by chemical synthesis to retard the growth of other microorganisms. Nowadays an extensive variety of antibiotics are available, and all are used to eradicate or retard the development of disease-causing microbes (Zimdahl 2015). These drugs, especially antibiotics, carried us from powerless sufferers to strong conquerors that could ward off life-threatening diseases. However, the overuse, abuse, and misuse of these medications have contributed to the emergence of multidrug resistance (MDR), a major global problem (Malik et al. 2020). Multidrug-resistant pathogens are typically unaffected by these antibiotics; this means that the current antibiotics are no longer effective (Hasan and Al-Harmoosh 2020). In the current situation, advancement of antibiotic resistance has evolved into a life-threatening trend which has already elevated the

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risk of morbidity and mortality caused by infectious disease (Chan et al. 2012). On the other hand, the discovery of antimicrobial drugs has slowed down dramatically over the previous two decades, which is a major issue. Using the same method to find the identical libraries leads to the similar treatment, which seems to be useless in the long run (Kirienko et al. 2019). Multidrug resistance is progressively habitual development, and bacteria have grown to fight with antibiotics for centuries. We can reduce antibiotic use by limiting antibiotic consumption and raising awareness about multidrug resistance among prescribers and the general public. Usage of antimicrobial agents in agriculture should also be limited. It is anticipated that the production of new antibiotic agents must take footstep with the improved resistance, so there should be a global intervention and expenditures on the part of the public. Several novel strategies are being developed based on a re-evaluation of resistance, sickness, and avoidance. Whole genome sequencing (WGS) is a process that enables for the speedy discovery of resistance pathways and bacterial resistance regulation. Another strategy is the quorum-quenching (QQ) method, which prevents infection by cooperating with bacterial cells. Bacteriophages are also promising alternatives to antibiotics. Humanized monoclonal antibodies are also there to treat infections. In 2019 the World Health Organization reported that bacterial resistance is accountable for the deaths of 700,000 people. However, by 2050 it is assumed that the number will reach up to 20 million globally. Consequently, it has become a prime issue, causing significant risk to our survival and wealth (Uddin et al. 2021). Tuberculosis infectious agent Mycobacterium tuberculosis is the supreme cause of death. In 2018 about ten million people were infected globally. As indicated by the World Health Organization, 27% of the worldwide tuberculosis patients are from India. However, India also reported 27% of the universal load of rifampicin-resistant TB2 (Shivekar et al. 2020). Clearly, we must look out alternate therapy modalities to bridge this widening divide. As a result, there is a growing interest in non-traditional techniques to treat drug-resistant microorganisms. In this chapter, a variety of these tactics are discussed.

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Antibiotic Failure due to Multidrug Efflux Pumps

Multidrug efflux pumps belong to various groups of transporters and contain numerous important efflux pumps. These pumps play a major role in the active efflux of drugs, which leads to multidrug resistance and treatment failure (Nikaido 2009). There are mainly five families of efflux pump proteins which significantly leads to the multidrug resistance such as the major facilitator superfamily (MFS), the multidrug and toxic-compound extrusion (MATE) family, ATP-binding cassette (ABC) superfamily, the resistance nodulation division (RND) family, and the small multidrug resistance (SMR) family (Piddock 2006). Many sectors of medicine, such as surgery, premature newborn care, cancer chemotherapy, and transplantation, may be adversely affected if new techniques to combating multidrug resistance microbes are not developed. MDR bacteria,

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according to the World Health Organization, are one of the top three health hazards to humans. Development of new antibiotics is one of the options for treating MDR bacterial illness. However, in the recent decades, merely two new classes of antibiotics have been launched commercially, neither of which is particularly effective against Gram-negative bacteria. Combining two or more antimicrobial medications during a therapy program is another way to combat MDR infections (Worthington and Melander 2013). Currently, combination therapy provides a significant scope to fight with multidrug resistance in different types of bacterial infections. For the cure of multidrug-resistant infections, it has been suggested that combination therapy is preferable than monotherapy (Malik et al. 2020). Beyond blocking or killing infections with small compounds, non-traditional approaches involve a variety of other ways to control the disease.

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Anti-virulence

The fundamental goal of this strategy is to stop virulence factors (VFs) from producing and acting. Variability can be seen across or within bacterial species, and VFs are strain-specific or even species-specific. The expression of virulence genes is impacted by different factors, including the site of infection, the environment, and the duration of a pathophysiological process. Many discoveries, preclinical studies, and development phases are currently underway that are entirely focused on anti-virulence strategies, and these efforts have progressed to the clinical trials of few anti-virulence drugs, with some drugs that significantly block exotoxins being approved for treatment.

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Toxins

Pathogenic bacteria are the primary producers of exotoxins, and without them, microorganisms are unable to cause symptoms in the infected person. As a result, anti-virulence therapies are the best targets. Some antibodies against the toxins of Clostridium botulinum, Clostridium difficile, and Bacillus anthracis have been approved for clinical usage. Bezlotoxumab, a human monoclonal antibody (mAb), was recently licensed by the FDA and the European Medicines Agency (EMA) for the prevention of acute Clostridium difficile infection in adults.

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Microbiome-Modifying Therapies

It is a recent advancement in metagenomic research that is based on the human microbiome and helps understand how it interacts with the host. This treatment involves modifying and engineering the human microbiome, and it’s a promising way to avoid illness. The USFDA has yet to approve a microbiome-modifying treatment (Theuretzbacher and Piddock 2019).

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Nanomedicine: A Novel Approach

In today’s world, a novel nanomedicine has the ability to overcome multidrug resistance, and the main goal is to create new ways for targeted drug delivery for MDR. Solid lipid nanoparticles, liposomes, polymeric nanoparticles, organic nanoparticles, inorganic nanoparticles, and hybrid particles are all examples of nanomedicines. These particles have been widely employed for medication, physical encapsulation, or chemical conjugation, and there are multiple preclinical studies that have demonstrated the efficacy of the unique technique, as well as several clinical studies are currently underway. Folate receptor-targeted, pH-sensitive cleavable PEGylated liposomes carrying doxorubicin and imatinib have been found to successfully deliver the intracellular drugs and overcome MDR with increased efficacy in breast cancer patients, in a study (Su et al. 2021).

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Non-traditional Approaches

Due to the ever-increasing number of drug-resistant bacteria, as well as the scientific limitations of finding new antibiotics, a non-traditional approach to MDR has evolved (Theuretzbacher and Piddock 2019). These non-traditional approaches will not fully replace the necessity of antibiotics, but they can be effective when used in combination with existing medications (Czaplewski et al. 2016). Although these approaches are not totally substitutes for antibiotics, they do allow us to consider additional options for treating a disease outside pathogen inhibition/killing via small molecules. In the future, these non-traditional approaches could open plenty of new possibilities for treating bacterial diseases. Here, we have attempted to explain the most recent advancements, applications, and challenges that these approaches face right now, in the hope of providing some new insight into non-traditional approaches that could deliver safe and effective therapies in the future (Fig. 3.1).

7.1

Anti-virulence Approaches

Virulence factors (VFs) are pathogen-produced compounds that boost the pathogen’s ability to cause disease. Invasion, transmission, toxicity, colonization, and battling host defenses are all things that VFs are involved in (Korves and Colosimo 2009). The virulence variables involved, and many other intrinsic mechanisms such as the amount of microbial entities supplied into the primary body, the method of regulation, and the host-specific defensive system, define the cause of pathogenesis generated by the microbial entities (Khan et al. 2010). In order to produce proteins or other biomolecules like polysaccharides or lipid mediators through a complicated biosynthetic pathway, pathogenic bacteria that cause disease have evolved virulence mechanisms and a large variety of virulenceencoding factors or genes. The particular adherence of bacteria to host tissue cells,

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Fig. 3.1 Various types of non-traditional approaches to combat multidrug resistance

mediated by microbial adhesions, is one of the most critical virulence determinants for microbial pathogenesis (Theuretzbacher and Piddock 2019). Anti-virulence approaches try to stop virulence factors (VFs) from producing or acting; however they usually have no impact on the microbe in vitro growth. VFs are frequently species- or even strain-specific, and they differ in their conservation between and/or within bacterial species. Blocking bacterial pathogenicity without killing or reducing bacterial growth is a unique method to creating medicines for infectious diseases (Rasko and Sperandio 2010). Several anti-virulence methods are currently in the development or preclinical expansion process (Garland et al. 2017).

Targeting Toxins The pathogenic bacteria produce exotoxins which are a major source of symptoms that an infected individual shows during infection. Exotoxins are the main focus of therapy in this approach, with the main goal being to avoid the negative effects of the toxins. This is accomplished by adding soluble analogues to the receptor to inhibit the toxin’s binding to its cognate receptor (Rasko and Sperandio 2010). Most of the bacterial toxins released in the environment are surface-localized structures that provide a good target to monoclonal antibodies and hence can be treated with antibody therapy. So far, the antibodies which are approved for clinical use can act against the toxins generated by Clostridium botulinum, Bacillus anthracis, and Clostridium difficile. Bezlotoxumab (Merck) is a human monoclonal antibody (mAb), recently approved by the FDA and EMA that binds to the toxin B of Clostridium difficile and is supposed to prevent the recurring Clostridium difficile infection (CDI) in adults at risk. Suvratoxumab is another mAb that binds and neutralizes Staphylococcus aureus toxin (Yu et al. 2017). Tocilizumab and canakinumab are two human monoclonal antibodies (mAbs) that are now being tested in clinical trials for the handling of SARS-CoV-2 infections (Ucciferri et al. 2020). The majority of mAbs are still in different stages of clinical trials, such as suvratoxumab, which is in phase 2, and aridis, which is in phase 3. As a result,

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passing all clinical trial phases is critical for the successful use of mAbs for targeting toxins under the anti-virulence method of non-traditional approach. Only a few mAbs have survived to the final rounds of clinical trials so far (Theuretzbacher and Piddock 2019). These exotoxin-targeting therapies are one of the most advanced non-traditional approaches in the present day.

Adhesins and Biofilms To systematically colonize and express the disease, bacteria need to attach to the host cells. Most microbes have a narrow host range and will just infect hosts with certain bacterial adhesion factor receptors on their cell surfaces (Ofek et al. 2003; Proft and Baker 2009). In another case P. aeruginosa expresses the surface polysaccharide alginate. It enhances the biofilm preparation and virulence to leukocyte mortality. Inhibitors of Specialized Bacterial Secretory Systems Despite the fact that there are many different varieties of Type III secretion systems (T3SSs), there are only a few strategies to stop them. They work as a direct pathway for virulence effector protein translocation into eukaryotic host cells (Hueck 1998). Using high-throughput screening (HTS) of small-molecule libraries, a number of small-molecule inhibitors of bacterial T3SS have been discovered. Chlamydia spp. are bacteria that live only inside cells. The bacteria encode a T3SS that secretes peptides into the infected host cell’s inclusion membrane and is required for the pathogen’s intracellular survival. The acylated hydra-zones of salicylaldehydes, a chemical class of small-molecule inhibitors, have been found to impede secretion through the T3SS of Chlamydia spp. (Bailey et al. 2007). Another study discovered a new class of compounds including INP0400 that inhibited Chlamydia trachomatis and Yersinia pseudotuberculosis secretion systems (Muschiol et al. 2006). VF Secretion Type III secretion systems (T3SSs) are essential virulence devices for different Gram-negative bacteria’s inner and outer membranes that are utilized to transport virulence effector proteins into host cells and help in the formation and spread of infections (Anantharajah et al. 2016). PcrV, a P. aeruginosa T3SS needle-tip protein, is targeted by a bispecific mAb (to disrupt T3SS-mediated injection of toxins in host cells and Psl exopolysaccharide to avoid microbes adhering to epithelial cells) (DiGiandomenico et al. 2014). Inhibitors of Organism-Specific Cell-to-Cell Signaling or Bacterial Communication QS is a molecular communication mechanism that affects in bacterial gene appearance and cell physiology by synchronizing the expression of certain genes. A large range of signaling molecules could be used as appealing and possibly broadspectrum anti-virulence targets (Rémy et al. 2018). Several QS-interfering agents (also known as quorum quenchers) have been identified, as well as natural and synthetic chemicals, enzymes, and antibodies that target every step of the QS system. These have undergone in vitro and in vivo testing (Defoirdt 2018). In both acute and

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persistent disease models, the P. aeruginosa QS passageways LasR-LasI, MvfR, IQS, and RhlR-RhlI have been investigated (Dickey et al. 2017). Other techniques, known as quorum quenching, employ enzymes that weaken the quorum-sensing signal. Lactonases are enzymes that break down the homoserine ring of acylhomoserine lactones (AHLs) and are found in bacteria such as Bacillus spp. Bacillus lactonase-expressing transgenic plants are resistant to quorum-sensing-dependent bacterial infection (Dong et al. 2001).

Counteracting Immune Evasion Many bacteria use their various components to avoid being detected by or evading the immunological response of the host. As a result, solutions to counteract such methods are being developed. MAbs targeting microbial shell epitopes are thought to improve microbial clearance via boosting antibody-dependent phagocytosis and additionally supplement intervened bactericidal or by bacterial death without the need of the immune system (Wang-Lin and Balthasar 2018). Antiviral strategies won’t be able to substitute antibiotics; thus they’re unlikely to help with the resistance problem or the lack of antibiotic pipelines. Nonetheless, they may help antibiotics work better; however, high-quality clinical trials are needed to show that they are beneficial to patients. This profession might benefit from more open debate and analysis of unsuccessful drug studies.

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Microbiome-Modifying Therapies

The fundamental idea is that restoring the microbiome following illness or conserving the microbiome to avoid infection will result in clinical advantage. Clinically successful transfer of full natural microbiota in the form of feces from healthy donors restores a balanced microbiome (fecal microbiome transplantation [FMT]). This treatment has an average cure rate of 90% (Cammarota et al. 2017). FMT reintroduces a complete, stable gut microbial ecosystem (Bakken et al. 2011; Borody and Campbell 2012). And it’s the most developed sort of microbiota treatment (Ooijevaar et al. 2018).

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Phage Therapy

Although bacterial diseases have been treated with microbial viruses (bacteriophages, widely known as phages) for about a century in Eastern Europe, interest in phage research and development has only grown in the previous 10–15 years as a result of the growth of multidrug-resistant pathogens (Kortright et al. 2019). One of the hottest topics in medicine today is the use of bacterial viruses (bacteriophages or phages) in the fight against AMR (phage treatment [PT]). There are an increasing number of publications indicating its viability, and relevant surveys tended to the subject of PT are published essentially consistently.

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The field of phage research has been resurrected thanks to synthetic biology and other current techniques. They allow for phage modification, as well as the identification and thorough screening of genes coding for toxins and VFs, as well as their elimination to prevent the spread of toxins and VFs from one bacterium to the next. Phages are generally thought to be safe since they do not infect mammalian cells (Pires et al. 2016). Liu et al. have evaluated PT reports of medical and animal research issued between January 1, 2008, and March 28, 2021 (Liu et al. 2021). A therapeutic technique involving phages is being pursued by at least 30 companies. Infections caused by P. aeruginosa, S. aureus, and E. coli are the subject of recent and current trials. As a result, antibody reactions to PT appear to be limiting its efficacy in immunocompetent individuals receiving extended phage treatment. In addition, phages have been demonstrated to have immunomodulatory effects ranging from stimulation to inhibition (Górski et al. 2017). Because of their anti-inflammatory, immunomodulatory, and antiviral properties, phages could be an effective treatment for COVID-19 (Górski et al. 2020).

9.1

Phages as Carriers

Non-replicative phages have been genetically modified to serve as specialized nanodelivery vehicles and contain payloads that have antibacterial activity in addition to cell destruction (Krom et al. 2015). Synthetic biology techniques allow a broad range of gene expression schemes to be used to target bacteria. Even though phage delivery systems are typically precise, the therapeutics delivered has a thin spectrum and is pathogen unambiguous. The supplied genes could be DNA sequence self-governing and have a quick bactericidal impact (Phico) or use CRISPR RNA-guided genome editing techniques to generate novel activities (Theuretzbacher and Piddock 2019). Due to the fact that phage vehicles are not self-replicating, the amount required to target bacteria in an infection must be extremely large.

9.2

Phage-Derived Products

Notwithstanding the way that phage-determined enzymes have been contemplated since the last part of the 1990s, renewed consideration has arisen to address contemporary medication obstruction challenges. The most well-investigated peptidoglycan-degrading enzymes are endolysins. To allow the progeny phage to be released within infected bacterial cells, phages encode them (Fischetti 2018). This causes fast osmotic lysis and bacterial cell death. Regardless of bacterial resistance to conventional antibiotics, endolysins are highly selective for a particular bacterial species or genus and are bacteriolytic in contact (Fernandes and São-José 2018). Pure, recombinant lysin-enabled enzymes can act from the outside, while endolysins typically carry out their functions inside the cell.

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Immunotherapy

Small-molecule medicines and proteins are used in host-directed therapies to target essential host signaling enzymes used by bacteria for intracellular assault, replication, dissemination, and virulence (Chiang et al. 2018). Immunomodulating treatments could include a wide range of medication classes that target a range of biological mechanisms that alter host cell activity. The immune response’s intricacy complicates the choice of appropriate objects. Immunotherapeutics are growing in other remedial fields, but bacterial infections remain relatively unexplored (Baker et al. 2018). A convincing association between the immunomodulating medicine and the clinical result of bacterial contamination, like other non-traditional methods, must be demonstrated.

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Other Bio-antibacterial Approaches

11.1

Antimicrobial Peptides (AMPs)

AMPs are short, positively charged peptides that play a function in many different organisms’ intrinsic immune systems. Because AMPs target membrane processes, it is hypothesized that they eliminate microbial infections directly. Tragically, they are harmful to the cells, restricting their clinical utility. Clinical trials of AMPs as novel anti-infective, immunomodulators, and wound healing boosters are under underway. According to Zharkova et al. (2019) (Zharkova et al. 2019), mammalian AMPs can synergize with different traditional antibiotics, including fluoroquinolones, polyketides, aminoglycosides, and β-lactams. This brings down how much of AMP required for powerful treatment while also reducing the likelihood of side effects. Badri et al. (2021) discovered and described the AMP isolated from necrophagous larvae, Sarconesiopsis magellanica (Badari et al. 2021). Sarconesin, as it was termed, appeared to boost permeability of target membranes as well as binding to DNA in mammalian cells with minimum damage. Moussouni et al. (2019) reported a study that targets MgtC, a virulence component needed for Salmonella and Mycobacterium species to survive intracellularly (Moussouni et al. 2019), using a normally planned synthetic version of MgtR. Belon et al. discovered that synthesized MgtR (based on the Salmonella form of the protein) inhibits intracellular survival and biofilm production in P. aeruginosa (Belon et al. 2015).

11.2

Bacteriocins

Bacteriocins are also naturally occurring chemicals secreted by microorganisms to reduce competition in their natural atmosphere. These peptides are commonly utilized in chemicals fighting against firmly related species, implying that they may have therapeutic use. Ghequire et al. (2018) investigated the therapeutic potential of Gram-negative proteobacteria’s lectin-like bacteriocins or Llps.

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Dissimilar to most bacteriocins, Llps appear to kill on the cell surface rather than inside the cell. If it’s utilized to treat pseudomonads, which have active export pumps that can lessen the effectiveness of significant toxins, this feature will be beneficial (Ghequire et al. 2018). Corre et al. (2019) utilized an analogous approach to uncover natural chemicals that inhibit Legionella pneumophila growth. After investigating over 250 culturable bacterial isolates, they uncovered a remarkable diversity of species that effectively prevented L. pneumophila development. Although this strategy, which takes advantage of natural competition in the bacterial environment, is not new, it has proven to be highly useful in the hunt for more targeted medications than previously sought (Corre et al. 2019).

11.3

Peptide Nucleic Acid (PNA)

Równicki et al. (2018) reported the development and testing of a mazEF-hipBA toxin-antitoxin system-based peptide nucleic acid (PNA)-based treatment in E. coli. They show that antisense PNAs can actually stop antitoxin interpretation, leading to bacterial demise. They also showed that antisense PNAs may be utilized to boost the declaration of the toxin-antitoxin system in the first place, boosting the treatment’s efficiency. Albeit the impact of PNA-mRNA cross breeds on the enactment of the interferon reaction, the difficulties of conveying PNAs into cells, and the straightforwardness with which antimicrobial opposition can arise against a nucleotide hybridization-based treatment remain concerns, antisense PNAs didn’t seem to strongly affect mammalian cells to show a lot of cytotoxicity (Równicki et al. 2018).

11.4

Nanoparticles

Nanoparticles range in size from 1 to 100 nanometers and have a variety of simultaneous mechanisms of action against Gram-positive and Gram-negative bacteria. Antibacterial coatings for implantable devices and pharmaceutical materials, wound dressings, bone cement, dental materials, and vaccines have been used for many years (Wang et al. 2017). For drug conveyance and expanded release shapes, a few kinds of nanoparticles (especially liposomes) are as of now accessible (Kwon et al. 2017). Nanoparticles have been explored as poison folios in different illnesses, including cholera (Das et al. 2018). One organization is working on liposomes that imitate toxin-targeted domains, neutralizing a variety of toxins such as phospholipase C, pore-forming toxins, and T3SS, and can be utilized to treat a variety of illnesses (Combioxin, phase 1/2) (Azeredo da Silveira and Perez 2017).

11.5

Antisense RNA

Antisense antimicrobial therapies are series-specific synthetic oligomers that mute the appearance of certain genes, as well as essential, non-essential, and resistance

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genes. Extensive functional genes are being studied as potential targets, but they must still be confirmed. Several chemical configurations have been investigated, but they all require a delivery method in order to reach microbial cells. Coupling antisense oligomers to cell-penetrating peptides is the most prevalent method (Sully and Geller 2016). Clinical trials for such conjugates have not yet begun. They confront numerous obstacles, including target selection, potential resistance emergence, and carrier and translational concerns, but advanced research could help them develop better ways (Stach and Good 2011).

11.6

Resistance Modulation and Removal of Drug-Resistant Plasmids

Studies on how to turn off drug resistance without hindering bacterial growth or preventing horizontal gene transfer between bacteria have been prompted by resistance. CRISPR-Cas or synthetic oligomers are two methods for silencing drugresistant genetic materials that have been identified (Stach and Good 2011; Levy et al. 2015). These techniques work by inactivating or deleting particular genes in order to restore the bacteria’s antibiotic susceptibility. The transport of the hereditary construct to and within bacteria is the most difficult challenge (Vila 2018). Phages, cell-penetrating peptides, nanoparticles, and transmissible plasmids are all examples of delivery mechanisms.

12

Utilizing Emerging Drug Targets in Drug Development

The majority of currently available antibacterial medications target three primary molecular classes for antibacterial therapy: (a) cell envelope biogenesis suppression, (b) DNA and RNA homeostasis, and (c) protein synthesis (Stokes et al. 2019). However, due to raise in the number of multidrug-resistant bacterial infections, new tactics and drugs to control and treat MDR bacterial infections are urgently needed. Antibacterial agents such as antibodies, bacteriophages and phage-derived enzymes, microbiome-modulating agents, immunomodulating agents, and several other strategies that involve the inhibition of exotoxin production or activity, bacterial adhesion, biofilm formation, and bacterial communication inhibition are all part of the non-traditional approach (Butler et al. 2022). Apart from these approaches, there are a number of new emerging drug targets that may aid future drug discovery efficiency (Mdluli et al. 2015). These targets include:

12.1

Targeting Iron Acquisition and Storage

The entire living species, together with harmful bacteria in an infection, require iron, and the mammalian-resistant system plays a range of techniques to prevent pathogens from obtaining it (Doherty 2007). Thus, pathogenic bacteria’s

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manufacture, transport, and usage of siderophore (a chemical that binds and transports iron in microorganisms) could be suitable targets for a novel medication.

12.2

Targeting Membrane Protein Large

Mycobacterial membrane protein large (Mmpls) are known to aid in mycobacterial proliferation, pathogenicity, and heme absorption (Jain and Cox 2005), making those interesting targets for TB medication research.

12.3

Targeting ClpP Protease

Previous research has shown that acyl depsipeptide (ADEP) has an antibacterial activity by binding to the ClpP (cutinase-like proteins, chaperon-linked proteases, or caseinolytic proteases) protease of Bacillus subtilis (Brötz-Oesterhelt et al. 2005), and these ClpP proteases have significance as therapeutic targets.

12.4

Targeting Central Carbon Metabolism

Many dangerous bacteria, such as mycobacteria, change their metabolism in response to their surroundings (Rhee et al. 2011). As a result of their multifunctionality, numerous metabolic enzymes have been confirmed as therapeutic targets.

12.5

Targeting Energy Generation Which Further Inhibits Respiratory Chain and ATP Synthesis

ATP synthesis and PMF production are among the greatest validated Mtb therapeutic targets (Andries et al. 2005), due to the experimental effectiveness of the ATP synthase inhibitor TMC207 (bedaquiline, Sirturo) (Diacon et al. 2009) and the TB medicine pyrazinamide, which blocks the PMF (proton motive force) pathway. The complexity of ATP synthase makes it difficult to target it, yet cell-based or membrane particle-based screens for ATP synthase inhibitors may find new classes of inhibitors (Zhang et al. 2003; Mak et al. 2012).

12.6

Targeting ROS and NOS

Innovative approaches for creating ROS/NOS at more enough concentrations should be observed as a latent new tuberculosis treatment, provided they do not produce oxidative damage to the host cells (Domann 2013).

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Drug Repurposing: Combination Therapies of Multiple-Drug Resistance (MDR)

13.1

Drug Repurposing

One of the primary areas of research for academia and small corporate research teams, which will play an increasingly significant role in drug development, is drug repurposing (Oprea et al. 2011). Drug repurposing (DR) is also known as drug re-tasking, drug reprofiling, drug rescue, drug recycling, drug redirection, and therapeutic switching. It is described as the discovery of novel pharmacological indications from old/investigational/already marketed/FDA-authorized medications/pro-drugs, as well as the application of newly produced drugs to diseases other than the drug’s original therapeutic purpose (Park 2019). It entails discovering new therapeutic applications for previously approved, discontinued, abandoned, and experimental medications. Conventional drug development is a time-consuming and high-risk procedure. Drug repositioning is a cutting-edge method of drug discovery that has the potential to replace conventional drug discovery programs since it lowers the high cost, extended development period, and higher risk of failure. It decreases the likelihood of disappointment in conventional medication revelation programs, which have a disappointment pace of 45% because of security or harmfulness issues, as well as setting aside to 5–7 years in normal medication improvement time (Corsello et al. 2017; Parvathaneni et al. 2019). As of late, the prescription repositioning system has built up momentum, with reused drugs representing about 33% of all new medication endorsements, producing around 25% of the pharmaceutical industry’s yearly revenue. More ways for speeding up the process of medication repositioning were created after roughly a century of development. Minoxidil, sildenafil, anti-inflammatory medicine, valproic acid, methotrexate, and different prescriptions that have developed from the DR technique incorporate sildenafil, minoxidil, ibuprofen, valproic acid, and methotrexate. For example, sildenafil, which was initially made to treat hypertension and angina pectoris, is presently used to treat erectile dysfunction (Breckenridge and Jacob 2019).

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Difference Between Traditional Drug Discovery and Drug Repurposing

The standard approach of drug discovery, known as de novo identification and creation of novel molecular entities (NME), involves five stages: disclosure and preclinical, security assessment, clinical examination, FDA survey, and FDA postmarket well-being checking. It’s a tedious and costly method with a high disappointment rate. Drug repositioning, then again, has just four phases: compound number,

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compound procurement, improvement, and FDA post-market well-being observing. Because of the development of bioinformatics and cheminformatics approaches as well as the accessibility of massive biological and structural databases, drug repositioning has significantly decreased the time and cost of drug development while also lowering the risk of failure. As of late, the use of in silico approaches, as well as structure-based drug design (SBDD) and artificial intelligence (AI), has ascended in prevalence (Mayr and Fuerst 2008). However, employing authorized medications for novel therapeutic purposes has shown to be a successful repositioning method, as evidenced by past serendipitous observations. This technique to drug discovery is clearly superior to the standard drug discovery program outlined below. For example, sildenafil (Viagra), a phosphodiesterase-5 (PDE5) inhibitor originally developed by Pfizer (1985) for the treatment of coronary artery disease (angina), has been repurposed to treat erectile dysfunction. It has the ability to lower development costs by shortening development time (Baily 1972; Comanor 1965). Metformin (Glucophage), an oral anti-diabetic medicine commonly used to treat type 2 diabetes, has been developed as a cancer treatment and is currently being tested in phase II/phase III clinical trials. When compared to standard drug discovery methods, drug relocation has various advantages. When evaluated to traditional drug discovery programs, there is a considerable reduction in R&D time. The development of a new medication takes 10–16 years in the traditional strategy, whereas it takes 3–12 years in the DR technique. A drug repositioning method costs only $1.6 billion to create, whereas the standard drug development costs around $12 billion. Furthermore, it takes only 1–2 years for researchers to uncover fresh therapeutic targets, while it takes an average of 8 years to produce a repositioned medicine. A repositioned medication skirts the 6–9 years of standard medication improvement and goes directly to preclinical testing and clinical preliminaries, bringing down the general gamble, time, and cost of improvement. As per reports, reused prescriptions require 3–12 years to get FDA as well as European Medications Agency (EMA) endorsement, and they cost 50–60% less. Since the up-and-comer drug has proactively gone through beginning phases of medication improvement like underlying enhancement, preclinical or potentially clinical preliminaries, notwithstanding the chance of the competitor drug being a supported medication with its clinical viability and security profile, a scope of preclinical (pharmacological, toxicological, and so on) and clinical adequacy and well-being data is as of now accessible toward the beginning of a repositioning project. Thus, there is a decrease in the dangers of beginning phase improvement disappointments, which are high in conventional methodologies, as well as an impressive expense decrease with the likely ascent in clinical security and consequently a high achievement rate (Grabowski et al. 1978; Grabowski and Vernon 1990).

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Drug Repurposing Strategies

The two basic DR approaches are on-target and off-target. The proven pharmacological action of a medicinal substance is used to a novel therapeutic application in on-target DR. The therapeutic molecule’s biological target is the same in this procedure, but the ailment is different. An on-track profile is seen in the repositioning of minoxidil (Rogaine), for instance, on the grounds that the medication works on a similar objective and creates two distinct helpful impacts. Minoxidil was made from an antihypertensive vasodilator to treat hair loss. Minoxidil, as an antihypertensive vasodilator, enlarges veins and opens potassium channels, permitting more oxygen, blood, and supplements to arrive at the hair follicles. This pharmacological impact helps its use in the treatment of male pattern hairlessness (androgenic alopecia) (Jensen 1987; Joglekar and Paterson 1986).

The pharmacological mechanism in the off-target profile, on the other hand, is unidentified. Drugs and drug candidates act on novel targets for new therapeutic indications that are outside of their original scope. As a result, both the targets and the signals are novel. The off-target profile of aspirin (Colsprin) is a good example. Aspirin has long been used as an NSAID to treat a variety of pain and inflammatory conditions. It also inhibits platelet activity, which decreases blood coagulation (clot formation) (antiplatelet drug). As a result, it is used to treat heart attacks and strokes. Another new usage of aspirin for prostate cancer treatment has been described.

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Drug Repurposing Approaches

The trial-based technique and the in silico-based approach are two unique and free ways to deal with drug repositioning. The analysis-based strategy is otherwise called activity-based repositioning, and it allows to the utilization of experiment assay to evaluate unique medications for new pharmacological indications. It involves protein target-based and cell-/creature-based screens in in vitro or potentially in vivo sickness models without the requirement for target protein structure data. A few examples of trial-based repositioning approaches are the target screening strategy, the cell assay approach, the animal model approach, and the clinical approach. In silico repositioning, then again, utilizes computational science and bioinformatics/cheminformatics tool to perform virtual screening of public data sets of enormous medication/synthetic libraries. The revelation of conceivable bioactive mixtures is achieved utilizing this strategy, which depends on the molecular communication between the therapeutic molecule and the protein target (Elias et al. 2006; Pammolli et al. 2011; Kola and Landis 2004; DiMasi et al. 2010).

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Mechanisms of Microbial Drug Resistance

Microbial bacteria exhibiting mutations at various locations in their genome are the main cause of antimicrobial resistance. This is because of the enormous hereditary adaptability of numerous unstoppable microorganisms, especially tiny organisms, which enable them to survive in various hosts and environments. The advanced systems of microorganisms that coexist naturally with species that create antimicrobials allow them to thrive in the presence of potent antitoxins that would otherwise kill them or disrupt their reproduction cycle. Because of gained blockage and fantastic hereditary adaptability, bacteria can thrive even in environments with strong anti-infection focuses, such as medical clinics, rural initiatives, and ecological examples.

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The Use of Combination Therapy to Counter Multidrug Resistance

Numerous microorganisms’ unexpected adaptation, resistance, and transformation rates have aided in their persistence and replication despite effective antibacterial experts. Undoubtedly, these characteristics have helped a number of clinically relevant unstoppable diseases adapt to strong anti-infection defenses, trigger genetic modifications, acquire safe properties, and ultimately flourish and spread to additional human and nonhuman hosts (Munita and Arias 2016). This is essential from a therapeutic perspective, as diseases caused by infections with these drug-resistant bacteria do not respond to conventional treatments (Liu et al. 2018; Lu et al. 2018).

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Future Perspective

The risk of antimicrobial resistance is extensive, and a lot of work is being done to overcome this problem. Though many non-traditional therapies are available for MDR, very few have reached preclinical development stage. There is a need to search and develop new non-traditional therapeutic approaches (anti-virulence, drug repurposing, combination therapy, non-conventional bactericides, and phage-based approaches) against MDR. Furthermore, several awareness programs must be conducted to demonstrate the proper use of antibiotics to reinvent the dominance over MDR.

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Treatment Strategies to Combat Multidrug Resistance (MDR) in Bacteria Bisma Jan, Rafia Jan, Suhaib Afzal, Mehrose Ayoub, and Mubashir Hussain Masoodi

Abstract

In the developing world, antibiotic failure is becoming a major issue in the healthcare system. The world is presently facing global catastrophic problems as multidrug resistances are enormously developing, resistances are occurring in almost all clinical isolates of bacteria, and repetitive infections are being caused which antagonize the success of the treatment process. In this context, new strategies are demanded by the time against multidrug resistance (MDR). Several attempts have been employed to tackle the MDR, but still none of them proved to be completely satisfying. To tackle with the entire certainty, a novel approach is to be employed. Nanotechnological approaches to combat MDR are proving to be of worth. Nano-drugs have unique properties, modes of action, and activity against multidrug resistance. Several other non-traditional methods like the combination approach include a combination of two or more antibiotics, antibiotic/adjuvant combination, and screening of previously approved drugs. The present chapter describes various non-traditional approaches to combat MDR like antimicrobial peptides, anti-virulence compounds, and drug repurposing antiinfectives. Also, further research is needed before a definitive statement can be made on any of these approaches. Keywords

MDR strategies · Anti-virulence · Drug repurposing B. Jan · S. Afzal · M. Ayoub · M. H. Masoodi (✉) Department of Pharmaceutical Sciences, University of Kashmir, Srinagar, Kashmir, India e-mail: [email protected] R. Jan Defense Research and Development Organization (DRDO), Hospital Khonmoh, Srinagar, Kashmir, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. Y. Wani, A. Ahmad (eds.), Non-traditional Approaches to Combat Antimicrobial Drug Resistance, https://doi.org/10.1007/978-981-19-9167-7_4

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Background

The disclosure of penicillin in 1928 was trailed by the revelation and business creation of numerous different anti-toxins. We presently underestimate that any irresistible sickness is treatable by anti-toxin treatment. Anti-toxins are produced at an expected size of around 100,000 tons every year around the world, and their utilization significantly affected the existence of microorganisms on the planet. More strains of microbes have become anti-infection safe, and some have become impervious to numerous anti-toxins and chemotherapeutic specialists, the marvel of multidrug opposition. Without a doubt, a few strains have become impervious essentially to the regularly accessible specialists as a whole. A famous case is the methicillin-resistant Staphylococcus aureus (MRSA), which is safe not exclusively to methicillin (which was created to battle against penicillinase-delivering S. aureus) yet normally likewise to aminoglycosides, macrolides, antibiotic medication, chloramphenicol, and lincosamides. Such strains are additionally impervious to sanitizers, and MRSA can go about as a significant wellspring of medical clinic-obtained diseases. An old anti-toxin, vancomycin, was restored for the treatment of MRSA contaminations. Nonetheless, adaptable protection from vancomycin is presently very normal in Enterococcus and discovered its direction at last to MRSA in 2002, albeit such strains are as yet uncommon (de Lencastre et al. 2007). A much more genuine danger might be the development of Gram-negative microorganisms that are impervious to the accessible specialists as a whole (Livermore 2004). The examination had the opportunity to respond to the danger of MRSA. Along these lines, there are recently evolved specialists that are dynamic against vancomycin-resistant MRSA, for example, linezolid and quinupristin/dalfopristin. In any case, the rise of “skillet safe” Gram-negative strains, prominently those having a place with Pseudomonas aeruginosa and Acinetobacter baumannii, happened all the more as of late, after most significant drug organizations halted the improvement of new antibacterial specialists. Subsequently, there are practically no specialists that could be utilized against these strains, where an external film hindrance of low porousness and a variety of effective multidrug efflux siphons are joined with a large number of explicit obstruction instruments. Multidrug opposition in microbes happens by the aggregation, on obstruction (R) plasmids or transposons, of qualities, with each coding for protection from a particular specialist, as well as by the activity of multidrug efflux siphons, every one of which can siphon out more than one medication type. Continuing impulsive use of drugs such as antibiotics is one of the driving forces for the development of multidrug resistance. It resulted not only in the emergence of multidrug resistance but also in extremely drug resistance (XDR) (Andersson and Hughes 2010). According to the WHO, antimicrobial resistance is presently regarded as a principal threat to the global population mainly due to the spread of MDR bacterial pathogens (Pacios et al. 2020). The increase in the resistance of microbes toward many antimicrobials has proven to be a major threat to the global population. Common infectious diseases are not treated with an entire efficiency due to the increment in the development of MDR, and because of this, common

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infectious diseases lead to illness for a longer time (Tanwar et al. 2014). After the outbreaking discovery of antibiotics, greater positive results were accumulated in lesser time, but with the increased use of these antibiotics, the resistances offered by bacteria also got increased significantly. Some of the deadly bacterial pathogens such as Klebsiella pneumoniae and Acinetobacter baumannii were much resistant to currently available drugs (Boucher et al. 2009; Slavcovici et al. 2015). Due to this reason, the Infectious Disease Society of America proposed the acronym ESKAPE to standardize this group of pathogens with MDR (Rice 2008). Due to the fatal effects of MDR bacteria, ECDC, DSA, and WHO encouraged pharma industries to make investigations and perform research extensively to tackle the problem of MDR (Boucher et al. 2009). But despite these efforts, there still lie some areas where there is a need for more research and investigation to develop new strategies.

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MDR

During the most recent couple of years, the occurrence of microbial diseases has expanded drastically. Nonstop arrangement of antimicrobial medications in treating contaminations has driven the development of obstruction among the different strains of microorganisms. Multidrug opposition MDR is characterized as a lack of care or obstruction of a microorganism to the managed antimicrobial medications (which are irrelevant and have distinctive atomic focuses) regardless of prior affectability to it (Popęda et al. 2014). As per the WHO, these safe microorganisms (like microscopic organisms, growths, infections, and parasites) can battle assault by antimicrobial medications, which prompts insufficient treatment bringing about tirelessness and spreading of contaminations. Albeit the advancement of MDR is a characteristic wonder, the broad ascent in the number of immunocompromised conditions, similar to HIV-disease, diabetic patients, people who have gone through organ transplantation, and serious consumption patients, make the body an obvious objective for medical clinic-obtained irresistible sicknesses, in this way adding to the additional spread of MDR. Studies from WHO report have shown exceptionally high paces of obstruction in microorganisms, for example, Escherichia coli against anti-infection agents as cephalosporin and fluoroquinolones, Klebsiella pneumoniae against cephalosporin and carbapenems, Staphylococcus aureus against methicillin, Streptococcus pneumoniae against penicillin, nontyphoidal Salmonella against fluoroquinolones, Shigella species against fluoroquinolones, Neisseria gonorrhoeae against cephalosporin, and Mycobacterium tuberculosis against rifampicin, isoniazid, and fluoroquinolone causing normal contaminations (Nikaido 2009). A restricted number of antifungal medications are accessible for the treatment of persistent parasitic contaminations. Protection from medications, for example, polyene macrolides (amphotericin B), azole subordinates (ketoconazole, fluconazole, itraconazole, and voriconazole), DNA and RNA union inhibitors (flucytosine), and 1,3-β-glucan synthase inhibitors (echinocandins) exists in detaches of Candida spp., Aspergillus spp., Cryptococcus neoformans, Trichosporon beigelii, Scopulariopsis spp., and Pseudallescheria boydii (Loeffler and Stevens 2003). Delayed medication

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openness and relentless viral replication result in the appearance of different safe strains and tirelessness of diseases regardless of treatment. This has made antiviral opposition a question of worry in immunocompromised patients. Results of antiviral medication obstruction were seen in immunosuppressed transfer beneficiaries and oncology patients tainted by one or the other cytomegalovirus (CMV), herpes simplex infection (HSV), varicella-zoster virus (VZV) (Ullman 1995), human immunodeficiency infection (HIV), flu an infection, hepatitis C (HCV), or hepatitis B infection (HBV) (Strasfeld and Chou 2010; Margeridon-Thermet and Shafer 2010). Parasitic multidrug opposition has been dissected in separates of Plasmodium, Leishmania, Entamoeba, Trichomonas vaginalis, schistosomes (Ullman 1995; Greenberg 2013), and Toxoplasma gondii (McFadden et al. 2000; Nagamune et al. 2007; Doliwa et al. 2013) against medications such as chloroquine, pyrimethamine, artemisinin, pentavalent antimonials, miltefosine, paromomycin, and amphotericin B (Vanaerschot et al. 2014; Mohapatra 2014) just as atovaquone and sulfadiazine. One of the most perfect representations of infection inclined to MDR is intestinal sickness, brought about by Plasmodium falciparum (Yang et al. 2011). Another protozoan parasite, Entamoeba spp., causes amebiasis which is likewise a significant general well-being danger in numerous tropical and subtropical nations (Bansal et al. 2006). A worldwide well-being danger of schistosomiasis is likewise thought to be like that of intestinal sickness and other ongoing infections (Greenberg 2013). This survey article underlines the meaning of MDR, different components adding to its turn of events, and what’s more, issues related to MDR and its potential cures.

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Global Incidence

Anti-toxin obstruction has turned into a worldwide issue, with 700,000 passings inferable from MDR happening every year. The Centers for Disease Control and Prevention (CDC) shows quickly expanding paces of disease because of antimicrobial-safe microscopic organisms. The point of the review is to depict the frequency of MDR, broadly drug-safe (XDR) and skillet drug-safe (PDR) in Enterococcus spp., Staphylococcus aureus, K. pneumonia, Acinetobacter baumannii, P. aeruginosa, and Enterobacter spp. (ESCAPE) microorganisms in youngsters conceded to Dr. Hasan Sadikin Hospital. All pediatric patients having blood cultures attracted from January 2015 to December 2016 were reflectively examined. Information incorporates the number of drawn blood cultures, number of positive outcomes, kind of microbes, and affectability design. Global standard definitions for gained obstruction by ECDC and CDC were utilized as definitions for MDR, XDR, and PDR microbes. From January 2015 to December 2016, 299 from 2.542 (11.7%) blood culture was positive, with Staphylococcus aureus, Enterococcus spp., Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter spp., separately 5, 6, 24, 5, and 20 with absolute 60 (20%). The MDR and XDR microorganisms found were in 47 and 13 patients, individually (Adrizain et al. 2018).

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Anti-infection opposition is considered a critical worldwide issue, and the US Centers for Disease Control and Prevention (CDC) has revealed that around 2,000,000 US people are contaminated by antibiotic-resistant microorganisms every year, subsequently bringing about 23,000 passings. The CDC likewise detailed that roughly the greater part of internal patients get an anti-infection and around 33% get an expansive range of anti-toxin during their stay (Kuehn 2014). Besides the high rate of antimicrobial opposition diseases announced by the CDC, people, in general, are requested to focus on the high-assessed passings in which an expected yearly 700,000 passings overall are inferable from contaminations by MDR microorganisms. It is assessed that continuously in 2050, around ten million individuals around the world will kick the bucket because of this issue (Hampton 2015). These diseases are brought about by methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), fluoroquinolone-safe Pseudomonas aeruginosa, ESBL-delivering E. coli and Klebsiella sp., Klebsiella pneumonia and E. coli creating carbapenemase, Acinetobacter baumannii, and Pseudomonas aeruginosa overall. These antimicrobial-safe microorganisms include Enterococcus faecium, Staphylococcus aureus, K. pneumonia remembered for Enterobacteriaceae, Acinetobacter baumannii, P. aeruginosa, and Enterobacter sp. which are by and large alluded as “ESKAPE” (Boucher et al. 2009; Borghesi and Stronati 2015). We meant to concentrate on the occurrence of MDR, widely drug-safe (XDR) furthermore and dish drug-safe (PDR) in ESKAPE microbes in kids conceded to Dr. Hasan Sadikin General Hospital, Bandung.

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Economic Impact

MDR is a widely known misanthropic issue and a substantial public health threat (Lee et al. 2007). Antimicrobial resistance (AMR) has emerged as a major concern for public health, as it poses obstacles to the effective prevention and treatment of long-lasting diseases. Despite several measures adopted in recent decades to address this issue, worldwide MR trends show no indications of abating. Numerous studies have exposed the devastating economic effects of AMR, which include skyrocketing healthcare costs as a result of an increase in hospitalizations and drug usage. Infection with AMR leads to serious illnesses, lengthier hospital stays, greater healthcare expenses, increased second-line drug costs, and treatment failures. It is projected that antibiotic resistance results in annual expenses of more than nine billion euros only in Europe. Additionally, the Centers for Disease Control and Prevention (CDC) estimates that antibiotic resistance increases direct healthcare expenditures in the United States by 20 billion dollars per year, excluding the 35 billion dollars in productivity losses (Dadgostar 2019). Various analyses have concluded that by the year 2050, the yearly cost of AMR might range anywhere from $300 billion to more than a trillion dollars across the globe (Dadgostar 2019; Ahmad and Khan 2019). The CDC in the United States reports that 23,000 individuals in the country die annually as a direct result of illnesses. Therefore, the economic burden

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caused by antibiotic resistance in the United States is projected to be $55 billion, with $20 billion in healthcare expenses and $35 billion in lost productivity per year. The escalating elevation in the number of people who were affected by the MDR bacteria showed an increased impact on the elevated death rates, prolonged hospitalization, and costly medication compared to the infection induced by the antibioticsusceptible bacteria (Acar 1997). An augmenting increase in the cost of hospitalization and prolonged time period was outlined for patients who were colonized by the MDR bacteria (Holmberg et al. 1987). In addition, MDR demands the use of different kinds of drugs in combination and the usage of various new technologies of medical management which attributes to the extra costs as compared to the nonmultidrug-resistant bacteria (Ried 2011). The impact of resistance on mortality, hospital costs, and length of stays was studied, and it was found that in various Gram-positive and Gram-negative-resistant bacteria like E. coli and K. pneumonia, MDR P. aeruginosa and acinetobacter spp., various direct and intangible costs were reported which included elevated rate of mortality, longer duration of hospital stays, and augmenting hospital costs (Giske et al. 2008).

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Currently Available Therapies

The main currently available treatments for MDR-GNB patients are discussed below:

5.1

Polymyxins

Polymyxin was identified from the Gram-positive soil bacteria Bacillus polymyxa (afterward recategorized as Paenibacillus polymyxa in 1993) in 1947. The polymyxins are small lipopeptide molecules with a molecular mass of 1200 daltons and consist of a polycationic peptide ring with a short projecting peptide coupled to a hydrophobic fatty acid tail. The cationic ring helps retain the solubility in aqueous solutions, whereas the entry into bacterial settings is encouraged by the hydrophobic acyl chain. All of the polymyxin share the same bactericidal properties and are effective against Gram-negative organisms and a few Gram-positive microorganisms (Velkov et al. 2010). Polymyxins were phased out of clinical use in the 1960s due to nephrotoxicity and the discovery of very well-tolerated drug moieties from other antibiotic drug categories (Nation et al. 2015). After its initial widespread application was curtailed due to toxicity concerns, polymyxins found very limited utility in the treatment of persistent infections in patients with cystic fibrosis, ocular conjunctivitis infections, and over-the-counter topical antibiotic ointments (Trimble et al. 2016). Despite toxicity concerns, the use of polymyxins has increased recently due to rising resistance to other medications. Currently, they are the drug of choice for the management of serious MDR Gram-negative bacterial infections especially those prompted by MDR Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii for which they have become the

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last-resort treatment for infections resistant to other available antibiotics (Nation et al. 2015; Vaara 2019). The resistance to polymyxin arises due to increased drug efflux, capsule formation, reduction in porin pathway, and alterations to reduce the net negative charge or fluidity of lipopolysaccharide (LPS) (Trimble et al. 2016).

Mechanism of Action Polymyxin binds to LPS that constitutes the outer membrane of Gram-negative bacteria and is then taken up by the “self-promoted uptake” pathway. The polycationic peptide ring adheres to the outer membrane, dislodging the calcium and magnesium bridges that keep the LPS stable and result in disruption of the membrane (Zavascki et al. 2007). Polymyxins increase porousness of cell film of the Gram-negative microorganisms by dislodging Ca2+ and Mg2+ from PO4 3of the bacterial cell film through an electrostatic association between αγ-Dab+ of polymyxins and PO43- of the bacterial cell film; lastly, bacterial cell passing takes place as shown in Fig. 4.1 (Hasan 2019).

5.2

Aminoglycosides

Aminoglycosides are either natural or semisynthetic antibiotics obtained from actinomycetes. They are very primary antibiotic agents implemented for widespread clinical use, and many have received approval and are considered safe for human consumption. Aminoglycosides are potent antibiotics with a broad spectrum of activity and exert their antimicrobial effect by inhibiting the synthesis of proteins. During the past few years, many other members of the class were discovered and developed. These members include neomycin (1949, from S. fradiae), kanamycin (1957, from S. kanamyceticus), gentamicin (1963, from Micromonospora

Db-Dab+

(A) Electrostatic interaction between Db-Dab+ and PO43-

Polymyxin

(C) Lipopolysaccharide (LPS) is destabilized

(D) Increased permeability of the bacterial membrane

(E) Leakage of cytoplasmic contents and cell death

Fig. 4.1 Mechanism of action of polymyxins

PO

3-

Gram-negative bacteria

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Ca2+/Mg2+

2+

2+

(B) Displacement of Ca /Mg

Gram-negative bacteria

Gram-negative bacteria

Died Bacteria

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purpurea), netilmicin (1967, derived from sisomicin), tobramycin (1967, from S. tenebrarius), and amikacin (1972, derived from kanamycin) (Krause et al. 2016). Aminoglycosides are one of the main antibiotic classes and present highly bactericidal action against Gram-negative bacterial agents such as Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Unlike fluoroquinolone, beta-lactam, and cephalosporin antibiotics, these medications have not experienced the same rise in bacterial resistance. Despite the outstanding broad-spectrum antimicrobial activity, the utilization of traditional aminoglycosides is restrained by dosedependent toxicity (nephrotoxicity, ototoxicity). Clinicians need to weigh the benefits of using aminoglycosides against the risks of nephrotoxicity and ototoxicity in patients (Rosenberg et al. 2020). Aminoglycoside resistance takes many different forms including enzymatic modification, target site modification via an enzyme or chromosomal mutation, and efflux (Ramirez and Tolmasky 2010) (Fig. 4.2). Due to a considerable understanding of aminoglycoside resistance, there has been a resurgence of interest in the development of new aminoglycosides such as arbekacin and plazomicin (Krause et al. 2016). The plazomicin is a next-generation semisynthetic aminoglycoside and acts in the same way that all aminoglycosides do, by blocking the process of protein synthesis in bacteria. The stability of plazomicin against various strains of bacteria produces aminoglycoside-modifying enzymes allowing it to be effective against multidrug-resistant Enterobacteriaceae (Zhanel et al. 2012). The major benefit of using this antibiotic is its ability to kill bacteria in a dosedependent manner that includes Gram-negative bacteria, such as a wide range of Enterobacteriaceae (including CRE (carbapenem-resistant Enterobacteriaceae), ESBL (extended-spectrum beta-lactamases), and MDR isolates) regardless of resistance to the aminoglycosides that are currently on the market (Livermore et al. 2011). Typical Uptake

Reduced Uptake Increased ef flux

Modification of ribosomal target

Modification of aminoglycosides

Active aminoglycosides Inactivated aminoglycosides

Fig. 4.2 Depiction of the various mechanisms of aminoglycoside resistance

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Tigecycline

Tigecycline belonging to the class of glycylcycline is a broad-spectrum novel antibiotic agent with characteristics that enable them to conquer many prevalent mechanisms of action and are therefore useful against many severe and lifethreatening infectious diseases for which other antibiotics are no longer beneficial (Rossi and Andreazzi 2006). The tigecycline limits the growth of bacteria by attaching to the bacterial 30S ribosomal subunit. This prevents amino-acyl transfer RNA molecules from entering the ribosome’s A site, which is essential for bacterial survival. Tigecycline has been utilized in combination with different specialists for the treatment of extreme CRE and CRAB contaminations (Rao et al. 2016; Tumbarello et al. 2012).

5.4

Fosfomycin

Fosfomycin is a phosphonic acid derivative isolated from the Streptomyces species. Fosfomycin exhibits bactericidal activity against susceptible organisms and exerts action by preventing peptidoglycan formation. It exerts its effect by blocking an enzyme known as uridine diphosphate-N-acetylglucosamine enolpyruvyl transferase (MurA), which disrupts the initial stages of the formation of peptidoglycan. It is effective against a wide range of Gram-positive and Gram-negative bacteria, but it must be combined with another antibiotic to slow down the development of resistance strains (Dinh et al. 2012). According to in vitro experiments, fosfomycin was effective against more than 80% Enterococcus faecium, K. pneumoniae, Staphylococcus aureus, and ESBL-producing Escherichia coli. However, its effectiveness was significantly lower against carbapenem-resistant (CR) K. pneumonia (Vardakas et al. 2016). MDR pathogens, including extended-spectrum beta-lactamase (ESBL), vancomycin-resistant Enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and carbapenemase-producing Enterobacteriaceae, are vulnerable and prone to the bactericidal action of fosfomycin. Nevertheless, a number of other resistance mechanisms have been identified. Many bacteria, which include Chlamydia spp., Vibrio fischeri, and Mycobacterium tuberculosis, can continue growing in vitro despite being exposed to high doses of this drug. It has been demonstrated that mutations in the MurA gene can lead to resistance to fosfomycin. This resistance is caused by a structural substitution of cysteine with aspartate in the active region of the MurA, which hinders fosfomycin from binding to the MurA protein (Falagas et al. 2019).

5.5

Ceftolozane/Tazobactam

Ceftolozane/tazobactam (C/T) is a combination of ceftolozane, novel oxyiminocephalosporin, and a well-explored b-lactamase inhibitor. Ceftolozane is structurally related to ceftazidime (Popejoy et al. 2017). It is now being investigated in

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combination with the beta-lactamase inhibitor, tazobactam, for the management of complicated infections such as urinary tract infections (cUTIs), ventilator-associated bacterial pneumonia (VABP), and complicated intra-abdominal infections (cIAIs). Ceftolozane showed enhanced action against Gram-negative bacilli which also includes those harboring classical b-lactamases (e.g., TEM-1 and SHV-1); however like other cephalosporins notably ceftazidime and ceftriaxone, ceftolozane is susceptible to the action of extended-spectrum b-lactamases (ESBLs) and carbapenemases. The addition of tazobactam expands the ceftolozane activity to cover the majority of ESBL-producing organisms and other anaerobic species (Zhanel et al. 2014).

5.6

Ceftazidime/Avibactam

Ceftazidime/avibactam is a newly approved agent combining ceftazidime and a novel β-lactamase inhibitor, and the combination can fight against multidrugresistant Gram-negative bacteria (Zasowski et al. 2015). Avibactam, a novel non-b-lactam, b-lactamase inhibitor, replenishes the action of ceftazidime against the majority of b-lactamases (ESBLs and carbapenemases, including KPCs, Ambler Class A; AmpC, Class C; and oxacillinase OXA-48, Class D) that lead to an extended wide range of MDR bacteria (Kaye and Pogue 2015; van Duin and Bonomo 2016). Ceftazidime/avibactam is endorsed for the management of infections caused by aerobic Gram-negative organisms in adult patients who have restricted therapeutic approaches. These infections include complicated intraabdominal infections (cIAIs), complicated urinary tract infections (cUTIs), hospital-acquired infections, and ventilator-associated pneumonia (HAP/VAP) (Sader et al. 2017). Ceftazidime/avibactam showed excellent activity against a majority of Enterobacteriaceae species or genera including Enterobacter spp., Proteus mirabilis, E. coli, and K. pneumoniae (Shirley 2018). It is a significant, successful, and right now accessible choice for the treatment of carbapenem-resistant Enterobacteriaceae (CRE) (Alraddadi et al. 2019). Gram-negative microorganisms have the potential to acquire resistance to CAZ-AVI via two primary mechanisms: (A) enzymatic resistance, which results in the inactivation of the antibiotics, and (B) chemical alteration of the antibiotic targeting site.

5.7

Eravacycline

Eravacycline (TP-434 or 7-fluoro-9-pyrrolidinoacetamido-6-dimethyl-6deoxytetracycline) is a novel fluorocycline with structural similarity with tigecycline and also exhibits the same antibacterial spectrum as that of tigecycline. It is effective against Gram-negative bacilli such as extended-spectrum beta-lactamase (ESBL) and Klebsiella pneumoniae carbapenemase (KPC) producing Enterobacteriaceae and Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) (Bassetti and Righi 2014;

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Abdallah et al. 2015). Eravacycline proved effective against bacteria that had developed resistance to multiple types of antibiotics, including those that had evolved mechanisms for resistance to carbapenems (Sutcliffe et al. 2013). Eravacycline was developed to circumvent tetracycline-specific efflux pumps (Sutcliffe et al. 2013).

5.8

Meropenem/Vaborbactam

Meropenem-vaborbactam is a fixed-dose combination product of a carbapenem antibiotic and a cyclic boronic acid β-lactamase inhibitor (Patel et al. 2018). Veborbactam, which was formerly known by the name RPX7009, is a boronic acid, a non-β-lactam β-lactamase inhibitor that does not possess any antibacterial action. The combination of vaborbactam with meropenem reestablished the action of meropenem against KPC-producing Enterobacteriaceae because of its powerful inhibitory effect on serine proteases. Meropenem/vaborbactam showed remarkable in vitro efficacy against Gram-negative strains notably KPC- and ESBL-producing Enterobacteriaceae. In clinical settings, meropenem/vaborbactam combination surpassed the piperacillin/tazobactam in terms of overall effectiveness (Dhillon 2018).

5.9

Linezolid

Linezolid is a synthetic antimicrobial agent and the main individual drug from the oxazolidinone class to get endorsement by the US Food and Drug Administration (FDA). It is essentially indicated for all Gram-positive cocci, a couple of Gramnegative anaerobes, and a few mycobacteria (Jones et al. 1996; Ashtekar et al. 1991; Zurenko et al. 1996; Kaatz and Seo 1996). Linezolid uniquely inhibits bacterial protein synthesis by preventing the formation of the 70S initiation complex (Shinabarger et al. 1997). Linezolid was approved by the FDA for the treatment of MRSA strains or Streptococcus pneumoniae including multidrug-resistant strains, complicated skin and skin structure infections (SSSIs), diabetic foot infections (DFIs), community-acquired pneumonia caused by S. pneumoniae, including cases with simultaneous bacteremia, or methicillin-susceptible S. aureus (MSSA). Moreover, the pharmacoeconomic aftereffects of that study show that the utilization of an oral specialist to treat MRSA contamination as opposed to utilizing intravenously managed vancomycin is an alluring and financially savvy choice (Li et al. 2001). Indeed, even in the humane use program, which was led before the medication’s endorsement, clinicians found the utility of the orally managed specialist, as shown by the huge level of patients (46.1%) who got orally controlled linezolid eventually in their treatment (Birmingham et al. 2003).

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Iclaprim

Iclaprim is a diaminopyrimidine with a 20 times higher potency to inhibit dihydrofolate reductase (DHFR) than trimethoprim while keeping up with the symbiotic effect with sulfamethoxazole that is exceptional to DHFR inhibitors (Laue et al. 2007; Oefner et al. 2009). The in vitro range of antibacterial action of iclaprim encloses the majority of strains of MDR S. aureus, including vancomycinintermediate, methicillin-safe S. aureus (MRSA), macrolide-, quinolone-, and trimethoprim-resistant strains and vancomycin-resistant strains (Laue et al. 2007). It additionally tucks up many strains of MDR Streptococcus pneumoniae, as well as those which are resistant to trimethoprim/sulfamethoxazole, levofloxacin, erythromycin, and penicillin (Peppard and Schuenke 2008; Sader et al. 2009). What’s more, iclaprim has antibacterial action against vancomycin-resistant Enterococcus (VRE) strains (Sader et al. 2009). In comparing the potency and safety of iclaprim in comparison to vancomycin for ABSSSIs by REVIVE-I and REVIVE-II, two riffled double-blind phase III clinical tests established that when 80 mg of iclaprim was delivered intravenously every 12 h is no smaller than vancomycin 15 mg/kg which is also regulated intravenously every 12 h for 5 to 14 days for accomplishing beforehand clinical reaction initially in 48–72 h (Holland et al. 2018).

5.11

Daptomycin

Daptomycin is a cyclic lipopeptide-possessing in vitro bactericidal activity against a broad spectrum of Gram-positive pathogens, including multidrug-resistant strains of staphylococci and enterococci (Rybak et al. 2013). In time kill analysis it was observed that daptomycin accomplised 99.9% killing of MRSA in 8h, which was more prominent than the kill rates for either linezolid or quinupristin-dalfopristin (p < 0.05). In an examination of four anti-infection agents, daptomycin, at a grouping of 2 mg/L (the conditional breakpoint at the hour of the review), brought about bactericidal action against 92% of strains tried staphylococcal separates, including MRSA and vancomycin-halfway S. aureus; different rates found were 72% for vancomycin, 46% for quinupristin-dalfopristin, and 7% for linezolid at their breakpoint focuses (Friedman et al. 2016; Fuchs et al. 2002; Rybak 2006).

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Non-traditional Approaches

6.1

Anti-Virulence Factors

Virulence effectors are regarded as the common denominator in resistant, severe, and complicated infections (Friedman et al. 2016; Rello et al. 2019). Various virulence factors that are produced by pathogenic microorganisms are also responsible for the damage caused to the host. The discovery and development of therapeutic chemicals that target bacterial virulence rather than bacterial growth is an important factor to

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alleviate the resistance emergence. Complete mitigation of disease is largely possible by treatments comprising one or more anti-virulence or anti-pathogenic factors. The primary aim of anti-virulence drugs is to prevent and treat complications associated with infections; they do not target bacteria, nor do they eradicate the pathogen. The efficacy of traditional antibiotics can be potentiated by anti-virulence compounds, and their action is complementary to that of antibiotics and can act as alternatives or adjuncts. Targeting quorum sensing systems and biofilms are major anti-virulence approaches.

6.2

Targeting Biofilms

Biofilms can be defined as a community of microorganisms encapsulated in a dense polymeric matrix produced extracellularly by them (Davies 2003; Piechota et al. 2018; Craft et al. 2019). By the mechanism of bacterial adherence, these microorganisms (biofilms) attach to the surfaces. Various environmental factors such as phagocytosis, antimicrobial agents, and opsonization result in development of biofilm mechanisms (Aslam 2008). Under infection conditions, bacteria in their growth phase become 10–1000 times more resistant to antibiotics, and in approximately 65% of infections, bacteria grow as biofilms (Hanke et al. 2013). Infections originating from medical devices such as pacemakers, catheters, shunts, heart valves, and contact lens have become an emerging challenge to the healthcare system (Hanke et al. 2013; Günther et al. 2017; Flemming et al. 2016; El-Azizi et al. 2005). Biofilms result in the development of bacterial resistance mainly by two mechanisms (Hall-Stoodley et al. 2004; Schulte et al. 2021): (a) physiological changes in the bacteria in the biofilms, it is believed that the dense nature of biofilm matrix creates a condition of hypoxia in the deeper biofilm layers and also the differences in metabolic activity of bacterial cells between different biofilm layers (b) by preventing drug penetration through protective biofilm matrix to reach the bacterium are considered a cause of the poor activity of antibiotics; subsequently, a resistant phenotype is developed by bacteria, altering drug targets for antibiotic activity (Stewart and Costerton 2001). Modern biofilm targeting can be divided into two classes: (de Lencastre et al. 2007) physico-mechanical approach: focused on removing and disrupting the biofilm and (Livermore 2004) prevention of biofilm by the use of antimicrobials or antibiotics on the matrix (Mistry et al. 2016). Therefore concentrations of antibiotics above minimum inhibitory concentration (MIC) are required to eradicate bacterial colonies in biofilms. A class of broadspectrum biofilm inhibitors is antibiofilm peptides (Pletzer and Hancock 2016). Peptide 1018 can be used as a new class of antibiotic adjuvants that not only possess broad-spectrum activity (de la Fuente-Núñez et al. 2014) but can also be synergized with frequently used antibiotics like ciprofloxacin, ceftazidime, tobramycin, and imipenem (Reffuveille et al. 2014).

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Targeting Quorum Sensing

Quorum sensing is a cell-cell communication mechanism controlling phenotype manifestations, such as virulence, and it controls population growth in many bacterial pathogens (Rutherford and Bassler 2012). Changes in cell population density result in the production of autoinducers which are QS signaling molecules. The population density of bacteria increases after colonization and accumulation of autoinducers occur in the microenvironment, and as a mechanism to modulate gene expression, bacterial cells detect these changes in population size (Abisado et al. 2018). QS is a constant secretion of signal molecules called autoinducers secreted by each bacterium. When a defined concentration of this molecular messenger reaches a threshold, the QS-controlled process is activated. QS influences many virulence aspects, and for this reason detection of molecules that can interfere with such mechanisms is presently a field of great concern. QS has a great significance in many pathogenic species and also plays a role in biofilm formation. It has been identified in a wide range of both Gram-positive and Gram-negative bacteria (Antunes et al. 2010). QS is believed to control bacterial biofilm formation (Li et al. 2017), sporulation (Slamti et al. 2014), toxin secretion (Sheng et al. 2013), bioluminescence (Kaur et al. 2018), virulence factor expression (Ha et al. 2014), and motility (Rader et al. 2007). Gram-negative bacteria use N-acyl homoserine lactones (AHLs) as signal molecules, while as Gram-positive bacteria have peptides as signal molecules, and they have a slight difference in chemical structure, as well as other autoinducers. Due to these similarities, bacteria can communicate between species through this mechanism, and this is helpful for bacterial co-infections (Wagner et al. 2016). It was studied in clinical trials that azithromycin interferes with the quorum sensing pathways of Pseudomonas aeruginosa which does not have bactericidal effect on it (Wagner et al. 2016). In their study, various virulence factors of methicillin-resistant Staphylococcus aureus (MRSA) were inhibited by 4-(1,3-dimethyl-2,3-dihydro-1H-S-benzimidazol-2-yl) phenol (BIP). This inimitable bacterial communication makes anti-quorum sensing compounds favorable for controlling bacterial virulence (Table 4.1).

Table 4.1 Drugs acting through quorum sensing Repurposed drug Raloxifene

Original indication Breast cancer

Niclosamide

Anthelminthic drug

Metformin

Anti-diabetic

Mechanism of action Raloxifene binds to PhzB2 in P. aeruginosa and inhibits the production of blue-colored pigment pyocyanin, which plays an important role in infection Niclosamide strongly inhibits the P. aeruginosa QS response and production of acylhomoserine lactone QS signal molecules Quorum sensing inhibitor in Pseudomonas aeruginosa

References (Ho Sui et al. 2012) (Imperi et al. 2013) (Abbas et al. 2017)

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Drugs Repurposed as Antibiotics

Drug repurposing also known as drug repositioning or reprofiling, drug reprofiling or drug recycling or drug rescuing or therapeutic switching, or drug re-tasking is a method of exploring the new therapeutic use of already-approved or investigational drug beyond the original medical use (Pushpakom et al. 2019; Rudrapal et al. 2020). Conventional drug development is a lengthy, tedious, time-consuming, extremely costly, and risky approach. Drug repositioning is an innovative strategy with the potential to replace the standard drug discovery process due to its ability to reduce the high financial costs, lengthy drug development times, and higher chance of failure associated with traditional drug discovery (Rudrapal et al. 2020). Drug repurposing is playing a critical role in bridging the gap in the search for new antibiotic candidates. With the development of resistance to several antibacterial treatments, bacterial infections have become a major public health problem worldwide. This highlights the critical need for the rapid discovery of novel antibiotics that can successfully combat bacterial illnesses resistant to several antibiotics (Konreddy et al. 2019). The ongoing need to develop new antimicrobial drugs that are effective against multidrug-resistant pathogens has spurred the research community to invest in various drug discovery strategies, one of which is drug repurposing (Farha and Brown 2019). Drug repurposing which is also known as repositioning of already existing non-antibiotic compounds is of great use as these drugs are approved by the FDA, and their pharmacological characteristics in preclinical and clinical trials are widely available. It has been reported that anthelminthic drugs possess activity against Gram-positive and Gram-negative bacteria (Chen et al. 2018). Antiseptics such as salicylanilides are thought to act by uncoupling oxidative phosphorylation, thereby impairing the motility of parasites. Rajamuthiah and colleagues described the efficacy of niclosamide against vancomycin, daptomycin, linezolid, daptomycin, and methicillin-resistant S. aureus isolates, and they concluded that these compounds possibly damage the bacterial membrane. They stated that niclosamide possesses bacteriostatic activity, whereas oxyclozanide exhibited antibacterial activity (Rajamuthiah et al. 2015). Furthermore, various anti-cancer agents revealed antimicrobial activity. The various repurposing treatments against multidrugresistant bacteria are summarized in Table 4.2.

7

Conclusion

Antibiotic resistance has rapidly spread over the past half a century and emerged as a significant threat to public health. Commonly studied resistance mechanisms include acquired resistance genes, increased expression of genes encoding cellular efflux pumps, and spontaneous mutations in target genes. It has contributed to the deaths of tens of thousands of people and driven up the cost of treatment to billions of dollars. The development of new drugs is time-consuming and expensive as witnessed by the paucity of antibiotics that has been granted approval by the FDA in recent years. It is crucial to enhance currently available antibiotics to counteract the increase in

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Table 4.2 Drugs repurposed as antimicrobial agent Drug Disulfiram

Earlier indication Alcohol abuse

Tamoxifen

Breast cancer

Ibuprofen, diclofenac, paracetamol Amitriptyline

Antipyretic drugs Antidepressant

Doxepin

Antidepressant

Zidovudine

Antiviral drug

Colchicine

Gout

Tramadol

Opioid

Chloroquine

Antimalarial

E. coli

In vitro

Amiloride

Diuretic

P. aeruginosa

In vitro

Ketoconazole

Antifungal

MRSP strain

In vitro

Efavirenz

Antiviral

The strain of B. subtilis

In vitro

Fluorouracil

Anti-cancer

In vitro

Indomethacin

Antipyretic

Carprofen

Antiinflammatory drug Expectorant

P. aeruginosa, A. baumannii, E. coli Strains of B. cereus, S. aureus, E. coli, P. aeruginosa, C. albicans MRSP strain

N-acetyl-Lcysteine

New indication Multidrug-resistant Staphylococcus aureus A. baumannii, P. aeruginosa, and E. coli Strains of A. baumannii, E. coli, P. aeruginosa, S. aureus, S. epidermidis Strains of E. coli, K. pneumoniae, P. vulgaris, P. aeruginosa, S. maltophilia, A. baumannii Strains of B. subtilis and P. aeruginosa E. coli, Bacillus subtilis E. coli, P. aeruginosa, P. mirabilis, S. aureus, E. faecalis, B. subtilis E. coli, S. aureus, S. epidermidis, P. aeruginosa

E. faecalis

Type of study In vitro In vitro and in vivo In vitro

In vitro

In vitro In vitro In vitro

In vitro

In vitro

References (Long 2017) (Miró-Canturri et al. 2020) (Zimmermann and Curtis 2017) (Laudy et al. 2017)

(Tazehkand 2018) (Shilaih et al. 2018) (Ozçelik et al. 2011) (TamanaiShacoori et al. 2007) (Jagadeesh et al. 2014) (Treerat et al. 2008) (Brochmann et al. 2016) (Shilaih et al. 2018) (Domalaon et al. 2019) (Sukul et al. 2014)

In vitro

(Brochmann et al. 2016)

In vitro

(Quah et al. 2012)

antibiotic resistance. However, virulence-attenuated bacteria may efficaciously be defeated by the host immune system or antibiotics. In conclusion, microbiome manipulation, drug repurposing, combination therapies, and through reformulation of already existing antibiotics are current promising alternative therapies for

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bacterial infection. Also, synthetic attempts, like the discovery of new compounds, rationally mutated bacterial toxins, or some molecules designed on virtual docking screens may help in the formulation of a viable long-term strategy to overcome antibiotic resistance.

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Alternative Therapy Options for Pathogenic Yeasts: Targeting Virulence Factors with Non-conventional Antifungals Obinna T. Ezeokoli, Ntombikayise Nkomo, Onele Gcilitshana, and Carolina H. Pohl

Abstract

Pathogenic yeasts constitute a significant part of the global fungal disease burden, particularly in immunocompromised individuals. Currently available antifungal drugs have limited efficacy against pathogenic yeasts such as Candida albicans, Cryptococcus and other non-Candida albicans spp., necessitating the development of alternative treatments and drug targets. Studies have shown great promise in targeting factors that facilitate the colonization, host immune evasion and infection by pathogenic yeasts. This chapter aims to provide an overview of current anti-virulence factor-mediated therapeutic targets, including biofilms, morphogenesis (yeast-hyphae transition), hydrolytic enzymes, prostaglandin E2 and other virulence macromolecules secreted by principal pathogenic yeasts. Furthermore, we provide a succinct overview of alternative and promising therapeutics such as antimicrobial peptides, probiotics, fatty acids, immunotherapeutics, antifungal vaccines and nanoparticles. Targeting virulence factors with non-conventional therapeutics are indeed promising alternative therapy options; however, the paucity of safety and efficacy evaluations in clinical trials and other technical hurdles impede their potential utility in the clinical setting. Keywords

Alternative antimicrobials · Candida · Pathogenic yeasts · Antifungals · Virulence factors

O. T. Ezeokoli · N. Nkomo · O. Gcilitshana · C. H. Pohl (✉) Pathogenic Yeast Research Group, Department of Microbiology and Biochemistry, University of the Free State, Bloemfontein, South Africa e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. Y. Wani, A. Ahmad (eds.), Non-traditional Approaches to Combat Antimicrobial Drug Resistance, https://doi.org/10.1007/978-981-19-9167-7_5

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Introduction

1.1

Global Burden of Yeast Infections and Associated Risk Factors

Yeast infections such as candidiasis (etiological agent: Candida spp., mostly C. albicans) and cryptococcal meningitis (etiological agent: mostly Cryptococcus neoformans) constitute a significant proportion of the global fungal disease burden (Bongomin et al. 2017; Firacative 2020; GAFFI 2020). Annually, more than 750,000 individuals are estimated to be affected by invasive candidiasis, with over 350,000 resulting in mortality (~40% mortality). According to estimates by the Centers for Disease Control and Prevention, more than 223,000 patients living with HIV/AIDS are affected by cryptococcal meningitis annually, with more than 180,000 estimated deaths worldwide (>50% mortality in developing countries) (Bongomin et al. 2017; GAFFI 2020; Rajasingham et al. 2017). Pathogenic yeast infections are a growing problem worldwide due to their resistance to multiple antifungals, environmental persistence and high fatality rate (Bongomin et al. 2017). The emergence of multidrug resistance yeast such as Candida auris has further exacerbated the burden of yeast infections, posing a challenge to the management of patients in intensive care units (Rudramurthy et al. 2017). Since the emergence of C. auris in 2009, it has been reported in over 32 countries on six different continents, necessitating an urgent search for alternate pharmacological therapies (Cortegiani et al. 2019; Satoh et al. 2009). Several risk factors predispose individuals to yeast infections. These include prolonged hospital stay, recent invasive procedures and/or surgery, diabetes, broad-spectrum antibiotic treatments, and immunocompromisation resulting from AIDS, cancer, organ transplants, prolonged corticosteroids use, and postsurgical care (Ezeokoli et al. 2021; Friedman and Schwartz 2019). Additionally, global warming, socioeconomic and geoecological factors and exposure to a large inoculum of fungi are contributory factors to the incidence and prevalence of yeast infections around the world (Bongomin et al. 2017).

1.2

Antifungal Resistance and Need for Alternative Therapies

Unfortunately, the rise in the global burden of pathogenic yeast infections has coincided with the emergence of species resistant to currently used antifungals, thereby contributing to increased yeast-associated morbidity and mortality. There are five classes of antifungal drugs, but only three main classes of systemic antifungal drugs are currently available to treat yeast infections (Table 5.1). Factors which contribute to antifungal resistance are numerous and include host and microbial factors (Hokken et al. 2019). The microbial factors include mutations of genes encoding antifungal targets, metabolic bypass, overexpression of efflux pumps, activation of stress pathways and adjustment of membrane homeostasis (Cannon et al. 2009; Hagiwara et al. 2016; Kelly et al. 1997).

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Table 5.1 Five main classes of current antifungals, mechanisms of action and limitations Class Echinocandins

Class members Caspofungin, anidulafungin, micafungin

Mechanism of action Hinder fungal cell wall biosynthesis by inhibition of 1,3-β-glucan synthase Fks1p or Fks2p

Azoles

Fluconazole, voriconazole, Posaconazole, ketoconazole, isavuconazole Amphotericin B, nystatin, natamycin

Ergosterol biosynthesis

Polyenes

Allylamines

Terbinafine

Nucleoside analogues

Flucytosine

Ergosterol biosynthesis; targets 14α-sterol demethylase Inhibit the squalene epoxidase enzyme, thereby hampering the ergosterol biosynthesis pathway Inhibits nucleic acid synthesis

Limitation Relatively costly, oral formulations are lacking, and minimal or no activity against emerging pathogens Drug-drug interactions, limited absorption in the absence of gastric acid

Low oral bioavailability and high toxicity Lack of standardized susceptibility testing

Severe toxicity; high incidence of acquired resistance

Given the limited array of antifungal drugs at our disposal and the issues around their narrow activity spectrum, host toxicity and drug resistance, alternative antifungal therapy options are required to tackle current and potentially emerging pathogenic yeasts. In this chapter, the concept of alternative strategies refers to treatment options that do not aim to inhibit the pathogen directly but rather aim to limit the ability of the pathogen to thrive in the host, withstand antifungal therapeutics and/or express their virulence attributes.

2

Virulence Factors as Alternative Therapeutic Targets in Pathogenic Yeasts

2.1

Virulence Factors: Overview and Therapeutic Concepts

In the context of fungal infection, virulence factors are features through which a pathogen may infect the hosts, obtain a competitive advantage for nutrients, evade the host immune system, and/or resist antifungal compounds (Rajendran et al. 2016). Virulence factors of pathogenic yeast are varied and can be specific for certain pathogens (Table 5.2). Since virulence factors differ among different pathogenic yeasts, the virulencemediated strategies must vary according to the target species. From an antimicrobial standpoint, the specificity of virulence attributes is advantageous since any actions aimed at the virulence factor of a pathogen may not act against other non-target

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Table 5.2 Some virulence factors of Candida spp. and other pathogenic yeasts Virulence factors Biofilm formation

Role in pathogenesis Protection and resistance to antifungal drugs and environmental factors

Adhesins

Crucial in colonization and biofilm formation Facilitates invasion and damage of host tissues, iron acquisition, escape from phagocytes, dissemination in the bloodstream, adaptation to low-nutrient conditions Promote biofilm formation, morphogenesis, colonization and competitive fitness in the host

Morphogenesis (yeast-tohyphae transition; dimorphism)

Eicosanoid (e.g. prostaglandin E2) production from exogenous arachidonic acid Secreted aspartyl proteinases (saps) Capsule formation

Phenotypic switching (white to opaque colonies) Phenotypic switching (changes in the polysaccharide capsule and cell wall) Melanin production

Species Candida albicans, C. auris, C. dubliniensis, C. krusei, C. glabrata, C. tropicalis, C. parapsilosis, Cryptococcus neoformans, cryptococcus gattii Candida species, cryptococcus spp. Candida species, cryptococcus spp.

Candida albicans, C. parapsilosis, cryptococcus neoformans

For adhesion and invasion

C. albicans

Anti-phagocytic role, alteration of antigen presentation, inhibits the migration of leucocytes into infected sites, affects cytokine and is likely responsible for complement depletion in the host Influence antigenicity, mating behaviour and biofilm formation Adaptation to a constantly changing microenvironment in the host and contributes to the persistence of infection Protects against oxidantinduced injury, phagocytic killing and antifungals

Cryptococcus neoformans

Candida albicans, C. tropicalis, C. dubliniensis Cryptococcus neoformans

Paracoccidioides brasiliensis, Cryptococcus neoformans, cryptococcus gattii

beneficial microorganisms. Furthermore, an anti-virulence-mediated strategy will help ensure that a specific opportunistic pathogen is kept at bay instead of completely wiping out the entire microbial population together with their benefits— a demerit of potentially beneficial microbial probiotics and broad-spectrum antimicrobials (Jacobsen et al. 2014). Such a strategy is particularly significant given that it leads to fewer side effects, minimizes selective pressure on non-essential targets and subsequently reduces the incidence of microbial resistance (Jacobsen et al. 2014; Shareck and Belhumeur 2011).

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Virulence factor-mediated strategies for combating pathogenic yeasts include those aimed at inhibiting biofilm formation, hyphal elongation, morphological switching from yeast to hyphae growth and the production of virulence macromolecules such as secreted aspartic proteinases (Saps), phospholipases and prostaglandin E2 (Fig. 5.1). The inhibition of virulence attributes of pathogenic yeasts may be achieved through three strategies: discovering novel antifungal compounds, repurposing currently known drugs, and enhancing antifungal activity through combination of known and potentially active drugs (Combination therapy; Fig. 5.1). Nevertheless, one or more of these strategies may be applied to understand the full range of capabilities and/or maximize the antifungal action of a compound. Most studies on alternative therapeutics for pathogenic yeasts are focused on Candida albicans because of the yeast’s prevalence and common association with the human microbiome. In the next subsections, some of the explored alternative strategies for combating major virulence factors of Candida albicans in superficial and systemic infections are discussed. Where studies are available on other pathogenic yeasts, particularly non-Candida albicans spp. and Cryptococcus spp., these are discussed as well.

2.2

Inhibition of Biofilm Formation

Pathogenic yeasts exist within host tissues and on medical devices or implants (e.g. catheters, cardiac pacemakers and prosthetic heart valves) as a complex but structured community called biofilms (Mochochoko et al. 2021). This structured biofilm serves as a mechanical barrier, protecting the underlying microbial community from external influences. Pathogenic yeasts, including C. albicans, C. auris and Cryptococcus neoformans, are known as biofilm formers, although the nature of the surface may also influence their ability to form biofilms (Atriwal et al. 2021; Hawser and Douglas 1994; Martinez and Casadevall 2015; Sherry et al. 2017). The process of biofilm formation involves several regulated stages. For C. albicans, four stages, namely, adhesion, proliferation (or initiation), maturation and dispersion of mature biofilms, are involved (Lohse et al. 2018). In the initial stage, planktonic yeast cells adhere to the tissue surface or medical devices via several adhesins (de Groot et al. 2013; Martin et al. 2021). Thereafter, biofilm formation is initiated and entails the formation of a basal layer that consists of yeast-form, pseudohyphal and hyphal cells, which enable attachment to the surface. In the next (maturation) stage, a complex but structured biofilm community develops with an increase in the formation of an extracellular matrix (ECM) which encapsulates the cells (Alim et al. 2018; Atriwal et al. 2021). The outer ECM comprises extracellular polymeric substances, including carbohydrates, glycoproteins, lipids and nucleic acids, which shields the cells from external influences, including antimicrobial agents and host immune responses (Alim et al. 2018; Atriwal et al. 2021; Kim et al. 2018). Following maturation, biofilms are dispersed into body fluids or the environment; thus, biofilms are a source of

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Fig. 5.1 Overview of alternative strategies and therapeutics

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Alternative Therapy Options for Pathogenic Yeasts: Targeting. . .

Anti-biofilm strategy

Stage

5

Stage 1 & Stage 4 Dispersal and Adherence

• Blocking adhesins • Modify surface properties to reduce adhesion • Destabilize cell membrane and hydrophobicity

Stage 2 Proliferation (Initiation)

• Interfere with qurorom sensing and cell-cell communication • Inhibit exopolysaccharide production • Block morphogenesis (yeast to hyphae formation)

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Stage 3 Maturation

• Distrupt biofilm via altering ECM integrity by shear force • Induce dispersal by utilizing dispersal agents (e.g., nitric oxide)

Fig. 5.2 Potential antibiofilm strategies for combating pathogenic yeasts. Stages 1 and 4 are combined since biofilm formation is a cycle in which adherence follows the dispersion of cells

persistent systemic infections in the host and propagation in the environment (Wall et al. 2019). The various stages of biofilm formation are potential therapeutic target points (Fig. 5.2). For instance, the initial adhesion step can be targeted by adhesininhibiting compounds, while surfaces of medical implants can be modified to prevent adherence of cells and consequently biofilm formation (Martin et al. 2021). In the case of mature biofilms, therapeutics aimed at disassembling biofilms via altering the outer ECM can be employed, as has been demonstrated in vitro for polyhexamethylene biguanide (an antiseptic agent) against preformed biofilms of C. albicans (Zheng et al. 2021). The dispersed cells (or disassembled biofilms) could become more susceptible to antifungals than the intact biofilms and can then be combated using co-administered antifungals. Importantly, because the dispersal or disassembly of biofilms in vivo may lead to fungemia, care must be taken first to determine the susceptibility of the dispersed cells (Wille and Coenye 2020). Indeed, more research is needed with respect to biofilm dispersal-based therapies before they can be employed in the clinical setting. At present, there are numerous experimental studies on the antibiofilm properties of several compounds, either alone or in combination with approved antifungals (Kovács et al. 2016; Uppuluri et al. 2008; Table 5.3). In one in vitro study, the combination of farnesol (an isoprenoid quorum-sensing molecule that inhibits the yeast-to-hypha transition in C. albicans), and echinocandins such as micafungin and caspofungin, showed a 4- to 64-fold decrease in the minimum inhibitory concentration compared to each echinocandin alone against Candida parapsilosis (Kovács et al. 2016). Furthermore, the combinations of farnesol and echinocandins significantly inhibited the metabolic activity of C. parapsilosis biofilms. Although in vivo studies are required to test these results, the observed positive results from the combination of farnesol and echinocandins are due to the anti-yeast-to-hyphae

Echinocandins + farnesol

Fluconazole + calcineurin inhibitors

Amphotericin B + stearidonic acid

Fluconazole/ itraconazole + palmatine

Biofilm/ morphogenesis

Biofilm

Biofilm

Biofilm

C. albicans, C. tropicalis, C. parapsilosis, C. krusei, C. glabrata

C. albicans, C. Dubliniensis

C. albicans

C. parapsilosis

In vitro and rat catheter model of the central venous system In vitro

In vitro

Target virulence Model and factor Therapeutics Species method Combination therapy (synergy between two or more compounds) Biofilm Fluconazole + calcium C. albicans In vitro channel blockers

Strong synergism against biofilms

Increased reactive oxygen species and apoptosis Probably due to inhibition of efflux pumps

Via direct calcineurin inhibition

Gao et al. (2013)

The combined antifungal mechanism is associated with calcium ND

Obvious enhancement of susceptibility by a combination of three drugs Four- to 64-fold decrease in the minimum inhibitory concentration compared to fluconazole alone; significant inhibition of the metabolic activity of biofilms Excellent sensitivity to the combinations compared to individual drugs Synergistic interaction at low concentrations

Wang et al. (2018)

Thibane et al. (2012)

Uppuluri et al. (2008)

Kovács et al. (2016)

References

Mechanism of action

Observation

Table 5.3 Studies on the inhibition of virulence factors of Candida albicans and other pathogenic yeasts

108 O. T. Ezeokoli et al.

Fluconazole + pseudolaric acid A (PAA)

C. albicans

In vitro

Antimicrobial peptide VLL-28 from archaea

Various long chain polyunsaturated fatty acids

Biofilm

Biofilm

C. albicans, C. Dubliniensis

C. albicans, C. parapsilosis, C. glabrata, C. krusei In vitro

In vitro

Novel compounds (discovery of new antifungal compounds or derivatives) Biofilm/ Helja, a lectin from C. albicans In vitro morphogenesis Helianthus annuus

Biofilm/ morphogenesis

VLL-28 reduced viability of the species with greater effect compared to C. krusei and C. tropicalis Fatty acids inhibited mitochondrial metabolism and biofilm formation. C. Dubliniensis was more susceptible than C. albicans. Stearidonic acid was the most effective

Inhibition of yeast-tohyphae transition and reduction in biofilm mass and coverage area

PAA (4 μg/ mL) + fluconazole (0.5 μg/mL) reduced biofilm adhesion and inhibited morphogenesis

Accumulation of intracellular reactive oxygen species leading to apoptosis

Possibly due to interaction with mannoproteins of C. albicans cell wall, disruption of the cell integrity and induction of oxidative stress. ND

Probably due to downregulation of genes involved in adhesion (e.g. ALS1, ALS2 and ALS4) and morphogenesis (e.g. ECE1 and PRA1)

(continued)

Thibane et al. (2012, 2010)

Roscetto et al. (2018)

Del Rio et al. (2019)

Zhu et al. (2022)

5 Alternative Therapy Options for Pathogenic Yeasts: Targeting. . . 109

Biofilm and secreted hydrolytic enzymes

Biofilm/ morphogenesis, hydrolytic enzymes (secreted aspartic Protease, lipase and phospholipase) Morphogenesis

Target virulence factor Biofilm/ morphogenesis

Secondary metabolites of streptomyces chrestomyceticus strain ADP4

2-alkylaminoquinoline derivatives

Therapeutics Medium-chain fatty acids (lauric acid, undecanoic acid, decanoic acid, nonanoic acid, octanoic acid, and heptanoic acid) Undecylenic acid

Table 5.3 (continued)

C. albicans

C. albicans

C. albicans

Species C. albicans

In vitro

In vitro and mouse oral mucosa

In vitro

Model and method In vitro and C. elegans model

2-[Piperidin-1-yl] quinolone and 6-methyl-2-[piperidin1-yl]quinoline were the most effective in inhibiting yeast-tohyphae transition Crude and partially purified metabolite extract effectively inhibited virulence

Observation Inhibition of biofilm formation and hyphal growth at 2 μg ml-1. Nonanoic acid reduced virulence in C. elegans with only slight cytotoxicity Effective inhibition of biofilm and morphogenesis at above 3 mM and 4 mM, respectively

Probably due to action of alkaloids, flavonoids, polyphenols, terpenoids and triterpenes in the extracts

Interference with hypha-specific genes in the cyclic AMP-protein kinase A and mitogenactivated protein kinase signalling pathways

ND

Mechanism of action ND

Srivastava et al. (2018)

Meng et al. (2019)

Shi et al. (2016)

References Lee et al. (2021)

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Nitric oxide (NO) + silver sulfadiazine

C. albicans

Biofilm, PGE2 production

Phenothiazine

C. albicans

Drug repurposing (known drugs repositioned as antifungals) PGE2 Anti-inflammatory C. albicans production, cyclooxygenase (COX) biofilm, inhibitors (aspirin, Morphogenesis diclofenac, ketoprofen, tenoxicam, ketorolac) Morphogenesis CGS 12066B, C. albicans disulfiram, prochlorperazine, ticlopidine and trifluoperazine Biofilm Tricyclic C. albicans, antidepressants C. utilis, (doxepin, imipramine, C. krusei, nortriptyline) C. glabrata Nitric oxide-releasing C. albicans Biofilm/ morphogenesis aspirin

Biofilm formation

In vitro

In vitro

In vitro

In vitro

In vitro

In vitro

Inhibition of hyphae and biofilm formation as well as the killing of cells in mature biofilms Significantly inhibited adhesion, biofilm formation and filamentation Reduced biofilm metabolic activity and biomass as well as inhibited PGE2 production

Inhibited biofilm formation and activity of mature biofilms; significant reduction in hyphae formation Inhibition of hyphal morphogenesis

attributes of the pathogen Combination of NO and silver sulfadiazine was synergistic against C. albicans biofilms

Likely due to inhibition of prostaglandin synthesis and release of nitric oxide ND

Probably due to cell lysis for nortriptyline

Inhibition of cyclooxygenase isoenzymes and subsequently prostaglandin synthesis Interaction with the endocytic pathway

ND

Alternative Therapy Options for Pathogenic Yeasts: Targeting. . . (continued)

Ells et al. (2013)

MadariagaVenegas et al. (2017)

Caldara and Marmiroli (2018)

Bar-Yosef et al. (2017)

Abdelmegeed and Shaaban (2013)

Privett et al. (2010)

5 111

Inhibitors derived from pepstatin A

Antimannoprotein and pepstatin A

HIV proteinase inhibitors (indinavir and saquinavir)

Saps

Saps

Therapeutics Moxifloxacin

Saps

Target virulence factor Biofilm/ morphogenesis

Table 5.3 (continued)

C. albicans

C. albicans

C. albicans, C. tropicalis, C. parapsilosis, C. lusitaniae

Species C. albicans

In vitro

Murine vaginitis model

In vitro

Model and method In vitro Observation Significant inhibition of developing and mature biofilm formation as well as altered expression of genes involved in hyphal formation Subnanomolar inhibitors derived from pepstatin A showed greater inhibition of Candida spp. Saps compared to pepstatin A Vaginal fluids from rats clearing a primary C. albicans infection showed significant protection against vaginitis. Postinfectious administration of pepstatin A accelerated the clearance of C. albicans from the rat’s vagina A dose-dependent significant reduction in proteinase activity

Pichová et al. (2001)

De Bernardis et al. (1997)

Cassone et al. (1999); Korting et al. (1999)

ND

ND

References Jadhav et al. (2017)

ND

Mechanism of action Multitargeting signalling pathways

112 O. T. Ezeokoli et al.

Pepstatin A

Highly active antiretroviral therapies (HAARTs) containing HIV protease inhibitors; non-nucleoside reverse transcriptase inhibitors (NNRTIs)

Sodium diclofenac, aspirin

Saps

Saps

PGE2 production and morphogenesis In vitro

Mucosal candidiasis

C. albicans

C. albicans

Murine model of disseminated candidiasis

Human epithelial cells

C. albicans

C. albicans

NA not applicable, ND Not determined, PGE2 Prostaglandin E2

HIV protease inhibitors (ritonavir, indinavir, saquinavir)

Saps/adhesion

A dose-dependent inhibition of adhesion to epithelial cells with ritonavir being the most effective inhibitor compared to indinavir and saquinavir Pre-treatment with pepstatin A showed strong dose-dependent protection against a subsequent lethal dose of an aspartic proteaseproducing C. albicans strain The anti-Sap effect of the proteinase inhibitorcontaining HAART was associated with clinical resolution of oral candidiasis but not with late and inconstant recovery of anticandidal cellular immunity 5.1–12.5% and 45–85% reduction of filamentation by sodium diclofenac and aspirin, respectively Cassone et al. (2002)

ND

Rusu et al. (2014)

Fallon et al. (1997)

ND

Inhibition of cyclooxygenase isoenzymes

Bektić et al. (2001)

ND

5 Alternative Therapy Options for Pathogenic Yeasts: Targeting. . . 113

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transition properties of farnesol on C. parapsilosis and the consequent blocking of biofilm formation (Kovács et al. 2016). Calcium homeostasis is vital for the growth of C. albicans and its physiological processes such as cell development, antifungal drug resistance and virulence (Uppuluri et al. 2008). Calcium homeostasis is mediated by calcineurin, a Ca2+calmodulin-activated serine/threonine-specific protein phosphatase. Thus, the sequestration of Ca from the extracellular compartment may inhibit the growth of the fungus. Utilizing both in vitro and in vivo (rat catheter model) experimental models, Uppuluri et al. (2008) demonstrated that the combination of fluconazole and calcineurin inhibitors such as cyclosporine A (CsA) and FK506 elicited excellent sensitivity in C. albicans cells within a biofilm compared to either fluconazole or calcineurin inhibitors alone. Furthermore, C. albicans strains lacking the 12-kDa FK506-binding protein (FKBP12) or expressing a dominant FK506-resistant calcineurin mutant subunit formed biofilms that were susceptible to fluconazole and cyclosporine A combination (fluconazole-CsA) but resistant to fluconazole and FK506 combination (fluconazole-FK506), demonstrating that the fluconazoleFK506 or fluconazole-CsA synergism is mediated through the inhibition of calcineurin (Uppuluri et al. 2008). Elsewhere, Gao et al. (2013) investigated the effects of a combination of fluconazole and doxycycline, as well as the effect of this combination in conjunction with calcium channel blockers such as benidipine and nifedipine, on C. albicans biofilm formation in vitro. For the fluconazole and doxycycline combination, the authors observed a reduction in the fluconazole sessile minimum inhibitory concentration from 64–512 mg L-1 to 1–16 mg L-1 over 12 hours. Furthermore, on the inclusion of calcium blockers, distinct enhancement of antifungal effects was observed for the combinations. Altogether, these observations suggest a synergistic effect of the fluconazole and doxycycline combination and that such synergistic effect is related to calcium availability. The formation of biofilms on the surfaces of medical implants is a significant risk factor for invasive fungal infections in patients (Martinez and Fries 2010). Therefore, the development of novel antibiofilm agents as coatings on surfaces of implants will help mitigate such risks associated with biofilm formation on medical implants. To this end, Privett et al. (2010) reported the ability of nitric oxide-releasing xerogelcoated silicon rubber coupons in impeding adhesion, viability and biofilm formation of C. albicans. Further exploration of a combined nitric oxide and silver sulfadiazine coating indicated a synergistic effect against C. albicans biofilm, with the killing of cells and biofilm inhibition higher than the nitric oxide-only treatments. In another study, the combination of nitric oxide and aspirin showed synergistic anti-adhesive action against C. albicans on abiotic surfaces (Madariaga-Venegas et al. 2017). Other studies exploring the use of novel compounds, combination therapy and repurposed drugs for inhibiting biofilm formation of pathogenic yeasts are summarized in Table 5.3. However, these studies demonstrate that biofilm-inhibiting compounds can be viable alternative therapeutic agents for combating pathogenic yeasts.

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2.3

115

Inhibition of Morphogenesis: Yeast-to-Hyphae Transition

Several pathogenic yeasts are polymorphic: they grow as yeasts, hyphae or pseudohyphae under certain environmental cues (Jacobsen et al. 2014; Lee et al. 2012). Such environmental cues include temperature, carbon starvation, neutral or alkaline pH, presence of serum, oxygen concentration and secreted compounds such as pheromones and proteins (Fu et al. 2013; Shareck and Belhumeur 2011; Zhao and Lin 2021). Morphogenesis in C. albicans can also be influenced by quorum sensing, a mechanism of microbial communication that is dependent on cell-population density (Zhu et al. 2022). It is known that most C. albicans mutants defective in the ability to switch forms (‘locked’ in either yeast or hyphae forms of growth) exhibit significantly reduced virulence (Lo et al. 1997; Rocha et al. 2001; Saville et al. 2006). Several small molecules, including cell cycle inhibitors, farnesol, geldanamycin, histone deacetylase inhibitors, lipids, disulfiram, prochlorperazine, ticlopidine, trifluoperazine and rapamycin, can modulate the yeast-to-hyphae transition in C. albicans (Bar-Yosef et al. 2017; Shareck and Belhumeur 2011). However, it is pertinent to note that many of these small molecules not only inhibit morphogenesis but also reduce viability and the biofilm-forming ability of C. albicans (Akerey et al. 2009; Honorato et al. 2022; Kovács et al. 2016). Therefore, inhibiting yeast-tohyphae transition may serve as a double-edged sword in combating yeast colonization on host tissues and physical surfaces. Nevertheless, these compounds’ relative contribution of cytotoxicity and inhibition of filamentation to protection against candidiasis require further investigations (Jacobsen et al. 2014).

2.4

Inhibition of Secreted Aspartic Proteinases

Candida albicans produces a family of ten Saps (encoded by ten SAP genes) that are considered a major virulent attribute of the pathogen (Aoki et al. 2012; Hoegl et al. 1996; MacDonald and Odds 1983; Naglik et al. 2003). Saps are hydrolytic enzymes that degrade human structural and immunological defence peptides (Pichová et al. 2001). Apart from C. albicans, strains of C. tropicalis, C. rugosa, C. lipolytica, C. lusitaniae and C. parapsilosis also secrete aspartic proteinases, with the more virulent species (C. albicans, C. tropicalis and C. parapsilosis) producing the most proteinases in vitro (Banerjee et al. 1991; Hoegl et al. 1996; Rüchel 1992). Immunological evidence, such as heightened anti-proteinase antibody levels in serum of patients with systematic Candida infections, points to the role of Saps in candidiasis (Rüchel et al. 1988). Saps also play a role in adhesion, colonization and invasion by C. albicans in mucosal infections (Colina et al. 1996; Hoegl et al. 1996; Naglik et al. 2003) and may contribute to host phagocytes evasion by C. albicans (MacDonald and Odds 1983). Saps may also play a role in HIV-related mucosal candidiasis and may be linked to resistance to azole drugs (Ollert et al. 1995).

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Interestingly, Candida Saps are associated with other virulence traits such as adherence and phenotypic switching (Naglik et al. 2003), and as such, the inhibition of Saps is potentially an effective strategy for combating multiple virulence attributes of Candida spp. and/or Candida infections (Table 5.3). Thus, it is now widely recognized that the inhibition of Saps is a promising drug target for combating Candida spp. (Bar-Yosef et al. 2017; Gauwerky et al. 2009). Commonly used Sap inhibitors include pepstatin A (a well-known aspartyl proteinase inhibitor) (Colina et al. 1996; Fallon et al. 1997), HIV proteinase inhibitors (Cassone et al. 2002; Korting et al. 1999; Pichová et al. 2001) and computer-assisted structurebased designed inhibitors (Stewart and Abad-Zapatero 2012). Collectively, these studies show that Sap inhibitors are promising antifungal therapeutic agents, especially in combating pathogenic yeasts in immunocompromised individuals.

2.5

Inhibition of Prostaglandin E2 Production

Prostaglandin E2 (PGE2) is an immunomodulatory eicosanoid that can either promote or suppress the release of pro-inflammatory T-helper cells in infected mammalian tissues (Kashem et al. 2015). During infections in mammalian cells, PGE2 is produced from arachidonic acid that is generated from the cleavage of mammalian membrane phospholipids by cyclooxygenase (COX) isoenzymes (Alem and Douglas 2004). Pathogenic yeasts also produce PGE2 through COX-like enzymes but depend on exogenous (mammalian-derived) arachidonic acid (Noverr et al. 2001; Tan et al. 2019). PGE2 production is considered a virulent factor of pathogenic yeasts as it promotes biofilm formation (Alem and Douglas 2004), morphogenesis (Noverr et al. 2001), intestinal colonization (Tan et al. 2019) and modulation of the host’s immune response (Kashem et al. 2015; Noverr et al. 2001). Thus, studies have focused on inhibiting PGE2 synthesis as an alternative therapeutic target for combating virulence traits of pathogenic yeasts, including biofilm formation and morphogenesis (Table 5.3). In a study by Alem and Douglas (2004), aspirin and non-steroidal anti-inflammatory drugs (etodolac, diclofenac, celecoxib, nimesulide, ibuprofen and meloxicam), which inhibit cyclooxygenases and thus PGE2 synthesis, were shown to significantly inhibit in vitro biofilm formation by C. albicans. Furthermore, the authors observed that physiological concentrations (75 to 200 μM) of aspirin reduced the activity of mature biofilms by 20%–80%. Elsewhere, Mochochoko et al. (2021) showed that biofilms of C. albicans mutants deficient in certain iron-acquisition genes had lower metabolic activity and produced significantly lower PGE2 compared to the wild-type strain, suggesting that the inhibition of iron acquisition may serve as an indirect means to inhibiting PGE2 production in C. albicans.

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2.6

117

Inhibition of Polysaccharide Capsule and Other Virulence Factors of Cryptococcus Spp.

Cryptococcus spp. have unique virulence attributes such as a protective polysaccharide capsule, melanin secretion, phospholipases and proteases (Table 5.2). The capsule is considered the primary virulence factor of the yeast and is a highmolecular-weight polysaccharide consisting of mostly glucuronoxylomannan (Yauch et al. 2006). One approach to combat the polysaccharide capsule of Cryptococcus spp. is the use of monoclonal antibodies (mAb) (Casadevall et al. 1992, 1998; Iyer et al. 2021; Shapiro et al. 2002). Casadevall et al. (1992) designed a serotype A glucuronoxylomannan-tetanus toxoid conjugate that, on inoculation in mice, generated mAb which had a similar specificity as those of the native cryptococcal infection. In a follow-up study (Casadevall et al. 1998), one of the isolated mAbs (mAb 18B7) showed biological activity against four Cryptococcus neoformans serotypes (A–D). In addition, mAb 18B7 rapidly cleared serum cryptococcal antigen and deposition in the liver and spleen following inoculation in infected mice. Further histological studies showed no reactivity of mAb 18B7 with normal human, rat or mouse tissues (Casadevall et al. 1998). The use of mAb for the inhibition of Cryptococcus neoformans’ melanin has also been investigated. Rosas et al. (2001) generated a melanin-binding mAb, which showed reduced fungal burden and prolonged survival in mice infected with Cryptococcus neoformans. Potentially, melanin-binding mAb may also be utilized as a broad-spectrum therapeutic against other pathogenic microorganisms that use melanin for growth and virulence (Coelho and Casadevall 2016). Although several preclinical studies in animal models have shown the efficacy and safety of certain mAb against Cryptococcus neoformans (Casadevall and Pirofski 2005, 2012; Iyer et al. 2021), no clinical trials have been successfully completed. Our search of the ClincalTrials.org (https://clinicaltrials.gov/) on August 20, 2022, returned two terminated randomized placebo-controlled phase 2 trials (NCT00324025 and NCT00847678) that aimed to evaluate the efficacy and safety of Efungumab (Mycograb®, a humanized genetically recombinant mAb against fungal heat shock protein 90 [HSP90]) as adjunctive therapy for cryptococcal meningitis in patients with AIDS. Such lack of successfully clinical trials indicates that certain technical constraints still need to be overcome for the development of an anticryptococcal immunotherapy drug. With regard to inhibiting other virulence macromolecules, Mor et al. (2015) identified two synthetic compounds, namely, 3-bromo-N-(3-bromo-4hydroxybenzylidene)benzohydrazide and N-(3-bromo-4-hydroxybenzylidene)-2methylbenzohydrazide, that inhibited the sphingolipid glucosylceramide produced by Cryptococcus neoformans and other pathogenic yeasts.

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Alternative Therapeutics for Pathogenic Yeasts

In the previous section, we focused on the anti-virulence strategies of promising antifungals. Here, we provide short overviews of the different alternative therapeutics that have been investigated for their anti-virulence activity. These therapeutics include antimicrobial peptides, microbial probiotics, nanoparticles/nanocomposites, phytochemicals, plant essential oils, fatty acids, photodynamic therapy and antifungal vaccines.

3.1

Natural Antifungal Peptides

Antimicrobial peptides (AMPs) are key components of innate immunity, capable of killing multiple pathogens, including bacteria, viruses and fungi. Thus, they play a crucial role in providing immediate and nonspecific protection against infections (Bradshaw 2003). Research shows that AMPs are attractive candidates for developing new antifungal agents due to their broad spectrum of activity, low level of induced resistance and immunomodulatory properties (Bradshaw 2003; Wang et al. 2016). To date, a total of 1257 antifungal peptides (AFPs) have been reported in the Antimicrobial Peptide Database (https://aps.unmc.edu/database/anti, accessed August 20, 2022). AMPs can be classified according to origin into three categories: natural, semisynthetic or synthetic (Wang et al. 2015). Families of naturally occurring AFPs are, in turn, categorized according to origin (Table 5.4). These compounds can be further classified according to the mechanism of action, those that form pores in membranes and nucleic acid inhibitors and those that target the cell wall (Mahlapuu et al. 2020). Empirical evidence has shown that some synthetic antifungal peptides, such as omiganan, novexatin (synthetic AMP), hLF (1–11), demegel and ETD151, demonstrate activity against Candida spp., including drug-resistant strains (Browne et al. 2020). Additionally, it has been shown that gomesin, a cationic antimicrobial peptide purified from haemocytes of the spider Acanthoscurria gomesiana, is highly effective against C. albicans (Rossi et al. 2012). Although antimicrobial peptides are promising alternative therapeutics to current antifungal and immunomodulatory therapies, further research is needed to ascertain their safety and efficacy (Vallabhaneni et al. 2016).

3.2

Probiotics

Probiotics are promising alternative therapeutics for combating pathogenic yeasts together with their virulence factors (Dube et al. 2020; Mailänder-Sánchez et al. 2017). The majority of probiotics are lactic acid bacteria of the genera Lactobacillus, Streptococcus, Bifidobacterium, and Propionibacterium (Krzyściak et al. 2017; Sharma and Srivastava 2014; Xie et al. 2017). Probiotics act against pathogenic yeasts by outcompeting them for adhesion sites and nutrients such as glucose,

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Table 5.4 An overview of the natural anti-Candida peptides Peptide VLL-28

Syringomycin E, syringostatin A, syringotoxin B

Source Archaea (archaeal transcription factor) Pseudomonas syringae

Mechanism of action Targets cell wall

Chemical characteristic Cationic

Small cyclic lipodepsipeptides

Sinden et al. (1971)

Peptide nucleosides Peptide nucleosides Cyclic hexapeptides with N-linked acyl lipid side chains Contains five unusual amino acids, 4-methylproline (MePro), 2-amino-6hydroxy-4methyl8oxodecanoic acid Helical, amphiphilic

McCarthy et al. (1985) Suzuki et al. (1965) De Lucca and Walsh (1999); Eschenauer et al. (2007) Abe et al. (2018); De Lucca (2000)

Nikkomycins X and Z Polyoxins A, B, and D Echinocandins, pneumocandins, aculeacins, mulundocandins, WF11899 Leucinostatins A, B, D, H, and K

Streptomyces tendae Tu 901 Streptomyces cacaoi Glarea lozoyensis

Forms voltagesensitive ion channels, alters protein phosphorylation Inhibits chitin biosynthesis Inhibits chitin biosynthesis Inhibits glucan synthesis

Purpureocillium lilacinum

Uncouplers mitochondria

Magainin 2

Xenopus laevis

Skin-PYY

Phyllomedusa bicolor

Lysis by dissipating ion gradient in cell membranes Membrane disruption

RsAFP1, RsAFP2, SPE10, NaD1

Raphanus sativus

Defensins, drosomycin, Thanatin

Sarcophaga peregrina

Membrane pore formation, ion efflux, induction of reactive oxygen species and programmed cell death Cell lysis

References Roscetto et al. (2018)

De Lucca and Walsh (1999)

Similar to neuropeptide NPY and gastrointestinal peptide PYY, C-terminal α-helix domain conserved Small, highly stable, cysteinerich peptides

Vouldoukis et al. (1996)

Hairpin-like betasheet structure

Dimarcq et al. (1998)

Bondaryk et al. (2017)

(continued)

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Table 5.4 (continued) Mechanism of action Cell lysis

Chemical characteristic Cyclic peptides and lipid residues

Peptides from aquatic sources

Unknown

Laxaphycins A, B, D, and E Tachycitin, ‘big defensin’ HNP-1, HNP-2, HNP-3, NP-1, NP-2, NP-3, and NP-4

Peptides from aquatic sources Peptides from aquatic sources Rabbit granulocytes

Unknown

Glycopeptide with unusual amino acids Cyclic peptides

Chitin binding

β-Sheet

Cell lysis

Tracheal antimicrobial peptide: gallinacin1, gallinacin1α, gallinacin-2

Human, bovine, chicken

Cell lysis

Protegrins 1, 2, and 3

Porcine leukocytes

Pore formations and lysis

Histatins 1, 3, and 5

Human saliva

Induction of cell death, osmosis stress

β-Sheet with cysteines forming intramolecular disulphide bonds β-Sheet with cysteines with a disulphide motif different from α-defensins. Amino terminals are blocked with a pyroglutamyl residue Cationic, cysteine-rich β-defensin Basic and neutral helical peptides

Peptide Aciculitins A–C

Source Peptides from aquatic sources

Theonegramide

References Bewley and Faulkner (2002) Bewley and Faulkner (2002) Bondaryk et al. (2017) Bondaryk et al. (2017) De Lucca and Walsh (1999)

De Lucca and Walsh (1999)

De Lucca and Walsh (1999) Koshlukova et al. (1999)

producing secondary metabolites that inhibit growth and subsequently modulate virulence attributes, as well as by stimulating the host’s immune system (Barbosa et al. 2016; Mailänder-Sánchez et al. 2017; Ribeiro et al. 2020) (Fig. 5.3). The probiotic Lactobacillus crispatus has been shown to prevent the virulence of C. albicans hyphae and enhance the immune response in an in vitro experiment (Niu et al. 2017). Similarly, the yeast Saccharomyces boulardii can stimulate the levels of anti-inflammatory cytokines, interleukin-4 (IL-4) and IL-10, as well as reduce the levels of pro-inflammatory cytokines IL-1 upon C. albicans infection (Fidan et al. 2009). One notable application of probiotics is their use as adjuvants in treating vaginal candidiasis (Xie et al. 2017). However, because probiotics have broad-spectrum antifungal activity (not just against pathogenic yeasts of interest) (Matsubara et al. 2016), their use may upset the balance of other functionally beneficial

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Fig. 5.3 Potential modes of action of probiotics against pathogenic yeast

microorganisms and lead to dysbiosis and occurrence/recurrence of microbial infections in certain body organs as have been reported for conventional antimicrobials (Han et al. 2021; McDonnell et al. 2021; Rannikko et al. 2021; Shukla and Sobel 2019); thus, the discovery of narrow-spectrum probiotics against specific pathogenic yeasts are most desirable and a fitting area for active research.

3.3

Nanoparticles and Antifungal Drug Delivery

Nanoparticles (NPs) are tiny materials ranging from 1 to 100 nm in size. The exploration of NPs and/or composites of at least two NPs (referred to as nanocomposites) as antifungal therapeutics or for drug delivery is a relatively new development. In addition to their high surface-area-to-volume ratio, the relatively low cost of nanoparticles makes them desirable antifungal therapeutics. Several in vitro studies have shown that metallic nanoparticles and nanocomposites are capable of inhibiting growth and virulence factors of pathogenic microorganisms (Di Giulio et al. 2018; Li et al. 2013) as well as have good applicability for effective delivery of antifungal drugs (Nami et al. 2021; Sousa et al. 2020). Nanoparticles of different materials, including silver (Ag), magnesium (Mg), iron (Fe), copper (Cu) and chitosan, have been reported to exhibit antimicrobial activities against human pathogenic yeasts. In one in vitro study, Fe3O4-NPs inhibited C. albicans, C. parapsilosis, C. krusei, C. tropicalis and C. lusitaniae (Salari et al. 2018). Interestingly, Fe3O4-NPs showed a greater reduction in biofilm formation in

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C. albicans and C. parapsilosis than fluconazole. The antifungal mechanism of action of NPs generally includes cell membrane structure disruption, generation of superoxide radicals which induce oxidative stress, disruption of osmotic balance, inhibiting the normal budding process of yeasts and prevention of DNA replication (Chen et al. 2020; Ing et al. 2012; Kim et al. 2009; Kumari et al. 2019). In one study on C. albicans, silver nanoparticles were found to affect multiple cellular targets crucial for drug resistance and pathogenicity, including fatty acids such as oleic acid, which is important for morphogenesis (Radhakrishnan et al. 2018). In recent years, the use of nanoparticles for antifungal drug delivery has been gaining interest. Nanoparticles are capable of increasing the solubility and stability of drugs, thereby improving the pharmacokinetic and pharmacodynamic properties of antifungal drugs (Sousa et al. 2020). Furthermore, nanoparticles can preserve the drug’s shelf-life and promote the slow and sustained stable release of antifungals, thereby maximizing the effect of antifungals (Macherla et al. 2012; Nami et al. 2021; Sharma et al. 2011). The effective delivery of such drugs eliminates the need for a high drug dosage, thus reducing the toxicity of conventional antifungals (Sousa et al. 2020). Presently, several novel nano-based drug delivery systems have been developed for topical, ocular, pulmonary, vaginal, oral, intravenous and intravital administration of several conventional antifungal drug classes (Sousa et al. 2020). Topical applications appear to be the most common route of administration of antifungalnanoparticle formulations, with polymeric- and lipid-based nanogels and nanohydrogels being the common carriers (Mohammed et al. 2013; Sharma et al. 2011; Sousa et al. 2020; Waghule et al. 2020). The antimicrobial effect of several antifungal nanoparticle-based formulations against pathogenic yeasts has been demonstrated, including the antifungal activity of a Carbopol proniosomal topical gel of miconazole nitrate against C. albicans (Mohammed et al. 2013) and voriconazole-loaded lipid-based nanoparticles against C. glabrata (Khan et al. 2020). Khan et al. (2020) also showed that novel methylglyoxal-conjugated chitosan nanoparticles effectively eliminated fluconazole-resistant C. albicans from both macrophages and infected mice. According to Sousa et al. (2020), nanoparticles can overcome the development of resistance mechanisms due to their multiple mechanisms of actions; the possibility of packaging multiple antifungals within a given nanoparticle; the ability of some nanoparticles to overcome decreased uptake and efflux by cells; and low doses are required since they have high delivery efficiency to the target site. Presently, several nano-antifungal drug formulations are undergoing or have undergone clinical trials for the treatment of certain types of candidiasis (see ClinicalTrials.gov Identifiers NCT03666195 and NCT02971007 at https://clinicaltrials.gov/ct2/home). While the safety and metabolic fate of nanoparticles within the human body still needs to be fully established, these clinical studies indicate progress towards their application in combating pathogenic yeasts.

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Phytochemicals and Essential Oils

Plants have a wide range of bioactive compounds with activities against pathogenic yeasts. These compounds include flavonoids, alkaloids, phenols, quinones, saponins, lectins and terpenoids (Arif et al. 2009) (Fig. 5.4). Their mechanism of action is related to cell wall disruption, apoptosis-like programmed cell death through the overproduction of reactive oxygen species (ROS), inhibition of adhesin expression and morphological transition and inhibition of budding or germ tube formation (Da et al. 2019; Li et al. 2015; Phumat et al. 2020). Several studies have investigated the effect of plant extracts and essential oils against mixed species in cariogenic biofilms (Table 5.5). Septiana et al. (2020) evaluated the inhibitory effect of Cajuputs candy, an Indonesian functional food comprising bioactive Melaleuca cajuputi essential oil, against dual-species biofilm comprising C. albicans and S. mutans—two microorganisms commonly associated with early childhood caries. As a functional food, Cajuputs candy is used to promote oral cavity health. Their study found that Cajuputs candy significantly inhibited biofilm formation with the mechanism of inhibition related to the interference of morphological transition of C. albicans and

Fig. 5.4 Some classes of bioactive compounds against pathogenic yeasts. Phenol (1), alkaloids (2), flavonoids (3–6), coumarins (7), steroidal alkaloid glycoside (8) and xanthones (9)

Green propolis

Rhamnus prinoides (gesho) Curcumin

Cranberry

Schinus terebinthifoliua

Melaleuca cajuputi

Plant Piper beetle

PA ethyl alcohol extract

Crude root extract

Stem ethanolic extract

Melaleuca cajuputi essential oil Hydroalcoholic (HA) and dimethylsulfoxide (DMSO) Crude *organic extract

Active compound/ extract 4Allylpyrocatechol

Single species on orthodontic materials

Dual species

Mixed biofilm. Salivacoated hydroxyapatite discs (s-HA) mounted on the high-throughput Amsterdam active attachment model Mono- and dual-species

Single species

Dual species

Biofilm model Mixed biofilm

Candida spp.

S. mutans-C. albicans

S. mutans-C. albicans

S. mutansC. albicans

C. albicans, streptococcus Mutans

Pathogens Streptococcus intermedius, S. mutans, and C. albicans S. mutansC. albicans

–

Downregulation of glucosyltransferase and quorum-sensing-related gene expression of S. mutans and agglutinin-like sequence family of C. albicans Inhibition of adhesion and biofilm formation

Reduction in polysaccharide production

2.5 μg/mL

Bezerra et al. (2020)

Campbell et al. (2020) Li et al. (2019)

500–1000 μg/ mL

0.5 mM curcumin

Philip et al. (2019)

–

–

Disruption of biofilm structural architecture

Septiana et al. (2020) Barbieri et al. (2014)

References Phumat et al. (2020)

–

Inhibitory concentration 400 μg/mL

Inhibition of morphological transition

Mechanism of inhibition Bactericidal and fungicidal

Table 5.5 Selected studies on plant extracts and essential oils against microbes associated with the oral cavity

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Essential oils

Allium sativum

Single species; topical mice treatment; ex vivo analysis of tongue samples Single species (in vitro)

–, not investigated; *, extraction solvent not disclosed

*pure extract

Glycyrrhiza spp. Candida albicans, C. glabrata, C. tropicalis, C. krusei

Candida albicans

625 μM

603.1– >1000 μg/ mL

Decreased biofilm formation and viability –

Seleem et al. (2016) MendozaJuache et al. (2017)

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the suppression of insoluble glucan production by S. mutans. Interestingly, not all phytochemicals have an antibiofilm effect on cariogenic biofilms. For example, the alkaloid nicotine found in tobacco has been shown to promote S. mutans-C. albicans mixed biofilms (Liu et al. 2017). Plant extracts with antifungal and/or antibiofilm properties can be incorporated into toothpaste, mouthwashes and other oral hygiene products to prevent oral biofilms or treat dental caries. Also, the bioactive molecules could serve as precursors for developing more efficient antifungals. To conclude, although plant extracts are one of the oldest therapeutics and the source of several drug compounds, their use in combating virulence factors of pathogenic yeasts have been largely limited to topical applications and the maintenance of oral hygiene.

3.5

Antifungal Free Fatty Acids and Derivatives

Naturally occurring fatty acids and their derivatives (Fig. 5.5) are being explored as alternative therapeutic options for combating the virulence attributes of pathogenic yeasts (Pohl et al. 2011; Shi et al. 2016; Thibane et al. 2010) (Table 5.3). Their modes of antifungal action include the alteration of the cell membrane fluidity via the destabilization of the phospholipid bilayer. Consequently, the leakage of the intracellular components occurs, leading to cell death (Ells et al. 2009; Mishra et al. 2014). The antifungal action of fatty acids generally increases with chain length;

Fig. 5.5 Some fatty acids and fatty acid derivatives with activity against virulence factors of pathogenic yeasts

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however, higher hydrophobicity of longer chain fatty acids may negatively impact their solubility and dispersal in an aqueous medium as well as their bioactivity (Pohl et al. 2011). Moreover, polyunsaturated fatty acids (PUFA) inhibit fungal growth more effectively than unsaturated long-chain fatty acids (Gołebiowski et al. 2014). Fatty acids and their derivatives are particularly advantageous due to their environmental biodegradability, high specificity and less likelihood of antifungal resistance development, particularly when combined with conventional antifungals (Gonzalez et al. 2002; Pohl et al. 2011). Ells (2008) showed that arachidonic acid (AA) (20:4) increased the susceptibility of Candida spp. biofilms to amphotericin B. Similarly, AA increased the susceptibility of Candida spp. biofilms to subinhibitory concentrations of either fluconazole or terbinafine (Mishra et al. 2014).

3.6

Antifungal Photodynamic Therapy

Antifungal photodynamic therapy (aPDT) is the use of photon energy from visible light and a non-toxic photoactive dye (also known as a photosensitizer) to inactivate pathogenic yeasts. The photoactive dye binds to the target cell and is activated by light of a specific wavelength. The activation of the photoactive dye by light leads to the production of cytotoxic oxygen species such as free radicals and singlet oxygen (Fumes et al. 2018; Soukos and Goodson 2011). Several photosensitizers, including toluidine blue ortho (TBO), methylene blue, synthetic tetracationic porphyrin, rose bengal, indocyanine green (ICG), endodontic irrigants (3% NaOCl, 17% EDTA and 2% CHX) and the chlorophyll derivative, Zn (II)chlorin e6 methyl ester (Zn (II)e 6Me), have been explored in aPDT against developing or mature biofilms of pathogenic yeasts (Diogo et al. 2018; Fumes et al. 2018; Mardani and Kamrani 2021; Tavangar et al. 2021). In one study by Pinto et al. (2018), aPDT with TBO as the photosensitizer inhibited biofilm formation and viability of C. albicans biofilms. Furthermore, the authors observed that the aPDT decreased the number of both cells and filamentous cells in the biofilm. Diogo et al. (2018) compared the efficacy of Zn (II)e6Me and TBO as photosensitizers in aPDT against a mixed biofilm of E. faecalis and C. albicans in a root canal infection model. The authors found that Zn(II)e6Me was superior to TBO, the reference photosensitizer for root canal disinfection. In addition, Zn(II)e6Me was not cytotoxic to human apical cells, suggesting that it is biocompatible for root canal disinfection. More recently, Mardani and Kamrani (2021) reported that aPDT with ICG significantly reduced the formation and metabolic activity of biofilms of two C. albicans strains, including one fluconazole-resistant strain. Although aPDT is a promising option for combating pathogenic yeast biofilms, particularly those formed on intravenous prosthetics or indwelling catheters, robust randomized clinical studies are required to determine the efficacy and safety of aPDT against in situ biofilms. This is particularly important because successful in vitro experimental results do not always translate to successful in vivo outcomes. For example, in one study consisting of both in vitro and randomized controlled trials, the viability of biofilms formed in vitro was significantly reduced by aPDT with

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TBO but was ineffective against biofilms formed on intraoral devices worn by study volunteers.

3.7

Antifungal Vaccines and Immunotherapeutics

The development of antifungal immunotherapeutics and vaccines may decrease the reliance on conventional antifungals (Portuondo et al. 2014). Therapeutic and prophylactic strategies that have been developed to elicit an immune system response against fungal antigens include passive antibody therapy, dendritic cell immunotherapy, dendritic cell vaccination, attenuated/killed vaccine, recombinant protein (subunit) vaccine and conjugate vaccines (Santos and Levitz 2014). The advantages and disadvantages of these strategies as well as key considerations for their design are highlighted in the excellent review by Santos and Levitz (2014). The common strategy of the ‘active immunization’ vaccine candidates is the combined administration of a live but weakened strain of the yeast (antigen) and an adjuvant (e.g. molecules, compounds or macromolecules) that helps elicit an appropriate immune response (Portuondo et al. 2014; Way 2012). Importantly, it is crucial that the correct antigen-adjuvant combination in conjunction with the immunopathology of the specific fungi be selected (Portuondo et al. 2014). This will ensure that both the minimal reactogenicity of the vaccine and the desired protective response for the specific fungus are achieved (Batista-Duharte et al. 2014). Central to the design of an effective fungal vaccine is the elicitation of a range of T cell immune responses, which is typical of adaptive antifungal immunity. In particular, T-helper 1 (Th1) cell responses driven by interleukin-12 (IL-12) are necessary for protective immunity to fungi (Carvalho et al. 2017; Lin et al. 2009b). Several vaccine candidates are being developed with promising antifungal immune responses demonstrated in mice (Table 5.6). Of these vaccine candidates, two anti-Candida vaccines, namely, NDV-3 and PEV-7, have undergone clinical trials (De Bernardis et al. 2012; Schmidt et al. 2012). NDV-3 is a live attenuated vaccine comprising the N-terminal portion of the Candida albicans agglutinin-like sequence 3 protein (Als3p) and the adjuvant, aluminium hydroxide, in phosphatebuffered saline (Schmidt et al. 2012). In preclinical studies in mice, the vaccine antigen was highly immunogenic and provided protection against oropharyngeal, vaginal and intravenous challenge with C. albicans and certain Candida species as well as protection against intravenous, skin and soft tissue infection with Staphylococcus aureus (Ibrahim et al. 2006; Lin et al. 2009a, 2009b; Spellberg et al. 2008). In completely randomized human trials involving 40 healthy adult subjects, no side effects were observed in subjects who received one of two doses (30 or 300 μg) of NDV-3 (Schmidt et al. 2012). Furthermore, increased immunogenicity was observed, with anti-Als3p antibodies reaching peak levels 14 days post-vaccination. The increased immune response suggests that the vaccine provided protection against C. albicans. The other anti-Candida vaccine, PEV-7 (Sap2p), comprises a truncated, recombinant Candida albicans aspartyl proteinase-2 incorporated into influenza virosomes (De Bernardis et al. 2012; Sandini et al. 2011). The vaccine

Recombinant Recombinant

Recombinant/ subunit Live attenuated Recombinant/ subunit Subunit Subunit Recombinant/ subunit Heat-killed, whole

Enolase (rEno1p) Enolase

Mannan

Dendritic cells

Dendritic cells Dendritic cells Aluminium hydroxide in phosphate-buffered saline –

Alum

–

Human serum albumin

Freund’s adjuvant Interleukin-12

Alum

Adjuvant Dioctadecyldimethylammonium bromide: Monoolein (DODAB:MO) liposomes MF59 (an oil-in-water emulsion of squalene oil) Lipid bilayer of the influenza virosomes

Clemons et al. (2014); Liu et al. (2011)

Stuehler et al. (2011)

Th1

Xin (2016) Cutler et al. (2011); Xin (2016) Schmidt et al. (2012)

Paulovičová et al. (2007); Xin et al. (2008) Martínez-López et al. (2008); Saville et al. (2009) Luo et al. (2011)

qing Li et al. (2011) Montagnoli et al. (2004)

Bromuro et al. (2010); Rachini et al. (2007); Torosantucci et al. (2005) De Bernardis et al. (2012); Sandini et al. (2011) Shukla and Rohatgi (2020)

References Carneiro et al. (2016)

Antibodies Antibodies Th1, Th17, antibodies Th1, Th2, Th17

T cells, antibodies Antibodies

Antibodies, Th1, Th2, Th17 Antibodies Antibodies, Th1 Th1, antibodies

Antibodies

Mechanisms of protection Antibodies, TH17 Antibodies

Alternative Therapy Options for Pathogenic Yeasts: Targeting. . .

* Phase I clinical trials have been completed, Th T-helper cells, –, not reported/applicable

Heat-killed Saccharomyces cerevisiae (HKY) Crfl

Met6 peptide Fba peptide NDV-3 (rAls3p-N)*

rHyr1p-N

Recombinant

Recombinant

Sap2-pp

Whole cell

Recombinant

Subunit

Type of vaccine Subunit

Laminarin (algal β-glucan) PEV-7 (Sap2p) *

Vaccine candidate(s) Cell wall surface proteins

Table 5.6 Vaccine candidates against Candida spp

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prevented vaginitis caused by C. albicans and was both effective and tolerated in humans. However, despite the well-recognized function of the immune system to control systemic fungal infections, to date, there are no approved immunotherapeutics or vaccines for the prevention and/or treatment of fungal infections. This has been partly due to a lack of sufficient understanding of the host-fungus interaction dynamics and mechanisms that govern heterogeneous immune responses to immunotherapy (Carvalho et al. 2017). Furthermore, active immunization may not provide long-term protection in immunocompromised individuals, who are the most susceptible to fungal infections (Carvalho et al. 2017; Way 2012). Therefore, to ensure that molecular mechanisms which play role in the regulation of vaccine-related immune responses are not dissipated after immunization, novel vaccine formulations containing potent adjuvants aimed at eliciting protective responses are needed (Carvalho et al. 2017; Levitz and Golenbock 2012).

4

Conclusions and Future Considerations

Virulence factors of pathogenic yeasts are indeed viable alternative therapeutic targets for combating superficial and systemic fungal infections, as demonstrated by the numerous studies reviewed in this chapter. Similarly, several therapeutics compounds have shown great promise to serve as alternatives to conventional antifungals, with a few therapeutics such as plant phytochemicals, antimicrobial peptides and nano-based products already being used in some traditional and local settings. As the body of knowledge is constantly expanding and with advancements in functional genomics, it is hoped that additional drugs and drug targets will be discovered to boost our current antifungal arsenal further. Additionally, the prospects of the first anti-Candida vaccine are exciting as it offers the opportunity for fungal prophylaxis, which is more advantageous than curative treatment, especially in certain immunocompromised patients. Nevertheless, for antifungal vaccines and the majority of the alternative therapy options discussed herein, several technical considerations and administrative hurdles need to be resolved before they can be authorized for clinical trials and approved for use in the clinical setting. These include among others (1) the demonstration of their efficacy in treating several yeast infections in different body sites; (2) establishing their safety and metabolites’ fate in vivo; (3) determining their mode of administration and dosage; (4) determining the economics of their production, including the cost-effectiveness of large-scale production and market affordability; (5) establishing their specificity or spectrum of activity; and (6) establishing ecological fate and potential risks associated with their (or metabolites) release into the environment.

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Thibane VS, Kock JLF, Ells R, Van Wyk PWJ, Pohl CH. Effect of marine polyunsaturated fatty acids on biofilm formation of Candida albicans and Candida dubliniensis. Mar Drugs. 2010;8: 2597–604. Thibane VS, Kock JLF, Van Wyk PWJ, Ells R, Pohl CH. Stearidonic acid acts in synergism with amphotericin B in inhibiting Candida albicans and Candida dubliniensis biofilms in vitro. Int J Antimicrob Agents. 2012;40:284–5. Torosantucci A, Bromuro C, Chiani P, De Bernardis F, Berti F, Galli C, Norelli F, Bellucci C, Polonelli L, Costantino P, Rappuoli R, Cassone A. A novel glyco-conjugate vaccine against fungal pathogens. J Exp Med. 2005;202:597–606. Uppuluri P, Nett J, Heitman J, Andes D. Synergistic effect of calcineurin inhibitors and fluconazole against Candida albicans biofilms. Antimicrob Agents Chemother. 2008;52:1127. Vallabhaneni S, Mody RK, Walker T, Chiller T. The global burden of fungal diseases. Infect Dis Clin North Am. 2016;30:1–11. Vouldoukis I, Shai Y, Nicolas P, Mor A. Broad spectrum antibiotic activity of skin-PYY. FEBS Lett. 1996;380:237–40. Waghule T, Sankar S, Rapalli VK, Gorantla S, Dubey SK, Chellappan DK, Dua K, Singhvi G. Emerging role of nanocarriers based topical delivery of anti-fungal agents in combating growing fungal infections. Dermatol Ther. 2020;33:e13905. Wall G, Montelongo-Jauregui D, Vidal Bonifacio B, Lopez-Ribot JL, Uppuluri P. Candida albicans biofilm growth and dispersal: contributions to pathogenesis. Curr Opin Microbiol. 2019;52:1–6. Wang S, Thacker P, Watford M, Qiao S. Functions of antimicrobial peptides in gut homeostasis. Curr Protein Pept Sci. 2015;16:582–91. Wang S, Zeng X, Yang Q, Qiao S. Antimicrobial peptides as potential alternatives to antibiotics in food animal industry. Int J Mol Sci. 2016;17:603. Wang T, Shao J, Da W, Li Q, Shi G, Wu D, Wang C. Strong synergism of palmatine and fluconazole/itraconazole against planktonic and biofilm cells of Candida species and effluxassociated antifungal mechanism. Front Microbiol. 2018;9:2892. Way, R., 2012. Fighting infectious fungi with vaccines [WWW Document]. Microbiol. Soc. URL https://microbiologysociety.org/blog/fighting-infectious-fungi-with-vaccines.html. Accessed 26 September 2021. Wille J, Coenye T. Biofilm dispersion: the key to biofilm eradication or opening Pandora’s box? Biofilms. 2020;2:100027. Xie HY, Feng D, Wei DM, Mei L, Chen H, Wang X, Fang F. Probiotics for vulvovaginal candidiasis in non-pregnant women. Cochrane Database Syst Rev. 2017;2017:CD010496. Xin H. Active immunizations with peptide-DC vaccines and passive transfer with antibodies protect neutropenic mice against disseminated candidiasis. Vaccine. 2016;34:245–51. Xin H, Dziadek S, Bundle DR, Cutler JE. Synthetic glycopeptide vaccines combining β-mannan and peptide epitopes induce protection against candidiasis. Proc Natl Acad Sci. 2008;105: 13526–31. Yauch LE, Lam JS, Levitz SM. Direct inhibition of T-cell responses by the Cryptococcus capsular polysaccharide glucuronoxylomannan. PLoS Pathog. 2006;2:1060–8. Zhao Y, Lin X. Cryptococcus neoformans: sex, morphogenesis, and virulence. Infect Genet Evol. 2021;89:104731. Zheng Y, Wang D, Ma LZ. Effect of polyhexamethylene biguanide in combination with undecylenamidopropyl betaine or PSLG on biofilm clearance. Int J Mol Sci. 2021;22:1–12. Zhu B, Li Z, Yin H, Hu J, Xue Y, Zhang G, Zheng X, Chen W, Hu X. Synergistic antibiofilm effects of Pseudolaric acid a combined with fluconazole against Candida albicans via inhibition of adhesion and yeast-to-hypha transition. Microbiol. Spectr. 2022;10:e0147821.

6

Role of Bacteriophages as Non-traditional Approaches to Combat Multidrug Resistance Koushlesh Ranjan, R. A. Siddique, M. K. Tripathi, M. K. Bharti, and Akshay Garg

Abstract

Bacteriophages are viruses affecting bacteria which are usually harmless to humans, plants, and animals. Most of the microorganisms are surviving for thousands of years by their competence to counter the antimicrobial agents. Currently, multidrug resistance (MDR) has emerged as a serious medical problem. To control the MDR microbes, several strategies may be employed including phage therapy. Conventionally, naturally occurring phages are used to infect the site of infection in the host and lyse the bacteria. However, with the advancement in molecular techniques, other strategies such as applications of purified phage lytic proteins and bioengineered phages have also been used as a supplemental or alternative therapy to conventional antimicrobial therapy. Phages may also be used for development of vaccine and diagnostic procedure against several pathogens. Apart from the several possible benefits, phage therapy may possess some of the limitations such as narrow antibacterial spectra of phages, activation of host immunity against phages and horizontal gene transfer to bacterial populations. However, these disadvantages of phage therapy may be overcome using the latest approaches of synthetic biology such as phage and phage product

K. Ranjan · R. A. Siddique (✉) · M. K. Bharti Department of Veterinary Physiology and Biochemistry, College of Veterinary and Animal Sciences, Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut, Uttar Pradesh, India M. K. Tripathi Agro Produce Processing Division, ICAR-Central Inst. of Agri. Eng, Bhopal, Madhya Pradesh, India A. Garg Department of Veterinary Microbiology, College of Veterinary and Animal Sciences, Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut, Uttar Pradesh, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. Y. Wani, A. Ahmad (eds.), Non-traditional Approaches to Combat Antimicrobial Drug Resistance, https://doi.org/10.1007/978-981-19-9167-7_6

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engineering and the development of phage genome resources. Thus, the latest development in molecular sciences has opened an alternate dimension in applications of phages as a new antimicrobial agent for therapeutic purposes. Keywords

Bacteriophage · Multidrug drug resistance · Phage therapy · Phage engineering

1

Introduction

1.1

Multidrug Resistance and Development of Multidrug Resistance

Multiple drug resistance (MDR) is a type of antimicrobial resistance exhibited by microorganisms to at least one group of antimicrobial drugs out of three or more antimicrobial classes (Magiorakos et al. 2012). The MDR bacteria may resist multiple classes of antibiotics and are a great threat to public health (Hussain 2015). Based on the drug resistance, the MDR bacteria can be classified into different categories, such as extensively drug-resistant (XDR) which denotes the non-susceptibility of bacterial species to all the known antimicrobial agents except for two or fewer antimicrobial agents. However, pan-drug-resistant (PDR) microbes exhibit non-susceptibility to all the antimicrobial agents in all the antimicrobial classes (Magiorakos et al. 2012). Many bacteria now exhibit multidrug resistance, including Streptococci, Staphylococci, Gonococci, Salmonella, Enterococci, Mycobacterium tuberculosis, etc. In most cases, bacteria gain antibiotic resistance capability through horizontal gene transfer, which is mediated through cell-cell conjugation. Through this mechanism, bacteria can transfer antibiotic resistance genes to even distantly related microbes (Hussain 2015). Most microorganisms are surviving for thousands of years because of their ability to adapt to an environment containing several antimicrobial agents. Microorganisms undergo several mechanisms to gain the capability to counter and make antibiotics ineffective (Bennett 2008). They acquire multidrug resistance through numerous mechanisms including spontaneous mutation in the microbial genome due to stress response leading to global cell adaptations (Munita and Arias 2016) and chemical alterations of the antibiotic by enzymatic modifications leading to inhibition in protein synthesis (Wilson 2014) through phosphorylation (chloramphenicol, aminoglycosides), acetylation (chloramphenicol, aminoglycosides, streptogramins) and adenylation (lincosamides, aminoglycosides) process. Other mechanisms include the destruction of the antibiotic molecule by the action of a microbial enzyme (D’Costa et al. 2011), reduced permeability of antibiotics to bacterial cells (Pagès et al. 2008), quick removal of antibiotics from the bacterial cell through efflux mechanisms (Li and Nikaido 2009), protection of target sites of antibiotic via modification or mutation of the target site (Mendes et al. 2014), etc.

6

Role of Bacteriophages as Non-traditional Approaches to Combat. . .

1.2

143

Classification of Bacteriophages

Bacteriophages are the classes of viruses that infect bacteria and replicate within them via injecting their genome into bacterial cytoplasm. Bacteriophages are usually simple in structure and comprise proteins that encapsulate the viral genome (DNA or RNA). The bacteriophage genome may possess a few to hundreds of different genes. Bacteriophages are ubiquitous and found wherever bacteria exist in nature (McGrath and van Sinderen 2007). One estimate reports that there are over 1031 bacteriophages may exist on the planet earth, which is higher than the combined number of all other organisms (LaFee and Buschman 2017). Based on morphology and nucleic acid composition, bacteriophages are classified into distinct orders and families by the International Committee on Taxonomy of Viruses (ICTV) (Table 6.1).

1.3

Genome Structure of Bacteriophages

There is a significant diversity in terms of genome size, and its organization and nucleic acid composition have been reported in the millions of different bacteriophages. The smallest genome of RNA phage (MS2) comprises only a few kilobases to larger genome of DNA phage (T4) which may possess hundreds of genes. The size and shape of the bacteriophage capsid also vary according to the size of the genome (Black and Thomas 2012; Al-Shayeb et al. 2020). Bacteriophage genomes are usually highly mosaic, i.e. bacteriophage genomes appear to comprise many individual modules which may be found in other bacteriophages with different arrangements. The bacteriophages infecting Mycobacterium spp., i.e. mycobacteriophages, comprise several host genome segments which enter phage genome due to genetic assortment from repeated occasions of sitespecific recombination and illegitimate recombination. This phenomenon is otherwise termed as phage genome acquisition of bacterial host genetic sequences (Morris et al. 2008). However, it is established that based on virion structure, type of nucleic acid and mode of the viral life cycle, the molecular mechanisms involved in the evolution of bacteriophage genomes may vary in different phage families (Krupovic et al. 2011). For a specific type of function, bacteriophages may also contain an unusual type of nucleotide in their genome. For example, some marine Roseobacter phage may contain deoxyuridine (dU) instead of deoxythymidine (dT) in their genome which may play an important role in mechanism to evade bacterial defence mechanisms including CRISPR/Cas systems and restriction endonuclease systems which may recognize and cleave the invading phage genome sequences and inactivate them. Similarly, several other bacteriophages have also been reported with unusual nucleotides including Bacillus phage having dU substituting dT in its genome (Takahashi and Marmur 1963) and cyanophage containing 2-aminoadenine (Z) instead of adenine (A) (Kirnos et al. 1977).

8

16

25

–

6

12

Turriviridae

Chaseviridae

Demerecviridae

Drexlerviridae

Guenliviridae

Rountreeviridae

Salasmaviridae

Belfryvirales

Caudovirales

1

Family Tectiviridae

Order Kalamavirales

Genera reported 5

They possess dsDNA genome of 52–56 kbp and G+C content 39.3–52.5%. They possess Head diameter of 48 ± 5 nm, Tail length of 121 ± 8 nm and Tail diameter of 10 ± 3 nm They are small viruses with genome size of 43 to 52 kb and G+C content 42–50% Linear dsDNA with contractile tail They are non-enveloped virus with head and tail. The icosahedral head has 54 nm in diameter. The tail is non-contractile. They possess linear genome of 20kb in length. They are non-enveloped with a head and tail. The tail is non-contractile with a collar and twelve appendages. They possess linear genome of around 20kb in length.

Morphological feature Nonenveloped, isometric, 60 nm, flexible lipid vesicle, pseudo-tail Enveloped, isometric

– Staphylococcus virus 66, Staphylococcus virus 44AHJD

Bacillus virus phi29, Bacillus virus PZA, Streptococcus phage Cp1

Linear dsDNA

Linear dsDNA

Escherichia phage RTP, Escherichia phage FP

Sulfolobus turreted icosahedral virus 1 Escherichia phage vB_EcoM-4HA13, Escherichia phage ST32, SP76, Vibrio phage Thalassa

Example PRD1

Linear dsDNA

Linear dsDNA

Linear dsDNA

Linear dsDNA

Linear dsDNA

Viral genome structure Linear dsDNA

Table 6.1 ICTV classification of bacteriophages (McGrath and van Sinderen 2007; Ackermann 2009; https://www.ncbi.nlm.nih.gov/Taxonomy)

34

33

–

148

108

13

2

Members reported 19

144 K. Ranjan et al.

Halopanivirales

Durnavirales

1

4

Podoviridae

Matshushitaviridae

133

Autographiviridae

1

7

Siphoviridae

Simuloviridae

33 6

Herelleviridae Myoviridae

3

10

Ackermannviridae

1

7

Zobellviridae

Picobirnaviridae (Proposed) Sphaerolipoviridae

48

Schitoviridae

Linear dsDNA

Linear dsDNA

Enveloped, isometric

Linear dsDNA

Linear dsRNA

Linear dsDNA

Linear dsDNA

Linear dsDNA

Linear dsDNA Linear dsDNA

Linear dsDNA

Linear dsDNA

Linear dsDNA

Enveloped, isometric

Enveloped, isometric

Nonenveloped, short noncontractile tail Nonenveloped, isometric

Nonenveloped, contractile tail Nonenveloped, icosahedral head, contractile tails Nonenveloped, long noncontractile tail Nonenveloped, short noncontractile tail

These viruses have liner genome size of 59–80 kb and they possess three RNA polymerase genes, They have linear dsDNA genome with icosahedral symetry Non enveloped, contractile tail

3229

λ, T5, HK97, N15

Role of Bacteriophages as Non-traditional Approaches to Combat. . . (continued)

2

2

7

3

– Haloarcula virus HCIV1, Haloarcula californiae icosahedral virus 1 Haloterrigena jeotgali icosahedral virus 1, Natrinema versiforme icosahedral virus 1 Thermus virus IN93, Thermus virus P23-77

771

373

Synechococcus phage S-CBP42, Agrobacterium phage Atu_ph02 T7, T3,Φ29, P22

63

12

92 1320

115

Achromobacter phage JW Alpha, Enterobacter phage EcP1 Vibrio virus VpV262, Salinivibrio phage CW02 Salmonella virus ViI, Escherichia virus ECML4 Bacillus virus SPO1 T4, Mu, P1, P2

6 145

Microviridae

Blumeviridae Steitzviridae

Inoviridae Paulinoviridae

Timlovirales

Tubulavirales

2 2

30 117

4

185 25

Fiersviridae Solspiviridae

Petitvirales

56 7

Atkinsviridae Duinviridae

7

Rudiviridae

Norzivirales

4

Lipothrixviridae

Ligamenvirales

Genera reported 3

Family Pleolipoviridae

Order Haloruvirales

Table 6.1 (continued)

Linear ssRNA Linear ssRNA

Circular ssDNA Circular ssDNA

Nonenveloped, filamentous Nonenveloped, filamentous

Circular ssDNA

Linear ssRNA Linear ssRNA

Linear ssRNA Linear ssRNA

Linear dsDNA

Viral genome structure Circular ssDNA, circular dsDNA or linear dsDNA Linear dsDNA

Nonenveloped, isometric, icosahedral head, virion size 27 nm, with 12 capsomeres Nonenveloped, isometric Nonenveloped, isometric

Nonenveloped, isometric Nonenveloped, isometric

Enveloped, rod-shaped capsid, lipids Nonenveloped, rod-shaped straight uncoated rods, TMV-like Nonenveloped, isometric Nonenveloped, isometric

Morphological feature Enveloped, pleomorphic

AVE020, AVE016 ssRNA phage SRR5467091_11, ssRNA phage Zoerhiza.4_21 M13 Thermus phage OH3, Thermus phage phiOH3, Propionibacterium phage B5

TTV1, Acidianusfilamentous virus 1 Sulfolobus islandicusrodshaped virus 1 (SIRV 1) SRR7976325_2, ESE020 Acinetobacter phage AP205, ssRNA phage SRR5995670_1 MS2, Qβ ssRNA phage SRR5466727_6, ssRNA phage SRR7976300_9 ΦX174

Example Haloarcula virus His2, Halorubrum virus HRPV1

67 3

34 412

140

471 36

94 7

20

12

Members reported 21

146 K. Ranjan et al.

2

1

1

1

1

2

1 1

2

Tristromaviridae

Cystoviridae

Corticoviridae

Ampullaviridae

Globuloviridae

Portogloboviridae

Plasmaviridae

Autolykiviridae

Finnlakeviridae Bicaudaviridae

Fuselloviridae

Primavirales

Mindivirales

Vinavirales

Unassigned

1

1

3

Plectroviridae

Nonenveloped, isometric Nonenveloped, lemon-shaped virions, 120x 80 nm, long tails Nonenveloped, lemon-shaped, tapered capsid with short spike end, lipids

Enveloped, pleomorphic 80 nm, with no capsid, lipids Nonenveloped, isometric

Enveloped, spherical, icosahedral head, 70–80 nm, lipids Nonenveloped, isometric, 63 nm in size, complex capsid, lipids Enveloped, bottle-shaped virion, 230 nm in length Isometric spherical virions, 70–100 nm, lipid-containing envelope Enveloped, isometric

Enveloped, rod-shaped

Nonenveloped, filamentous

Vibrio phage 1.249.A. _10N.261.55.B9, Vibrio phage 1.020.O._10N.222.48. A2 FLiP ATV SSV1

Circular ssDNA Circular dsDNA Circular dsDNA

Linear dsDNA

Circular dsDNA

Sulfolobus polyhedral virus 1, Sulfolobus polyhedral virus 2 MVL2

PSV

Linear dsDNA

Circular dsDNA

ABV

PM2

Acholeplasma phage MV-L51, Spiroplasma phage SVTS2 Pyrobaculum filamentous virus 1, Pyrobaculum filamentous virus 2 Φ6

Linear dsDNA

Circular dsDNA

Linear dsRNA

Linear dsDNA

Circular ssDNA

Role of Bacteriophages as Non-traditional Approaches to Combat. . . (continued)

11

1 1

21

5

2

1

1

3

3

3

5

6 147

Order

Genera reported 1 1 1

1

1

Family Halspiviridae Thaspiviridae Guttaviridae

Clavaviridae

Spiraviridae

Table 6.1 (continued)

Nonenveloped, rod-shaped

Morphological feature Nonenveloped, lemon-shaped Nonenveloped, lemon-shaped Nonenveloped, ovoid, dropletshaped Nonenveloped, rod-shaped

Circular ssDNA

Circular dsDNA

Viral genome structure Linear dsDNA Linear dsDNA Circular dsDNA Aeropyrum pernix bacilliform virus 1 Japan/ Tanaka/2005 Aeropyrum coil-shaped virus

Example His 1 virus Nitmarvirus NSV1 SNDV

1

1

Members reported 1 1 1

148 K. Ranjan et al.

6

2

Role of Bacteriophages as Non-traditional Approaches to Combat. . .

149

Culture and Characterization of Bacteriophages

Bacteriophages are propagated and maintained using classical bacterial culture techniques with certain modifications that facilitate the optimal production of phage particles. For phage culture and propagation, several parameters are taken in consideration, including optimal growth parameters for the host bacterium, multiplicity of infection (MOI), time of co-culture of phage and bacteria, etc. (Pelzek et al. 2013). For optimum bacteria culture, specific bacterial culture media, streaking a culture on a plate, inoculation of cultures, viable plate count, colony PCR, etc. should be practised with certain modifications according to host-strain of choice for phage work. The sterile hood and aseptic bacteriological techniques may prevent the cross-contamination of phage stocks. The solutions which cannot be autoclaved, such as organic components of media, glucose, metal salts, vitamins, antibiotics, etc., should be filter-sterilized through a 0.22-μm membrane. However, during filter sterilization, care should be taken to avoid the contamination of non-autoclavable solution stocks because phages may easily pass through 0.22-μm filtration. All the biological materials should be autoclaved before discarding. The basic method of phage isolation by enrichment procedure and culture lysis has remained unchanged since it was developed (Van Twest and Kropinski 2009). Some researchers are also using the direct plating technique of environmental samples on isolation hosts without doing enrichment cultures (Gencay et al. 2017; Bhunchoth et al. 2015). However, this technique requires a comparatively higher concentration of phages in the environmental sample. A direct plating technique has been applied for phage isolation against Escherichia coli from sewage effluent (Debartolomeis and Cabelli 1991), stool samples (Chibani-Chennoufi et al. 2004) and phages from saliva infecting several species of oral bacteria (Bachrach et al. 2003) and phages from dental plaques (Tylenda et al. 1985). The multiplicity of infection (MOI) is also an essential parameter for phage propagation, which denotes the ratio of the number of detectable infective phage particles to the number of bacterial cells present during the initial phase of bacteriophage infection culture (Pelzek et al. 2013). The MOI may be optimized according to the host-phase combination. Most of the bacteriophages may infect ≥95% of bacteria at MOI of ≥3 (Carlson 2005). Bacteriophages can be isolated from various types of samples, including sewage water, human skin, etc. For the isolation of phages from anaerobic bacteria, some of special arrangements such as airtight jars and commercially available anaerobic pouches are required to suit the growth of anaerobic bacteria. Similarly, some of the specific phages, such as thermophilic phages, archaeal phages and cyanophages, also require special consideration and protocols for laboratory propagation (Fulton et al. 2009; Millard 2009). Phages infecting photosynthetic planktonic bacteria have been isolated from photic seawater (Li et al. 2016), and phages of mammalian intestinal bacteria from sewage, faecal material, etc. (Khan et al. 2015; Karumidze et al. 2013; Jakhetia et al. 2013). Similarly, phages from bacteria infecting human skin have been isolated from the skin (Liu et al. 2015), throat secretions (Ronda et al. 1981), wound exudates

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(Rasool et al. 2016), etc. In one experiment, eight different phage genomes were identified from the human skin microbiome on Staphylococcus epidermidis culture. Out of which six phage sequences were reported unique and spanned within major staphylococcal phage families including Siphoviridae, Podoviridae and Myoviridae (Valente et al. 2021). For the maintenance of appropriate biosafety level, host bacterial strains should be chosen carefully. The waste materials of phage culture experiment are considered potentially dangerous as they may contain pathogenic microorganisms. Moreover, phage genomes may also encode virulence factors and antigens which may be transferred to the suitable bacterial hosts in the laboratory condition leading to production of virulent bacteria. Therefore, the biological waste material should be properly autoclaved before disposal according to the host organism. For pathogenic hosts, bacterial surrogates should be used as far as possible for phage culture and propagation. The surrogate hosts are especially recommended for those laboratories which are not equipped to handle pathogenic microorganisms. However, during isolation and propagation of phage, surrogate hosts should be selected carefully because bacteriophages may not infect surrogate hosts with equal efficiency as pathogenic bacterial strains. In one of the experiments, researchers used the Mycobacterium smegmatis in place of Mycobacterium tuberculosis for phage isolation (David et al. 1980). Phages for phase therapy can be characterized on the basis of culture lysis, plaque morphology, spot testing, temperature, pH susceptibility, host range, etc. (Table 6.2).

3

Phage Therapy against Multidrug Resistance Pathogens

Bacteriophages have been tried as an alternative to antibiotics in several countries since the twentieth century. Bacteriophages have the potential to be used as a possible therapeutic agent against multidrug-resistant strains of several bacterial species (Keen 2012; Kortright et al. 2019; Gordillo Altamirano and Barr 2019; González-Mora et al. 2020). Phages interact with the immune system primarily via two mechanisms, viz. by influencing the host innate immunity followed by bacterial clearance and phage-encoded protein expression by host bacteria (Popescu et al. 2021). Phage therapy has been poorly investigated in the medical field. Most of the work in phage therapy against multidrug resistance has been done in recent years. Phage therapy data suffered from several lacunae, such as scarce knowledge of immune response, pharmacodynamics, tolerance, pharmacokinetics, etc. However, phage therapy research has now been in progress in several countries (Table 6.3) and is included as one strategy to control antibiotic resistance in the USA (Reardon 2014). Despite the presence of phages in several environments, the identification of a specific phage against a particular bacterium is a laborious task. The sewage water is a potential source of bacteriophage against several antibiotic-resistant bacteria (Mattila et al. 2015). In one experiment, phages infecting antibiotic-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa, E. coli and Salmonella were found

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151

Table 6.2 Methods for detection and characterization of bacteriophages Method Culture lysis

Description Bacteriophage filtrate is incorporated to broth culture of bacterial. Bacterial cell lysis is appreciated through loss of culture turbidity

Advantages • Bacterial viability and its metabolic activity can be easily detected by application of metabolic dyes. • This technique also allows automation through spectrophotometry. • The technique is ideal for bacteria growing in broth.

Spot testing

Small drop of bacteriophage filtrate is placed on host bacteria on agar plate and allowed for the incubation. The zone of lysis on agar plate indicates the presence of bacteriophage phage

• It is simple to perform. • Multiple bacteriophage samples can be tested on same culture plate.

Plaque testing

The solution of serial dilutions of bacteriophage filtrate mixed with bacterial cells is poured on culture plate surface. The soft agar is overlayed, incubated and observed for the development of plaques Phage lysates are serially diluted to produce just less than confluent lysis on a plate

• The plaque size may give indication about the size of bacteriophage due to diffusion effects since larger bacteriophages diffuse slowly. • It confirms the presence of productive phage infection. • This technique is used for bacteriophages which are unable to produce distinct plaques. • Confirmatory tests for

Routine test dilution

Molecular tests (PCR,

Bacteriophagespecific nucleic

Disadvantages • It may give false-positive result due to non-productive lysis. • The cellular debris of lysed bacterial cells in early phase of infections may inactivate bacteriophage and may inhibit the subsequent phase of infections. • It may show false-positive result due to bacterial inhibition by other reasons including phage binding without causing productive infection and inappropriate media components. • Bacterial host must grow to confluence lawn on culture plate. • Some of the bacteriophages are unable to form plaques due to low productivity or limited diffusion in agar.

• May show falsepositive result when media components are properly not diluted. • Bacteriophagespecific primers

References Sullivan et al. 2003; McLaughlin 2007

Oliveira et al. 2017

KęsikSzeloch et al. 2013; Pallavali et al. 2017

Douglas and Elberg 1976; Thomas and Corbel 1977

Brussow et al. 1994; (continued)

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Table 6.2 (continued) Method

Description

Advantages

Disadvantages

References

real-time PCR, nucleic acid sequencing, etc.)

acid sequences (DNA/RNA) are amplified using specific primers

identification of bacteriophage.

are required. • Amplification relation is dependent on thermal condition, purity and integrity of nucleic acid.

Edelman and Barletta (2003); Owen et al. 2021

frequently in sewage water, whereas phages infecting Acinetobacter baumannii and vancomycin-resistant Enterococcus (VRE) rarely and phages infecting methicillinresistant Staphylococcus aureus (MRSA) were found very infrequently (Mattila et al. 2015). Some researchers have shown that the probability of finding a specific phage against a particular bacterium for phage therapy is rare. Therefore, the specific type of source samples such as sewage coming from a hospital (Nikkhahi et al. 2017) or in patients themselves should be screened on a priority basis (Rasool et al. 2016). It is also assumed that phage for at least some strains of every bacterial species can be isolated from the environment if samples are screened properly. However, this may not be true for all the combinations of bacteria and phage types.

4

Phage Engineering and Antimicrobial Activity

4.1

Phage Engineering and its Role in Widening the Antimicrobial Spectra

Phages act as natural antibacterial agents against target-specific bacteria according to their host range. Researchers have reported that most of the phages infect specific bacterial strains within a species (Kasman and Porter 2021). However, some phages may infect a broad range of bacteria of several species and genera (Matilla and Salmond 2014). The narrow range of host specificity may have developed because of the long interaction of phage bacteria, making the phage a specific antibacterial agent (Antonovics et al. 2013). However, the host specificity of phages creates a problem in phage therapy, as it is sometimes difficult to search for a particular type of phage acting against a specific bacterial pathogen. To overcome this issue, a cocktail combination of different phages acting against different host ranges is in practice for a wider spectrum of bacterial pathogens (Jault et al. 2019) (Fig. 6.1). Phage infection to bacterial cells starts with the adsorption of phage to the bacterial cell which is governed by phage components, such as phage structural proteins or tail fibres, which allow the binding of phage cell to the target cell receptors. Molecular biology tools can create phages that can infect multiple bacterial species.

Study model Rat model

Mouse model

Mouse model

Mouse model in neutropenic mice

Mouse model

Mouse model

Mouse model

S. n. 1

2

3

4

5

6

7

Infection

Sepsis

Pneumonia

5x107 CFU MDR ST258 strain of K. pneumoniae 2x108 CFU K24 Carbapenemresistant 533 strain of K. pneumoniae

1.7 x 108 PFU of vB_KpnS_Kp13 phage IP

IP Pharr and FKpNIH-2 phages at various MOIs and times

Carbapenemresistant strain of A. baumannii

Pneumonia

Carbapenemresistant strain of A. baumannii

IP inoculation of 109 PFU of vB_AbaP_PD-6A3 1 h postinfection SH-Ab15519 phage as IN inoculation 1 h post-infection at MOIs of 0.1, 1 and 10 and 2 h post-infection at 10 MOI BФ-C62

109 CFU/mL of A. baumannii

Pneumonia

Sepsis

Bacteriophage used for therapy vB-GEC_Ab-M-G7

Cocktail of 109 PFU of PBAB08 and PBAB25 phages injected

Bacterial pathogens 5x108 CFU/mL of A. baumannii

108 CFU of MDR strain of A. baumannii

Disease condition Wound

Table 6.3 In vivo studies using phage therapy against bacterial pathogens

Pathogen was not found in lungs, and improvement of histological damage was observed 3 days posttreatment Survival of animal was found more dependent on time than on dose. Good survival results were observed at 1 h post-infection 100% survival of animals in phage treatment after 10 min and 12.5% of survival after 1 h

Result Treated rats showed reduced symptoms and load of bacterial infection by 5-log showing the efficacy of treatment Phage-treated mouse showed 100-fold reduction in lungs in comparison to control group. An increase of 20% of IgE was observed in IP administration An increase of survival rate of 60% was observed in comparison to control group 90% of survival rate was observed in mice treated after 1 h and 66.7% survival in mice 2 h post-infection

Role of Bacteriophages as Non-traditional Approaches to Combat. . . (continued)

Horvath et al. 2020

Hesse et al. 2021

Jeon et al. 2016

Hua et al. 2018

Wu et al. 2019

Cha et al. 2018

Reference Kusradze et al. 2016

6 153

Study model Mouse model

Zebrafish

G. Mellonella

Mouse model

Mouse model

Mouse model

Rabbit model

G. Mellonella

Mouse model

S. n. 8

9

10

11

12

13

14

15

16

Table 6.3 (continued)

Pneumonia

–

Ileal loop infection

Pneumonia

Sepsis

Gut infection

–

–

Disease condition Pneumonia

Bacterial pathogens 109 CFU of K. pneumoniae as IN 103 CFU of K. pneumoniae IM ST258 KL106 and ST23 K1strain of K. pneumoniae 106 CFU of enteroaggregative strain of E. coli 108 CFU/mL of E. coli 536 bio-luminescent and VAP strain of E. coli as IN 0.5 mL of 108 CFU/mL of O157:H7 E. coli 20 μL with 108 CFU/mL 2x106 CFU/mL of P. aeruginosa IN 20 μL of 104 PFU/mL of ec311 phage at every 6 h PELP20 phage Administration 24, 36, 48, 72, 144 and 156 h Post-infection IN

Cocktail of 106 PFU/mL of PAH6 and P2BH2 phage

5 x109 CFU/mL of phage cocktail IP 536_P7 and 536_P1 phage with MOI of 0.3, 3 and 10

vB_KpnP_KL106ULIP54 and vB_KpnP_KL106ULIP47 4x108 PFU of PDX phage

108 PFU/mL 2 h post-infection IM

Bacteriophage used for therapy VTCCBPA43 phage as IN 2 h post-infection

Reduce production of liquid in ileal loop and decrease number of bacterial loads Three subsequent doses achieve 100% survival Complete clearance of bacteria was observed in 100% of mice, where phage was administered at 24, 36, 48 and 72 h, whereas 70% of clearance in mice treated at 144 and 156 h was observed

The reduction of the target bacteria was observed in murine faeces without dysbacteriosis 100% of survival in mice after 100 h of phage administration 100% of survival observed in mice

Similar type of survival was observed for both strains

Result Mice exhibited reduction in bacterial load and lesions after 48 h of infection 77% reduction of bacterial load

Manohar et al. 2018 Waters et al. 2017

Alam et al. 2011

Kaabi and Musafer 2019 Dufour et al. 2015

Cepko et al. 2020

Thiry et al. 2019

Sundaramoorthy et al. 2021

Reference Anand et al. 2020

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Mouse model

G. Mellonella and mouse

G. Mellonella and mouse

Preventive mouse model

17

18

19

20

109 CFU/mL PAK-lumi in larvae And 107 in mouse as IN of P. aeruginosa 2.5x107 CFU/mL Of P. aeruginosa IN

–

Infection

–

2.5x106 CFU FADDI-PA001 strain Of P. aeruginosa intratracheal 105 CFU/mL YMC11/02/R656 Strains of P. aeruginosa

Pneumonia

1.2x 109 PFU/mL cocktail of phage 48 h prior to Infection IN

Cocktail phages of E217, PYO2, E215, DEV, PAK_P4 and PAK_P1 (IN IN mice)

BФ-R656 and BФ-R1836 at MOIs of 100, 10 and 1 (IN IN Mice)

2x107 PFU/mg Intratracheally aerosolized PEV20 phage

Treatment with BФ-R656 and BФ-R1836 treatment increased 66 and 83% survival in mice and 50 and 60% of survival in larvae, respectively MOI of 8 increased survival from 17% to 49% and MOI of 25 increased to 63% after 20 h in larvae 100% of survival with 0.05 and 1 MOIs observed in mice >70% of pre-treated mice cleared the bacterial infection 24 h of post challenge. Remaining 30% mice harboured up to 20 CFU/mL of infection

Reduction in bacterial count in treated mice from 1.3x1010 CFU to 6x104 CFU were observed in lungs

Pabary et al. 2015

Forti et al. 2018

Jeon and Yong 2019

Chang et al. 2018

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Fig. 6.1 Strategies for application of bacteriophages against antimicrobial resistance

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The filamentous phage fd usually infects Escherichia coli having F plasmid as the phage receptor and receptor-binding domain of enterophage Ike, gene 3 protein (g3p), that binds. with N or I pili. To make the chimeric phage, the receptor-binding domain of enterophage Ike was grafted to the orthologous g3p region in the phage fd. Thus, the resulting chimeric phage could infect Escherichia coli having either N pilus or F pilus (Marzari et al. 1997) (Fig. 6.1). Similarly, another chimeric phage fd having the g3p fused with the orthologous protein (pIIICTX) of vibriophage CTX was created where the resultant chimeric phage showed the capability to infect both E. coli and Vibrio cholerae (Heilpern and Waldor 2003). Lin et al. (2012) created the T3-based recombinant tailed phage (T3/T7), where tail fibre gene 17 resulted from recombination between the tail fibre gene of T3 and T7. The resultant hybrid T3/T7 phage was characterized by enhanced adsorption efficiency and a wider host spectrum than both T3 and T7 phages (Lin et al. 2012).

4.2

Phage Engineering and Reduced Host Immune Response

Apart from the problem associated with a narrow host range, phage therapy is also affected by the patient’s immune system. The patient’s immune system may recognize phages as immunogens and may inactivate the phages (Gorski et al. 2012). The lytic phages may release materials by bacterial lysis, which may initiate the inflammatory responses in human hosts (Abedon et al. 2011). Therefore, to overcome these issues, certain approaches such as a reduction in immunogenicity of phages and reduction in collateral immunogenicity because of bacterial-derived material generated by phage lysis should be utilized. In one experiment in mouse models, the serial passage technique was used to identify the phage variants, which can avoid entrapment by the reticuloendothelial system and may remain in a host’s circulatory system for a longer period and show enhanced antibacterial efficacy compared to wild-type phage. Such phage variants are characterized by a common substitution mutation at 158th residue, i.e. from lysine to glutamic acid (K158E) in major phage head protein E. Vitiello et al. (2005) have shown that this specific substitution mutation might be responsible for longcirculating phenotype. Apart from the above-mentioned strategy, there is still the possibility of the release of toxic bacterial components which may lead to stimulation of the immune system. In one experiment, mice treated with lysis-deficient phage using phage T4-derived mutant (LyD) exhibited reduced bacteraemia and improved survival chances in a E. coli-infected murine peritonitis-sepsis model (Matsuda et al. 2005). Similarly, recombinant Staphylococcus aureus temperate phage (P945), with its inactivated endolysin gene, exhibited lysis-deficient bactericidal activity against lethal methicillin-resistant S. aureus (MRSA) infection in in vitro experiment (Paul et al. 2011). The filamentous inophages can be used as an ideal tool for the generation of synthetically engineered lysis-deficient phage. The filamentous inophage M13 is

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engineered to generate M13R and M13S105 for enhanced antibacterial activity by integrating of S105 Holin gene and the gene responsible for the production of BglII restriction enzyme. Further study revealed that phage M13S105 produced the non-specific membrane lesions, whereas M13R phage caused the nonrepairable double-strand breaks in the bacterial chromosome by restriction enzyme (Hagens and Blasi 2003). Both the phages exhibited a similar level of killing efficiency and are comparable to the lytic variant of the phage.

4.3

Phage Engineering and Enhanced Antimicrobial Efficacy

Phages can also be used as a tool for the enhancement of the antibiotic spectrum of an antibiotic. The previous study showed that phages can be used as an adjuvant to augment the bioactivity of currently used antibiotics and show a great deal of promise (Lu and Collins 2009). The overexpression of LexA gene is responsible for the suppression of SOS response, resulting in the inactivation of DNA damage-inducible repair followed by random emergence of antibiotic resistance in bacteria (Cirz et al. 2005). In one experiment, the M13mp28 phage was engineered with the capability of overexpression of LexA3 gene, which was shown as a cleavage-resistant variant of the LexA repressor gene (Lu and Collins 2009). It was observed that M13mp23 exhibited the enhanced antibacterial efficacy of antibiotics (ofloxacin, ampicillin and gentamicin) in murine infection model. M13mp23 also showed an enhanced killing efficacy against antibiotic-resistant cells and cells inside structured biofilms (Lu and Collins 2009). The other mechanism which may lead to a reduction in bacterial growth is the insertion of heterologous phage genes leading to inhibition of bacterial growth. The engineered M13 phages having toxin genes (gef and chpBK) are considered to involve in cell death during nutritional deficiency (Engelberg-Kulka and Glaser 1999; Westwater et al. 2003). The experiment suggests that bacteria expressing Gef and ChpBK because of infection by engineered M13 phage infection showed a reduction in colony-forming units (CFUs) by 948- and 1579-fold, respectively. The study also suggested that these phages exhibited a significant level of antibacterial efficacy against intraperitoneal infection by E. coli in the murine model (Westwater et al. 2003).

4.4

Bacteriophages and CRISPR-Cas System

The combination of a bacteriophage and a CRISPR-Cas system is a part of phage engineering (Fig. 6.1). In natural conditions, a clustered regularly interspaced short palindromic repeats-associated (CRISPR-Cas) system provides sequence-based adaptive immunity to bacteria and archaea against the viruses (Barrangou et al. 2007). Based upon the precise site interference of CRISPR-Cas system and the high infection efficiency of phage, the CRISPR-Cas system along with phage can be

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specifically delivered to target bacterial genomic DNA (or RNA) to eliminate the pathogenic bacteria and restrict the evolution of bacterial resistance (Greene 2018). For the target bacteria having an active endogenous CRISPR-Cas system, a miniCRISPR array is included in the phage genome to express the artificial crRNA corresponding to the drug-resistant bacterial genome. The system is designed in such a way that the endogenous CRISPR-Cas system is utilized by bacteriophage to kill the host cell (Li and Peng 2019). In one experiment, the endogenous CRISPR-Cas3 in C. difficile is reprogrammed as an antimicrobial to kill the bacterial cells through the phage infection (Selle et al. 2020). However, in pathogens without the CRISPR-Cas system, an additional exogenous CRISPR nuclease is included for target bacterial killing. A previous study showed that S. aureus phage NM1 was used to deliver an exogenous CRISPR-Cas system to control the pathogenic host (Bikard et al. 2014). The study also revealed that some bacterial genomes also encode anti-CRISPR (Acr) proteins to inhibit the CRISPR-Cas system in several ways (Pawluk et al. 2016). The bacteriophage in association with CRISPR-Cas system can be programmed to kill only antibioticresistant bacteria leading to control of the emergence of resistant bacteria (Yosef et al. 2015). Thus, CRISPR-Cas-associated phages can be used for the prevention and treatment of antibiotic-resistant bacteria.

5

Phage-Based Proteins as Antibacterial Agents

Researchers have developed several techniques for phage engineering and phage therapy. Still, these techniques have several shortcomings, such as narrow antibacterial spectra, horizontal gene transfer, initiation of host immunity, etc. By increasing the knowledge of the phage’s life cycles, their genomics, structural components including capsid proteins, etc., the antibacterial efficiency of phages can be improved. The phage proteins are broadly categorized into two classes: structural proteins (tails and capsids) and non-structural proteins (polymerases and enzymes). The structural proteins play an important role in receptor switching (e.g. pyocins), whereas the non-structural proteins can be engineered to phagederived non-structural enzymes (e.g. endolysins) (Fig. 6.1).

5.1

Polysaccharide Depolymerases (PSDs)

Some of the bacteriophage species encode polysaccharide hydrolase, which can degrade the carbohydrate molecules in the host bacterial envelope (Fig. 6.1). The PSDs of phages degrade the bacterial exopolysaccharides (EPS), capsular polysaccharides (CPS) and lipopolysaccharides (LPSs) and allow the phage to reach the adsorption receptors on host cell surfaces (Knecht et al. 2020; Dennehy and Abedon 2021). Most of the bacteriophages having the capability of degrading the host polysaccharides belong to the category of virulent bacteriophages. The three families

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Myoviridae, Siphoviridae and Podoviridae constitute the tailed bacteriophages and are placed under the order Caudovirales (Ackermann 2006). The characteristic feature of Caudovirales is to recognize the surface receptor and absorb to the host surface through host surface polysaccharide via tailed-associated protein. Bacteriophages possess different polysaccharide depolymerases, including endolysins or lysins, endorhamnosidase, alginate lyase, endosialidase, hyaluronidases, etc. (Yan et al. 2013). Researchers have used the bacteriophage polysaccharide depolymerase against antimicrobial resistance because of its potential biomedical applications including activity as diagnostic kits, bacterial biofilm disruptants, novel antibiotics, adjuvants for antibiotics, etc. (Yan et al. 2013). The efficacy of phage PSDs has been tested against several bacterial species including Pseudomonas aeruginosa (Olszak et al. 2017), Klebsiella pneumoniae (MajkowskaSkrobek et al. 2016), E. coli (Lin et al. 2017), Pasteurella multocida capsular serogroup A (Chen et al. 2018), etc.

5.2

Virion-Associated Peptidoglycan Hydrolases (VAPGHs)

VAPGHs are phage-encoded lytic enzymes having the capability of degrading the peptidoglycan (PG) component of the bacterial cell wall during phage infection (Fig. 6.1). They help form a small hole in the cell envelope through which phage genetic material ejects at the starting point of the infection cycle (Rodríguez-Rubio et al. 2013). VAPGHs degrade the bacterial peptidoglycan layer locally without the lysis of bacterial cells (Rodríguez-Rubio et al. 2013). Researchers have reported the antimicrobial activity of VAPGHs through the phenomenon of ‘lysis from without’, where bacterial cell wall disruption was reported before the phage production (Abedon 2011). Because of specific characteristics such as modular organization, extraordinary thermostability and high specificity, VAPGHs can be recognized as a new antibacterial agent against antibiotic-resistant microbes in human and veterinary therapy, as biocontrol agents against harmful bacteria in agriculture and as bio-preservatives in the food industry. The recombinantly manufactured VAPGHs can lyse and kill the bacteria upon direct contact of the molecule with the cell walls including multidrug-resistant bacteria (Basit et al. 2021; Heselpoth et al. 2021). PGHs can also selectively target the specific species of bacteria without causing harm to commensal host microflora (Rahimzadeh et al. 2018; Heselpoth et al. 2021). They have shown efficacy against several bacterial species including P. aeruginosa (Guo et al. 2017), Bacillus anthracis (Park et al. 2018), Clostridium spp. (Swift et al. 2019), S. pneumoniae (Silva et al. 2020), S. epidermidis (Muharram et al. 2020), etc. They have also been applied to mucosal surfaces and used for the treatment of biofilms (López et al. 1997; Landlinger et al. 2021). Researchers have also supplemented the PGHs with other antibacterial agents, including antibiotics or antibacterial enzymes to control the bacterial growth (Letrado et al. 2018; Blasco et al. 2020).

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5.3

161

Endolysins

The application of phage therapy now has been extended up to phage enzyme engineering termed enzybiotic engineering (Nelson et al. 2001) which is considered a first-in-class antimicrobial biologic. The properties of enzybiotic materials as antibacterial were shown for the first time in group A streptococcal infection (Fischetti 2010). Among the various phage proteins, endolysins and ectolysins have been most extensively studied for lysin engineering. Both endolysins and ectolysins are phage-derived peptidoglycan (PG) hydrolases that play an important role in the degradation of bacterial PG and act as potent antibacterial agents (Donovan 2007). The ectolysins act as phage structural hydrolases and facilitate the phage genome entry into bacterial cells, whereas endolysins act as non-structural hydrolases and are essential for the release of phages from bacteria (Channabasappa et al. 2018). Thus, almost all the lytic phages possess their endolysins, and therefore, for lysin engineering, phage endolysins are mostly targeted. The structure of endolysins comprises two functional domains, along with a linker in-between. The N-terminal domain is termed an enzymatically active domain (EAD), and C-terminal domain is called as cell wall-binding domain (CBD). The EADs are responsible for antibacterial activity, and CBD determines the specificity of action (Fig. 6.1). In one experiment, four chimeric endolysins (Cpl-117, Cpl-177, Cpl-711 and Cpl-771) were engineered by domain shuffling of pneumococcal phage lysin (Cpl-1) along with an engineered lysin (Cpl-7S). The study showed that Cpl-711, possessing the EAD from Cpl-7S and the CBD from Cpl-1 along with linker, demonstrated the maximum lytic activity (Díez-Martínez et al. 2015). Similarly, in another, it was shown that the stability of endolysin can be extended by fusion with the albumin-binding domain (ABD), with no change in its antibacterial activity (Seijsing et al. 2018). Thus, the endolysins engineering may improve the antibacterial activity of phages via domain shuffling through an augmentation in catalytic efficiency and broaden the specificity and enhancement in vivo (serum) stability.

5.4

Holins

The bacteriophages produce small proteins termed Holins. Holins play an important role in the degradation of the host cell wall in the lytic cycle (Fig. 6.1). Holins are passively accumulated in the inner cell membrane, and, after reaching a specific concentration, they trigger the formation of a hole in the membrane. From the pores, in the host cell membrane, lysins enter the cell and destroy the cell wall by degradation of peptidoglycan. Thus, Holins are responsible for generalized permeabilization of the bacterial cell membrane. However, they cannot lyse the bacterial cell alone (Roach et al. 2017). Holins have the capability to regulate the timing of host cell lysis with great precision (Wang et al. 2000). However, the timing of Holin triggering is allele-specific because its effect can either be improved or retarded by missense mutations leading to fine-tuning in phage lysis times

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(Gründling et al. 2000). The potential of antibacterial activity of Holins is studied together with lysins (Sui et al. 2021).

5.5

Pyocin

The R-pyocins are directly not produced by phages. However, they are considered a potent antibacterial agent because of their efficiency to disrupt bacterial membranes (Strauch et al. 2001). R-pyocins are a type of bacteriocins found in P. aeruginosa isolates. It provides competitive growth advantages to P. aeruginosa in mixed population culture (Heo et al. 2007). They are composed of long tube cores connected to a baseplate, which is connected to receptor-binding proteins (RBPs). The structure of R-pyocins is similar to that of the tail of phage (Ge et al. 2015). R-pyocins create pores to disrupt membrane potential, resulting in rapid cell death (Scholl 2017). Researchers have done several scientific studies on pyocin engineering to improve the antibacterial spectrum of R-pyocins. In one study, engineered chimeric pyocins were created by the fusion of C-terminus of P2 phage tail fibre and N-terminus of R-pyocin tail fibre. The resultant chimeric pyocin inhibited the growth of E. coli and lost its inhibition effect against P. aeruginosa (Williams et al. 2008). Thus, the pyocin engineering shifted the target from P. aeruginosa to E. coli by the substitution of the tail fibre (Williams et al. 2008). Subsequently, similar strategies were applied to kill the highly virulent enteroaggregative strain of E. coli O104:H4 and non-O157 STEC strain along with Shiga toxin-producing E. coli strains including O157:H7 and EHEC (Scholl et al. 2012). The R-pyocin engineering has also proved its potential against intestinal pathogens through oral administration to kill and remodel the intestinal microbiome (Scholl 2017). The R-pyocin engineering is similar to endolysins because of the similar modular structures of both the entities. R-pyocin possesses unique features which are distinct from both lysins and phages and can reduce the growing incidence of antibiotic resistance. However, R-pyocins possess more disadvantages in comparison to engineered endolysins because of their enormous size. The size of R-pyocins is comparable to phages and also possesses several epitopes that may elicit more host immunity than endolysins.

6

Phage-Based Pathogen Detection

6.1

Identification of Bacterial Pathogens Using Antibody-Based Method

Immunomagnetic separation (IMS) is a technique used for the efficient isolation of cells out of body fluid or cultured cells. This technique uses antibody-coated magnetic beads to capture, concentrate and isolate the bacteria from samples (Madonna et al. 2003). The IMS assay along with bacteriophage-dependent assays

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can be used for the detection of several bacterial species (Favrin et al. 2001; Madonna et al. 2003). In one of the experiments, Salmonella serovar enteritidis was detected in broth, using bacteriophage SJ2. The detection limit of the assay was found less than 104 CFU/ml in 4–5 h using either optical density or fluorescence measurement (Favrin et al. 2001). In another study, Salmonella enteritidis and E. coli O157:H7 was detected in food samples by bacteriophage SJ2 and bacteriophage LG1, respectively, using IMS and phage-based assay (Favrin et al. 2003). The assay could detect 2 CFU/g of E. coli and 3 CFU of Salmonella enteritidis in 25 g or 25 ml food samples in 20 h (Favrin et al. 2003). Bacteriophages have the capability to bind with bacterial hosts with high specificity like antibodies. However, they have possessed the advantages of cheaper and large-scale production and less sensitivity to pH and temperature in comparison to antibodies (Naidoo et al. 2012).

6.2

Labelled Phages

The host-bacteriophage binding can also be identified through the application of variously labelled bacteriophages. The Shiga toxin-producing E. coli was examined using HRP labelled bacteriophages 56, AR1 and CBA120. The assay was able to detect E. coli with a threshold of approximately 105 CFU/ml to 1 CFU/ml (Willford et al. 2011). In another study, combined immuno-separation of E. coli O157 was done where the bacteriophage-host binding was identified with the help of flow cytometry-based identification methods (Goodridge et al. 1999a). In this study YOYO-1 dye-stained bacteriophage LG1 was used with a detection capability of 104 CFU/ml in 8 h (Goodridge et al. 1999a). Moreover, the same assay was also able to detect E. coli O157 in ground beef at 2.2 CFU/g in 7 h and raw milk at 10 to 100 CFU/ml in 12 h (Goodridge et al. 1999b).

6.3

Ice Nuclease Reporter Bacteriophages

The super-cooled water may remain liquid at sub-zero temperatures. However, super-cooled water can rapidly undergo a chain reaction of freezing just after the introduction of a nucleating agent to it. Several microorganisms including Erwinia, Pseudomonas and Xanthomonas have the ability to act as nucleating agents for super-cooled water. In one of the experiments, the recombinant bacteriophage P22 was constructed with ice nucleation as a reporter signal by incorporating of inaW gene from Pseudomonas fluorescens (Wolber and Green 1990). The inaW gene product generates ice nucleation at temperatures below -9.3 °C. The assay was found sensitive enough to specifically detect Salmonella with a lower limit of 2 CFU/ml in mixed bacterial populations without the need for an enrichment step (Wolber 1993). The ice nucleation-based assay possesses the advantage that most of the background bacteria do not contain similar genes to inaW, thus making the assay specific.

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Phage-Based Biosensors

Bacteriophages have the potential to act as a bio-recognition probe for bacteria since it offers numerous benefits in rapid bacterial diagnosis including host specificity (Balasubramanian et al. 2007), resistance to harsh environmental conditions such as high temperature, a wide range of pH (Bárdy et al. 2016), capability to discriminate live and dead bacteria as they grow only in live bacteria (Altintas et al. 2015), etc. The phage-based biosensor devices contain whole phage or phage constituents and target bacterial cells. The phage-host binding ultimately led to the production of signals which can be measured via colorimetric-, electrical luminescent- and fluorescent-based biosensing system. For phage-based biosensors, phages are used with different mechanisms. The reporter bacteriophages are genetically modified phages having specific genes which can be inserted into the genome of the target bacteria in such a way that it expresses them in the host bacterial cell, and bacteria are identified easily as optical colorimetric or as a fluorescent marker (Burnham et al. 2014). The insertion of several genes including the inaW gene, firefly luc or bacterial lux gene, green fluorescent protein (GFP) gene, etc. has been used for the creation of reporter phages for the detection of Gram-positive and Gram-negative bacteria (Sharp et al. 2015). Another approach is bacterial detection through lytic phages. The lytic phage infection results in cell bursts leading to the release of descendant phages and cell lysis materials which can be used to recognize the target bacterium (Burnham et al. 2014). The adenosine triphosphate from the released cell component can be used for bacterial identification through bioluminescence (Blasco et al. 1998). The amount of released progeny phages from a particular phage is directly proportional to the number of lysed cells which can be used for bacterial sensing (Cox et al. 2015). Moreover, the progeny phages can be easily enumerated by various detection mechanisms including plaque assays, PCR or qPCR, isothermal nucleic acid amplification (ITNAA), etc. (Brovko et al. 2012). The stained phages with different fluorescent dyes can also be used for the detection of target bacteria using fluorescence sensing tools (Bhardwaj et al. 2017). In one of the experiments with the help of fluorescent quantum dots (QDs) tagged phages, E. coli was detected at 20 CFU/ mL in water samples within 1 h (Yim et al. 2009). The immobilized phages on a solid matrix can also be used for capturing specific bacterial cells from a complex sample (Ullah et al. 2017). Bacteriophages possess functionally active groups like carboxyl group (-COOH), hydroxyl group (OH), aldehyde group (-CHO), etc., on their exterior surfaces which can interact with surface receptor molecules to capture the bacteria (Ullah et al. 2017). Previous studies have shown that phages have been successfully used to capture specific bacterial cells from complex samples (Anany et al. 2018). Some of the phage components such as receptor-binding proteins (RBPs) display natural magnetism against the host cell, for example, receptor-binding proteins (RBPs). Phage tail consists of RBPs which assist to bind with the host bacterium cell surface with the help of a specific polypeptide or polysaccharide present on the

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cell surface (Casjens and Molineux 2012). In one of the experiments, RBP protein Gp047 of phage NCTC12673 was used to detect the Campylobacter bacteria from milk and chicken samples (Poshtiban et al. 2013).

7

Phage-Based Vaccine Development

Bacteriophage-based vaccines might be a potent alternative to overcome the limitations of traditional vaccines. The bacteriophage possesses inherent properties to enhance the stability and immunogenicity of antigens and induction of both cellular and humoral immunities (Adhya et al. 2014; Nicastro et al. 2014). These properties of bacteriophages predispose them to an excellent candidate for the vaccine. The major approaches for phage-based vaccines are phage display vaccines and bacteriophage DNA vaccines (Nicastro et al. 2014). However, the combination of these two strategies has led to the development of a hybrid phage vaccine. Phages have several applications in biological sciences including biosensor development, phage therapy, drug delivery and vaccine delivery systems (Adhya et al. 2014; González-Mora et al. 2017). Many of these applications are based on phage display technology which is a result of the manipulation of bacteriophages to present bacterial antigens on their surface. This technique has been used for the development of phage display vaccines against several diseases such as parasitic, fungal cancer, viral drug abuse, etc. (Gu et al. 2013; Asadi-Ghalehni et al. 2015). The vaccines acting against drug abuse display the antibodies on phage particles with the capability to block the effects of certain drug compounds (Carrera et al. 2004). DNA vaccines consist of antigen encoding foreign DNA molecule which is used for direct administration to induce the host immunity. The bacteriophage DNA vaccine consists of eukaryotic expression cassettes encoding a specific antigen (Bazan et al. 2012). The expression cassettes are designed in such a way that it consists of all the necessary regulatory sequences responsible for correct gene expression of antigen followed by protein folding. In phage DNA vaccines, the phage particles serve as passive carriers to transfer the DNA-encoded antigen to eukaryotic cells (Adhya et al. 2014). The phage DNA vaccines are more stable and convenient for transport, storage and administration. For phage-based vaccine production, lambda-phage as well as filamentous phages have been tested (Hashemi et al. 2010). The filamentous phage DNA vaccines may efficiently contain multiple gene copies, which shows an additional advantage to allowing immunization against several epitopes in a single delivery vector (Hashemi et al. 2010). Further study shows that phage DNA vaccines may induce better immune-protection in comparison to naked DNA vaccines (Nicastro et al. 2014). The hybrid bacteriophage vaccines are made through the combination of strategies of phage display and phage DNA vaccines. This kind of vaccine reveals the capability to successfully induce the cellular and humoral immune responses (Bazan et al. 2012). Researchers have developed a double-hybrid filamentous bacteriophage fd with the capability to co-display the peptides which are recognized by major histocompatibility complex (MHC) class I and class II receptors along with

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epitopes from the antigen MAGE (melanoma antigen gene) to enhance the antitumour immune activity through CTL responses (Sartorius et al. 2008). The adequate CTL response of hybrid phages proved that a hybrid phase vaccine may act as a potent tool for the development of effective anti-cancer vaccines.

8

Future Prospects

The world of phages is very vast and diverse. But still it is completely not explored. In the coming future, bacteriophages may be used for several biological research and product formation including control of antimicrobial resistance.

8.1

Bacteriophages as an Emerging Tool to Control Antimicrobial Resistance

Bacteriophages have the potency to control the growth of species-specific microbes. The antibacterial capability of bacteriophages can be upgraded via combination with other agents, especially against the complex biofilm communities (Koo et al. 2017). The simultaneous application of phages and antibiotics shows the synergistic effects against the planktonic cells (Jansen et al. 2018; Yazdi et al. 2018) and old biofilms (Chaudhry et al. 2017; Akturk et al. 2019) (Fig. 6.1). Researchers have shown that individual prolonged treatment with phages may augment the biofilm production. However, the combined application of antibiotics and phages assisted in the eradication of biofilm (Henriksen et al. 2019). In another approach, the phage genome can be modified to improve the outcomes of phage therapy. The phage genomes can be manipulated and engineered through the latest tools of synthetic biology (Pires et al. 2016; Kilcher et al. 2018). Synthetic biology tools can be applied to engineer the host range of phage to enhance the host specificity to prevent the targeting of beneficial bacteria. The tailor-made phage with swapping of receptor-binding protein genes of different phages may improve the host range of the phage (Ando et al. 2015). The host range augmentation of phages can also be done by the fusion of the heterologous receptor-binding domain to the receptor-binding protein of a phage, leading to an increase in the phage host range (Heilpern and Waldor 2003). Moreover, engineered phages can also be used to deliver specific cargo to augment the antimicrobial activity of phages. In one of the experiments, phage T7 was engineered to deliver the enzymes lactonase and dispersin B to enhance the phage activity against biofilms (Lu and Collins 2007; Pei and Lamas-Samanamud 2014). Thus, the synthetic biology and phage engineering approaches may improve the antimicrobial properties of phages and show an alternate path to fight against bacterial infections.

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Bacteriophages and their Role in One Health Approach

The One Health concept discusses animals, humans and environmental health together. Currently, it is a popular term for discussion among researchers, scientists and policymakers. With the indiscriminate use of antibiotics, horizontal gene transfer and genetic mutations, microorganisms are evolving with resistance to antibiotics. Therefore, to mitigate the emergence of antimicrobial resistance under One Health concept, effective alternative therapeutics are required. Bacteriophages have shown their potential to control microbial proliferation in several microbiomes, including humans (Pires et al. 2020), animals (Oliveira et al. 2010), environmental settings (Withey et al. 2005), etc. They can also be used in food processing and to the improvement of the shelf life of food products (Alves et al. 2019). Thus, bacteriophages may play a major role in the One Health approach including animals, plants, etc. by minimizing the use of antibiotics and the subsequent emergence of antibiotic resistance in the human population (Kittler et al. 2017).

8.3

Possibility of Microbial Resistance against Bacteriophages

With the increase in the use of phage therapy, the discussion started on the possibility of the development of resistance against phage similar to antibiotic resistance. However, it is a rare possibility because phages are usually not the first line of treatment against bacterial infections. The phase therapy can be expected only in cases of patients with antibiotic resistance and treatment failure. Phage therapy preparations are expected to be prepared in a personalized way which may delay the emergence of bacterial resistance to phages. Moreover, the co-evolution of phages and bacteria in nature is a continuous process. The study showed that due to the evolution process bacteria have developed certain molecular mechanisms to counter the phage infection. However, bacteriophages have also evolved with counter-strategies to avoid these bacterial defence mechanisms (Samson et al. 2013). Therefore, the evolutionary race between bacteria and phages will never be stopped, and some of the phages will find the mechanism to infect and kill the bacteria. Apart from evolutionary race, the strategies of combined therapies with antibiotics and phage genome engineering may also prevent the emergence of phage resistance.

9

Conclusion

Antibiotic resistance is a progressing threat to the future of mankind on this planet. The threat of antibiotic resistance needs immediate action. With the failure of antibiotics against bacteria, phage therapy can be used as a part of the alternative strategies to control bacteria. Most phage therapies are effective and specific to their host. With the advancement in phage engineering, phages with improved hosts range have been generated. Although the field of phage therapy is rapidly advancing, the

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gaps in knowledge must be fulfilled before the use of phage as a common therapeutic method. However, it is predicted that with the emergence of antibiotic resistance, phage therapy may bring commercial societal and economic benefits to mankind. Acknowledgements The authors are thankful to SVP University of Agriculture and Technology, Meerut, Uttar Pradesh, for providing the facility to prepare the manuscript.

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Drug Repurposing: An Approach for Reducing Multidrug Resistance Ruchi Khare, Sandeep Kumar Jhade, Manoj Kumar Tripathi, and Rahul Shrivastava

Abstract

Drug repurposing is paving ways to develop new therapeutic uses of old drugs which can be helpful in treatment of common and rare diseases. Multidrug resistance (MDR) is a major health issue for the treatment of infectious diseases. MDR is a medical condition where pathogen present in the human body acquires insusceptibility or resistance against the drugs. The combination of different old drugs has shown beneficial upshots during the treatment of MDR diseases. The treatment of such diseases like tuberculosis becomes problematic when the bacterium becomes drug resistance. So, the new approach of drug repurposing which is the process of finding new uses for existing drugs is playing an important role in treatment of MDR diseases with old drugs, which reduces the cost of treatment, cost of drug discovery, and time for treatment of disease. The biggest advantage of drug repurposing is that a lot of information about the characteristics and safety profile of existing drug molecules is already known, which makes it easier for pharmaceutical firms to save valuable time and resources, in repurposing a drug. This chapter deals with the art of repurposing existing drugs for reducing multidrug resistance, the mode of actions of old drugs used, the different approaches of drug repurposing, and advantages of drug repurposing over existing drugs.

R. Khare · S. K. Jhade · R. Shrivastava (✉) Department of Biological Science and Engineering, Maulana Azad National Institute of Technology, Bhopal, Madhya Pradesh, India M. K. Tripathi ICAR-Central Institute of Agricultural Engineering, Bhopal, Madhya Pradesh, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. Y. Wani, A. Ahmad (eds.), Non-traditional Approaches to Combat Antimicrobial Drug Resistance, https://doi.org/10.1007/978-981-19-9167-7_7

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Keywords

Drug repurposing · Drug repositioning · Multidrug resistance

1

Background

The complex process of drug discovery and development is comprised of identification of the potential molecular entities for the treatment of any of the many diseases. Basically, it is the process by which existing medicines used for the treatment of diseases are reprofiled for other diseases. This entire process revolves around the role of gene/protein/metabolite/bioactive molecule and their potency in the mitigation of diseases. In the traditional drug discovery and development, pharmacological studies provide the insight of the drug development phase involving their molecular pathways, mode of actions, beneficial attributes, and the side effects. After the testing of these molecular entities at early stage, they are processed further to attain clinical information of its absorption, distribution, mechanism of action, and its excretion pathways. It is highly expensive, laborious, and time-consuming process as it accounts for approximately 10–16 years study for the development of a new drug involving high risk of failure. . This includes five stages: • • • • •

Discovery and preclinical analysis of drug Safety review Clinical research FDA review FDA post-market safety monitoring (Hughes et al. 2011)

With the constant usage of medicinal drugs in human therapies, it has led to the development of bioaccumulation in the human body. Many strains of bacteria, fungi, and other pathogens have acquired the characteristics of multidrug resistance because of global misuse and overuse of drugs. According to the European Centre for Disease Prevention and Control, developed economies of the world are spending their large amount of wealth on population health as this “antibiotic pollution” has led to evolution of multidrug resistance in pathogenic species resulting in high mortality rate per year (Kraemer et al. 2019).

2

Multidrug Resistance (MDR): The Problem

Multidrug resistance (MDR) is defined as potential resistance acquired by a microorganism (bacteria/viruses/fungi) against the administered antimicrobial medicines/ antibiotics. An antibiotic is a microbial chemical product used to inhibit or to destroy microorganisms causing mitigation of diseases. Development of MDR is a natural phenomenon, which leads to high mortality rates and high medical costs and affects

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the potency of antimicrobial agents (Tanwar et al. 2014). In general, MDR microorganisms possess resistance by two ways, either firstly by accumulation of resistance gene or plasmid in the host cell or secondly by effluxion of the antibiotics out of the host system, which makes them resistant to more than one antibiotic. The coding of RND (resistance-nodulation-division) superfamily efflux pumps in chromosomal gene and its overexpression in Gram-negative bacteria have made them the area of research interest to study multidrug resistance (Nikaido 2009). Worldwide increase of MDR among the pathogenic strains is an alarming situation, as it signifies the scarcity of better antimicrobial agents to treat infections. It is estimated that by 2050, there will be no effective antibiotic available, if no new drug is developed. Tuberculosis (TB) disease caused by Mycobacterium tuberculosis is one of the major public health concerns from decades, as it is a contagious disease attacking the pulmonary or laryngeal tissues leading to necrosis of the cells. So now, the treatment has become a new challenge with the development of MDR-TB strain, which can be reduced by drug repurposing. The MDR-TB strains have reported resistance against two first-line drugs – rifampicin and isoniazid. This raises the need to search for alternative methods of controlling multidrug-resistant pathogens (Palomino and Martin 2014). In view of this problem, repurposing of available drugs has emerged as the best alternative, where the old drugs can be used for the treatment of other or same diseases which can reduce time, as well as the cost of drug discovery and development.

3

Drug Repurposing: A New Alternative Approach

Drug repurposing or drug repositioning means the deployment of an existing drug to a new disease with minimized costs, time, and labor. It is a process of finding new therapeutic uses of existing drugs. A schematic diagram explaining the process of drug repurposing is given in Fig. 7.1. There are some examples of reprofiled drugs showing high efficacy in treatment of other diseases. For instance, in 1957, a sedative drug thalidomide caused skeletal abnormalities in infants, when consumed by pregnant women in their early trimesters; later it was withdrawn from the market. But in 2017, the same drug and its derivatives lenalidomide (Revlimid, Celgene) were found highly effective against multiple myeloma with commercial success (Urquhart 2018). These examples have accelerated the research on developing an effective strategy in reprofiling existing drug molecules with new therapeutic indications (Dey 2019). Repurposed drugs are revealing beneficial characteristics to address the global MDR challenges. They may target some genes and surface proteins that are not covered by existing antimicrobials (Miró-Canturri et al. 2019). Repurposing of drugs is growing as a common practice in the pharmaceutical laboratories where already existing drugs are reprogrammed in a new way. This is beneficial mainly because these drugs are already cleared for human use and thus may skip straight to phase II clinical trials, which offers significantly less risk and costs compared to developing

Fig. 7.1 Schematic diagram for repurposing of drugs

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new drugs. Drug repurposing could be a promising approach to improve the therapeutic resource against multidrug-resistant (Vivas et al. 2019).

3.1

Mechanism of Repurposing

The repurposing of drug is based on two principles: One is “drug specific” and the other is “disease specific.” In drug-specific approach, the drugs effective against one disease may also possess efficacy against any other interrelated disease, while in disease-specific approach, a drug candidate can work against different targets or pathways involved in the same disease. Table 7.1 describes the example of drugs repurposed for treatment of TB. Drug repurposing ropes the detection of novel “drug-disease” link with two types of approaches, biological experimental approach and computational approach or mixed methodologies (Jarada et al. 2020).

Table 7.1 Examples of repositioned drugs for reducing MDR S. No. 1.

Drug name Aspirin

Earlier utility Analgesic and antipyretic

2.

Sanfetrinem cilexetil

Antibacterial activity on both Gramnegative and Grampositive bacteria

3.

Disulfiram

Treat chronic alcohol addiction

4.

Metronidazole (nitroimidazole)

Broad-spectrum antibiotic to treat gastrointestinal infections

5

Tolcapone and entacapone

Catechol-Omethyltransferase inhibitors used in the treatment of Parkinson’s disease

Repurposed for treatment of TB Antiplatelet aggregation drug; antituberculosis drug in initial phase Used as potential betalactam against Mycobacterium tuberculosis. It targets the cell wall of bacteria by inhibiting the formation of peptidoglycan Potent antimycobacterial activity against clinical isolates, MDR, and XDR strains Showed efficacy on pulmonary MDR-TB Also used as effective against parasitic infections like amebiasis, giardiasis, and trichomoniasis Entacapone and tolcapone could evade the KatG activation associated with isoniazid resistance in many resistant strains so both might be a possible treatment of MDR-TB

Reference (Maitra et al. 2016) (Vilchèze 2020; RamónGarcía 2020)

(Maitra et al. 2015; Dalecki et al. 2015) (Carroll et al. 2013; Dingsdag and Hunter 2018)

(Kinnings et al. 2009; Maitra et al. 2015)

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Biological Methods Biological experimental methods are also termed as “experiment-based approach” or “activity-based repositioning” which refers to screening of drugs for new therapeutic indications through experimental assays like protein target-based and cell-based screenings. Data extracted from clinical studies, animal model studies, cell assays, and target screening tests are precedents of biological experimental methods (Lionta et al. 2014; Oprea and Overington 2015). In one such study, in vitro competition binding assay was used to evaluate 38 kinase inhibitors against 317 human protein kinases; it was found that non-kinase targets of small molecules originally designed to inhibit protein kinases were identified as leading molecules to treat antibioticresistant microorganisms (Sun et al. 2016). Assays like column chromatography explain the affinity of particular molecules toward their receptors based on their biochemical characteristics. Same way, by exploiting multiple biological methods like animal cell line-based assay followed by mass spectrometry, an anticancer drug imatinib was found to be effective against tyrosine kinase of BCR-ABL in chronic myelogenous leukemia (CML) which is repurposed as to treat stromal tumors (Blanke et al. 2008). Computational Methods However, the computational or in silico method involves computational biology and bioinformatics tools for screening of large public databases of chemical libraries facilitating the study of drug molecules and their potential cellular/metabolic/protein target leading to the identification of potential bioactive molecules (Talevi 2018). Reduced time, cost of development, and low risk of failure are some of the advantages of computational repurposing over biological experimental method (Rosa and Santos 2020). The signature matching, computational molecular docking, and genome-wide association studies (GWAS) are the most common computational methods for drug repurposing. Signature matching methods are comprised of comparison of specific characteristic of a particular drug for different disease. To examine such molecular entities, proteomics, metabolomics, and genomic studies are performed utilizing electronic databases. In transcriptomic matching, gene profiling of tissues or cells is executed before and after drug treatment to unveil the likeliness between drug and disease or drug-drug interaction (Dudley et al. 2011; Iorio et al. 2013), whereas computational molecular docking is based on binding affinity of drugs with protein targets. A group of researchers performed molecular docking studies between 3671 FDA-approved drugs against 2335 human protein crystal structures isolated from international database. Their study suggested the mebendazole, an anti-parasitic drug, has high affinity to bind with vascular endothelial growth factor receptor 2 (VEGFR2) and can act as potential inhibitor for angiogenesis (Dakshanamurthy et al. 2012). However, GWAS studies accelerated after the completion of Human Genome Project, which provided insight to genome of humans and many other organisms. This paved a pathway to computational identification of new drugs for the treatment of diseases, and many of them are “bio-farmable” as a part of molecular farming, although

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prediction of human genome is still under process as there are many genes undiscovered which coded by introns of human genome (Willyard 2018). Moreover, the validation of such studies is supported by in silico studies followed by preclinical biological experiments and clinical studies, which are termed as mixed or combinatorial approach. These are robust, logical and reliable methods for the discovery of new drug-disease link offering opportunities for many conventional drugs to develop as repositioned drugs showing high efficacy and rapid treatment (Turanli et al. 2018).

4

Database Resources for Repurposing

The drug data repositories have a lot of information related to the different properties of drug entity and its vast effect on genomics, proteomics, metabolomics, etc. A chemical property of drug reveals a lot of information required for scheming chemical-based approaches. Global databases of chemical structures hold enormous information such as 2D topological fingerprints and 3D conformations, which can be employed for predicting novel drug structures to find new indications for drugs having similar structures. Pharmacological databases include information regarding drug intervention in the biological system and the interactions between drugs and different intracellular and extracellular entities. Pharmacological data is submitted in a range of data banks that establishes various computational approaches. Some cases are given below: (a) Drug Bank: It is a bioinformatics and cheminformatics resource that combines detailed drug data with comprehensive drug target information. Proteomics database holds information related to proteome of every organism which is eventually a collection of data reported in studies. Such kind of database is exploited for building homogenous protein-protein interaction (PPI) networks and heterogeneous networks like drug-protein-disease networks. (b) PubChem: This database contains information-related drugs, their other chemical names, functions, safety, toxicity, and physical properties. It can be used to construct chemical networks based on similarities and dissimilarities of chemical entities. PubMed is the most widely used biological literature database that establish information regarding old drugs which can be repurposed for different diseases. This includes citation of literatures like MEDLINE, science citationindexed journals, and publisher websites, although the data may include with full text contents or the abstracts of the studies. The quality of data extracted from the computational libraries is dependent upon the query searches, the keywords, and proper approach to mine the knowledge out of the vast repositories (Xue et al. 2018). The information gathered from such sources decreases the time, cost, and manpower, as with increase in information of the databases and medical literature, the proposed approach for repositioning of drugs becomes unidirectional and simpler.

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Fig. 7.2 Data resources for drug repurposing

A picture depicting the possible strategy for information collection from databases for drug repurposing of drug is given in Fig. 7.2.

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Chemical Structure and Molecule Information Strategy

According to genome drug-based strategy, the drugs which have common characteristics entitle them to conduct comparative studies related to transcriptional responses of old drugs and their new target protein of disease that may affect genes, proteins, and other biological moieties. Drugs with similar profiles are considered as good target for repositioning as they exhibit identical chemical structure and molecule information. Studies suggested that screening of enormous proteins with similar drug targets through molecular docking have revealed thousands of unexpected associations, in which few of them were tested and confirmed experimentally. The binding affinity between drugs and protein targets can explain the side effects of existing drugs, by predicting the pathways accountable for activation/deactivation of cellular proteins through drugs. This approach helps to study the molecular activity and other biological information required to screen new associations and potential off-target effects for approved and investigational drugs (Jarada et al. 2020).

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Challenges in Drug Repurposing

Drug repurposing is a multidisciplinary study performed in coordination with research institutes, hospitals, and pharma companies. The regulatory considerations, organization hurdles, and marketing strategy of pharma companies play a major role in successful completion of drug repurposing projects. Research conducted in organizations regarding repurposing of drugs are lacking in clinical data to validate the study because such collaborations involve sharing of their intellectual property rights. However, it has challenges related to intellectual property protection afforded to such medicinal products, which can reduce their return on investment and discourage companies from developing them. As repositioned drugs are considered as a new chemical entity for patenting, the subsequent medicines containing the same entity can only be protected by a new application patent, possibly backed up by a new formulation process. Patents on applications of therapeutic uses of repurposed drugs are limited than those for new drugs. For example, they cannot always prevent generic products containing the same drug being prescribed off-label for the patented application. They are also weaker, particularly in the face of a potential legal challenge on the basis that the new indication was predictable from data in the scientific literature. Along with the financial support funded by the government of different developed countries, their research organizations and academic institutions are working in collaboration with companies like AstraZeneca and Pfizer which are working on several discontinued drugs in the market.

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Rationale for Drug Repurposing

In the past three decades, a lack of antibiotics for treatment multidrug-resistant bacteria has been observed. Even with high doses of antibiotics, it has become difficult to mitigate the disease having MDR. In case of productivity of such MDR treating drugs, an ever-growing gap is seen within the pharma sector. Drug repurposing is broadly considered as an alternative to ‘drug crisis” for treating MDR. A huge amount of capital has been invested on the research and development programs of different developed countries; a rise in studies related to human diseases coupled with clinical trials has also been observed. However, all of this is giving minimal output to the government of different countries as many compounds exhibiting high efficacy at preclinical phase are unsuccessful in subsequent clinical trials. Escalating developmental expenses, crashed success rates, and time period spent on clinical trials to bring molecule from bench to approval have changed the pharma industry into an unappealing asset (Pushpakom 2022).

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Advantages of Drug Repurposing

The advantages of drug repurposing consist of the simplification in the regulatory procedures for introducing a previously approved drug in the market. This procedure utilizes data previously acquired, regarding the drug safety and toxicity, which can make the initial phases of development for a repositioned drug considerably faster and therefore cheaper and increases the chances of introducing it to the market. Drug repurposing has real economic advantages as it offers new pathways or targets to study novel perspectives for curing diseases.

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Conclusion

In the diseased state of the body, immune system lacks the ability to generate responses against pathogens, it remains compromised, and the MDR pathogens remain untreated because of their high resistance toward drugs. Multidrug resistance acquired by the pathogens has led the world to move back to use conventional drugs, as designing of new antibiotic, antimycotic drugs are a big challenge in today’s world. Drug repurposing has emerged as a smart strategy to overcome the issue of multidrug resistance. The drug repurposing is a unique method by which one drug can be exploited for the treatment of diseases depending upon their severity. A blend of biological- and computational-based approaches has shown promising results for single drugs (such as protein inhibitors) in identification of multiple protein targets active in different diseases. This chapter has focused on many examples of repurposed drugs along with their strategies respectively. In the future, we hope for rise in opportunities in this field, along with better financial aid and mutual collaboration between organizations and pharmaceutical companies.

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Quorum Sensing as an Alternative Approach to Combatting Multidrug Resistance Aimee Piketh, Hammad Alam, and Aijaz Ahmad

Abstract

Due to the continuous rise and spread of multidrug-resistant bacteria, additional therapeutic techniques, including antimicrobial medicines, are needed. Cell-tocell communications, also known as quorum sensing (QS), and microbial biofilm development are two promising approaches. Microbial biofilms are clumps of cells adhering to a substrate and encased in a matrix of self-produced extra polymeric material (EPS). Biofilm production and quorum sensing are currently being investigated as potential new targets for antimicrobial therapy to combat multidrug-resistant diseases. Antimicrobial resistance (AMR), in a larger sense, continues to adapt and spread across all boundaries. Infectious infections have grown increasingly difficult, if not impossible, to manage as a result, leading to a rise in morbidity and mortality. Due to the growth of resistance to conventional antimicrobials, the use of antimicrobial compounds for the treatment of bacterial infections has become a serious problem in recent years. Nowadays, quorumsensing inhibitors (QSIs) are considered as the best alternatives to these antimicrobial drugs since they work as natural immune boosters and disrupt disease resistance without putting bacterial pathogens under selection pressure. Several antimicrobial agents known as quorum-sensing inhibitors have been found and

A. Piketh · H. Alam Department of Clinical Microbiology and Infectious Diseases, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa A. Ahmad (✉) Department of Clinical Microbiology and Infectious Diseases, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa Division of Infection Control, Charlotte Maxeke Johannesburg Academic Hospital, National Health Laboratory Service, Johannesburg, South Africa e-mail: [email protected]; [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. Y. Wani, A. Ahmad (eds.), Non-traditional Approaches to Combat Antimicrobial Drug Resistance, https://doi.org/10.1007/978-981-19-9167-7_8

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are known to be useful in lowering resistance since they do not kill germs but rather impair their communication. In the microorganism community, quorum sensing (QS) is a crucial communication system that controls survival and pathogenicity. The relevance of QS in bacterial infections has prompted researchers to look for QS inhibitors (QSIs) to help fight germs. Keywords

Multidrug-resistant · Quorum sensing · Cell-to-cell communications · Biofilm

1

Introduction

Microorganisms are microscopic organisms such as bacteria, viruses, fungi, and parasites which have the potential to cause disease and even death in humans. Bacteria are prokaryotic single-cell organisms which are omnipresent (Bhunia 2018; Méthot and Alizon 2014). Additionally, these organisms can be found in and on various parts of the human body as they form part of our natural flora. These symbiotic bacterial species are essential to the survival of humans (Hooper et al. 2002). It is only when an individual’s immune system and subsequent immune response is compromised or when an individual is infected with bacteria from the external environment that these organisms have the potential to become pathogenic (Round and Mazmanian 2009; Monack et al. 2004). Antimicrobial substances are agents which are capable of either terminating or inhibiting the growth of microorganisms. Various types of these agents have over time been discovered and used by the human species to combat disease and death which occurs as a result of the pathogenic nature of some microorganisms (Zindel et al. 2011; Junaid et al. 2013). The increased and sometimes inappropriate use of antimicrobial agents such as antibiotics, however, has led to acquired antimicrobial resistance in many microorganism species. Antimicrobial resistance can be defined as the mutations and changes which occur in bacteria, viruses, fungi, or parasites over time which ultimately leads to antimicrobial agents no longer preventing the growth and infection of these pathogens within a host (Hughes and Andersson 2015; Coates et al. 2002). Additionally, antimicrobial resistance in 2019 was listed as one of the top ten threats to global health by the World Health Organization (Thangaraju and Venkatesan 2019). As a result of this, it is of utmost importance that influential stakeholders such as doctors, medical scientists, laboratories, and pharmaceutical companies continue to research and develop new and alternative methods which can help combat multidrug resistance in the pathogens posing the highest burden to global healthcare systems; one example of such an alternative is quorum sensing (Vogenberg et al. 2010). Quorum sensing (QS) can be defined as the extracellular signalling moleculemediated communication which occurs within species-specific bacterial colonies (Decho et al. 2010; Helman and Chernin 2015). These signalling molecules are called autoinducers. Additionally, recent studies have found a family of molecules,

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called autoinducer-2 (AI-2). It is thought that these AI-2 molecules have the ability to facilitate interspecies communication among bacteria (Federle 2009; Xavier et al. 2007). QS is density dependent on bacterial cell population density, which means that QS systems are triggered by greater concentrations of extracellular autoinducer chemicals caused by increased bacterial cell density (Darch et al. 2012; Swift et al. 2001). Bacterial colonies grow in growth through a process known as binary fission, in which a single bacterial cell duplicates its genetic material and then divides into two identical daughter cells via cytokinesis and the resulting daughter cells are clones of the parent (Angert 2005; Oliferenko et al. 2009). Bacteria can divide quickly when the environmental circumstances for bacterial growth are optimal (abundant availability of nutrients and suitable temperature). Bacteria such as Escherichia coli can divide every 20 min, which means that one bacterial cell can produce approximately two million new bacterial cells in 7 h (Rosenberg et al. 1965; Roostalu et al. 2008). Quorum sensing thus relies on bacteria’s ability to produce, release, and detect the presence of autoinducer molecules capable of diffusing across their cell membranes (De Kievit and Iglewski 2000; Li and Tian 2012). Bacterial cells have receptors on their cell membranes and within their cytoplasm that allow them to recognise these compounds. When a certain concentration of autoinducer is reached, these receptors are activated (Federle 2009; Liu et al. 2020a). QS molecules are then able to bring about changes in gene expression and physiological characteristics within the bacterial cell (Cornforth et al. 2014). These signalling molecules have been discovered to cause synchronisation within a bacterial colony. QS controls processes like as bioluminescence, sporulation, competence, antibiotic synthesis, biofilm formation, and virulence factor secretion (Banerjee and Ray 2017). Virulence is defined as a microorganism’s ability to cause injury to host cells and tissues. It is through virulence factors that bacterial cells can attack and infect a host. These virulence factors can be traced back to corresponding genes which are responsible for the expression of certain pathogenic traits. QS therefore operates by activating the genes which bring about the physiological processes associated with the implementation of these virulence factors (Ghosh et al. 2022). Some examples of virulence factors are toxins, surface coats that prevent phagocytosis, or even surface receptors which are capable of binding to host cells (Bryers 2008; Amin et al. 2020; Rosman et al. 2021). There are various bacterial pathogens which are of great clinical importance, which utilise QS to modulate virulence (Castillo-Juárez et al. 2015; Piewngam et al. 2020). It is very unlikely that a single bacterial cell that had entered a host’s body would be capable of launching an attack that could overwhelm the immune system. It is for this reason that information on the size of a bacterial colony is important for the colony itself, as the bacteria hope to improve their chances of survival by working together (synchronisation) (Cronenberg et al. 2021). This means that after colonisation, bacterial cells can ‘fly under the radar’ of a host’s immune system until they attain an acceptable population density. Once the bacterial cell population density threshold is met, the beginning of virulence factors begins (the bacterial population is now considered ‘quorate’)

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(Williams et al. 2007). This tries to increase the likelihood of a host’s defences being overpowered, infection occurring, and bacterial colony survival.

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Quorum-Sensing Regulation Mechanism

Considering that it is the QS communication systems which facilitate the onset of virulence in bacterial colonies, manipulation or interference of these systems could present an opportunity for an alternative means of treatment and perhaps even a solution to the development of resistance (Haque et al. 2021; Muhammad et al. 2020). Preventing QS will allow a host’s immune system the opportunity to clear the infection before any major tissue damage can occur (Amin et al. 2020). Understanding these processes as well as being able to create and implement mechanisms to prevent QS from occurring is therefore of great clinical importance. Processes which seek to prevent QS from taking place have been called ‘quorum quenching’. It is believed that when compared to treatment with antibiotics, blocking bacterial virulence without killing or inhibiting bacterial growth will result in decreased evolutionary pressure which is responsible for the development of acquired resistance (Rasko and Sperandio 2010; Bhardwaj, and A., Vinothkumar, K.,, and Rajpara, N. 2013). Some antivirulence strategies include the inhibition of: 1. 2. 3. 4. 5.

Toxins Molecules and substance which facilitate bacterial adhesion to host cells Secretory systems Virulence gene expression Quorum sensing (intercellular communication)

Bacterial QS systems are classified into three groups based on various selfinducible chemicals. One is the QS system, which is found in Gram-negative bacteria and uses acyl-homoserine lactone (AHL) as the self-inducible molecule (Zhao et al. 2020; Raju et al. 2022; Kobayashi et al. 2004). The oligopeptides are self-inducing QS systems that are found in Gram-positive bacteria. The other types of quorum-sensing systems, which may be found in both Gram-positive and Gramnegative bacteria, utilise furan borate diesters as self-inducing chemicals (Zhao et al. 2020; Monnet and Gardan 2015). Signal molecules represented by short molecule oligopeptides, known as autoinducing oligopeptides (AIOP), are predominantly used as quorum-sensing signal molecules in Gram-positive bacteria. The ATP-binding cassette (ABC) transporter is used to carry precursor molecules across the body for extracellular secretion (Zhao et al. 2020). Bacteria begin to manufacture a huge number of virulence factors as cell density increases, increasing their pathogenicity. This is an oligopeptide signal molecule response to regulate gene expression and stimulate cells (Banerjee and Ray 2017). Most Gram-negative bacteria use the quorum-sensing signal molecules represented by acyl-homoserine lactones, represented by autoinducer-l (AI-1). Acyl-homoserine lactones as a synthetic product in the LuxR-LuxI system found in Gram-negative bacteria can readily

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flow into and out of bacterial cells and accumulate in the surrounding environment (Zhao et al. 2020; Zhang and Li 2016; Papenfort and Bassler 2016).

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Quorum Sensing in Gram-Positive Bacteria

Quorum-sensing processes in Gram-positive bacteria for the most part utilise oligopeptides as intercellular signalling molecules. These quorum-sensing systems involve signalling peptides whose cognate receptor proteins are membrane-bound, two-component histidine kinase receptors or they bind directly to their corresponding intracellular receptors (Fig. 8.1). Gram-positive QS systems operate by translating a peptide signal precursor locus into a precursor protein (process represented by B) that is divided to generate a processed peptide autoinducer signal (Structure C). These peptide molecules are then transported to the exterior of the cell by means of ABC transporters (Structure A). ABC transporters are ATP-binding cassette transporters which are essential membrane proteins, responsible for the ATP-powered translocation of various substances across the cell membranes (Zhao et al. 2020). The extracellular concentration of autoinducer molecules grows with the size of a bacterial colony’s population. Once the extracellular concentration of signalling molecule has reached the minimum threshold, it can then be detected by a histidine sensor kinase protein (these are two-component adaptive response proteins) (De Kievit and Iglewski 2000; Miller and Bassler 2001). Upon stimulation, a peptide ligand (a ligand can be

Fig. 8.1 The quorum-sensing inhibitor mechanism is depicted schematically

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described as a molecule capable of forming a coordination complex by donating a pair of electrons to the central atom) will initiate a phosphorylation cascade (process represented by P) which results in the phosphorylation of an associated response regulator protein. The response regulator is phosphorylated on a conserved aspartate residue (Structure D). Subsequently, DNA can now bind to the response regulator protein which modifies transcription and results in the expression of the target quorum-sensing genes. In Gram-positive bacteria, the basic signalling mechanism remains largely the same; however, variations in regulating and the timing of these processes have been discovered. These variations arise to increase the effectiveness of signal transduction within specific environments. The different quorum-sensing systems in Grampositive bacteria include: 1. Streptococcus pneumoniae ComD/ComE competence system (Martin et al. 2010) 2. Bacillus subtilis ComP/ComA competence or sporulation system (Nakano and Zuber 1991) 3. Staphylococcus aureus AgrC/AgrA virulence system (Srivastava et al. 2014)

3.1

Streptococcus pneumoniae ComD/ComE Competence System

In some cases, recipient bacterium must first become ‘competent’ before they are able to take up exogenous DNA molecules. For bacterial species such as S. pneumoniae and B. subtilis to achieve this state of ‘competence’, there is first a sequence of complex processes, partially controlled by QS, which must be completed (Martin et al. 2010; Nakano and Zuber 1991). In S. pneumoniae, a peptide signal called competence stimulating peptide (CSP) is produced from a precursor ComC amino acid compound and is needed to achieve the previously described state of competency (Baig et al. 2021). This processed CSP is then secreted by a ComAB and ABC transporter apparatus. Subsequently, once the bacterial cell density is high, a ComD sensor kinase protein is then capable of recognising the accumulated CSP (Miller and Bassler 2001). This stimulates the autophosphorylation of ComD that precedes a phosphoryl group being transferred to the ComE response regulator (Ween et al. 1999). The products of autophosphorylation (Phospho-ComE) are then responsible for activating the ComX gene transcription. The development of competence is then achieved through the transcription of structural genes stimulated by the presence of ComX. In S. pneumoniae, however, this state of competence is brief, and its ability to take up exogenous DNA is lost once exponential growth ends (Martin et al. 2010).

3.2

Bacillus subtilis ComP/ComA Competence/Sporulation System

B. subtilis is a soil bacterium that grows vegetatively when nutrients are readily available. However, when nutrients become scarce, the bacteria adapt to overcome

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Fig. 8.2 The signal transduction network for sporulation competency

environmental pressure. These bacteria do so by developing motility, swarming, forming biofilms, developing competence, resorting to cannibalism, producing antimicrobials, or forming spores. Quorum sensing in B. subtilis is facilitated by two peptides, namely, ComX and CSF (competence and sporulation factor) both of which are secreted and accumulate as cell density increases (Kalamara et al. 2018; Schultz et al. 2009). It is thought that the concentration of CSF is responsible for determining which of these two states will be favoured. More specifically, lower concentrations of internal CSF promote competence development, while high internal CSF concentrations prevent competence and encourage sporulation (Fig. 8.2).

Competence A very small proportion of a B. subtilis cell population become competent. Additionally, competence is realised at a later phase of growth. Consequently, due to cell lysis and increased cell density, there is an increased concentration of exogenous DNA available for the competent bacterial cell population (Spoering and Gilmore 2006). This fact ensures that B. subtilis cells inherit the DNA of their own species, and competence serves as a mechanism of gaining new genetic material (VerdugoFuentes et al. 2020). There are various theories as to the purpose of this DNA uptake, one of which is that the exogenous DNA is utilised for the restoration of damaged or mutated chromosomes. At high concentrations ComX is recognised by means of a two-component ComP/ComA sensor kinase/response regulator pair (Kalamara et al. 2018). The response regulator, phospho-ComA, regulates the expression of the comS gene, and ComS then inhibits the ComK protein from being degraded by enzymatic proteases (Auchtung et al. 2006). ComK can be described as a transcriptional activator, regulated by a transcriptional autoregulatory loop which encourages

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the continued commitment to the expression of structural genes which are essential to the development of competence. Additionally, CSF is a pentapeptide and the second B. subtilis QS signalling molecule. At low intracellular concentrations, CSF binds to and impedes RapC (ComA-specific phosphatase) resulting in increased levels of phospho-ComA and by extension competence development (Pottathil and Lazazzera 2003).

Sporulation As a last resort strategy, when environmental conditions are unfavourable and nutrients are scarce, B. subtilis cells favour the sporulation mechanism. Dormant and environmentally resistant spores are formed by asymmetrical cell division. Spore production is poor at low cell density, and at high cell density, sporulation is, in part, controlled by a quorum-sensing mechanism. The mechanism by which sporulation is activated is analogous to the CSF mechanism for competence development. As the extracellular concentration of CSF increases with increased cell density, it is moved into the cell by an ABC-type oligopeptide transporter called Opp (Quentin et al. 1999). High concentrations of intracellular CSF then inhibit the expression of the comS gene, increase proteolysis of ComK, and favour sporulation (Pottathil and Lazazzera 2003). Additionally, sporulation is stimulated when CSF inhibits RapB (a phosphatase) that dephosphorylates a response regulator (Spo0A) (Pottathil and Lazazzera 2003; Rai et al. 2015). Preventing RapB phosphatase activity leads to an increase in phospho-Spo0A concentrations which results in a shift in commitment from competence to the sporulation pathway (Rai et al. 2015).

3.3

Staphylococcus aureus AgrC/AgrA Virulence System

Staphylococcus aureus is a clinically relevant bacterium, causing a wide variety of diseases in human hosts, especially so in those who are immunocompromised (van Belkum et al. 2009). A variety of virulence factors required for successful host infection are regulated by QS systems. The agrBDCA operon has partial control over the regulation of RNAIII levels in the cell. RNAIII then regulates cell population density-dependent virulence factors (Lindsay and Foster 1999). AgrC is the sensor kinase, and AgrA is the corresponding response regulator which plays an important role in increasing concentrations of RNAIII (Lindsay and Foster 1999). Several other signalling molecules, genes, and peptides are responsible for the production of the processed autoinducing peptide (AIP) which is essential for signalling activity. The AIP molecules vary widely among the many S. aureus strains, and this diversity is responsible for the highly specialised interaction with a specific AgrC sensor kinase (Lindsay and Foster 1999; Vasquez and Blackwell 2019). Every AIP unique to a particular strain of S. aureus is capable of inhibiting the Agr response in other S. aureus groups all while stimulating its own Agr virulence cascade (Lindsay and Foster 1999). It is thought that this ability to interfere with the virulence factors of other S. aureus strains serves as a mechanism to outrival strains which attempt secondary invasion and QS circuit development.

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Quorum Sensing in Gram-Negative Bacteria

As previously stated, despite both Gram-positive and Gram-negative bacteria being capable of QS, variations arise in signalling mechanisms, the type of chemical signals, the corresponding receptors, methods of signal transduction, as well as the phenotypic traits that are consequently expressed. N-Acylated-L-homoserine lactones (AHLs), signalling molecules produced by AHLs synthases, are the most extensively researched and often used autoinducers in QS by Gram-negative bacteria (Liu et al. 2020b; Wang et al. 2017). The three main components of AHL stimulated QS systems include: (i) LuxI-type synthase (this produces the signalling molecule) (ii) AHL signalling molecule (iii) LuxR type receptor protein (this facilitates binding to the signalling molecule) The Gram-negative quorum-sensing systems include: 1. 2. 3. 4.

Vibrio fischeri LUXI/LUXR bioluminescence system Pseudomonas aeruginosa LasI/LasR-RhlI/RhIR virulence system Agrobacterium tumefaciens TraI/TraR virulence system Erwinia carotovora ExpI/ExpR-CarI/CarR virulence/antibiotic system

4.1

Vibrio fischeri LUXI/LUXR Bioluminescence System

Quorum sensing was originally discovered by John Woodland Hastings and his postdoctoral candidate Kenneth Nealson. Hastings was a Professor of Molecular and Cellular Biology at Harvard University (Greenberg et al. 2014). Before 1994, quorum sensing was known as ‘autoinduction’. In the 1970s, this phenomenon was originally discovered in the Gram-negative marine organism Vibrio fischeri (Boettcher and Ruby 1995). Experiments conducted on cultures of V. fischeri illustrated induced bioluminescence. For the first time, the autoinduction of luminescence in V. fischeri was described as a transcriptional level process that was controlled by extracellular autoinducers (Boettcher and Ruby 1995). Vibrio fischeri are motile, Gram-negative rods that survive by forming interdependent relationships with marine life like the bobtail squid. This squid has evolved to forage for food at night and utilises the V. fischeri bacteria’s ability to emit light as a mechanism of protection from predation (Fig. 8.3). Vibrio fischeri LuxI/LuxR quorum-sensing circuit utilises two regulatory genes (luxR and luxI) and five luciferase structural genes (luxCDABE) to achieve light emission or bioluminescence (Urbanowski et al. 2004; Kimbrough and Stabb 2013). The genes can be found adjacent, divergently transcribed units. The LuxI protein synthesises the HSL autoinducer N-(3-oxohexanoyl)-homoserine lactone. With an increase in bacterial cell population density, this is also an increase in autoinducer concentrations both within and outside of the cell. Once a threshold concentration is

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Fig. 8.3 Vibrio fischeri bacteria displaying bioluminescence capabilities on a bobtail squid

reached, the autoinducer and LuxR protein become bound together (Kimbrough and Stabb 2013). The newly formed LuxR-autoinducer complex product is now able to activate transcription of the operon by binding at the luxICDABE promoter. This leads to increased transcription of luxI and luxCDABE resulting in the increased emission of light (Bazhenov et al. 2021).

4.2

Pseudomonas aeruginosa LasI/LasR-RhlI/RhIR Virulence System

Pseudomonas aeruginosa is a highly pathogenic bacterium responsible for a high burden of healthcare facility-acquired infections, fatal infections in immunocompromised populations, and chronic infections in patients suffering from cystic fibrosis (Rossi et al. 2021). The virulence factors modulated in part by QS mechanisms include: 1. Those involved in acute infections which are either secreted or are found on its surface. Exotoxin A causes tissue necrosis, phospholipase C causes red blood cell lysis, and a number of proteases cause bleeding and tissue necrosis. The pili exoenzyme S plays a role in bacterial adherence to host cells (Elleboudy et al. 2013; Basso et al. 2017). 2. Those involved in chronic infection include siderophores which facilitates the replication of cells even with no ferrous ions present. P. aeruginosa utilise a well-stablished network of quorum-sensing receptors and regulators. The quorum-sensing systems in P. aeruginosa consist of LasR-, LasI-,

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Fig. 8.4 Pseudomonas aeruginosa LasI/LasR-RhlI/RhlR quorum-sensing system

RhlR-, and RhlI-type systems (de Kievit et al. 2002). Additionally, a PqsRcontrolled quinolone system and the IQS system are involved. These systems are arranged with the LasR protein at the top of the cascade hierarchy. These proteins function as transcriptional activators. Activation of these virulence factors requires the following QS processes to be realised. Pseudomonas aeruginosa utilises two LuxI-/LuxR-like autoinducer-sensor pairs for quorum-sensing regulation (de Kievit et al. 2002). In Fig. 8.4 the homoserine lactone signalling molecule N-(3-oxododecanoyl)-homoserine lactone (Structure A) is generated by the LasI protein. N-(butryl)-homoserine lactones (Structure B) on the other hand are synthesised by the RhlI protein. When the LasR protein has accumulated to a threshold concentration outside of the cell, it binds to the LasI-dependent autoinducer. Next, a wide range of virulence factor promotors are stimulated after the LasR-autoinducer complex has bound to them. Furthermore, the LasR-autoinducer complex can stimulate a second QS circuit through transcription. Once RhlR binds to RhlI, the products can activate the transcription of virulence and target genes (Mukherjee et al. 2018).

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Quorum Sensing in Fungi

Fungi are eukaryotic cells, with membrane-bound organelles and a defined nucleus. Along with the fact that they lack chlorophyll and have distinct structures and physiological processes, they have been characterised in a kingdom separate to that of plants. Their primary means of vegetative growth and nutrient absorption methods further distinguish them from other living organisms. Furthermore, fungi grow from filaments known as hyphae and are comprised of a mycelia body. Nutrients are also externally digested before being absorbed by the mycelia. Fungi, similar to bacteria, can be found in a wide variety of environments in the atmosphere, soil, bodies of water even on and within the plants, and the bodies of animals. An additional similarity that fungi share with bacteria is that both organisms play a role in the decomposition of dead plant and animal material, a function essential for releasing nutrients back into the surrounding environment (Fig. 8.5). Fungi are clinically relevant organisms. In 1928, Alexander Fleming discovered that Penicillium notatum was capable of releasing penicillin, one of the first antibiotics used to treat bacterial infections in humans (Tang et al. 2014). This is just one example of the many of fungi which can be used for medicinal purposes. Another characteristic that fungi share with bacteria is that they are both capable of utilising quorum sensing as a means of intercellular communication. QS regulation was discovered in fungi very soon after it was recognised in bacteria. In 1969, studies described a filament regulation system, controlled in part by cell density (Tian et al. 2021). QS mechanisms in fungi have been described to regulate processes such as sporulation, secondary metabolite production, morphological transition, and enzyme secretion. Additionally, there are eukaryotic-specific behaviours which are not associated with pathogenicity, which are regulated by QS systems; these include sexual development, meiosis, and apoptosis. Below is a timeline depicting major discoveries associated with quorum sensing in fungi (Fig. 8.6).

Fig. 8.5 Fungi in their complex form (mushrooms)

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Fig. 8.6 A few milestones in research and discoveries relating to fungal quorum-sensing models

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The Role of Quorum Sensing in Fungal Adaptation Strategies

Much like bacteria, fungi have processes in place which facilitate changes and strategies for adaptation which help them to respond to unsuitable environmental conditions.

6.1

QS Regulation of Fungal Morphology

Fungi are capable of transitioning between morphotypes. These transitions are brought about external stimuli, either as a response to unfavourable environmental conditions or as a mechanism for infecting a host (Tian et al. 2021). Fungi which are capable of transitioning between the yeast and hypha morphologies during their lifecycles are called dimorphic. Dimorphism is a clinically relevant phenomenon as it is closely related to pathogenicity in various fungal species (Tian et al. 2021). Dimorphism, however, can be observed in both pathogenic and saprophytic fungi (fungi that feed on dead organic matter). Additionally, these morphological changes are regulated by QS molecules. Understanding these processes therefore presents us with opportunities for the development of treatments of dimorphic fungal infections. QS regulation of these morphological transitions was first studied in C. albicans, which utilises farnesol as its signalling molecule (Tian et al. 2021). Of course, variations in signalling molecules and mechanism of action arise between different fungal species. This is also true for fungi that infect and cause disease in plants. The

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farnesol cascade is activated by external stimuli and involves at least five coordinated pathways (Tian et al. 2021). It is important to note, however, that fungi are also capable of morphotype heterogeneity. This means that in a single fugal population, the morphology of subpopulations can be heterogeneous (diverse in character or nature). This is yet another trait regulated by QS systems and offers fungal populations increased chances of survival in volatile environments. C. albicans can once again be used as an example of this ability during biofilm formation processes (Tian et al. 2021). This ability is useful, as both morphotypes are beneficial at different stages in biofilm development. Hyphae offer an increased ability to secrete adhesion proteins along with structural strength and stability, characteristics best suited to the promotion of biofilm maturity (Tian et al. 2021). Yeast, on the other hand, promotes cell dispersion from the well-developed (mature) biofilm when the accumulation of nutrients is necessary. C. albicans utilises tyrosol and farnesol as signalling molecules during the various stages of biofilm development (Tian et al. 2021). During initial and intermediate phases, when hyphae are needed for biofilm maturation, yeast cells are transformed into hyphae when stimulated by tyrosol. Farnesol works in opposition to tyrosol to bring about increased yeast populations, where they offer the most benefit during the later stages of biofilm formation (Tian et al. 2021).

6.2

QS Mechanisms Associated with Inter- and Intraspecies Communication

Quorum-sensing molecules can also facilitate communication between different fungal species and even between entirely unique organisms like bacteria who share the same types of signalling molecules. Additionally, QS molecules can be used by microorganisms to disrupt the survival processes or prevent growth in species competing for the same environmental resources (Tian et al. 2021). Farnesol is an example of such a QS molecule; it is capable of interfering with morphogenesis and even possess antifungal abilities. Once again, C. albicans is one example of a fungus capable of degrading N-acyl-homoserine lactones, an integral QS autoinducer used in Gram-negative bacteria (Tian et al. 2021). This therefore means that it is able to quench QS in certain bacterial species.

6.3

Quorum Sensing and Fungal Infections

Fungal pathogenicity is regulated by QS molecules in the following ways: 1. They behave as virulence factors that facilitate colonisation and infection. 2. They stimulate the manufacturing of virulence determinants to defeat host restriction. 3. They regulate processes associated with virulence, like morphogenesis, cell size, and functional modifications.

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QS Molecules and Virulence Factor Modulation

For a pathogen to successfully infect a host, virulence factors are produced. These virulence factors achieve this through agitation or reduction of host immune defences (Tian et al. 2021). Secreted QS molecules in fungi often act as virulence factors. For example, C. albicans produces a QS molecule called farnesol which is able to integrate into the cell membrane of mammals, can behave as a virulence and suppression immune system of a host, prevent monocytes from differentiating into fully matured dendritic cells, as well as impair the stimulation of the T cell response (Tian et al. 2021). Apart from acting as virulence factors themselves, QS signalling molecules in fungi are also capable of activating the genes which express other virulence factors. A conserved virulence determinant in many human fungal pathogens is cell wall remodelling (Tian et al. 2021). This process facilitates the onset of infection and disease by allowing pathogen evasion and overstimulation of a host’s innate immunity (Tian et al. 2021). Additionally, this is another process modulated by QS mechanisms. An example of this would be the role that the Qsp1 QS signalling peptide plays in controlling the production of melanin in C. neoformans (Tian et al. 2021). Melanin actively participates in cell wall remodelling, an integral function during host cell infection with C. neoformans (Tian et al. 2021). This is a significant virulence factor in this species of fungus, and Qsp1 influences the expression of multiple cell wall genes.

6.5

QS Molecule Control of Cell Shape, Size, or Physiological Status

QS signalling molecules also regulate pathogenicity through virulence-associated cell size modification. C. neoformans utilises Qsp1 and PA (pantothenic acid) in the transforming from normal yeast cells (5–7 μm) into titan cells (>10 μm) (Tian et al. 2021). Titan yeast cells are increasingly resistant to host immunity mechanisms such as phagocytosis. Qsp1 has a negative effect, but PA has a stimulatory role within a certain range of concentrations (Tian et al. 2021). Additionally, PA facilitates the reactivation of viable but nonculturable (VBNC) dormant cells into proliferative cells (Tian et al. 2021). This is clinically significant in cases where patients have reoccurring infections with the same organism (Fig. 8.7).

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Regulation of the Microbial Resistance by Quorum Sensing and Biofilm Inhibition

Chronic infections are caused by both quorum-sensing signalling and biofilm. They are linked to intricate regulatory switches that govern a huge number of genes, the activation of which affects the pathogen’s lifestyle in order to support long-term microbial persistence within a host. This contrasts with more stereotypically acute

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Fig. 8.7 Quorum sensing molecules modulate virulence factor-associated fungal cell size and metabolic state

virulence determinants, which result in outcomes for which microbial presence is less important. Alternative therapeutic approaches are urgently required to fight this ever-evolving bacterial war because of the worrisome rise in antibiotic resistance. Due to their numerous focused activities, combinatorial treatments have attracted attention. Antibiotic use is unavoidable, and antibiotic combinations have been used to treat illnesses with drug resistance. As key regulators of the pathogenicity of drugresistant infections, compounds that disrupt QS have emerged as potential for novel antimicrobial treatments. By acting as QS inhibitors, metal and metal oxide nanoparticles provide fresh solutions to the problem of antibiotic resistance in Gram-negative bacteria. Using qRT-PCR, the relative expression of QS-regulatory genes that govern virulence factors in P. aeruginosa was studied in order to determine the possible quorum quenching effect of ZnO nanoparticles. Significantly, ZnO nanoparticles confirm the phenotypic findings by drastically downregulating the relative expression of the QS regulatory genes lasI, lasR, rhlI, rhlR, pqsA, and pqsR. Similarly, another study established that silver nanoparticles decreased the expression of rhlI, rhlR, lasI, and lasR by preventing rhlR and lasR at the molecular level. ZnO the impact of nanoparticles could be comparable to that of silver. By blocking both lasR and rhlR, nanoparticle’s inactivation of QS circuits followed by disruption generation of virulence factors. Staphylococcus aureus, Escherichia coli, P. aeruginosa, Proteus mirabilis, and B. cereus were just a few of the bacteria that the AgNPs were capable of killing. Various studies have shown that AgNPs have antibiofilm and anti-QS properties. In a different work conducted by Jagtap et al., in 2013 silver nanowires showed anti-QS efficacy by preventing P. aeruginosa from forming biofilms and C. violaceum from producing violacein. Salmonella typhi, methicillin-resistant S. aureus, and E. coli are just a few of the microorganisms against which AuNPs have demonstrated antibacterial activity. In one study, gold

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nanoparticles (AuNPs-CA) were synthesised and described using Capsicum annuum aqueous extract. The AuNPs-CA were tested against the QS-controlled virulence factors and biofilms of P. aeruginosa PAO1 and Serratia marcescens MTCC 97. The ability of nanoparticles (NPs) to stop the proliferation of microbial cells and so combat harmful organisms makes them the most often used medicine in delivery system. The prevention of biofilm and microbiological growth by NPs is accomplished through a variety of ways. Numerous investigations were carried out to determine the most likely mechanism by which the NPs might prevent microbial development. It was discovered that ZnO NP can prevent S. aureus NorA efflux pump from functioning. Soha Lotfy Elshaer and Mona I. Shaaban (2021) observed that Au-NPs were more efficient than Se-NPs against the tested Pseudomonas strains in a different study that demonstrated the production of Au-NPs and Se-NPs utilising cell-free-Streptomyces S91 supernatant. The MIC for the investigated Pseudomonas strains ranged between 9.2 and 147.7 μg/mL and 4.6–1184.4 μg/mL. Additionally, the produced nanometals had powerful activity of Au-NPs compared to Se-NPs and strong anti-QS and antivirulent capabilities. Finally, both nanometals clearly showed their broad-spectrum anti-QS characteristics by dramatically inhibiting the synthesis of pyocyanin, elastase, and protease from the investigated P. aeruginosa strains. With particular emphasis on its toxicological effect on the host system and in vivo efficacy, more research on nanoparticles as QSIs must be conducted. It is anticipated that soon cellular communication inhibition by nanoparticles may become a more effective therapy option for microbial disease in humans and animals, particularly against drug-resistant strains.

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Biofilms

An accumulation of microorganism cells that are able to attach themselves to a surface can produce an extracellular polysaccharide matrix which forms a biofilm. This conditioning film covers the surface colonised by the cells, and its synthesis commences after plasma proteins become bound to a surface and the coagulation cascade and complement are subsequently activated (Wasserman 2021). Bacterial biofilms are for the most part ubiquitous and nearly all bacterial species are capable of producing one. The formation of these biofilms is beneficial to bacterial cells as they provide mechanisms of protection against harsh and unfavourable environmental conditions such as exposure to ultraviolet light, harmful concentrations of antimicrobial substances, as well as adverse pH levels (Wasserman 2021) (Fig. 8.8). Bacterial biofilms are of great importance to the human species. Depending on the context, biofilms can have either a favourable or damaging role when it comes to human activities. From a clinical point of view, however, biofilm formation and pathogenesis are closely related. These polysaccharide matrices can form on biological tissue surfaces or even on inanimate surfaces within clinical facilities (e.g. medical instruments) (Wasserman 2021). Biofilms make eradicating bacterial colonisation and infection more challenging as they offer bacterial cells’ augmented resistance capabilities to immune system attacks, antimicrobial agents, and

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Fig. 8.8 The cascade of events during the process of biofilm formation

antiseptics (Wasserman 2021). As a result of this, finding new means for combatting the formation of these structures or accelerating their degradation is extremely important. This is relevant in the context of quorum sensing, because, as mentioned earlier, biofilm formation is one of the bacterial functions or processes which is modulated by quorum sensing in many clinically relevant bacterial species (Wasserman 2021). Using synthetically manufactured molecules which mimic bacterial quorum-sensing signalling particles, therefore, presents an opportunity to prevent bacterial colonies from forming these films. Additionally, bacteria are highly adaptive as genes can be selectively expressed based on the varying environmental stress factors to which they are exposed (O’Toole et al. 2000). In different regions of the biofilm, they exist in varying metabolic states. For example, bacterial cells inhabiting regions of the biofilm where nutrients and oxygen are abundantly available can exist in a planktonic state. Bacterial cells in a planktonic state are free moving and under active metabolic processes, as opposed to those in the lower levels of the biofilm which are sessile and have decreased metabolic functions as a result of the decreased availability of nutrients and oxygen (O’Toole et al. 2000). These varying metabolic states which have corresponding variations in growth rates have a direct influence on the effectiveness of antimicrobial agents used to clear infection and bacterial colonisation. These agents are less effective at eradicating cells in the lower layers of a biofilm which remain more dormant; this therefore means that a biofilm’s increased

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resistance brings about reoccurring infections and decreased treatment success (O’Toole et al. 2000) (Fig. 8.9).

8.1

Biofilms in Gram-Positive and Gram-Negative Bacteria

The Role of Cell Wall Components in Biofilm Formation As previously mentioned, the production of biofilms is a multi-step, complex process. The first of them, which is a function of the features of the surface itself, is the adherence of cells to live or non-living surfaces and the attachment to various surfaces (Ruhal and Kataria 2021). For example, uncoated, abiotic surfaces require bacterial cells to secrete and utilise physiochemical agents. It is thought that these agents utilise binding strategies based on the hydrophobic and electrostatic properties of the cells and surface (Ruhal and Kataria 2021). An example of such a characteristic would be that bacteria are known to be negatively charged and as a result have increased success when binding to an abiotic surface with the opposite charge (Ruhal and Kataria 2021). On the other hand, biotic surface adhesion is facilitated by surface adhesin molecules. The agents responsible for providing the negative, surface charge on bacterial cells vary between Gram types. Teichoic acid (TA) is present in the cell wall of Gram-positive bacteria, whereas lipopolysaccharides (LPS) are essential components of the outer membrane of Gram-negative bacteria (Ruhal and Kataria 2021) (Table 8.1). An experiment was conducted to confirm the role of these cell wall components in the synthesis of biofilms. The Gram-positive, Bacillus subtilis bacterium was used, in which the tagA-O operon is responsible for the synthesis of teichoic acid. Researchers facilitated the complete deletion of tagD, which resulted in no teichoic acid synthesis and, subsequently, planktonic growth inhibition. Additionally, the removal of enzymes (glycosyltransferase) which are responsible for teichoic acid modification led to the ineffective formation of bacterial cell biofilms. This experiment, therefore, confirmed the role of these molecules in biofilm formation and established these structures as clinically important targets for antibiofilm action (Ruhal and Kataria 2021). The Role of Motility in Biofilm Formation For adhesion to occur successfully, bacterial cells need to be in close proximity to a surface. This is achieved through active or passive movements by cells, through a liquid medium. Gram-negative bacteria, for example, like Pseudomonas aeruginosa, achieve swimming or swarming motility by means of flagella (Ruhal and Kataria 2021). Additionally, they are capable of crawling along a surface by means of type IV-based pili twitching. Some non-flagellated Gram-positive bacteria like Staphylococcus aureus on the other hand utilise Brownian motion to approach surfaces (Ruhal and Kataria 2021). There are, however, other components which play a role in the adhesion of bacterial cells to surfaces, such as polysaccharide intercellular adhesins (PIA), environmental DNA (eDNA), and cell wall-anchored proteins (CWAP). These factors all work together and form an essential part of cell adhesion

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Table 8.1 The table below summarises the differences which arise in cell wall structure and components between Gram types which are significant for biofilm formation Gram-positive No outer membrane Produces a thick peptidoglycan in cell wall Teichoic acid plays an important role in bacterial cell colonisation and infection

Gram-negative Outer membrane present Cell wall of lipopolysaccharides Length and variation of lipopolysaccharides plays an important role in adhesion and subsequent biofilm formation

Table 8.2 The table below summarises the differences which arise between the Gram types, when it comes to means of transportation of substances across the cell membrane Gram-positive Membrane vesicles Transports active β-lactamase out of the cell, which contributes to antibiotic resistance Involved in the bacterial cell response to stress stimuli, biofilm formation, as well as immune regulation

Gram-negative Outer membrane vesicles Protective transport vesicles which carry and deliver toxins, enzymes, matrix proteins, and DNA Promotes bacterial cell survival when exposed to stress conditions

and have therefore been identified as potential targets for the inhibition of biofilm formation (Ruhal and Kataria 2021).

The Role of Biofilm Matrix Components in Biofilm Formation Matrix components fulfil a multitude of functions during the development of biofilms. The various matrix proteins and polysaccharides are altered into structural and functional higher orders in order to generate multicellular colonies and increase the microorganism populations’ chance of survival. The matrix contains biofilmassociated proteins (such as amyloid proteins, found in both Gram types) needed for biofilm initiation and maturation, their abilities to create channels within the biofilms to allow access to nutrients; they assist in immune evasion and facilitate dispersal of bacterial cells by means of the surfactant properties which they exhibit (Ruhal and Kataria 2021). Polysaccharide secretion has also been found to play a crucial role in the initial adhesion of biofilms and the preservation of mature biofilms. Additionally, both Gram-negative and Gram-positive bacteria require extracellular DNA (eDNA), and the investigations unequivocally showed that DNase therapy reduced biofilms (Ruhal and Kataria 2021). Further, eDNA may play a role in the resistance to antibiotics due to its electrostatic nature. Possible mechanisms of eDNA include autolysis, prophage-mediated death, and secretion (Table 8.2). These components, which remain unique to the biofilm matrix of bacteria, have also been proposed as target sites for action by therapeutics. Because biofilms are associated with chronic and recurring infections, inhibition of quorum sensing or enzymatic degradation of the biofilm matrix could result in biofilm dispersal and has been suggested as an infection eradication strategy (Ruhal and Kataria 2021).

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Biofilm-degrading enzymes could potentially improve the efficacy of certain antimicrobial agents against bacterial cells. However, as some new studies emerge which indicate that dispersed cells have the potential to exhibit increased virulence and antibiotic resistance, more research needs to be done before this mechanism can be confirmed as a therapeutic strategy (Ruhal and Kataria 2021).

8.2

Biofilms in Fungi

There are a variety of clinically significant fungi which are capable of producing biofilms. These include Candida, Aspergillus, Cryptococcus, Trichosporon, Coccidioides, and Pneumocystis (Kernien et al. 2018). Additionally, fungal biofilms are also capable of propagating on both biotic and abiotic surfaces. The biofilm structural intricacy, the production of extracellular matrix which accumulates as the biofilm matures and promotes cohesion, metabolic heterogeneity in metabolic stages among sessile and planktonic bacterial cells, as well as the ability of biofilms to generate antimicrobial resistance-associated efflux pumps are all factors which contribute to the increased resistance commonly associated with biofilms (Kernien et al. 2018). Similar to bacterial biofilms, fungal biofilms cause recurring infections and are notoriously difficult to eradicate. In this section, we will discuss the formation of biofilms in one of the most clinically relevant fungal species, Candida. Candida fungi are commensal microorganisms that colonise the gastrointestinal tract. These micro-organisms, however, in immunocompromised individuals can bring about severe disease with high mortality, especially those with implanted medical devices (Kernien et al. 2018). Candida biofilms can form and develop on a wide variety of abiotic surfaces, including on inserted medical devices, for example, central venous catheters or urinary catheters (Kernien et al. 2018). Biotic surfaces that this species is capable of colonising include both the vaginal and oral mucosal membranes (Kernien et al. 2018). Biofilm initiation and development happens in a similar sequence of events to that of bacterial biofilms. The initial stage of biofilm formation in this species of microorganism involves adhesion of yeast cells to a surface. This is then followed by the propagation of fungal cells to form a community, along with the construction of an extracellular matrix, embedded with yeast cells (Kernien et al. 2018). In Candida albicans, however, biofilm formation also involves the production of hyphae, with the extent of filamentation varying (Fig. 8.10).

Matrix Composition The biofilm matrix assembly and composition has been investigated, specifically in Candida albicans, and studies revealed that the mature biofilm matrix is made up of a wide variety of macromolecules, including proteins (55%), carbohydrates (25%), lipids (15%), and DNA 1111111%) (Kernien et al. 2018). It is important to note that the matrix constituents are vastly different from those that make up the cell wall. Additionally, it was established that more than 95% of the matrix-associated proteins

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Fig. 8.10 Biofilm formation in Candida albicans

within a fungal biofilm were of host origin, meaning that the host plays an important role in the construction of a biofilm (Kernien et al. 2018).

Host Immunity and Candida Biofilm Interactions When a host becomes infected with the Candida species, neutrophils are the primary leukocyte responders. When neutrophils encounter the Candida pathogen, various responses are initiated which are essential for infection control and eradication (Kernien et al. 2018). These processes include phagocytosis, degranulation, the production of reactive oxygen species (ROS), as well as neutrophil extracellular trap release (Kernien et al. 2018). Despite their usual ability to eradicate planktonic Candida cells, neutrophils are incapable of eradicating Candida biofilms. These biofilms have demonstrated a twoto fivefold increase in resistance to neutrophil activity. Studies have shown that Candida albicans biofilm’s resistance to neutrophil action is robust and varies by strain and species (Kernien et al. 2018). For the host’s immune response to achieve infection control and eradication, neutrophils must release neutrophil extracellular traps (NETs) (Kernien et al. 2018). NETs are well adapted to destroy large organisms and structures that otherwise cannot be ingested by means of phagocytosis, structures like hyphae which are a large component of Candida albicans biofilms (Kernien et al. 2018). However, recent investigations have revealed that neutrophils are incapable of releasing NETs and generating reactive oxygen species in the presence of Candida biofilms (Kernien et al. 2018). The manufacturing of the extracellular matrix seems to be closely associated with this phenomenon. This process is therefore an immune response evasion adaptation by Candida albicans biofilms.

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Additionally, a second evolutionary adaption and strategy for immune evasion by Candida albicans biofilms appears to have developed. Candida biofilms demonstrate a two- to threefold increase in resistance to action by host immune monocytes and macrophages as opposed to fungal planktonic cells (Kernien et al. 2018). These biofilms achieve this resistance by means of (i) inhibiting monocyte cell action and altering their cytokine profile, (ii) preventing the phagocytotic ingestion of biofilmassociated Candida albicans by peripheral blood mononuclear cells, and (iii) impairing the migratory capacity of macrophages (Kernien et al. 2018).

Aspergillus The Aspergillus species are present and capable of forming biofilms ubiquitously in the environment. Additionally, they release spores into the atmosphere to which humans are constantly exposed (Kernien et al. 2018). It is only in the immunocompromised that the immune system is incapable of clearing these spores after inhalation. It is in these individuals that severe disease development is of high risk. Aspergillus is clinically relevant and causes a variety of invasive, chronic, and allergy-associated disease (Kernien et al. 2018). Aspergillomas (fungal balls) are generated in the lung or sinus cavities during persistent infections with this microorganism because aspergillomas are composed of agglutinated hyphae that form a dense structure and conidial heads are rarely capable of growing within a biofilm (Kernien et al. 2018). One species that can result in invasive pulmonary aspergillosis is Aspergillus fumigatus. Matrix Production During biofilm formation, Aspergillus fumigatus produces an extracellular matrix which is composed of DNA, 40% protein, 43% carbohydrates, 14% lipids, and 3% aromatic-containing compounds (Kernien et al. 2018). The polysaccharide component of this matrix is responsible for cohesion and protection from the host immune system. An example of one of these polysaccharides (which is found in multiple aspergillus species) is galactosaminogalactan (GAG), without which biofilm formation would not be possible (Kernien et al. 2018). Additionally, deacetylation is a characteristic of GAG required for the facilitation of hyphal adhesion to separate anionic surfaces (like host cells). Clinically relevant Aspergillus biofilms also secrete a thicker extracellular matrix. Host Immunity and Aspergillus Interactions Immunocompromised people, particularly those with neutropenia, are more likely to develop severe and even fatal aspergillus infections because neutrophils play a crucial role in the immune response during the eradication of Aspergillus (Kernien et al. 2018). Phagocytosis is the process by which neutrophils destroy Aspergillus spore, neutrophils also capable of releasing NETs (neutrophil extracellular traps) when stimulated by the presence of hyphal elements. Additionally, the secretion of an extracellular matrix-containing GAG provides protection from neutrophil attack (Kernien et al. 2018). Aspergillus cells which have mutated and are lacking deacetylated GAG exhibit attenuated virulence. As a result of this, aspergillus

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infections that are treated with glycoside hydrolases show observable decreased growth. It is thought that the protection provided by GAG can be attributed to the electrostatic repulsion between this cationic polysaccharide and the cationic antimicrobial properties within the NETs themselves (Kernien et al. 2018). Additionally, GAG is capable of masking and altering recognition of fungal cells by the host immune system by impairing accumulation and stimulating apoptosis of neutrophils during biofilm development (Kernien et al. 2018).

Saccharomyces Cerevisiae Molecular Basis for Yeast Biofilm Formation The Saccharomyces species are seldom pathogenic to humans, and they produce a haploid biofilm. Additionally, S. cerevisiae can easily be manipulated on a genomic level and has therefore been utilised as a major eukaryotic model for research. Saccharomyces cerevisiae was the first eukaryotic genome to be sequenced, making it amenable to global genetic and phenotypic analysis (Bojsen et al. 2012). S. cerevisiae possesses a protein adhesin called FLO (flocculin). FLO proteins form an integral part of the plasma membrane which remains bound to the cell wall (Bojsen et al. 2012). This structural characteristic is responsible for its ability to facilitate cell-to-surface and cell-to-cell adhesion. Biofilm formation in S. cerevisiae as well as its regulation mechanisms, quorum sensing, and extracellular matrix components are widely conserved in opportunistic fungal pathogens like Candida (Bojsen et al. 2012). These similarities mean that we will explore biofilm development in the Saccharomyces species only very briefly as Candida has already been described above. Molecular Basis for Cell Surface Adhesion For S. cerevisiae to attach to external surfaces, the Muc1/Flo11 cell surface protein is required. Flo1, Flo5, Flo9, and Flo10 are homologues to Flo11, and each is made up of an A, B, and C domain (Bojsen et al. 2012). Adhesive qualities can be attributed to the N-terminal A domain, and Flo1,5,9 and 10 Flo5, Flo9, and Flo10 bring about cell to cell adhesion (Bojsen et al. 2012). The B domain on the other hand is comprised of tandem repeats which exhibit an abundance of serine and threonine residues. These residues can form N- or O-linked glycosylation with Flo1- and Flo11-glycosylated proteins. And lastly, in the C-terminal of the C domain are responsible for the mediation of Flo adhesins to the plasma membrane through sites available for covalent attachment of a glycosyl phosphatidylinositol anchor (GPI) (Bojsen et al. 2012). Quorum Sensing Quorum sensing in the Saccharomyces species has illustrated a regulatory function through control of FLO11 and therefore, by extension, controls the growth of biofilms. In S. cerevisiae, quorum-sensing regulatory molecules, also described as autoinducers, include ethanol and the aromatic alcohol tryptophol and phenyl

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ethanol (Bojsen et al. 2012). When the desired cell population density is achieved, threshold levels of ethanol and aromatic alcohols stimulate FLO11 expression (Bojsen et al. 2012). Ammonia therefore plays a role in the mediation of communication between S. cerevisiae subpopulations. In S. cerevisiae, intercellular communication is achieved with airborne ammonia molecules, and one cell produces and secretes these molecules while the other recognises and responds by stimulating cell development (Bojsen et al. 2012). Extracellular Matrix and Biofilm Resistance The S. cerevisiae extracellular matrix structure contains mono- and polysaccharides as well as various types of proteins. The matrix offers protection to cells from antifungal agents by decreasing their ability to move across the membrane, and the cavities found within the matrix are thought to serve as storage pods for nutrients and waste products within the biofilm itself (Bojsen et al. 2012). It has already been established that increased resistance to antimicrobial agents is a characteristic of biofilms which makes them notoriously difficult to eradicate. Studies done on S. cerevisiae biofilms have shown that in flocculating cell populations, cells are resistant to fungicidal agents even at exponentially increased concentrations, unlike their planktonic cell counterparts (Bojsen et al. 2012). Flocculating cells in the fungal Saccharomyces, like sessile cells in bacterial biofilms, exist in a state of decreased metabolic function (dormancy). The consequent decreased growth rates are thought to be a contributing factor, responsible for this increased resistance to antimicrobial agent commonality among the various microorganisms.

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Nanoengineering Approaches to Fight Multidrug-Resistant Bacteria Sahadevan Seena and Akhilesh Rai

Abstract

In the last few years, a high prevalence of multidrug-resistant (MDR) bacteria, mostly methicillin-resistant Staphylococcus aureus (MRSA), MDRmycobacterium tuberculosis, and carbapenem-resistant Enterobacteriaceae, has been reported globally. Infections caused by MDR bacteria are difficult to treat and eradicate as they develop resistance by employing novel mechanisms against antibiotics and other antimicrobial agents. Based on the current rate of antibiotic production and approvals by medical regulatory agencies, it is anticipated that approximately ten million people could die annually due to MDR pathogens by year 2050. Therefore, alternative materials, such as the nanoparticles (NPs), antimicrobial peptides (AMPs), and small cationic molecules, have been explored to formulate potent antimicrobial agents to replace antibiotics or reduce the burden of antibiotics from patients. Various compositions of NPs such as metallic, inorganic, organic, and lipid have been synthesized to prepare antimicrobial materials. Antibiotics, AMPs, and small molecules have been conjugated on the surface of NPs to enhance their antimicrobial activities and reduce the systemic cytotoxicity. This book chapter addresses the recent approaches, prospects, and challenges of nanotechnological tools for controlling the transmission and emergence of antibiotic-resistance bacteria.

S. Seena MARE - Marine and Environmental Sciences Centre, Department of Life Sciences, University of Coimbra, Coimbra, Portugal A. Rai (✉) Faculty of Medicine, University of Coimbra, Coimbra, Portugal CNC-Center of Neuroscience and Cell Biology, CIBB-Center of Innovation in Biomedicine and Biotechnology, University of Coimbra, Coimbra, Portugal e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. Y. Wani, A. Ahmad (eds.), Non-traditional Approaches to Combat Antimicrobial Drug Resistance, https://doi.org/10.1007/978-981-19-9167-7_9

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Keywords

Nanoengineering · Multidrug resistance · Nanoparticles (NPs) · Antimicrobial peptides (AMPs)

1

Introduction

The continuous emergence of MDR bacteria is one of the most important public health challenges as it jeopardizes the potency of drugs, leading to increased morbidity, mortality, and economic burden on healthcare systems. The cases of MDR bacterial infections are found in all places and are steadily increasing worldwide. It is reported that more than 2.8 million MDR-related infections were found in the USA in 2019, corresponding to 35,000 deaths (Gómez-Núñez et al. 2020). Notably, MDR bacterial infection might cause more than ten million deaths per annum globally by 2050. Most MDR bacterial infections are generally found in undeveloped and low economy countries as 43% of deaths in Thailand occur due to MDR bacteria (Lim et al. 2016). Not only humans but also 87% of farm animals and 90% seawater-originated bacteria are resistant to antibiotics (Gómez-Núñez et al. 2020). Bacteria that became resistant to multiple drugs are termed MDR or informally called superbugs. ESKAPE group of bacteria [Enterococcus faecium (E. faecium), Staphylococcus aureus (S. aureus), Klebsiella pneumoniae (K. pneumoniae), Acinetobacter baumannii (A. baumannii), Pseudomonas aeruginosa (P. aeruginosa), and Enterobacter spp.) are prone to develop MDR (Rice 2008). This acronym is used because these bacteria can escape the antimicrobial effect of antibiotics through well-developed mechanisms. The World Health Organization (WHO) and Centers of Disease Control and Prevention (CDC) consider MDR infections one of the main threats to public health globally. NDM-1 gene found in bacteria is responsible for the generation of super-resistance. The commercially available antibiotics control cell wall synthesis and DNA replication. Notably, the number of antibiotics approved by the Food and Drug Administration USA (FDA) is much less than the population of MDR bacteria especially difficult-to-treat MDR Gram-negative bacteria. It is reported that NPs act directly on the bacterial membrane and severely damage them without penetrating inside the cell; this phenomenon has raised the hope that (NPs) might be less prone to develop bacteria resistance (Gupta et al. 2017). Although the membrane composition of Gram-positive and Gram-negative bacteria differs, cationic NPs act strongly on both bacteria (Fig. 9.1) (Gupta et al. 2019). Moreover, the ability to easily fine-tune the physicochemical properties of NPs makes them attractive candidates as antimicrobial materials or effective cargos to deliver antimicrobial molecules such as antibiotics, (AMPs), and small cationic molecules. Importantly, alternative technologies such as computation and in silico tools have been explored to identify novel targets and design appropriate drugs/ biomolecules. Nanoengineering approaches have revolutionized bioengineering and medicine areas. It is believed that the advanced NPs can deliver drugs to the target

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Fig. 9.1 Schematic diagram showing (a) cell wall structures of Gram-positive and Gram-negative bacteria and (b) antimicrobial mechanism of NPs: (A) disruption of cell membrane resulting in cytoplasmic leakage; (B) binding and disruption of intracellular components; (C) disruption of electron transport causing electrolyte imbalance; and (D) generation of reactive oxygen species (ROS). The figure is adopted from Ref. (Gupta et al. 2019)

organ in a controlled manner without inducing side effects of drugs. Nanotechnology tools could be used for targeted drug delivery, diagnostics, imaging, and detections, which have revolutionized the healthcare areas. Given the multifunctional properties of NPs, they are extensively explored in combination with various antimicrobial agents in treating MDR and MRSA bacterial infections (Gupta et al. 2019).

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Advantage of Nanotechnology-Based Approach to Fight MDR Bacteria

NPs as defined by the FDA are materials with at least one dimension lesser than 100 nm. Owing to the unique physicochemical and biological properties of NPs such as smaller size, size-dependent internalization by cells, ability to conjugate multiple molecules, NPs have been extensively used to deliver drugs and other biomolecules for various biomedical applications (Rai and Ferreira 2021). The large surface area to volume ratio of NPs promotes the conjugation of a higher number of molecules and increases the contact area with bacteria. The surface of NPs can be modified with suitable ligands, which facilitate the conjugation of one or multiple molecules (Rai and Ferreira 2021). These properties make NPs unique to deliver several molecules in a single administration. Notably, the inherent antimicrobial properties of NPs are explored in combination with the conjugated antimicrobial molecules such as antibiotics and AMPs to develop potent antimicrobial formulations to eradicate nosocomial and MDR bacterial infections (Comune et al. 2021; Rai et al. 2022; Rai et al. 2010). NPs enhance the antimicrobial effect of antibiotics and maintain the biological activity of AMPs in the protease environment. Additionally, the NPs can

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Antimicrobials Efflux pump

Reduced uptake

Deactivating enzymes

Target modification

Inactive metabolic state

Deactivating enzymes

Adsorbed or trapped

Resistance gene transfer

Susceptible cell

Resistant cell

Persister cell

Fig. 9.2 The mechanisms of the generation of antibiotic resistant in bacteria. The figure is adopted from Ref. (Makabenta et al. 2021)

be functionalized with a suitable drug or antibody to selectively target the infection site with their higher accumulation, leading to reduced systemic toxicity. The synergistic antimicrobial properties of NPs and conjugated molecules help them to evade the common drug resistance including the increased efflux through overexpression of efflux pumps, alternation of target sites, decreased cell permeability, and enzyme inactivation. Bacteria cannot undergo multiple gene mutations simultaneously to develop multiple resistance mechanisms (Fig. 9.2) (Dakal et al. 2016; Lee et al. 2019; Makabenta et al. 2021). The biomolecules or antibiotic-conjugated NPs use multiple pathways to kill bacteria and, therefore, do not induce the generation of bacterial resistance (Lee et al. 2019). Moreover, photocatalysis, photothermal effects, reactive oxygen species (ROS) generation, and ability to bind nucleic acids make NPs potent antimicrobial agents compared to commercially available antibiotics. Various compositions such as inorganic, polymeric, liposomes, self-assembled, and lipids are used to prepare NPs, which have inherent antimicrobial properties and/or are conjugated with antimicrobial agents to treat various infections (Rai et al. 2022). The ability of NPs to make spatial-temporal release of antibiotics or other biomolecules upon exposure of stimuli such as magnetic field, light, pH, temperature, and enzymes

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enables consistent therapeutic dosing over a longer period, thus reducing the systemic toxicity.

3

The Mechanisms of Antimicrobial Resistance

The development of antimicrobial resistance (AMR) in bacteria is continuously increasing due to random and inappropriate use of antibiotics. The prolonged exposure to antibiotics causes the development of AMR in bacteria. For example, after introducing penicillin in the market, bacteria developed resistance against it within a few years (Davies and Davies 2010). Nevertheless, most antibiotics induce the bacterial resistance despite having different modes of antimicrobial action. Bacteria have used multiple mechanisms to develop resistance against antibiotics (Gómez-Núñez et al. 2020; Makabenta et al. 2021). The search for effective antimicrobial drugs and materials is imperative for combating AMR bacteria. There are multiple pathways for the generation of AMR. (i) ABC Transporters Most antimicrobial agents inhibit intracellular metabolic pathways in bacteria or damage the bacterial cell membrane. The amount of antimicrobial agent accumulated within the bacteria cytoplasm decides the survival fate. However, the efflux pumps in the cell membrane control the amount of drugs/ biomolecules required for bacteria by easily removing unwanted amounts (Alcalde-Rico et al. 2016). The efflux pumps are considered the first life of bacterial defense against antibiotics. The efflux action plays an important role in MDR resistance mechanisms and interplays with other mechanisms such as enzyme degradation and membrane permeability regulation (Makabenta et al. 2021). The efflux pumps are a family of transporter proteins which are energy dependent. The first energy-dependent efflux pumps were reported in the 1970s followed by the discovery of p-glycoprotein pumps in mammalian cells (Gottesman and Ling 2006). Later, the efflux pump (Tet proteins) in Escherichia coli (E. coli) against tetracycline was discovered. Importantly, MDR pumps discovered in P. aeruginosa and E. coli play a crucial role in developing drug resistance (Li et al. 1994; Nikaido 1994). There are five families of efflux pumps in bacteria classified based on energy and structure source: (i) ATP-binding cassette (ABC), (ii) multidrug and toxic compound extrusion (MATE), (iii) resistance-nodulation-cell division (RND), (iv) major facilitator superfamily (MFS), and (v) small multidrug resistance (SMR) (Reygaert 2018). ABC transporters known as classical membrane proteins are found in bacteria and eukaryotic cells and control nutrient homeostasis, immunity, and modulate resistance to antibiotics. ABC transporters are responsible for MDR resistance in bacteria and cancer cells. Two transmembrane domains (TMDs) and two cytosolic nucleotide-binding domains (NBDs) are the main components of ABC transporters (Lei and Karim 2021).

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(ii) Alternation in Drug Targets Antimicrobial agents target several components in bacteria to kill them. However, bacteria modify these targets to enable resistance against antibiotics and other antimicrobial agents. Vancomycin inhibits the synthesis of the cell wall of Gram-negative bacteria. The thick LPS layer in Gram-negative bacteria prevents the action of antibiotics and acts as an intrinsic resistance to antibiotics (Cox and Wright 2013). Vancomycin-resistance enterococci (VRE) and MRSA bacteria have van genes, which promote the modification of peptidoglycan precursor structure, causing a decrease in binding avidity of vancomycin (Cox and Wright 2013). Similarly, the mutations in genes (mprF; responsible for calcium binding) modify the charge of the cell membrane to positive; therefore, daptomycin does not bind to the bacterial membrane (Stefani et al. 2015; Yang et al. 2009). Penicillin-binding proteins (PBPs) are transpeptidases responsible for the synthesis of peptidoglycan in the cell wall. The structural change of PBP2a in S. aureus by the acquisition of the mecA gene inhibits the binding of drugs (Reygaert 2018; Reygaert 2009; Beceiro et al. 2013). Additionally, the ribosomal mutation (involving erm genes) inhibits the action of aminoglycosides and oxazolidinones, while ribosomal subunit methylation prevents binding of aminoglycosides and macrolides (Roberts 2004; Kumar et al. 2013). Similarly, the modifications in DNA gyrase (gyrA) and topoisomerase IV (grlA) genes cause a change in their structures, which decreases the ability of fluoroquinolones to bind to these targets (Redgrave et al. 2014). Mutations in enzymes (dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR)) prevent the binding of sulfonamides and trimethoprim to the active sites of enzymes (Vedantam et al. 1998). (iii) Drug Inactivation Bacteria inactivate drugs by the replacement of a chemical group of drugs or by drug hydrolysis. For instance, the β-lactamase enzymes (penicillinases and cephalosporinases) degrade β-lactam drugs, while tetX genes in bacteria are responsible for the inactivation of tetracyclines (Kumar et al. 2013; Blair et al. 2015). β-Lactamases open β-lactam ring, which cause the inactivation of β-lactam antibiotics, leading to their inability to bind to the target PBP proteins. Acetyl, adenyl, and phosphoryl groups are used to modify the drugs using acetylation, phosphorylation, and adenylation mechanisms. Acetylation is a common mechanism to alter aminoglycosides, chloramphenicol, and streptogramins (Schwarz et al. 2004). (iv) Modification in the Membrane Permeability Bacteria modify their outer membrane compositions to prevent the permeability of antibiotics. The reduced expression of outer membrane proteins such as diffusion porins (Omps and OccDs) is associated with AMR. Moreover, a small variation in other outer membrane proteins can also lead to AMR in Gramnegative bacteria (Lei and Karim 2021; Vila et al. 2007).

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Classification of Infections

Bacteria cause intracellular or extracellular infections depending on their interactions with hosts. Infectious diseases are clinical disorders resulting from the presence of microorganisms. (i) Intracellular infections Some bacterial strains such as Rickettsia spp., Ehrlichia spp., Anaplasma spp., and Orientia spp. are obligate, while Listeria monocytogenes (L. monocytogenes), Salmonella spp., and Legionella pneumophila are facultative intracellular infective bacteria. Mycobacterium leprae and Yersinia pestis are intracellular pathogens and caused the highest number of deaths. Deaths due to Mycobacterium tuberculosis (Mtb) infections are second to HIV/AIDS. During the infection, the immune system can detect some bacteria and recruit macrophages to eliminate them. However, bacteria can alter the autophagy process to reside in macrophages and protect from the host’s immune response (Bah and Vergne 2017). Bacteria can invade different cells such as keratinocytes, intestinal epithelial cells, phagocytes, neutrophils, and lung endothelial cells using zipper and trigger mechanisms. Bacteria express specific proteins on cell membranes, which interact with receptors of cells, triggering signaling cascades to reorganize the actin cytoskeleton (Ham et al. 2011). The binding of bacterial cell proteins with cell receptors promotes host-pathogen interactions, which facilitates the internalization of bacteria by cells. In the case of the trigger mechanism, bacteria secrete specific molecules in the cytoplasm to polymerize the actin cytoskeleton, promoting the formation of pseudopods to engulf bacteria within cells (Veiga and Cossart 2006). Moreover, bacteria use type III and type IV secretion systems to enter phagocytes. The type III and type IV secretion systems secrete proteins to mediate the bacteria internalization in cells (Wang et al. 2016). Some bacteria inhibit the fusion of phagosomes with lysosomes to protect themselves. The bacteria escape from phagosomes to the cytosol of cells. For example, Mtb resides in macrophages despite their unfavorable internal condition. The host cells provide a suitable environment for bacteria to survive and replicate (Yeh et al. 2020). Reticuloendothelial system (RES) cells engulf the bacteria and however become reservoirs for bacteria, allowing the bacteria to cause secondary infections (Bhavsar et al. 2007). L. monocytogenes, Mtb, and S. aureus infect circulating macrophages and leukocytes and then infect other organs (Drevets et al. 2004). It is very difficult to treat intracellular infections as bacteria reside in host cells. The therapeutic concentrations or minimum inhibitory concentration (MIC) of antibiotics do not reach host cells. Antibiotics such as tetracyclines, quinolones, sulfonamides, and β-lactams are commonly used to treat intracellular infections. For example, β-lactam antibiotics enter cells through passive diffusion and block the peptidoglycan synthesis in the infected bacteria. On the other hand, tetracyclines use an active transport pathway to enter cells and prevent bacterial protein synthesis (Bongers et al. 2019).

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(ii) Extracellular Infections Several bacteria strains such as S. aureus, S. pyrogens, E. coli, P. aeruginosa, K. pneumoniae, etc. cause extracellular infections in the skin, lung, urinary tract, and bone. Bacteria secrete virulence proteins or molecules to evade the immune defense system and phagocytosis. These pathogens multiply at the extracellular sites of the host cells. Moreover, these pathogens invade injury sites and multiply there in the presence of a suitable fluid medium. The extracellular pathogens use initial intracellular residence in vivo, which lead to typical extracellular infections (Silva 2012).

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Effect of Nanoparticles on the Bacterial Resistance

Various compositions of NPs have been developed as potent antimicrobial agents and new life of defense against MDR bacteria due to their unique antimicrobial mechanisms. It is believed that NPs can act as excellent carriers to antibiotics to reduce systemic toxicity and simultaneously induce antimicrobial activities. Moreover, NPs can fill the advanced features, which antibiotics frequently fail. Antibiotics have limited membrane penetration ability, and, therefore, could not effectively eliminate intracellular pathogens in host cells (Andrade et al. 2013). The smaller size of NPs leads to greater interactions with the bacterial membranes due to a larger surface area to volume ratio. The multifunctional properties of NPs combat bacteria using multiple mechanisms (Fig. 9.3) (Lei and Karim 2021). Generally, NPs severely damage bacteria membrane, and it is difficult for bacteria to modify the compositions of the membrane through the genetic mutations to develop resistance against NPs. Biofilms are harbors of bacteria proliferation and infections. The complex structures of biofilms protect bacteria from antibiotics and antimicrobial agents. Biofilm contributes for the development of bacteria resistance and provides a breeding ground to the exchange of mutated genes among bacterial cells (Khameneh et al. 2016). Different compositions of NPs such as Au NPs (Yu et al. 2016), Ag NPs (Mohanta et al. 2020), ZnO NPs (Rosenberg et al. 2020), CuO NPs (Padmavathi et al. 2019), Fe3O4 NPs (Alavi and Karimi 2019), NO-releasing Si NPs (Slomberg et al. 2013), and MgO NPs (Hayat et al. 2018) eliminate bacterial biofilm and subsequently kill bacteria. Additionally, rod-shaped NPs are more effective to destroy biofilms than spherical NPs (Slomberg et al. 2013). NPs can be loaded with multiple drugs that can effectively eliminate bacteria, and therefore, it is difficult for bacteria to generate resistance against them. Moreover, the hybrid NPs have been synthesized as potent antimicrobial agents compared to individual counterparts (Haddar et al. 2021). The hydrophilic property of a few antibiotics reduces their penetration through the mammalian cell membrane. Furthermore, antibiotics are accumulated in acidic endolysosomal compartments where they can be degraded. On the other hand, NPs protect drugs from degradation and increase their level in serum with the maintenance of potency. For example, vancomycin kills Gram-positive bacteria; however, it also affects the kidney and ears. Vancomycin-loaded mesoporous Si NPs detect

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Fig. 9.3 Schematic illustration of the antimicrobial mechanisms of NPs. The figure is adopted from Ref. (Lei and Karim 2021)

and kill bacteria without inducing any cytotoxic effect to macrophages (Qi et al. 2013). The surface of NPs can be modified with suitable ligands or antibodies to deliver at the target infection site to minimize systemic side effects. Active targeting mechanisms can be used to deliver drug-loaded NPs through the surface modification of NPs with suitable ligands or antibodies, which specifically recognize the receptors of cells. The active targeting approaches deliver a higher amount of drug at the infection site, thereby overcoming the bacterial resistance. In the active targeting approach, specifically, macrophages are targeted using antibiotic-loaded nanogel as bacteria are present inside macrophages at the infection site (Xiong et al. 2012). Intracellular microorganism such as Mtb resides in macrophages. NPs can be delivered to the infected cells through passive targeting to enhance the therapeutic efficacy of antibiotics or drugs in the intracellular environment. However, the passive targeting approach is unsuitable in treating intracellular infections as drugloaded NPs can be nonspecifically delivered to other sites and mostly cleared by RES.

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Can Nanoparticles Cause Bacterial Resistance?

It is believed that bacteria cannot generate resistance against NPs due to their multiple antimicrobial mechanisms to attack various targets in bacteria (NiñoMartínez et al. 2019). However, a few reports suggest that bacteria have developed some degree of resistance against metallic NPs through biofilm adaptations, mutations (Graves et al. 2015), formation of protective extracellular matrixes (Zhang et al. 2018a), activation of ion efflux pumps (Yang et al. 2012), and electrostatic repulsions. The detection of NP resistance is found in bacteria when they are repeatedly exposed to a lower amount of NPs in vitro conditions. Moreover, the usage of the very small size of NPs, which have a high surface area to volume ratio, can act potently to kill bacteria, thereby minimizing the possibility of the development of NP resistance (Agnihotri et al. 2014).

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Designing Nanoparticles as Antimicrobial Agents

7.1

Inorganic and Metallic Nanoparticles

Ag has been used as an antimicrobial agent for the treatment of infections in burns and wounds as well as water potable as early as 4000 BCE. Moreover, the antimicrobial activity of Ag is enhanced when bulk Ag is converted to nanometric size recently. Ag NPs cause coloration on the skin, mucosal membrane, and conjunctiva, which is their main drawback. Ag NPs have potential antimicrobial activity against a wide range of Gram-positive and Gram-negative bacteria. Ag NPs bind to cell membrane and alter permeability of E. coli and P. aeruginosa (Ramalingam et al. 2016). Ag NPs also form hole-like structures in E. coli as confirmed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) (Sondi and Salopek-Sondi 2004). The size and shape of Ag NPs determine the antimicrobial activity as the minimum inhibitory concentration (MIC) of Ag NPs below 25 nm size is 6.75–54 μg/mL while the above 25 nm size is 1.7–13.5 μg/mL against MRSA and S. epidermidis and vancomycin-resistant E. faecium and K. pneumoniae (Franci et al. 2015). The MIC of Ag NPs varies from 40 to 180 μg/mL with different strains of E. coli such as MTCC 443, MTCC 739, MTCC 1302, and MTCC 1687 (Ruparelia et al. 2008). Ag NPs can kill clinically isolated S. mutans, which cause dental caries (Pérez-Díaz et al. 2015). Moreover, Ag NPs exhibit potent antimicrobial activity against erythromycin-resistant S. pyogenes, MDR-P. aeruginosa, and ampicillin-resistant E. coli O157:H7 with MIC of 83.3 mM (Lara et al. 2010). CuO NPs have been synthesized using reverse micelles, thiol-induced reduction, laser irradiation, and biological sources (Rudramurthy et al. 2016). CuO NPs exhibit potent antimicrobial activity against Gram-positive S. aureus and Bacillus subtilis (B. subtilis) and Gram-negative E. coli bacteria (Ruparelia et al. 2008; Chatterjee et al. 2012). CuO NPs interacted strongly with –NH and –COOH groups of the cell membrane of B. subtilis and increased its permeability. The antimicrobial activity could be due to the adhesion of NPs on cell membrane and generation of ROS by

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Cu2+, leading to oxidative stress, and DNA and membrane damage (Pramanik et al. 2012). CuO NPs synthesized using Gloriosa superba plant extract inhibit the growth of E. coli, S. aureus, K. aerogenes, and P. desmolyticum. ZnO is considered generally recognized as safe (GRAS) by the FDA. Microemulsion, emulsion, precipitation, and mechanochemical approaches have been used to synthesize ZnO NPs. ZnO NPs exhibit antimicrobial activity against E. coli, L. monocytogenes, Salmonella, and S. aureus bacteria (Tayel et al. 2011; Liu et al. 2009). The MICs of 3.4 mM and 1 mM of ZnO NPs are able to kill E. coli and S. aureus (Reddy et al. 2007). ZnO NPs synthesized using the seed extract of Butea monosperma decreased the virulence factors such as protease, hemolysin, and pyocyanin of MDR P. aeruginosa at sub-MIC. ZnO NPs accumulated at high concentration inside P. aeruginosa (Ali et al. 2020). Similarly, ZnO NPs synthesized using Aristolochia indica against MDR bacteria isolated from diabetic foot ulcers (Steffy et al. 2018). ZnO NPs generate ROS and elevate membrane lipid peroxidation, leading to leakage of the cellular content of carbapenem-resistant A. baumannii (Tiwari et al. 2018). Titanium dioxide (TiO2) is widely used in pharmaceuticals, food colorants, toothpaste, and cosmetics. Moreover, TiO2 NPs exhibit antimicrobial activity by the generation of ROS. The photocatalytic activity of TiO2 is explored to eliminate biofilms and bacteria (Wu et al. 2010). The bactericidal effect of TiO2 is attributed to the production of free radicals upon light exposure, which affects the lipopolysaccharides (LPS), phospholipids, and peptidoglycan of the bacterial membrane. Wu et al. showed that TiO2 upon UV light exposure changed the morphology of E. coli from rod shape to elliptical and spheroplast, indicating the destructions of the outer membrane. However, such changes were not found when TiO2 or UV light alone was exposed to E. coli bacteria. TiO2 is doped with Ag2O and N2 to enhance the antimicrobial potential (Fig. 9.4) (Wu et al. 2010). Harun et al. prepared a hybrid TiO2/ZnO NP to demonstrate antimicrobial activity against MDR E. coli, S. aureus, and K. pneumoniae associated with nosocomial infections (Harun et al. 2020).

7.2

Antibiotic-Conjugated Nanoparticles

Au NPs have been used for various biomedical applications due to their unique physicochemical properties. For example, Verigene containing Au NPs is used for the detection of Gram-positive bacteria in the blood in vitro condition (Rai and Ferreira 2021). Verigene is approved by the FDA in 2012. Various shapes such as nanosphere, nanorod, nanocubes, nanotriangles, and nanocages are prepared with varying optical, catalytic, and photothermal and biological properties (Rai and Ferreira 2021). The bare Au NPs do not have antimicrobial activity, and therefore various antibiotics have been conjugated on their surfaces to prepare potent antimicrobial agents. Xu et al. designed vancomycin-conjugated AuNPs using [bis (vancomycin)cystamide] for the treatment of vancomycin-resistant Enterococci (VRE) (Gu et al. 2003a). Vancomycin was conjugated on Au NPs through Au-thiol interactions. 61 vancomycin molecules were loaded on 4 nm Au NPs.

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Fig. 9.4 TEM images of (a) untreated; (b) well-treated E. coli cells. Treatment is visible-light illumination upon Ag+/TiON powder for 2 h—dark granules are observed and cells have damaged cell membrane. The figure is adopted from Ref. (Wu et al. 2010)

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Importantly, vancomycin@Au NPs kill bacteria with MIC of 2–4 mg/mL, while vancomycin has MIC of 64 mg/mL. Vancomycin conjugated on biogenic Au NPs through electrostatic interactions kills vancomycin-resistant S. aureus; however, NPs are unstable in solution for a longer period (Mohammed Fayaz et al. 2011). Vancomycin is conjugated on magnetic NPs (Lin et al. 2005; Gu et al. 2003b), silica NPs (Qi et al. 2013), Fe3O4@Au nanoeggs (Huang et al. 2009), and Ag NPs (Sun et al. 2013) to prepare antimicrobial agents to kill pathogenic bacteria. In another work, aminoglycoside antibiotics-loaded Au NPs had potent activity against E. coli, while the bare antibiotics did not kill bacteria effectively (Nirmala Grace and Pandian 2007). We have developed cefaclor-reduced Au NPs with a potent antimicrobial activity against E. coli and S. aureus (Rai et al. 2010). Cefaclor@Au NPs attacked the cell membrane of both bacteria and caused the leakage of cell content. Atomic force microscopy (AFM) analysis indicated that bacterial cell membrane was severely damaged. Cefaclor@Au NPs led to the formation of holes in the bacterial membrane (Fig. 9.5). Several compositions of NPs and antibiotics have been used in combination to increase their antimicrobial potency compared to individual components. The synergistic actions of antibiotics (polymyxin B, ampicillin, vancomycin, erythromycin, ciprofloxacin, and ceftazidime) and Au, Ag, and ZnO NPs have been evaluated against E. coli, E. faecium, A. baumannii, P. aeruginosa, S. aureus, antibiotic resistance-A. baumannii, and VRE bacteria through the damage of bacterial membrane and interference in molecular pathways. Moreover, Fayaz et al. developed ampicillin-conjugated Ag NPs, which showed improved antimicrobial activity against several bacteria due to the polyvalent effect of ampicillin and Ag NPs on the bacterial membrane (Fayaz et al. 2010). Hydroxyl and amido groups of ampicillin chelate with Ag to form ampicillin@Ag NPs. Ampicillin binds to bacterial membrane, facilitating the better penetration of Ag NPs, which bind with DNA. The synergistic action requires a lesser amount of antibiotic-conjugated NPs compared to their counterparts (Hemeg 2017). ZnO NPs conjugated with clinically approved drugs (ceftriaxone, ampicillin, quercetin, naringin, and amphotericin B) had potent antimicrobial activity against MRSA, S. pneumoniae, and S. pyogenes by generating ROS and affecting bacterial cell integrity (Akbar et al. 2021). TiO2 NPs in combination with antibiotics such as penicillin, aminoglycosides, cephalosporins, glycopeptides, macrolides, lincosamides, and sulfonamides are effective against MRSA compared to antibiotics alone. In another work, TiO2 NPs combined with cefepime showed the potent killing of clinically isolated MDR P. aeruginosa. The authors did not study the effect of UV light on the antimicrobial potency of antibiotic-loaded TiO2 NPs (Ahmed et al. 2020). Interestingly, polyol-coated CuO, ZnO, and CuZn NPs act as efflux pump inhibitors and have the potent synergistic antimicrobial activity in combination with antibiotics meropenem and ciprofloxacin against MDR P. aeruginosa (Eleftheriadou et al. 2021). NPs at sub-MIC increase the intracellular accumulation of ethidium bromide (EtBr) as demonstrated through the EtBr cartwheel method. There was a little accumulation of EtBr in bacteria in the absence of NPs. These results indicate

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Fig. 9.5 Representative height mode AFM images of E. coli treated with cefaclor-reduced Au NPs (a), E. coli treated with cefaclor (b), E. coli treated with NaBH4-reduced Au NPs (c), and plain E. coli without the treatment (d). The corresponding phase mode AFM images (e–h) and crosssectional analysis (i–l) are shown. The figure is adopted from Ref. (Rai et al. 2010)

that the hybrid NPs with a high amount of organic content might act as an efflux pump inhibitor (Eleftheriadou et al. 2021). Notably, the antimicrobial potential of these antibiotic-conjugated NPs have been evaluated in vitro models; none of them has been tested in an animal model to validate their clinical efficacies. In one report, the bioaccumulation of gentamycinloaded Au NPs was studied in intramuscular infections of S. aureus in the thigh of mice after intravenous administration (Ahangari et al. 2013). It was shown that a large percentage of NPs was accumulated in the kidneys and blood with a small amount in the infected thigh but more than a noninfected thigh within 60-min postadministration. However, the antimicrobial efficacy of gentamycin-loaded Au NPs was not studied in the animal models (Ahangari et al. 2013).

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Small Molecule-Conjugated Nanoparticles

Small cationic molecules have been used as alternatives to antibiotics to eradicate bacteria. Jiang and coworkers have developed a library of 3-nm Au NPs conjugated with amino-substituted mercaptopyridine to evaluate antimicrobial potential against MDR clinical isolate bacteria (Zhao et al. 2010). From this library, 4,6-diamino-2pyrimidinethiol-conjugated Au NPs (DAPT-Au NPs, positively charged) showed the best antimicrobial efficiency against bacteria compared to other pyrimidineconjugated Au NPs. Authors proposed that DAPT-Au NPs strongly bind to bacteria membranes, leading to leakage of nucleic acid. DAPT-Au NPs compromise ATPase activity and disturb membrane potential, leading to bacteria death. Moreover, the proteomic study indicated that DAPT-Au NPs could bind with DNA and inhibit replication and protein synthesis. Notably, DAPT-Au NPs induce bacterial resistance slowly compared with gentamicin with resistance increasing after 50 passages for NPs. On contrary, bacteria developed tenfold resistance against gentamicin after ten passages. Negatively charged hydroxyl-substituted mercaptopyridine-conjugated Au NPs showed no antimicrobial activity against MDR bacteria. DAPT-Au NPs do not show cytotoxicity behavior to human umbilical vein endothelial cells (HUVECs) (Zhao et al. 2010). DAPT-Au NPs embedded in bacterial cellulose exhibited antimicrobial activity against E. coli and P. aeruginosa in wounds and promoted rapid wound healing (Fig. 9.6) (Li et al. 2017). DAPT-Au NP-coated orthodontic device inhibited the growth of Porphyromonas gingivalis (P. gingivalis) planktonic cells and biofilms (Zhang et al. 2020). Intermediary antibiotic components are unstable in solution; however, once conjugated on NPs, they show long-term stability. 7-Aminocephalosporanic acid (7-ACA), 6-aminopenicillanic acid (6-APA), and 7-aminodesacetoxycephalosporanic acid (7-ADCA) capped Au NPs electrospun with poly(ε-caprolactone) (PCL) and gelatin to produce wound dressings are capable of fighting MDR infections in wounds with enhanced skin regeneration in comparison with Ag mat or PCL/gelatin fibers (Yang et al. 2017). Rotello’s group developed a library of cationic Au NPs using molecules containing varying chain lengths with nonaromatic and aromatic features (Li et al. 2014). The structure-activity relationship showed that varying the hydrophobicity of ligands could modulate the antimicrobial activity of NPs. It is found that NPs having cationic and hydrophobic properties effectively kill 11 clinical MDR isolates at MICs ranging from 8 to 64 nm, by damaging the bacterial membrane and leaking cytoplasmic contents. Moreover, bacteria did not develop resistance against NPs after 20 passages at sub-MIC of NPs. The authors suggested that these NPs combined with antibiotics could be used to treatment of MDR bacterial infections. The synergistic action of sub-MIC of NPs and antibiotics requires 8- to 16-fold fewer antibiotics against MDR bacteria and MRSA (Gupta et al. 2017). The potent antimicrobial activity is due to the action of NPs as efflux pump inhibitors to increase the concentration of antibiotics inside bacteria. Proteomic analysis indicates the deregulation of efflux pump proteins of the outer membrane of bacteria. Importantly,

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NPs combined with antibiotics were biocompatible to mammalian cells (Gupta et al. 2017). The selectivity of NPs against Gram-positive or Gram-negative bacteria can be achieved by tailoring their surface chemistry with mixed charged ligands. NPs modified with 80:20 and 48:52 ratios of N,N,N-trimethyl(11-mercaptoundecyl) ammonium chloride (TMA) and 11-mercaptoundecanoic acid (MUA) selectively kill Gram-negative and Gram-positive bacteria, respectively (Pillai et al. 2016). Cationic ligand-coated NPs interact with bacteria membrane via electrostatic interactions, while anionic ligands with carboxyl functional groups interact through H-bonding interactions with the membrane, leading to disruption of the membrane

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integrity. In another work, NPs functionalized with a ligand having a positive charge in the outermost layer exhibited potent antimicrobial activity compared with NPs functionalized with the positively charged group inside the ligand termini (Huo et al. 2016). The surface functionalization of NPs with a mixture of ligands significantly reduces the MIC compared with NPs functionalized with individual ligand (Bresee et al. 2014). For example, 2-nm Au NPs functionalized with p-mercaptobenzoic acid (pMBA-Au), 3-mercaptopropylsulfonate, and 2-mercaptoethylamine completely kill E. coli with the MIC of 0.5 μM, while 2-mercaptoethylamine-Au NPs require 2 mM and 3-mercaptopropylsulfonate, and p-mercaptobenzoic acid-Au NPs shows no antimicrobial activity (Bresee et al. 2014).

7.4

Antimicrobial Peptide-Conjugated Nanoparticles

AMPs are considered alternatives to commercially available antibiotics due to growing concerns of increasing cases of MDR bacterial infections (Rai et al. 2022). So far, more than 5890 AMPs have been isolated from bacteria, fungi, insects, plants, and vertebrates or chemically synthesized according to the data repository of AMPs (DRAMP). AMPs have a short sequence of amino acids (5–50 residues) and are positively charged with amphipathic and hydrophobic properties. AMPs have different secondary structures such as α-helical, β-sheet, and extended random coil. AMPs strongly interact with negatively charged bacterial membrane, leading to damage of membrane and leakage of cellular content. Importantly, AMPs are more selective to damage the bacterial membrane than the zwitterionic or neutral mammalian membrane. A few AMPs are negatively charged; however, their antimicrobial mechanisms have been poorly described. Apart from antimicrobial properties, AMPs exhibit other biological activities such as skin regeneration, bone regeneration, immunomodulation, and anticancer properties, attracting them as potential drugs (Rai et al. 2022). Currently, numerous AMPs are under different phases of clinical trials for the treatment of venous leg ulcers, diabetic foot ulcers, skin infections, and oral mucositis. Surprisingly, no AMPs have reached to market despite being extensive research have been done in this area due to their instability in serum and poor therapeutic efficacy. AMP-capped NPs display potent antimicrobial and other biological activities along with prolonged stability and robust therapeutic efficacy in animal models (Rai et al. 2022). Recently, we have developed a one-pot synthesis approach preparing small, stable cecropin-melittin (CM)-conjugated Au NPs with potent antimicrobial activity against Gram-positive and Gram-negative bacteria and MDR bacteria in the presence of human serum and nonphysiological concentrations of proteases (Rai et al. 2016). Importantly, CM-conjugated Au NPs displayed an excellent antimicrobial activity and biocompatibility compared with soluble CM peptides. An animal study demonstrated that CM-conjugated Au NPs killed P. aeruginosa and MRSA bacteria in the wound infection model (Fig. 9.7) (Rai et al. 2016). Moreover, CM-conjugated Au NPs were able to reduce the bacterial population burden in the sepsis animal model (Fig. 9.7). Similarly, Comune et al. synthesized LL37 conjugated-Au NPs

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Fig. 9.7 (a) Antimicrobial activity of CM-SH-Au NPs in a murine chronic wound model. Mice were wounded and infected with 5–8 × 103 CFU PAO1 and 1–3 × 104 MRSA. Animals were euthanized on the following day. Wound tissue was harvested, homogenized, and plated for CFU quantification. Results are ean ± SEM (n . 6e9). *P < 0.05, **P < 0.01, indicates statistical significance between experimental groups. (b) Antimicrobial activity of CM-SH-Au NPs in a sepsis animal model. (B.1) Antimicrobial activity of Au NPs, CM-SH-Au NPs (in both cases, 1 mg of NPs resuspended in 200 mL of PBS) and CM-SH peptide (300 mg solubilized in 200 mL of PBS) injected in CLP mouse model. Results are mean ± SEM (n . 6e18). ***P < 0.001 indicates statistical significance between experimental group and control (PBS). (b.2, b.3, and b.4) Pro-inflammatory (IL-1b, TNFa) and anti-inflammatory (IL-10) cytokine measurement from blood after 24 h of NP injection in CLP mouse model. In b.2, b.3, and b.4, results are mean ± SEM (x . 5e11). **P < 0.01 and ***P < 0.001 indicate statistical significance between experimental groups. (c.1) Au content in different organs of CLP mice after 24 h of Au NPs or CM-SH-Au NP administration (in both cases, 1 mg of NPs resuspended in 200 mL of PBS), as quantified by ICP-MS. An inset of Fig. c.1 shows the amount of Au present in blood of CLP mice after 24 h of administration of NPs. (c.2 and c.3) Au content in different organs of CLP mice, after 24 h, administered by a single (1 dose, 1 mg per animal) (c.2) or multiple doses (3 doses, 3 mg per animal) (c.3) of CM-SH-Au NPs, as quantified by ICP-MS. (c.4) Au content in different organs of non-CLP mice, after 24 h, administered by multiple doses (3 doses) of CM-SH-Au NPs, as quantified by ICP-MS. In c.1, c.2, c.3, and c.4, results are mean ± SEM (n = 2–3). The figure is adopted from Ref. (Rai et al. 2016)

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(LL37-Au NPs) and demonstrated antimicrobial activity and higher skin regeneration as compared to the soluble LL37 peptides in vitro and in vivo models (Comune et al. 2017). LL37-Au NPs killed several strains of bacteria in human serum. Barun et al. prepared LL37-loaded mesoporous Si NPs (LL37-MSNs), which had potent antimicrobial activity against MDR E. coli in the presence of proteases due to preferential localization of LL37 in the pores of MSNs (Braun et al. 2016). Similarly, the modified human defensin peptides (T7E21R-HD) loaded in anionic MSNs are active against MDR E. coli and MRSA by the permeabilization of outer and inner membranes of bacteria (Zhao et al. 2019). T7E21R-HD peptides were aggregated on the surface of MSNs, increasing the local surface charge and hydrophobicity, which favored the severe rapture of the membrane of MDR E. coli bacteria. Several other AMPs such as indolicidin (Rahimi et al. 2019), VG16KRKP (Chowdhury et al. 2017), pediocin-AcH (Singh et al. 2018), Esc (1–21) (Casciaro et al. 2017), and polymyxin B (Singh et al. 2017) are used to prepare AMP-conjugated Au NPs to eradicate MRSA and MDR bacteria. It was found that Esc (1–21)-Au NPs killed motile and sessile forms of P. aeruginosa by damaging its membrane. TEM analysis indicated that NPs were mostly accumulated near the bacterial membrane (Casciaro et al. 2017). In another case, Odorranain-A1-conjugated Ag NPs have potent antimicrobial activity (MIC 5–15 μM) against MDR bacteria than the mixer of Odorranain-A1 peptides and Ag NPs (Pal et al. 2019). Chen et al. prepared surfactin (SFT) and 1-dodecanthiol-coated Au nanodots (Au NDs). SFT-Au NDs showed antimicrobial activity against MDR bacteria with lower cytotoxicity and hemolysis compared to free SFT peptides (Chen et al. 2015). Importantly, SFT-Au NDs promoted wound healing, fast epithelialization, and collagen deposition along with the killing of MRSA bacteria in the infected wound animal model (Chen et al. 2015). The photothermal therapy (PTT) approach has gained tremendous attention lately to eliminate bacteria. Thermal energy generated by NPs upon near-infrared light compromises the integrity of the bacterial membrane. However, the PTT approach is not so effective in completely eradicating bacterial infections. It is proposed that PTT in combination with AMPs could be lethal to bacteria. Using a similar concept, BF2b AMP-conjugated Au nanorods (Au NRs) killed MRSA due to the combined effect of AMP and photothermal energy (Chen et al. 2020). The lethal killing is attributed to the presence of a high density of AMP on the surface of NRs, which favors stronger interactions with bacterial membrane. Importantly, this approach killed MRSA in the in vivo infection model with an efficacy of 100 times more than the soluble BF2b AMP (Fig. 9.8) (Chen et al. 2020).

7.5

Antimicrobial Peptide-Conjugated Organic Nanoparticles

Due to the non-degradability of metallic and inorganic NPs in vivo models and the human body, it is difficult to get approval from the FDA as drugs or drug carriers. Organic or polymeric NPs are attractive candidates, which degrade in physiological conditions without inducing any significant cytotoxicity. Several compositions of

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organic NPs have been developed, which are used for the delivery of AMPs. AMPs, GIBIM-P5S9K (G17) and GAMO019 (G19) loaded in poly(D L-lactide-coglycolide) PLGA NPs, have antimicrobial efficiency of 90.5% against MRSA and E. coli O157:H7 (Gómez-Sequeda et al. 2020). The MIC of AMPs decreased significantly when encapsulated in PLGA NPs from 12.5 to 3.13 μM (G19NP) for E. coli O157:H7 and 0.7 μM against MRSA. Similarly, the MIC of G17NP decreased from 1.5 to 0.2 μM against MRSA. Yang et al. explored the photodynamic antimicrobial chemotherapy (PACT) approach to eradicate MRSA and P. aeruginosa WLBU2 (AMP) and temoporfin (as a photosensitizer) encapsulated in liposomes kill both bacteria upon 665-nm laser exposure. The formulation reduced the population of P. aeruginosa by three logs upon laser exposure compared to the dark condition (Fig. 9.9) (Yang et al. 2011). Interestingly, liposomes containing oleic acid, vancomycin, and AMPs (EKKRLLKWWR and KWWKLLRKKR) potently killed MRSA and S. aureus at pH 6 due to the rapid release of vancomycin. AMPs and oleic acid help to permeate the bacterial membrane, and vancomycin disrupts the biosynthesis of the cell wall. The liposomal formulation killed intracellular MRSA in human embryonic kidney cells without causing any cytotoxicity (Faya et al. 2020). Yang’s group modified azithromycinloaded liposomes with AMP DP7 to decrease its cytotoxicity and increase the antimicrobial activity against MRSA. The formulation controls the secretion of pro-inflammatory cytokines and chemokines to minimize inflammation (Zhang et al. 2018b).

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Fig. 9.9 (a) Scheme of the conjugation of WLBU2 with liposomes incorporating NHS PEG2000DSPE, including the structure of NHS-PEG2000-DSPE. (b) Dark toxicity and PACT efficiency of free temoporfin or temoporfin-loaded liposomes on P. aeruginosa DSM1117 after incubation at RT for 90 min or 180 min, respectively. The bars representing dark toxicity results are shaded gray (n = 2–3). The figure is adopted from Ref. (Yang et al. 2011)

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Mechanisms of Antimicrobial Activity of Nanoparticles

The physicochemical properties of NPs such as surface charge, hydrophobicity and surface functionality, and the membrane composition of bacteria determine the antimicrobial activity of NPs. The positively charged NPs interact strongly with negatively charged bacteria membrane through electrostatic interactions and

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severally damage the bacterial membrane. For example, positively charged CTABcoated Au nanorods (NRs) and NPs are found throughout the cell membrane of B. cereus. Mannose-encapsulated Au NPs selectively bind to mannose adhesion FimH of bacterial type-1 pilli as demonstrated by the TEM (Lin et al. 2002). Cationic Au NPs (2 nm) are able to lyse Gram-positive B. subtilis but do not kill Gramnegative E. coli (Hayden et al. 2012). On the other hand, 6-nm cationic Au NPs are nontoxic and aggregate on the surface of bacteria. These results defy the notion that all cationic NPs can damage the bacterial membrane (Hayden et al. 2012). Other interactions such as hydrophobic, van der Walls forces, and receptorligands facilitate the binding of NPs to the bacterial membrane (Wang et al. 2017). NPs can pass through the bacterial membrane and alter the activity of intracellular enzymes, ribosomes, and DNA, thereby disturbing the metabolic activity of bacteria, which lead to bacteria death (Rai et al. 2022; Wang et al. 2017). NPs also generate ROS and cause oxidative stress to kill bacteria. Ag NPs interact with bacteria membrane, and silver ions released from Ag NPs passed through the bacterial membrane using an electron transport chain and then bind to DNA to disrupt its activity (Dakal et al. 2016; Mohanta et al. 2020). TiO2, ZnO, and CuO NPs kill different strains of bacteria through the generation of ROS(Wang et al. 2017). Different ROS such as superoxide radical (O2–), hydrogen peroxide (H2O2), singlet oxygen (O2), and hydroxyl radical (.OH) are produced by various compositions of NPs, which produce different levels of dynamics and antimicrobial activity.. OH and O2 induce acute antimicrobial effects, while O2– and H2O2 cause mild activity. CaO and MgO NPs produce O2-, whereas ZnO NPs generate OH and H2O2. ZnO NPs change the spiral shape of Campylobacter jejuni to a spherical shape, causing cell damage and death. ZnO NPs split H2O into H+ and then react with dissolved O2 to generate H2O2, which penetrate bacterial membrane to kill them. Oxygen vacancies present on the surface of ZnO NPs also produce H2O2. Metal ions leached from metal NPs such as silver ions from Ag NPs interact with functional groups of proteins and nucleic acids such as amine (–NH), carboxyl (– COOH), and thiol (–SH), affecting metabolic activity, enzyme activity, respiratory chain, and cell membrane structures, which cause killing of bacteria (Dakal et al. 2016; Lemire et al. 2013). Ag NPs also have antimicrobial activity due to generation of ROS and strong interactions with the cell membrane of bacteria.

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Conclusions and Prospect

The continuous increase of patients infected with MDR and nosocomial bacteria significantly affected the socioeconomic burden globally, especially in developing countries. Although advanced detection tools and drugs are available readily, the emergence of MDR and XDR bacteria has inspired researchers to develop innovative formulations to combat them effectively without inducing bacterial resistance. Therefore, various compositions of organic and inorganic nano formulations have been developed, which are loaded with several antimicrobial agents such as antibiotics, small cationic molecules, and AMPs. Importantly, the formulations

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having target specificity and spatial-temporal release of cargos have been developed to treat infections without inducing adverse side effects in different organs. In the future, research will be directed toward understanding the molecular mechanisms and pathways of antimicrobial resistance in bacteria followed by the design of suitable drugs and nanocarriers to combat infections in various organs. The clinical application of NP-based formulations would be another challenge; NP-based formulations have failed in preclinical and clinical trials. The commercial translation of NP-based formulations has not been achieved so far. Moreover, the stringent protocol to validate in vitro and in vivo efficacies and large-scale production is required to facilitate the transition of NP-based formulation from the lab to the clinic. Acknowledgments SS and AR would like to thank the postdoctoral fellowship ((IT057-18-7254) and investigator grant (IF/00539/2015) from Portuguese Science and Foundation, respectively. AR would also like to thank the financial support of LightImplant project (PTDC/CTM-CTM/1719/ 2021).

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Quorum Sensing-Mediated Targeted Delivery of Antibiotics

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Mohmmad Younus Wani, Manzoor Ahmad Malik, and Irfan A. Rather

Abstract

Quorum sensing (QS) is an innate chemical mechanism many bacteria use to initiate group behavior. Among clinically relevant bacteria, it forms biofilms or produces virulence factors essential for establishing infection. Since bacteria produce small molecules that regulate quorum sensing, it is possible to develop chemical strategies to interfere with these signals and attenuate QS. Numerous QS inhibitors have also been developed to block different quorum sensing systems. Regardless of their potency, it is essential to note that QS inhibitors do not affect bacterial viability. Therefore, another strategy to kill the bacteria needs to be concomitantly utilized. An effective strategy could be the conjugation of the QS inhibitors with antibiotics to selectively deliver the antibiotics and at the same time disrupt the bacterial communication with minimal resistance. Keywords

Quorum sensing · Antibiotics · Targeted delivery · Drug resistance

M. Y. Wani (✉) Department of Chemistry, College of Science, University of Jeddah, Jeddah, Saudi Arabia e-mail: [email protected] M. A. Malik Department of Chemistry, Jamia Millia Islamia (Central University), New Delhi, India I. A. Rather Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. Y. Wani, A. Ahmad (eds.), Non-traditional Approaches to Combat Antimicrobial Drug Resistance, https://doi.org/10.1007/978-981-19-9167-7_10

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Introduction

Across the globe, antibiotic resistance is a growing problem. Compared to infections caused by nonresistant strains of the same bacteria, infections caused by drugresistant bacteria have worse clinical outcomes, death, and economic burdens (Frieri et al. 2017). According to a CDC report (2022 special), over 29,400 individuals died in the first half of the COVID-19 pandemic as a result of its impact on antibiotic resistance in the United States. The infection was acquired by nearly 40% of these individuals in the hospital. Therefore, the threat of antibiotic resistance is becoming more significant than the ability to control it. Besides, the pipeline of new antibiotics needed to stem the antimicrobial resistance (AMR) tide is dwindling. In the USA alone, antibiotic-resistant bacteria cause 2 million infections and 23,000 deaths annually (Ventola 2015). Since the discovery of penicillin in the 1940s, most of the microbial infections were effectively treated. In addition, the antibacterial effect of most antibiotics leads to the promotion and rapid spread of antimicrobial resistance among pathogens and non-pathogen microbes. In the current era, most pathogenic bacteria can develop resistance against most antibiotics currently in use. Of note, Klebsiella pneumoniae, Enterococcus faecium, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacter species, known as the ESKAPE pathogens, have developed resistance against all classes of antibiotics (Boucher et al. 2009; Dodds 2017; Rolain et al. 2016; Tommasi et al. 2015). Therefore, these bacteria often cause life-threatening infections in critically ill and immunocompromised people (Chellat et al. 2016; Khameneh et al. 2016). Drugresistant strains of common bacterial infections are estimated to kill nearly 700,000 people annually, and by 2050, the number will surpass ten million (Fig. 10.1) (Review on AMR 2014). Every year, nearly 60,000 newborns die from antibioticresistant infections in India (Laxminarayan et al. 2013). It is even becoming more difficult to treat infections with antibiotics of last resort, such as vancomycin, due to the development of bacteria resistant to the antibiotic, such as vancomycin-resistant S. aureus (VRSA). We, therefore, are in a critical situation where finding new antibiotics is inevitable. Moreover, it is imperative to develop new treatment strategies that can improve the efficacy of currently available antibiotics while limiting the spread of drug resistance. Infections caused by microbes can be treated with the help of a quorum sensing (QS) system that uses small signaling molecules to enable cell-to-cell communication (LaSarre and Federle 2013). The activation of this system has been demonstrated to regulate the expression of genes involved in pathogenic processes, such as the production of toxins or virulence factors, the formation of biofilms, and the invasion of host cells by many clinically relevant bacteria (Rutherford and Bassler 2012). Consequently, quorum sensing-modulating molecules have been identified as potential anti-virulence agents. The phenomenon has been observed typically in Gram-positive bacteria involving auto-inducing peptides (autoinducers) and small molecules like γ-butyrolactones—likewise, cyclic dipeptides, N-acylhomoserine lactones (AHLs), and quinolones in Gram-negative bacteria (LaSarre

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AMR in 2050

10 million Tetanus 60,000

Road traffic accidents

Cancer

1.2 million

8.2 million AMR now 700,000 (low estimate)

Measles

Cholera

130,000

100,000120,000

Diarrhoeal disease

Diabetes

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Fig. 10.1 Estimated deaths due to AMR and predicted deaths in 2050. Adapted from Refs. (Review on AMR 2014; Laxminarayan et al. 2013)

and Federle 2013; Rutherford and Bassler 2012; González and Keshavan 2006). The small molecules, once secreted, diffuse away from the cell and attach to specific receptors on the cell surface or inside the cell to interact with the same or different bacterial cell. Transduction occurs once a sufficient signal is detected, which triggers gene activation that controls numerous survival functions, such as toxic production, resistance to hosting defense mechanisms, etc. It has been sought to identify and develop chemical compounds and enzymes that inhibit QS by targeting signaling molecules to prevent and disrupt cell-to-cell communication (Bhardwaj et al. 2013; Kalia and Purohit 2011; Kalia 2013). These anti-QS molecules if conjugated to another toxic molecule, for example, an antibiotic, could be delivered to the target site, therefore imparting them target and site specificity, minimizing off-target toxicity and minimal chances of resistance.

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Bacterial Resistance to Antibiotics

In the case of bacteria, fungi, viruses, and parasites, antimicrobial resistance (AMR) can develop if they are overexposed to antimicrobial drugs such as antibiotics, antifungals, and antivirals. As a result, bacteria develop mutations or resistant mechanisms to combat the drug (Zaman et al. 2017; Brown and Wright 2016). Developing and underdeveloped nations are experiencing an increase in AMR incidents. Almost 490,000 people developed multidrug-resistant TB in 2016, complicating efforts to fight HIV, cancer, and other deadly diseases (Kumar et al. 2013; WHO 2018). Antibiotic resistance has developed faster than new antibiotics have been produced in recent decades, making it harder to treat bacterial infections. “Superbugs” could cause a public health crisis if they become as virulent as the untreatable bacteria already in existence. The use of excessive and misuse of antibiotics further accelerates and aggravates the development of antimicrobial resistance (Brown and Wright 2016). Another starling mechanism of resistance to antibiotics is that the bacteria evolve “dormancy” or sleep for most of the antibiotic treatment and transiently protect themselves and later evolve with higher resistance and tolerance to antibiotic treatment (Levin-Reisman et al. 2017). Staphylococcus aureus is already resistant to the first-line drugs, and it has been estimated that about 64% of the people die more likely due to the infection with MRSA (methicillin-resistant Staphylococcus aureus) than those infected with a nonresistant form (Chambers and DeLeo 2009). Resistance in Klebsiella pneumoniae to carbapenem antibiotics is widespread now. The bacterium K. pneumoniae is one of the most common causes of hospital-acquired infections, including pneumonia, bloodstream infections, and infections in newborns and patients in intensive care units. Approximately half of the people treated for K. pneumoniae with carbapenem antibiotics in some countries are ineffective due to drug resistance (Kidd et al. 2017). Globally, fluoroquinolone antibiotics are becoming increasingly resistant, and almost half of the patients no longer respond to this treatment (Collignon 2009). A total of ten countries (Austria, Australia, Canada, France, Norway, Japan, South Africa, Slovenia, Sweden, Northern Ireland, and the UK of Great Britain) have confirmed that the last resort of medicine (thirdgeneration cephalosporin antibiotics) for treating gonorrhea has failed (WHO 2018; Allen et al. 2013; Fifer et al. 2016). Colistin has proved to be a final option for treating life-threatening infections triggered by Enterobacteriaceae that are carbapenem resistant. In several countries and regions, resistance to colistin has recently been detected, making it tough to treat infections caused by such bacteria (Biswas et al. 2012). As many as 121 countries have been identified as having extensively drugresistant tuberculosis (XDR-TB), resistant to at least 4 of the most common antiTB drugs. According to estimates, 6.2% of people with multidrug-resistant TB have XDR-TB (Marwar et al. 2010; Prasad 2010). ECDC reported that antibiotic resistance in most bacteria under examination continued to increase in 2015. It has also been reported that combined resistance to carbapenems and polymyxins (e.g., colistin) has increased in K. pneumoniae from

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Table 10.1 Developing antibiotic resistance: a timeline of key events (Ventola 2015) Antibiotic resistance identified Penicillin-R Staphylococcus 1943

Tetracycline-R Shigella 1959 Methicillin-R Staphylococcus 1962 Penicillin-R Pneumococcus 1965 Erythromycin-R Streptococcus 1968 Gentamycin-R Enterococcus 1979

Antibiotic introduced Penicillin (1940) Tetracycline (1950) Erythromycin (1953) Methicillin (1960) Gentamycin (1967)

Imipenem and ceftazidime (1985) Ceftazidime-R Enterobacteriaceae 1987 Vancomycin-R Enterococcus 1988 Levofloxacin-R Pneumococcus 1996 Imipenem-R Enterobacteriaceae 1998 XDR-Tuberculosis 2000 Linezolid-R Staphylococcus 2001 Vancomycin-R Staphylococcus 2002 PDR Acinetobacter and Pseudomonas 2004–2005 Ceftriaxone-R Neisseria gonorrhoeae 2009 PDR Enterobacteriaceae Ceftaroline-R Staphylococcus 2011

Levofloxacin (1996) Linezolid (2000) Daptomycin (2003) Ceftaroline (2010)

PDR Pan drug resistance, R resistance, XDR extensively drug resistant

6.2% in 2012 to 8.1% in 2015. These two groups of antibiotics are usually considered the last treatment options for patients infected with resistant bacteria (European Centre for Disease Prevention and Control (ECDC) 2016) (Table 10.1). The CDC considered a 10-year projection of incidence, transmissibility, availability of effective antibiotics, and barriers to prevention when assessing antibioticresistant bacterial infections (Centers for Disease Control and Prevention (US) 2013). Each bacterium was assigned a threat level based on whether it was urgent, serious, or concerned (Table 10.2). Despite the dire need for new antibiotics, which are already in decline (Fig. 10.2), pharmaceutical companies tend to show little interest in antibiotics due to their lower profitability than drugs for chronic illnesses like diabetes, psychiatric disorders, asthma, gastrointestinal diseases, and cancer (Gould and Bal 2013; Piddock 2012; Bartlett et al. 2013) and partly because antibiotics cost less relatively and therefore lack economic appeal. Compared with cancer chemotherapy, which costs a hundred times more than antibiotic treatment, newer antibiotics are generally priced between 1000 and 3000 USD per course (Gould and Bal 2013; Piddock 2012; Bartlett et al. 2013). For detailed information about the factors that cause antibiotic development delays and other regulatory approaches that need to be taken to accelerate the development of antibiotics, authors are advised to read the paper by C. Lee Ventola (Ventola 2015).

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Table 10.2 CDC assessment of antibiotic resistance threats (Centers for Disease Control and Prevention (US) 2013)

3

Urgent Clostridium difficile (CDIFF) Carbapenem-resistant Enterobacteriaceae (CRE) Drug-resistant Neisseria gonorrhoeae Concerning Multidrug-resistant Acinetobacter Vancomycin-resistant enterococci (VRE) Multidrug-resistant Pseudomonas aeruginosa Methicillin-resistant Staphylococcus aureus (MRSA) Drug-resistant Streptococcus pneumoniae Drug-resistant tuberculosis Drug-resistant Salmonella typhimurium Beta-lactam-resistant Enterobacteriaceae Serious Vancomycin-resistant Staphylococcus aureus (VRSA) Erythromycin-resistant group A Streptococcus Clindamycin-resistant group B Streptococcus

Bacterial QS Systems

Several types of bacteria have developed unique cell-to-cell communication systems that produce and distinguish signaling molecules called autoinducers (Whiteley et al. 2017). The three major types of autoinducers studied are acylated homoserine lactones (AHLs), also termed as autoinducer-1 (AI-1), which are used by Gramnegative bacteria, peptide signals, which are used by Gram-positive bacteria. In addition, autoinducer-2 (AI-2) is used by both Gram-positive and Gram-negative bacteria. These classes of QS signals range from the Pseudomonas quinolone signal (PQS) to the diffusible signal factor (DSF) to the autoinducer-3 (AI-3).

3.1

QS in Gram-Positive Bacteria

Genetically encoded and ribosomally generated oligopeptides are generally used as autoinducer molecules by Gram-positive bacteria. The peptide molecules cannot permeate the cell membrane and are transported out by specialized transporters. In addition to their surface manifestation, these signaling molecules can also be detected intracellularly. The QS target genes are activated or repressed by membrane-bound sensor kinases, which switch on and off as needed. In S. aureus, the Agr system controls virulence factor production, toxin production, and biofilm formation. In Enterococcus faecalis, the FSR system prevents biofilm formation and virulence factor production. Figure 10.3 shows four types of autoinducing molecules (AIP-I, AIP-II, AIP-III, AIP-IV) used by S. aureus according to the agrgene type (agr-I, agr-II, agr-III, agr-IV). It sends activation signals to the cognate agrC receptor

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6 4

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Fig. 10.2 Approval of new antibiotics is in decline. Over the past three decades, there has been a steady decline in the number of new antibiotics being developed and approved. (Adapted and modified from Ref. (Ventola 2015)

Fig. 10.3 S. aureus agr system and the structures of the AIPs used for QS

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while inhibiting three other non-cognate receptors (Thoendel et al. 2010). FSR, on the other hand, uses GBAP, which triggers gelatinase production via sensing kinase FsrC (Nakayama et al. 2001). The extracellular presence of linear peptide-based autoinducers is also found in Streptococcus pneumonia, Enterococcus faecalis, and Bacillus thuringiensis (Rocha-Estrada et al. 2010; Havarstein et al. 1995; Dubois et al. 2012; Rocha et al. 2012).

3.2

QS in Gram-Negative Bacteria

The N-acyl-homoserine lactone (AHL) molecule is most commonly used as an autoinducer for QS in Gram-negative bacteria. There are generally 4–18 carbons in the acyl chain of these molecules, which contains a homoserine lactone ring. Aside from chain length, acyl chains can differ in their saturation and oxidation states at position 3 (Fig. 10.4). Except for a few bacterial species, AHL synthases belong to the LuxI family and utilize S-adenosylmethionine (SAM) as a substrate. AHL synthases generally produce a single kind of AHL, which spreads across the cytoplasm and accumulates locally. AHLs are also transported across cell membranes by some bacteria using transporters (Chan et al. 2007; Evans et al. 1998; Pearson et al. 1999). These AHLs are detected by membrane-bound sensor kinases, such as Lux N in V. harveyi. Each species of bacteria produces and responds to AHL through a cognate synthase/receptor pair. AHL molecules can be detected and responded to simultaneously by several bacteria species that utilize multiple synthase/receptor pairs (Jimenez et al. 2012a; Subramoni and Venturi 2009). It is believed that Gram-negative and Gram-positive bacteria use the same autoinducer molecule (AI-2), which diffuses freely through the membrane and accumulates in the extracellular space. This molecule is produced sequentially with the enzymatic activity of 5-methylthioadenosine and the metalloenzyme LuxS from the precursor S-adenosylhomocysteine (SAH). It has been discovered that many species of bacteria produce and respond to AI-2 and AI-2 receptors, including V. harveyi and Salmonella enterica serovar Typhimurium. V. harveyi detects a boric acid complexed form of AI-2 through a Luxp/LuxQ receptor-sensor complex, whereas S. typhimurium uses a non-borated form of AI-2 through LuxR regulator system (Fig. 10.5) (Chen et al. 2002; Neiditch et al. 2006; Taga et al. 2003) (Fig. 10.6).

Fig. 10.4 General structure of N-acyl-homoserine lactone (AHL)

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Fig. 10.5 Structure of AI-2 molecules used by V. harveyi and S. enterica

Fig. 10.6 Structure of PQS and HHQ mostly used by Pseudomonas

4

QS Antagonists and Agonists

There are three ways to target QS systems: signal molecules (AHL, AI-1, AI-2, etc.), receptors (Lux R, etc.), and signal generators. Of the three, the blockage of signal production has received the least attention. Degradation or inactivation of the signal molecule can be achieved by chemical degradation, enzymatic destruction, or metabolic processes. Invading QS bacteria are usually defeated by raising the pH above 7 to inactivate the AHL signal molecules, resulting in the alcoholysis-ring opening of the AHL (Yates et al. 2002). Lactonolysis of AHLs is possible through the action of enzymes. The enzyme, AiiA, produced by B. mycoides, B. cereus, and B. thuringiensis, belonging to the genus Bacillus, is specialized for the deterioration of AHLs (Dong et al. 2000; Dong et al. 2001a; Wang et al. 2004). Biologically, plants, bacteria, and mammals inactivate QS signal molecules to protect themselves from pathogens. An excellent strategy to obstruct QS involves blocking receptors analogous to the signal molecule or the destruction of the receptors.

4.1

Natural Product-Based and Natural Product-Derived QS Inhibitors

Natural products can hinder bacteria’s virulence factors by destroying the signaling molecules released by bacteria (Koh et al. 2013). In several Gram-negative bacteria, halogenated furanones produced by the alga Delisea pulchra interfere with the AHL regulatory system. Furanones can control bacterial colonization and foul on their surfaces in natural marine environments (Bauer and Teplitski 2001; Kjelleberg et al.

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Fig. 10.7 Structure of a furanone compound

Fig. 10.8 Structure of penicillic acid (PA) and patulin

1997). The ability of some bacteria to quench QS has been reported in several studies. For example, AiiA is an enzyme that hydrolyzes the lactone bond of AHL, a signaling compound found in the N-acyl-homoserine family of proteins (Dong et al. 2001b). In addition, paraoxonase (PON) enzymes also display interaction with QS systems in human airway epithelial cells (Chun et al. 2004). It is reported that furocoumarins inhibit AI-1 and AI-2 activity and biofilm formation from E. coli, S. typhimurium, and P. aeruginosa (Girennavar et al. 2008). Aside from biofilm formation, obacunone inhibits both AHL and AI-2 systems and EHEC virulence (Vikram et al. 2010a). The organism D. pulchra produces halogenated furanone compounds that interfere with QS-controlled motility, and thereby prevent macrofouling (Givskov et al. 1996). A number of bacteria are also sensitive to halogenated furanones, including V. fischeri, Vibrio harveyi, Serratia ficaria, and Proteus aeruginosa, while natural furanones are ineffective against these pathogens. To circumvent this problem, enormous libraries of compounds were developed to have different side chains, attachment points on the furanone ring, and substituents within the ring. P. aeruginosa’s two QS systems have been susceptible to synthetic derivatives without an acyl side chain (Manefield et al. 2002; Hentzer et al. 2003). Hentzer et al. (2003) found by DNA microarray analysis that the furanone compound significantly downregulated the expression of about 80% QS genes (Fig. 10.7). P. radicicola and P. coprobium have been found to produce penicillic acid (PA) and patulin (Fig. 10.8), targeting about 60%, and 45% of QS genes in P. aeruginosa, respectively, and disrupt the LasR and RhlR QS regulators (Rasmussen et al. 2005). It has been established that in P. aeruginosa, the metabolites like disulfides and trisulfides extracted from garlic inhibited LuxRbased QSI (Walker et al. 2004). In P. aeruginosa, rosmarinic acid from sweet basil reduced the expression of the elastase and protease, as well as an inhibition in the biofilm formation (Teplitski et al. 2000). It is also reported that in C. violaceum, the pea seedlings and root exudates inhibit pigment production, exochitinase activity, and protease activity (Daniels et al. 2002). The substances

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produced by Medicago truncatula, soybean, rice, and tomato all of which are ALH mimicking substances (Daniels et al. 2002; Vandeputte et al. 2010). A flavonoid (flavan-3-ol catechin) from the bark of Combretum albiflorum decreased the production of QS-mediated virulence factors – elastase, pyocyanin, and also the biofilm formation by P. aeruginosa PAO1 (Vikram et al. 2010b; Nakayama et al. 2007). Streptomyces sp. strain Y33-1 produces siomycin I, a peptide antibiotic that inhibits gelatinase and gelatine biosynthesis, activating pheromones, which disrupt Enterococcus faecalis biofilms (Tsunakawa et al. 1995). Gram-positive bacteria are selectively inhibited by siomycin, while Gram-negative bacteria are not affected by it (Bobadilla Fazzini et al. 2013). Pseudomonas reinekei MTI and Pseudomonas sp. B13 naturally produce protoanemonin, such as 4-methylenebut-2-en-4-olide, a catabolite that inhibits QS-mediated factors. Pyoverdine and pyochelin are siderophore molecules involved in iron starvation response in P. aeruginosa (Dobretsov et al. 2010). It has been reported that malyngolide and lyngbyoic acid isolated from Lyngbya majuscula inhibited the production of violacein in C. violaceum, as well as the production of elastase and pyocyanin in P. aeruginosa (Kwan et al. 2011; Kwan et al. 2010). In addition, two other compounds have been isolated from L majuscula – malyngamide C and 8-epi-malygamide – inhibiting QS activity in P. aeruginosa (Calfee et al. 2001).

4.2

Synthetic QS Inhibitors

Synthetic analogues of QS signals have clear commercial relevance, yet there is a scarcity of such antagonists. To synthesize QSI, researchers have targeted the biosynthetic pathway that leads to signal production, the substitution of signals, and changes in the chain length. As a result of a multistep reaction, anthranilate and keto fatty acids are combined to produce a Pseudomonas quinolone signal (PQS). It was found that P. aeruginosa (PAO1) did not grow well when exposed to a methyl anthranilate (an analogue of anthranilate) that inhibited the production of PQS and reduced elastase production without affecting its growth (Chhabra et al. 1993). AHL analogues with a different geometry had a strong effect on their QS activity. D-isomers did not show any activity; presence of unsaturation near the amide linkage stopped its binding to the receptor and also a 50% reduction in activity with an increase of one methylene unit and 90% loss with an increase of two methylene units in the acyl chain length (Zhu et al. 1998). Substitutions of carbonyl at 3 position with a methylene in the acyl chain of QS signal – 3OC8-HSL of TraR in A. tumefaciens – transformed it into an antagonist of equivalent activity (Ishida et al. 2007). It has been reported that QS functions are inhibited by AHL analogues N-acyl cyclopentyl amines (Cn-CPA) in P. aeruginosa and C10-CPA has been found to be most active QSI against Las and Rhl QS system in P. aeruginosa (Morohoshi et al. 2007). C9-CPA has been verified as the most effective QSI in S. marcescens (Koch et al. 2005a).

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N-(heptyl-sulfanyl acetyl)-L-HSL is a good antagonist that repressed LasRmediated QS, LuxR-controlled GFP expression, and 3OC6HSL-facilitated protease activity of Pectobacterium A2JM (Persson et al. 2005a; Wu et al. 2004). An AI-1 controlled pathogenicity inhibitor, (Z)-4-bromo-5-(bromomethylene)furan-2(5H)-one and (Z)-5-(bromomethylene)furan-2(5H)-one, has been employed to inhibit P. aeruginosa-infected mouse lungs. As a result, these compounds effectively prevent QS-mediated biofilm formation (Martinelli et al. 2004). 5-Hydroxy-3[(1R)-1-hydroxy-2,2- dimethylpropyl]-4-methylfuran-2(5H)-one was able to completely block the production of violacein at a concentration of 10-3 M, but was detrimental to the growth of a bacterium called C. violaceum (Kim et al. 2005). N-(Sulfanyl acetyl)-L-HSL and fimbrolide (F1) (Koch et al. 2005b) were also synthesized signals that inhibited QS differently from the QS signal 3-OC6HSL by increasing LuxR turnover instead of competing for the receptor binding site (Persson et al. 2005b). Among other things, brominated ylidenebutenolide [(5z)-4-bromo-5(bromo methlene-2(5H)-furanone] works as a biomimetic of 3OC12HSL and as an analogue of naturally occurring fimbrolide produced by D. pulchra (Lönn-Stensrud et al. 2008). Among the most promising synthetic furanones, (Z)-3-bromo-5(bromomethylene)furan-2(5H)-one was found to reduce bioluminescence in Staphylococcus epidermidis efficiently (identified as F206) and (Z)-5-(bromomethylene)furan-2-(5H)-one (identified as F202) (Muh et al. 2006). PD12, a tetrazole with alkyl chain of 12 C atoms, and V-O6-O18, a phenyl ring attached to 12 C alkyl chain, inhibited the production of a QS-mediated virulence factor – pyocyanin. The underlying mechanism was the antagonism of LasR receptor (Lowery et al. 2009). QS mediation by AI-2—(4S) dihydroxy-2,3-pentaedione (DPD) is noticed in most of the bacteria. Increased inhibitory activity was confirmed with the increase in the C chain length. In fact, hexyl-4,5-dihydroxy-2,3-pentaedione (hexyl-DPD-5) was found to be the most effective inhibitor, having EC50 value of 9.65 μM (Ganin et al. 2009). Such compounds possibly may inhibit DPD signal of S. typhimurium and unveil a coactive result on QS of V. harveyi (Lowery et al. 2008; Ren et al. 2004). Reports reveal that afimbrolide natural product -(5Z)-4-bromo-5(bromomethylene)-3-butyl-2(5H)-furanone I acted as an effective antagonist of equally AHL and AI-2 mediated QS number of bacteria, however not limited to bacteria (Ren et al. 2005). Apolipoprotein B, a structural protein sequesters QS signal AIP1 and inhibits Agr-dependent virulence in MRSA strains (Peterson et al. 2008). Several AIP analogues have been synthesized and explored to gain insights into structure-activity relationships within AIPs and the cell membrane receptors AgrCs, resulting in highly potent AIP-based agr modulators (Fig. 10.9) (Lyon et al. 2000; Stephenson et al. 2000; Wright et al. 2005; Gorske and Blackwell 2006). Recently a seminal study proved that AIP analogues having a lactam bridge rather than thiolactone were chemically stable and functioned as potent agr inhibitors. In addition, a recent study revealed that a non-AIP small molecule termed “savirin” disrupted QS in S. aureus but not in Staphylococcus epidermidis – an important skin commensal (Salam and Quave 2018). They also revealed that agr inhibition using savirin promoted host

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Fig. 10.9 Structure of designed universal inhibitors of QS system in S. aureus

defense, resulting in decreased virulence and improved clearance in murine skin infection models infected with MRSA. Furthermore, resistance was not developed after continual chemical agr inhibition both in vitro and in vivo.

5

QS Mediated Delivery: Design

QS systems play pivotal roles in many pathogenic events including the production of toxin/virulence factors, host cell invasion, and biofilm formation. Indeed, modulation of QS leads to suppression of virulence factor production, disruption of biofilms, and in some cases increased susceptibility against existing antibiotics (Galloway et al. 2012; Rasmussen and Givskov 2006; Raffa et al. 2005; Guo et al. 2013). In addition, QS systems are indispensable for survival, and thus QS modulators exert less evolutionary pressure on pathogenic bacteria than traditional antibiotics. As such, modulation of QS has evolved as a potential strategy for virulence control. Chemical approaches using synthetic modulators have been pursued, which has revealed that small structural variations in the signaling/autoinducer scaffolds significantly affect QS modulatory activity, modes of action (agonism/antagonism), and species specificity. Despite the therapeutic potential of QS modulation, QS modulators including synthetic AIP or AHL analogues do not affect viability of bacterial cells (Rasmussen and Givskov 2006; Guo et al. 2013; Reuter et al. 2016). Thus, another strategy for excluding or killing pathogens needs to be concomitantly utilized with the QS modulation strategy to achieve clinically effective treatment. This needs to be addressed before QS-based anti-virulent approaches become a clinically effective antimicrobial strategy that can augment our current treatment options for emerging

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antibiotic-resistant pathogens. An efficient strategy is to use the antiquorum sensing molecules (e.g., the truncated AIP in S. aureus) as a guiding or delivery vehicle to which any cytotoxic molecule or antibiotic can be attached through certain suitable linkers for efficient delivery to the target site. This design could allow for recognition of the target and accumulation on the target cell membrane through signal-receptor interactions. This targeted delivery of the antibiotic using the antiquorum sensing molecules will provide multiple benefits: attenuated virulence by QS modulation, enhanced antibacterial activity, selective killing of the bacteria, and reduced collateral cytotoxicity to other nontarget bacterial species and host cells. This strategy would minimize the off-target toxicity, suppress the biofilm formation, and kill the bacteria with lower doses of antibiotics. For maximum efficiency stable linkers that could allow for efficient drug release at the target site are required. A variety of linkers have been designed including non-cleavable thioether or maleimidocaproyl linkers and cleavable hydrazone, disulfide, or peptide linkers (Nolting 2013). Despite providing greater stability and tolerability, non-cleavable linkers can reduce drug release and efficacy. It has been shown that cleavable linkers made up of hydrazone or disulfide bonds are sensitive to oxidative and reductive environments, respectively, and such environments exist outside of the cells as well. Thus, most developed linkers target enzymes abundant near the infection site that selectively cleaves the linkers. Many hydrolytic enzymes recognize the sequences of peptides, making them ideal enzyme-cleavable linkers. With peptide-based linkers, such as valine-citrulline (Val-Cit) dipeptide linkers, the drug is released more rapidly into the target cell due to the excellent systemic stability (Dal Corso et al. 2017). Disulfide linkers have also been used in the construction of conjugates (Danial and Postma 2017). The antiquorum sensing molecule, which may be an AHL or AIP analogue, could be linked to the antibiotic of choice by use of the linkers as shown in Fig. 10.10. The antiquorum molecule will serve two functions: it will cut off the bacterial communication and simultaneously guide the antibiotic to the target bacteria. This will stop the bacteria from producing the virulence factors, and in the meantime the antibiotic will eradicate the bacteria from the site of infection.

6

Conclusions

Repeated antibiotic exposure is the leading cause of multiple drug resistance in bacteria. Within biofilms, bacteria exhibit a similar effect as their free-living counterparts. Biofilms provide them with protection by using exopolysaccharide materials. A disruption of biofilms could make bacteria more sensitive to antibiotic treatment, even at low doses. Interference with quorum sensing disrupts biofilmprotective measures, and a plethora of molecules have already been designed to disrupt or confuse the bacterial communication. But the major problem is that this disruption does not completely eradicate the bacteria or kill them although the production of virulence factors and toxin production are affected to a great extent. This problem can be addressed if an antibiotic is conjugated to the anti-QS molecule

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Fig. 10.10 Schematic showing quorum sensing-guided delivery of antibiotic (in this case S. aureus)

(AIP analogue in case of S. aureus) through different types of linkers for pinpoint delivery of the antibiotic to the target site. This magic bullet concept will serve two functions – disrupt the bacterial communication, exerting less evolutionary pressure on the target microbe, and concomitantly kill the microbe after confusing it. This strategy offers a novel route to tackle the antimicrobial drug resistance problem. Although several other important factors are to be considered to fight the antimicrobial drug resistance problem on a global scale which are listed below: – -As a result of preventing infections in the first place, antibiotics are used less frequently, and resistance is less likely to develop. – -Preventing the unnecessary and inappropriate use of antibiotics in humans and animals. – -Choosing the right antibiotic and administering it in a right way is highly important. This is often known as antibiotic stewardship. – -Because antibiotic resistance occurs naturally when bacteria evolve, it cannot be stopped. Therefore, development of new antibiotics is highly important to tackle the developing resistance. Acknowledgments Authors are thankful to the University of Jeddah, KSA, for providing online recourses and library facilities.

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Metal Chelation as a Promising Strategy to Combat Fungal Drug Resistance

11

Sandeep Hans, Zeeshan Fatima, and Saif Hameed

Abstract

Among various human fungal diseases, candidiasis caused by Candida albicans is predominant in immunocompromised individuals. Widespread and continuous consumption of antifungal drugs has led to emergence of multidrug resistance (MDR). The success of C. albicans to cause pathogenicity is owing to its ability to persevere in environments regardless of the healthy immune response by the host. MDR being a multifactorial occurrence is initiated by various unidentified mechanisms; therefore, dissecting new mechanisms for fighting MDR is currently required. Within the diverse host niches inhabited by C. albicans, it frequently encounters adverse conditions in the form of extreme pH, metabolic, oxidative, osmotic, and hypoxic stress conditions. Micronutrient availability is among one such stress conditions which is encountered by C. albicans for which it must adapt to cause infection. Magnesium (Mg2+) is one of the most plentiful divalent cations in existing cells that has a significant role in maintaining membranes and ribosomes, necessary as a cofactor in a range of many enzymatic reactions and in the neutralization of nucleic acids; hence, it is obligatory for all organisms to sustain its physiological levels to the best concentration. Since Mg2+ is not freely available for C. albicans, therefore harnessing the Mg2+-dependent pathways in C. albicans could be utilized as effective antifungal strategy. This chapter presents insights into the various Mg2+-dependent factors in C. albicans and how Mg2+ homeostasis can be exploited as potential drug targets to combat MDR.

S. Hans · Z. Fatima · S. Hameed (✉) Amity Institute of Biotechnology, Amity University Haryana, Gurugram, Haryana, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. Y. Wani, A. Ahmad (eds.), Non-traditional Approaches to Combat Antimicrobial Drug Resistance, https://doi.org/10.1007/978-981-19-9167-7_11

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Keywords

Candida · MDR · Metal homeostasis · Micronutrients · Magnesium

1

Introduction

Candida albicans is the main infectious agent responsible for vaginal and oral candidiasis (Wenzel and Gennings 2005). In general, Candida species are not a threat to normal hosts as host immune cells and antagonistic microflora prevent their uncontrolled proliferation and dissemination. However, conditions that weaken the immune response or those that lead to microbial dysbiosis favor the widespread infection of Candida within the host (Singh et al. 2015). Infections caused by C. albicans are referred to as candidiasis, which is a broad term encompassing local, systemic, invasive, and noninvasive forms of the disease. In some cases, Candida enters the bloodstream causing a life-threatening condition known as candidemia. HIV/AIDS patients are particularly prone to mucosal candidiasis. While usually benign, Candida can cause superficial thrush or infections of the genitourinary tract (Achkar and Fries 2010; Brown et al. 2012). More alarmingly, invasive candidiasis is the fourth leading cause of hospital-acquired bloodstream infections, with mortality rates of approximately 40% despite treatment (Brown et al. 2012). Over 90% of all Candida infections are caused by five species: C. albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, and Candida krusei. Among these, C. albicans is among the most prevalent, responsible for 40%–70% of disseminated infections (Pfaller and Diekema 2010; Pappas et al. 2009). Together, these opportunistic pathogens pose a serious threat to human health, demanding the development of novel therapeutic strategies to combat these infections. The exposure of resistance by an occurrence identified as multidrug resistance (MDR) in yeast has led to the reduced efficacy of various at present accessible drugs. Therefore, dissecting mechanism of resistance of antifungal at the molecular level is requisite, for the improvement of strategies to circumvent MDR in pathogenic yeast. Micronutrients are the essential elements required for the propagation and survival of both host and the pathogen. Thus, this competition between the host and pathogen for limited micronutrient availability forms the basis of the establishment of infection. Harnessing the micronutrients stress has been extensively reviewed as an alternative strategy to overcome fungal infections (Hameed et al. 2020). The most common micronutrients are magnesium, iron, zinc, copper, calcium, and manganese. Magnesium (Mg2+) is one of the most abundant divalent cations in living cells that has a critical role in stabilizing membranes and ribosomes, required as a cofactor in various enzymatic reactions and the neutralization of nucleic acids; hence, it is mandatory for all organisms to maintain its physiological levels to an optimum concentration. Deficiency of these nutrients leads to changes in cellular response, and yeast morphology thus represents a novel strategy in which metal homeostasis affects host-pathogen interaction. For instance, the impact of Mg2+ deprivation on C. albicans virulence and survival has already been demonstrated (Hans et al. 2019).

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Despite these roles, Mg2+ transport and homeostasis are poorly understood at the physiological level in C. albicans, and in further sections we attempt to describe the factors governed by Mg2+ availability in C. albicans.

2

Chemistry and Biology of Magnesium

Mg2+ is known to be essential for every living organism (Wacker 1993). It is an alkaline earth element in the periodic table with atomic number 12. Mg2+ exhibits +2 oxidation state all time due to loss of electron from the outermost shell (Hughes 1981). It exists in the solid form in the earth and eighth most abundant element in the earth (Maguire 1990). Mg2+ has high bioavailability for the living cells and its salts are highly soluble in water. It is one of the hardest and densely charged cations known which exhibits low polarizability, high electropositivity, and a tendency for electrostatic bonding (Maguire 1990; Gadd 1992). It is non-exchangeable and rarely substituted by any other cation in any biochemical reactions and physiological functions in cells (Gadd 1992). The significance of Mg2+ in genetic biology was first recognized due to its meditative role in the form of Epsom salts. Mg2+ completes the essential needs as a supplement in each species and phylum. It has an important role in many biological enzymatic activities including every reaction that requires ATP together with phosphorylation. It has many roles in biological process like RNA and DNA synthesis, plasma membrane transport, nerve conduction, cell division, and oxidative phosphorylation (Wacker 1993). Due to its similarity with some other metal ions and simple chemistry of Mg2+, there are conditions where Mg2+ competes for binding sites with numerous other ions. Mostly Mg2+ does not bind to bulk region of biological substrates, like mono- and di-carboxylates, sugar phosphate, amino acids, mononucleotides, sulfate, as the negative charge density on approximately all minute substrates with particular anion centers is too little to bind Mg2+ (Williams 1974). In contrast Ca is more suitable than Mg2+ in binding to the many anionic ligand groups (Martin 1990). The connections between Ca and Mg2+ are mainly due to frequent interactions, as these two metal ions are liable to act antagonistically toward each other (Williams 1976).

3

Significance of Magnesium in Yeast

Yeast, like any other living organisms, requires Mg2+ as an integral part of many of its housekeeping biological processes. In a normal cell, the overall Mg2+ content is about 50 mM; the cation is mostly bound by ATP and proteins, while the free Mg ion concentration is in the 1 mM range (van Eunen et al. 2010). Mg2+ does not appear to be stored in a specific storage compartment even though the cytosol is the only site in which we are able to reliably quantify free Mg2+ content, with dyes like Mag-fura2 (Grubbs 2002). In yeast cell Mg2+ requirement between 2 and 4 mM for the growth requirement cannot be compensated by other ions and majorly manifested as important metabolic and structural functions of yeast. High concentrations of other cations

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in various compartments interfere with the dye’s binding capacity to Mg2+, but this is generally not a problem in the cytosol as the Mg2+ concentration is greater than other cations (Grubbs 2002). While Mg is not tightly implicated in mediating cellular signals as other ions, for example, Ca, it is of vital importance in ATP energy transfer which is a critical part of catalysis in many enzyme active sites and maintains the structural integrity of many proteins.

4

Magnesium Channels

CorA proteins are present in abundance in prokaryotes and found to have homologues in yeast and humans. These proteins present a group of transporters for ions which mediate transportation of divalent metal ions across the biological membranes. Generally, they are involved in transport of Mg2+ ions, but some members of this family can transport cobalt and nickel ions (Niegowski and Eshaghi 2007). Structurally, these proteins possess two transmembrane domains which are partitioned by short loop oriented toward the membrane and YGMN/F motif present at the N-terminus end (Kolisek et al. 2007). The characterized homologue present in inner mitochondrial membrane present in mammals and yeast is MRS2/LPE10 type, whereas ALR/MNR type is present in plasma membrane (Ebel et al. 2002, Liu et al. 2002a, 2002b). In addition to the usual attributes of yeast that makes it an ideal model organism, it codes for several Mg2+ channels. S. cerevisiae encodes four CorA-family Mg channels that reside in different membranes of the cell: Alr1p and Alr2p in the plasma membrane (Graschopf et al. 2001), Mrs2p in the inner mitochondrial membrane (Kolisek et al. 2003), and Mnr2p in the vacuolar membrane (Pisat et al. 2009). Alr1p, a member of the CorA family, is the only highly active channel to move Mg2+ across the cell membrane.

5

ALR1 and ALR2

Fungal Alr1, Alr2, and Mnr2 (Fig. 11.1) and its homologue Alr2 are members of the CorA-Mrs2-Alr1 superfamily exclusively found in the plasma membrane of fungi (Graschopf et al. 2001). Their name is derived from their ability to award aluminum resistance to yeast when overexpressed, but not at normal expression levels. Aluminum is not transported by the Alr proteins but acts as an inhibitor of Mg2+ uptake. Overexpression of Alr1 and Alr2 likely alleviates this inhibitory effect by allowing enough Mg2+ influx to sustain yeast viability (MacDiarmid and Gardner 1998). Alr1 and Alr2 divide 69% amino acid series identity and similar lengths of 859 and 858 amino acids, respectively. In S. cerevisiae, Alr2 provides poorly to Mg2+ uptake, perhaps due to its low appearance in the yeast strains used (Johansson and Jacobson 2010; MacDiarmid and Gardner 1998). An amino acid exchange (E768R) in the periplasmic loops connecting the TM helices of Alr2 has also been tied to low channel movement (Wachek et al. 2006). Consequently, Alr1 showed the major Mg2+ uptake system and regulator of Mg2+ homeostasis in yeast (da Costa et al.

11

Metal Chelation as a Promising Strategy to Combat Fungal Drug Resistance Mg2+

Mg2+

275

Mg2+

Mg2+ Mg2+ Mg2+

Mg2+

1 Alr

2 Alr

Mg2+

Cytosol Mg2+ Mg2+

Mitochondria

Mg2+

Lpe10

Mg2+/Na+ Mg2+/H+ exchanger

Mrs2 Mg2+

Mnr2

ase TP A V

H+/Na+ Vacuole

Brg1 Nucleus

Fig. 11.1 Magnesium acquisition and homeostasis in C. albicans

2007). Other substrates of Alr1 include Co, Ni, Mn, and Zn, while Al serves as an inhibitor (MacDiarmid and Gardner 1998). Patch-clamp studies have categorized Alr1 as a channel, able of mediating both influx and efflux of Mg2+ (Liu et al. 2002a, 2002b). Alr1 expression and stability at the plasma membrane has been proposed to be controlled by Mg2+ (Graschopf et al. 2001). In this study, limiting Mg2+ concentrations induced Alr1 expression at the MPNA level, while high Mg2+ concentrations lead to internalization and degradation of the protein by endocytosis to the vacuole. Alr1 is regulated by cytosolic Mg similarly to the bacterial CorA (Lim et al. 2011). This study also proposed that Alr1 may be regulated by phosphorylation. S. cerevisiae Alr1 is more than double the length of TmCorA (859 versus 351 amino acids, respectively), and the two proteins share 24% amino acid sequence identity. Homology to TmCorA is localized at the C-terminus of Alr1 and includes the conserved GMN motif. In addition, Alr1 contains a long N-terminal extension (~400 amino acids) and a short C-terminal extension (~60 amino acids), the shorter bacterial CorA is able to partially rescue the Alr1 deficiency phenotype in yeast (Graschopf et al. 2001), and that truncation of the first 239 N-terminal amino acids of Alr1 does not significantly interfere with Mg2+ uptake by yeast cells suggesting that the N-terminal domain serves a role that is not directly related to the translocation of Mg2+ along the TM pore (Lee and Gardner 2006). The N-terminal extension is

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unique to fungal Alr1-related proteins, and its importance for Mg2+ homeostasis is unclear.

6

MRS2

MRS2 protein channel deals with Mg2+ transport in mammals and mainly influx in prokaryotes. It is also known for specially transport of Mg2+ in mitochondria in yeast, plants, and mammals (Zsurka et al. 2001). MRS2 channel was firstly discovered in mitochondria of S. cerevisiae (Zhou and Clapham 2009). CorA superfamily proteins in which Mrs2 is 50 kDa in size and nuclear encoded attached with two domains and glycine-methionine-asparagine (GMN) motif which is important for the function of the channel (Khan et al. 2013). Mrs2 protein is localized in the inner membrane of the mitochondria and essential for the regular mitochondrial Mg2+ homeostasis (Bui et al. 1999). In addition conserved sequence of GMN motif in outer pat of the TM1 α-helix is necessary for Mg2+ recognition (Worlock and Smith 2002). High-conductance channel of Mrs2 is Mg2+ selective, which maintains Mg2+ influx into mitochondria. Consequently, pharmacological agents inhibiting the ADP/ATP translocase are capable of adjusting Mrs2 action foremost to a significant decrease of the amplitude Mg2+ influx. A well-designed experiment performed on yeast mitochondria by (Kolisek et al. 2003) showed how Mrs2 overexpression induces a distinct enhance in free mitochondrial Mg2+, as well as the deletion of the gene eliminates elevated ability of influx. Contribution of Mrs2 protein which uptake mitochondria is known by the deletion of the gene that results in disturbance of mitochondria function and decrease in the Mg2+ amount in mitochondria leading to defect in RNA splicing. Furthermore, expression of the CorA protein in yeast and targeted to the mitochondria could partly overcome for these mutant effects by restoring wild-type levels of mitochondrial Mg2+ (Bui et al. 1999).

7

MNR2 and LPE10

Mnr2, a new member of the CorA-Mrs2-Alr1 superfamily that is directly related to the Alr proteins, derives its name from its ability to confer manganese resistance to yeast (Pisat et al. 2009). Similar to the Alr1 proteins, Mnr2 is much longer than its bacterial counterparts where the S. cerevisiae Mnr2 is 969 amino acids in length. The localization and activity of Mnr2 have been widely investigated in S. cerevisiae (Pisat et al. 2009). Mnr2 localization to the vacuole membrane, an organelle that is known to accumulate Mg2+ via a Mg2+/H+ exchanger (Borrelly et al. 2001), where it likely plays a role in the release of the vacuolar Mg2+, stores in response to low Mg2+ conditions. This model is supported by the fact that Mnr2 mutant cells accumulated more intracellular Mg2+ compared to wild-type yeast when grown on media supplemented with Mg2+. Noticeably, the improved intracellular Mg2+ content had little effect on the growth rate of these Mnr2 cells under Mg-free conditions, suggesting that while the overall intracellular content of Mg2+ was high, the cytosol

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became Mg2+ deficient. Thus, it emerges a model for Mg2+ homeostasis in yeast in which Alr1, and to a much lesser extent Alr2, transports extracellular Mg into the cell and serves to maintain its cytosolic pool. Any surplus Mg2+ is transported into the vacuole via a Mg2+/H+ exchanger and released via Mnr2 when the cytosolic Mg2+ pool becomes depleted in response to environmental conditions. Lpe10 a protein closely related to Mrs2 is also found in the yeast mitochondrial inner membrane where it plays a role in Mg2+ transport and group II intron splicing. However, Mrs2 and Lpe10 cannot completely substitute for one another, and instead Lpe10 appears to interact with Mrs2 and enhances its activity (Gregan et al. 2001; Sponder et al. 2010). Filamentation induced by Mg2+ is dependent on Ras1-PKA signaling pathway, in addition to the transcription factor Brg1 (Su et al. 2018).

8

Magnesium Exchange Mechanism

Mg2+ intrusion/extrusion in the vacuole is mediated by two different kinds of exchange mechanism, viz., Mg2+/Na+ and Mg2+/H+ pathways (Romani 2007). The stoichiometry of this exchange mechanism is not fully understood, but it has been observed that its cAMP-mediated activation is the leading cause (Liu et al. 2002a, 2002b).

9

Targeting Mg Homeostasis Against Candida

C. albicans has been known to adopt various strategies to avoid killing by the host defense system. Hence, it has become a priority to develop efficient therapeutics and strategies in order to stop Candida propagation and persistence. It has been discovered that Mg2+ deprivation leads to inhibition of the fungal hyphae development in C. albicans (Hans et al. 2019). Likewise, overexpression of Mg2+ transporter in S. cerevisiae causes aluminum ion resistance (Walker et al. 1984). Deletion or inactivation of the ALR1 leads to decreased Mg2+ uptake, and excess Mg2+ also leads to growth defect in C. albicans (MacDiarmid and Gardner 1998). In other study, it revealed that the antifungal activity of trypsin inhibitor BPTI and metal chelator DPTA to inhibit the development of C. albicans is via obstruction of cellular Mg2+ uptake (Bleackley et al. 2014; Polvi et al. 2016). It is also reported that Mg2+ deprivation inhibits the circuitry involving virulence and pathogenesis of C. albicans (Hans et al. 2019). Additionally, Mg deprivation inhibits the efflux pump activity of the CaCdr1p and CaCdr2p in C. albicans (Hans et al. 2021). Therefore, the potential of Mg2+ homeostasis as effective antifungal strategy should be exploited further by indulging in wide ranges of such studies.

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Conclusion

The effect of perturbed Mg2+ homeostasis on C. albicans pathogenicity is evident from wide ranges of studies but needs to be exploited further. Considering the growing prominence, metal chelation strategy may grasp huge attraction for enlightening new therapeutic strategies for life-threatening fungal diseases.

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Propolis: A Natural Antibiotic to Combat Multidrug-Resistant Bacteria

12

Deepak M. Kasote, Archana A. Sharbidre, Dayanand C. Kalyani, Vinod S. Nandre, Jisun H. J. Lee, Aijaz Ahmad, and Amar A. Telke

Abstract

Infections associated with multidrug-resistant (MDR) pathogens are becoming challenging due to the limited availability of antibiotics. Conversely, overuse of antibiotics is further leading to the development of MDR traits among the various pathogenic microorganisms. Considering these challenges, in recent times, extensive research about developing new antibiotics, including exploring the utility of different alternative and complementary medicines against a range of MDR pathogens, has been undertaken worldwide. Phytochemicals, plant extracts, and D. M. Kasote (✉) Herbal Medicine, Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth University, Pune, Maharashtra, India A. A. Sharbidre Department of Zoology, Savitribai Phule Pune University (Formerly University of Pune), Pune, Maharashtra, India D. C. Kalyani CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, India V. S. Nandre Department of Chemistry, Division of Biochemistry, Savitribai Phule Pune University (Formerly University of Pune), Pune, Maharashtra, India J. H. J. Lee Department of Plant Science and Technology, Chung-Ang University, Anseong, Gyeonggi-Do, Republic of Korea A. Ahmad Division of Infection Control, Charlotte Maxeke Johannesburg Academic Hospital National Health Laboratory Service, Johannesburg, South Africa A. A. Telke Norwegian Veterinary Institute, Oslo, Norway # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. Y. Wani, A. Ahmad (eds.), Non-traditional Approaches to Combat Antimicrobial Drug Resistance, https://doi.org/10.1007/978-981-19-9167-7_12

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natural products are receiving a great deal of research attention as resistance-modifying agents against MDR pathogens. Honeybees (Apis mellifera) and stingless bees have natural wisdom about selecting and collecting antimicrobial principles from plants. Using this inherited wisdom, honeybees produce propolis for their protection from microbes, viruses, and insects. Recent scientific studies also substantiated these ancient antimicrobial claims of propolis and proved its utility in treating MDR pathogens. In this book chapter, we have reviewed the antibacterial activity of different propolis samples against MDR pathogens, emphasizing their mode of action, and synergistic interaction with various antibiotics and other natural products against MDR pathogens. Keywords

Propolis · Multidrug resistance · Phytochemicals · Pathogens

1

Introduction

Antibiotics are the evolutionary drugs of the twentieth century. The discovery of antibiotics has still been considered one of the most significant discoveries of mankind, which saved countless lives from deadly microbial infections (Davies and Davies 2010). However, in the last few decades, the efficacies of some promising antibiotics have been threatened by the emergence of microbial resistance (Baym et al. 2016). The term “multidrug-resistant (MDR)” is routinely used to define microorganisms that are insensitive or resistant to more than one antimicrobial agent (Magiorakos et al. 2012). In general, the development of this sort of resistance among microbes, including clinical pathogens, is a natural phenomenon (Tanwar et al. 2014). However, rising resistance among clinical pathogens against available antibiotics can be one of the greatest threats to human health, considering the growing burden of untreatable infections (Blair et al. 2015). Over ten million people per annum will be estimated to be infected with these superbugs by 2050 (Shin et al. 2018). The infections associated with MDR pathogens is becoming challenging to treat due to the limited available number of antibiotics. Conversely, overuse of antibiotics is further leading to the development of MDR traits among the various pathogenic microorganisms. Considering these challenges, in recent times, extensive research about developing new antibiotics, including exploring the utility of different alternative and complementary medicines against a range of MDR pathogens, has been undertaken worldwide. Since antiquity, humankind has been using plants and natural products to cure infectious diseases (Cushnie and Lamb 2005; Rios and Recio 2005). Plants are rich in a wide range of antimicrobial phytochemicals such as phenolics, quinones, and alkaloids (Saleem et al. 2010). Phyto-components showed antimicrobial activity when used alone and function as synergists or potentiators of other antibacterial agents (Abreu et al. 2012; Chung et al. 2011). Moreover, these days,

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Propolis: A Natural Antibiotic to Combat Multidrug-Resistant Bacteria

283

phytochemicals, plant extracts, and natural products are receiving a great deal of research attention as resistance-modifying agents against MDR pathogens (Abreu et al. 2012). Honeybees (Apis mellifera) including stingless bees are collecting antimicrobial principles from plants, and used to produce propolis for their protection from microbes, viruses, and insects (Bankova et al. 2014; Kasote 2017). The word propolis is used in Ancient Greece as pro for in front of or at the entrance to and polis for city or community (Bulman et al. 2011). Since approximately 300 BC, human beings also recognized the therapeutic value of propolis. These ancient antimicrobial claims of propolis have been substantiated in recent studies, including its utility in treating MDR pathogens (Boukraâ and Sulaiman 2009; Onlen et al. 2007). In this book chapter, we have reviewed the antibacterial activity of different propolis samples against MDR pathogens have been reviewed, and emphasized their mode of action. In addition, recent information about the synergistic activity of propolis samples with various antibiotics against MDR pathogens has been summarized. Anti-biofilm formation and anti-quorum sensing activities of propolis samples in association with MRD pathogenesis have also been briefly discussed.

2

Propolis: A Natural Antibiotic

In several studies, extracts of different types of propolis were found to have promising activity against a range of pathogenic bacteria and fungi (Przybyłek and Karpiński 2019; Scazzocchio et al. 2006). Moreover, propolis extracts were also reported to enhance the efficacy of other antimicrobial drugs synthetically in numerous studies (Orsi et al. 2006; Stepanović et al. 2003). However, the antimicrobial potential of propolis predominantly depends on its chemical composition, which is highly complex and varies with the flora around beehives and, to some extent, on bee species (Smith 2019). In general, raw propolis consists of vegetable resin and balsam (4 mg/mL (MBC)

256 μg/mL (MIC)

11.3 μM (MIC)

1420 μg/mL (MIC)

Boisard et al. (2015) Nandre et al. (2021)

Astani et al. (2013)

Isla et al. (2012) Veiga et al. (2017) PamplonaZomenhan et al. (2011) da Cunha et al. (2020) Jingli and Feili (2008) Pepeljnjak and Kosalec (2004)

30 μg/mL (MIC) 246
3 μg/mL (MIC)

Reference Vera et al. (2011)

MIC/MBC/ZoI/MBL 65 ± 20 μg/mL (MIC)

286 D. M. Kasote et al.

Italy

Jamaica

Lebanon

Libya

Moroccan

Pacific region Palestine

9.

10.

11.

12.

13.

14.

15.

Iran

8.

Kirby-Bauer disk diffusion method

Agar dilution method

Disk diffusion method and cup cut diffusion Broth microdilution method

Kirby-Bauer disk diffusion method

Spot dilution assay

Broth microdilution method Microbroth dilution method

Broth microdilution method

2.5–5 mg/mL (MIC) 2.5–5 mg/mL (MBC) 1.25–5 mg/mL (MIC) 1.25–5 mg/mL (MBC) 0.31–2.5 mg/mL (MIC) 0.31–2.5 mg/mL (MBC) 1.25–5 mg/mL (MIC) 2.5–5 mg/mL (MBC)

0.36 mg/mL (MIC) 0.98–1.22 mg/mL (MBC) 8–32 mg/mL (MIC)

0.25–1.79- mg/mL (MIC) 12.5 mg/mL (MIC) 100 mg/mL (MBC) 6.25 mg/mL (MIC) 50 mg/mL (MBC) 1–7.5 mm (ZoI)

0.0143 mg/mL (MIC) 0.0286 mg/mL (MBC) 0.75 mg/mL (MIC) 1.5 mg/mL (MBC) 1000 μg/mL (MIC) 500 μg/mL (MBC) 125 μg/mL (MIC) >2000 μg/mL (MBC)

(continued)

Raghukumar et al. (2010) Daraghmeh and Imtara (2020)

El-Guendouz et al. (2018)

Shubar et al. (2018)

Williams et al. (2021) Chamandi et al. (2015)

Salami et al. (2016) De Marco et al. (2017)

Zeighampour et al. (2014)

Propolis: A Natural Antibiotic to Combat Multidrug-Resistant Bacteria

Streptococcus faecalis

Staphylococcus aureus

E. coli

P. aeruginosa

Ethanolic extract (70%)

Propolin C and D

S. aureus ATCC 6538 MRSA2, MRSA15, MRSA16, MRSA

MRSA

MRSA

Klebsiella pneumoniae

Imipenem-resistant Pseudomonas aeruginosa Drug-resistant biofilmforming Pseudomonas aeruginosa 21 Neisseria gonorrhoeae

Pseudomonas aeruginosa

Staphylococcus aureus

Ethanolic extract (70%)

Hexane and methanolic extract

Ethyl acetate extract and -(+)medicarpin Ethanolic extract (70%)

Ethanolic extract (85%)

Ethanolic extract (96%)

Ethanolic extract (90%)

12 287

Saudi Arabia Taiwan

Turkey

17.

18.

19.

Ethanolic extract

Ethanolic extract (95%)

Ethanolic extract (70%)

Extracts/components Ethanolic extract (absolute)

MRSA and VREF

S. aureus E. coli MRSA

Microorganism Acinetobacter baumannii Broth macro-dilution method Broth microdilution method Macrodilution broth method

Evaluation method Agar dilution method

Notes: MIC minimum inhibitory concentration, MBC minimum bactericidal concentration, ZoI zone of inhibition

Origin of propolis Pakistan

Sr. No. 16.

Table 12.1 (continued)

90%. In addition to the opportunistic pathogenic fungi, a limited number of fungi exist with a true pathogenic potential for healthy hosts and cause life-threatening infections (Groll and Walsh 2001). These pathogens, e.g., Histoplasma capsulatum, Paracoccidioides brasiliensis, Penicillium marneffei, and Coccidioides immitis, are therefore classified in biohazard class 3.

3

Current Antifungal Agents and Their Toxicity

The era of the antifungal drug effectively began with the discovery of amphotericin B (Amp B-deoxycholate) in 1958 by Squibb Laboratories, after developing orally bioavailable formulations of more than 200 polyene macrolides produced by the soil microorganisms (Actinomycete Streptomyces) (Dutcher 1968). Although Amp B was mainly used for the treatment of severe fungal infections for many years, due to adverse effects, mainly nephrotoxicity prompted the continued search for equally effective and less toxic alternatives. In the year 1990, the introduction of fluconazole showed effective for the treatment of oropharyngeal candidiasis in patients with AIDS. FLC quickly became one of the most widely prescribed antifungal agents for mucosal and systemic yeast infections. However, it showed a lack of antifungal activity against opportunistic molds (i.e., Aspergillus, Mucorales, and Fusarium species), and drug resistance among Candida species (C. glabrata and C. krusei) created a need for broader-spectrum alternative. Itraconazole (ITR; 1992) was a partial solution to the limitations of FLC because the drug had improved activity against endemic fungi and Aspergillus species. The introduction of the broaderspectrum new triazoles voriconazole (VOR; 2002) and posaconazole (POSA; 2006) transformed the management of invasive mold infections in severely immunocompromised patients. The antifungal drugs (VOR and POSA) were shown to be more effective than conventional Amp B for the treatment of invasive aspergillosis and fusariosis. The second-generation triazole (isavuconazole) showed a broad spectrum of activity against clinically important fungi and was approved in the USA and

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Polyenes (e.g. Amphotericin B)

Echinocandin (e.g. Caspofungin)

Candida spp. Cryptococcus spp. Aspergillus spp. etc.

Candida spp. Aspergillus spp.,

Azoles (e.g., Fluconazole)

etc.,

Others (e.g. Ciclopirox)

Antifungals and their applications

Candida spp. Cryptococcus spp. Aspergillus spp. etc.

Nucleoside Analog (Fluorocytosine) Candida spp. Cryptococcus spp. Aspergillus spp. etc.,

Candida spp., Dermatophytes,

Allylamine (e.g., Terbinafine) Few Candida spp. Dermatophytes, Aspergillus spp. etc.,

Fig. 13.1 Classification of antifungal agents and their activity against different human fungal pathogens

Europe. It is available in both intravenous and oral formulations and is used for the treatment of invasive aspergillosis and mucormycosis. The commonly reported side effect among isavuconazole recipients were gastrointestinal disorders (such as diarrhea, vomiting, and nausea). However, isavuconazole has several advantages as a new treatment option for invasive mold infections such as bioavailability, predictable pharmacokinetics, and no cross-reaction of oral formulation in the gastrointestinal tract (Ellsworth and Zeichner 2020) (Fig. 13.1) (Table 13.2). The echinocandins (caspofungin, micafungin, and anidulafungin) are semisynthetic lipopeptides that inhibit fungal growth by inhibiting the synthesis of β-1, 3-D-glucan synthase in susceptible fungi such as Candida spp. This enzyme is responsible for the formation of the fungal cell wall. The echinocandins have less toxic to mammalian cells due to the lack of glucan-rich cell walls in mammalian cells. These agents were predicted to be effective antifungal agents with very little collateral toxicity in mammalian cells which has been proven true in clinical trials of patients with invasive candidiasis and which the antifungal agents demonstrate efficacy. However, echinocandins still lack activity against some common opportunistic yeast (Cryptococcus species) and less common molds (i.e., Fusarium,

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Table 13.2 List of common antifungal drugs and their spectrum of activity and toxicity Class Azoles (fluconazole, itraconazole, posaconazole, voriconazole, ketoconazole, clotrimazole, miconazole, etc.) Polyenes (amphotericin B, nystatin, natamycin, etc.)

Echinocandins (caspofungin, micafungin, anidulafungin, etc. Nucleoside analogue 5-Flucytosine Griseofulvin

Fungal pathogens Yeasts Molds Antifungal Antifungal activity activity (fluconazole resistant against Candida glabrata) Antifungal activity

Antifungal activity

Antifungal activity (amphotericin B resistant against Mucorales, Fusarium species) No antifungal activity

Antifungal resistant

No antifungal activity

Dimorphic Antifungal activity

Antifungal activity

No antifungal activity No antifungal activity

Mode of action Cell membrane permeability (ergosterol biosynthesis pathway by targeting 14α-lanosterol demethylase) Cell membrane permeability (directly bind to ergosterol and leads to loss of membrane integrity) Inhibit cell wall components (inhibitors of glucan synthase) Intracellular inhibition of nucleic acids and cell cycle

Scedosporium, and Mucorales) and are less effective in the case of Aspergillus and Fusarium species. The scientific community continuously putting efforts underway to reformulate the POSA suspension into better oral and intravenous formulations could address many of the drug’s pharmacokinetic shortcomings. The significant milestone of antifungal drug discovery in the twentieth century was the identification and development of the echinocandin class of antifungal agents (Table 13.3). The toxicity profiles of antifungal drugs also play a major role in the treatment of fungal diseases. The systemic antifungal agents (Amp B) showed nephrotoxicity, and triazoles showed hepatotoxicity and also longer-term risks, inclusion malignancies. The oral itraconazole formulation can cause nausea and gastrointestinal (GI) problems. The itraconazole has also been described as causing a unique triad of hypertension, hypokalemia, and edema that may be related to the effect of the drug or adrenal suppression. Although the rash is reported with all antifungal classes in 5–15% of patients, VOR treatments in ambulatory patients have been associated with retinoid-like phototoxic problems. From a clinical perspective, drug resistance may be defined as the persistence or progression of infection despite appropriate drug therapy. The clinical outcome of treatment depends not only on the susceptibility of the pathogen to a given drug but also on many other factors including pharmacokinetics, drug interactions, immune status, and patient compliance, as well as several specific conditions such as the

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Table 13.3 Different classes of antifungals and their cytotoxicity against human Host toxicity Fever, anaphylaxis, nausea, gastrointestinal disturbance, phototoxicity, cardiomyopathy, chill, vomiting, headache, etc. Nephrotoxicity, anemia, toxicity, etc. Dark urine color, hepatic toxicity, etc. Gastrointestinal disturbance, anemia associated with decreased epoetin production, etc. Rash, photosensitivity, etc. Hepatic toxicity, rash, etc.

Class of antifungal drugs Azoles (fluconazole, itraconazole, posaconazole, voriconazole, ketoconazole, clotrimazole, miconazole, etc.) Polyene (amphotericin B, nystatin, natamycin, etc.) Echinocandin (caspofungin, micafungin, anidulafungin, etc.) Nucleoside analogues (flucytosine, griseofulvin, etc.) Allylamines (naftifine, terbinafine, tolnaftate, etc.) Others (aurones, ciclopirox, haloprogin, miltefosine, orotomide, etc.)

occurrence of biofilms on surfaces of catheters and prosthetic valves. Antifungal drug resistance has been classified as either primary, when a fungus is resistant to a drug before any exposure, or secondary when an initially sensitive fungus becomes resistant after exposure to the drug (Cannon et al. 2009).

4

Himalayan Medicinal Plants and Their Therapeutic Potential

The Himalayan region is one of the 17 mega-biodiversity countries in the world. The Himalayan biodiversities include India, Nepal, Bhutan, and Tibet. The medicinal plants of the Himalayan region have a diverse group of bioactive compounds with unique chemical compositions (Dias and Urban 2012). These properties of the flora are due to the great variation in climate and geographical habitat to be found in this region. There are found more than 8000 species of plants of which ~1748 are known for their medicinal properties in Himalayan regions (Hamilton and Radford 2007) (Table 13.4). In India, there are ~17,000 species of higher plants, and ~ 7500 are known for medicinal uses. So far, about ~8000 species of angiosperms, ~44 species of gymnosperm, and ~ 600 species of pteridophytes have been reported in the Indian Himalayas, of these 1748 species are known as medicinal plants (Kala et al. 2006). The Indian Himalaya is divided into three regions: (1) North-Western Himalaya; (2) Western Himalaya; and (3) Eastern Himalaya. About 25% of modern medicine is of plant origin, many of which have been derived from Himalayan medicinal plants (Bhutani and Gohil 2010). There are various parts of the medicinal

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Table 13.4 List of selected Himalayan medicinal plants having antifungal properties Medicinal plants/family Amomum subulatum Roxb. (Zingiberaceae) Artemisia indica Wild. (Asteraceae) Blumea lacera (Burm. F.) DC. (Asteraceae) Cassia fistula L. (Fabaceae) Chaerophyllum villosum Wall. Ex DC (Apiaceae) Cinnamomum camphora (L.) J. Presl. (Lauraceae) Cinnamomum glaucescens Hand- Mazz (Lauraceae) Curcuma longa L. (Zingiberaceae) Juniperus communis L. (Cupressaceae)

Matricaria recutita L. (Asteraceae) Mentha spicata L. (Lamiaceae) Morina longifolia Wall. Ex DC (Caprifoliaceae) Nardostachys grandiflora DC. (Caprifoliaceae) Pinus roxburghii Sarg. (Pinaceae) Tanacetum nubigenum Wall. Ex DC. (Asteraceae) Tanacetum longifolium Wall. (Asteraceae) Thymus linearis Benth. (Lamiaceae)

Antifungal activity (Reference) Aspergillus Niger (Satyal et al. 2012a) A. niger (Rashid et al. 2013) C. albicans, A. niger (Satyal et al. 2015a) C. albicans, A. niger (Satyal et al. 2012b) C. albicans, C. glabrata (Joshi 2013a) A. niger (Satyal et al. 2013a) A. niger (Prakash et al. 2013; Satyal et al. 2013a) A. niger (Essien et al. 2015) C. albicans, C. kefyr, Trichophyton mentagrophytes, T. rubrum (Pepeljnjak et al. 2005) C. albicans (Satyal et al. 2015b) A. flavus, Rhizopus solani (El Menyiy et al. 2022) Alternaria alternate, A. flavus, A. fumigates, Fusarium solani (Kumar et al. 2013) C. albicans (Joshi et al. 2016) A. niger (Satyal et al. 2013b) C. albicans, A. flavus (Haider et al. 2015) C. albicans, C. glabrata (Joshi 2013b) T. rubrum (Joshi et al. 2016)

plants used for the treatment of human diseases (Newman and Cragg 2007) (Fig. 13.2). According to the World Health Organization (WHO), 80% of people still rely on plant-based traditional medicine for primary healthcare (Ekor 2014; Farnsworth et al. 1985). At present, there are 125 clinically useful drugs of a known constitution which have been isolated from about 100 species of high plants. It has been estimated that about 5000 plant species have been studied in detail as a possible source of new drugs (Katiyar et al. 2012). The bioactive secondary metabolites such as alkaloids, phenolics, essential oils and terpenes, sterols, flavonoids, lignins, tannins, etc. are present in different parts of medicinal plants (Fig. 13.3) (Ramawat et al. 2009). It is estimated that 60% of anti-infectious drugs already on the market or undergoing clinical trials are of natural origin (Cragg and Newman 2005; Newman and Cragg 2020). The secondary metabolites are produced either as a result of the organism adapting to its surrounding environment or as an act of a possible defense

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Whole Plant Flower Root Leaf Fruit Stem Seed Bulb Bark 0

5

10

15

20

25

30

35

40

Fig. 13.2 Percentage of different medicinal plant parts used in the preparation of medicines Fig. 13.3 Percentage of medicinal plants used for medicine preparation

Climb Tree (5%) (10%)

Shrubs ( 15%)

Herbs (85% )

mechanism against predators. The biosynthesis of secondary metabolites is derived from the photosynthesis, glycolysis, and the Krebs cycle intermediates (Dias and Urban 2012). The route of administration of medicinal plants is commonly oral (70%), skin (25%), and nasal and other (5%) (Fig. 13.4).

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Fig. 13.4 The delivery method of medicinal plants

80 60 40 20 0 Oral

5

Dermal

Nasal and Others

Conclusion

The medicinal plants have been a significant source of a variety of bioactive compounds for many centuries and are largely used as crude as well as a purified form for treating several human diseases. Medicinal plant products have been known for many decades as a rich source of therapeutic agents for the treatment of pathogenic microbial infections. The Himalayan regions contain a diverse group of bioactive compounds with unique biological properties due to significant variations in climate and habitat. These Himalayan medicinal plants played a vital role in the discovery of new therapeutic agents for fungal pathogens including yeasts, molds, and dimorphic. In recent years, there is a growing need for medicinal plant products and other traditional remedies for the treatment of diseases caused by fungal pathogens to immunocompromised patients globally. Notably, these medicinal plants are several benefits such as being easily available, cost-effective, and less side effects. In the future, a detailed study of medicinal plants and their antifungal screening, purification of bioactive compounds, and chemical characterizations are needed for the development of potent bioactive agents which may be used as antifungal therapy against fungal pathogens including multidrug-resistant strains. Acknowledgments IKM greatly acknowledged CSIR, New Delhi, India for the CSIR-SRA fellowship.

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Antimicrobial Stewardship Programme: Why Is It Needed?

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Mohd Younis Rather, Ajaz Ahmad Waza, Yasmeena Hassan, Sabhiya Majid, Samina Farhat, and Mohammad Hayat Bhat

Abstract

Antibiotics have transformed medical practice by making previously lethal infections treatable and allowing for subsequent medical advancements such as cancer treatment and organ transplants. Antibiotics, like other medications, can have serious adverse effects; around 20% of hospitalised patients who get antibiotics develop severe complications. Patients who receive too many antibiotics run the risk of experiencing these unfavourable side effects with no additional benefit. Antibiotic medication research is sluggish, yet antimicrobial resistance is rising. Antimicrobial stewardship is more crucial than ever for maximising antibiotic usage to reduce the establishment of resistance and improve patient outcomes. Antibiotic-resistant strains of nosocomial infections are caused by overuse of antibiotics, especially wide-spectrum ones, and M. Y. Rather (✉) · A. A. Waza Multidisciplinary Research Unit, Department of Health Research, Government Medical College Srinagar, Srinagar, J&K, India Y. Hassan Mader-e-Meharban Institute of Nursing Sciences & Research (MMINSR), Sher-I-Kashmir Institute of Medical Sciences (SKIMS), Soura, Srinagar, J&K, India S. Majid Multidisciplinary Research Unit, Department of Health Research, Government Medical College Srinagar, Srinagar, J&K, India Department of Biochemistry, Government Medical College Srinagar, Srinagar, J&K, India S. Farhat Postgraduate Department of Pharmacology, Government Medical College Srinagar, Srinagar, J&K, India M. H. Bhat Department of Endocrinology, Government Medical College Srinagar, Srinagar, J&K, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 M. Y. Wani, A. Ahmad (eds.), Non-traditional Approaches to Combat Antimicrobial Drug Resistance, https://doi.org/10.1007/978-981-19-9167-7_14

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noncompliance with infection prevention and control procedures in hospital settings. Every year, illnesses brought on by microorganisms resistant to antibiotics claim thousands of lives. The antimicrobial stewardship programme (ASP) is defined as “the appropriate selection, dose, and duration of antimicrobial therapy for improved clinical outcome for the treatment or prevention of illness”. Antimicrobial stewardship has three objectives. The prevention of Clostridium difficile infection and the management of antibiotic confrontation are the main long-term objectives of antimicrobial stewardship. Antimicrobial resistant infections contributed to around 5 million deaths globally in 2019 and this number is believed to rise to ten million by 2050. Unfortunately, few institutions provide information technology people and financial support for pharmacists and doctors who specialise in infectious diseases, and most health systems lack adequate infrastructure to support antimicrobial stewardship. Making an institution’s antimicrobial stewardship programme successful requires strong leadership, dedication, and support from hospital administration. Keywords

Antimicrobial stewardship · Antibiotics · Antimicrobial resistance

1

Introduction

2

Antibiotic Resistance

Microorganism growth inhibition can be caused by a wide variety of substances, and these substances have been categorised using a wide range of terms. The phrases “antimicrobials”, “antibacterial”, and “antibiotics” are three that are frequently interchanged (Hall and Mah 2017). Antibiotics are compounds made by microbes that work against other germs. In a strict sense, substances created through chemical or biochemical synthesis are not considered antibiotics. Contrarily, “antimicrobial” refers to any substances that work against all varieties of microorganisms, including parasites, fungi, viruses, and bacteria (antibacterial, antiviral, and antifungal) (antiparasitics). The inability of a bacterium to be treated with an antimicrobial medication that was previously effective in treating diseases caused by that bacterium is referred to as antimicrobial resistance (AMR). Antimicrobial medications, such as antibiotics, antifungals, antivirals, and antimalarials, are unable to effectively treat resistant microorganisms, causing illnesses to persist and pose a greater risk of spreading to others. Resistant strain evolution is a natural phenomenon that happens when bacteria reproduce incorrectly or when resistant features are shared between them (Davies and Davies 2010). The widespread expenditure of antibiotics in veterinary, the healthcare, and agricultural industries have created and continue to create a significant selection pressure for resistant microorganisms (Davies and

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Davies 2010). Any phenomenon that modifies the behaviour and fitness of living creatures within a specific environment is referred to as selective pressure (the driving force of evolution and natural selection). Antibiotic overuse has also resulted in a buildup of these medications in various habitats, allowing antibiotic-resistant microorganisms to thrive. This has also led in the selection and spread of bacteria resistant to a variety of antibiotics (Davies and Davies 2010). Since the development of antibiotics, there have been 70% fewer deaths from bacterial infections. However, bacteria have developed strong defences against antibiotic exposure. A lot of microorganisms are naturally resistant to many antibiotics in addition to acquired resistance. As a result, antibiotic-resistant pathogens cause at least 700,000 fatalities annually worldwide (Lade and Kim 2021). In accordance with the World Health Organization (WHO), one of the top ten global health concerns confronting humanity is antibiotic resistance (Cantón and Morosini 2011). Because only a tiny number of new antibiotics with resistance-breaking capabilities are currently being found, and even fewer amalgams are efficaciously inflowing the arcade, the situation is improbable to alter dramatically in the calculable future. According to the Wellcome Trust’s analysis of AMR, more than ten million people could die each year from MDR by the year 2050 (Hasan and Yang 2019).

3

Mechanism of Antibiotic Action

Antibiotics work by interfering with crucial bacterial cell processes or structures (Lade and Kim 2021) (Fig. 14.1). This either eliminates the organism or inhibits its growth. An antibiotic is referred to either bactericidal or bacteriostatic based on these outcomes. Antibiotics are divided into various classes (Lade and Kim 2021). These may target entirely different strains of bacteria or may affect the same target at a different site. In general, bacteria have three primary antibiotic targets: 1. The membranes or cell walls that encircle the bacterial cell 2. The equipment used to create DNA and RNA, which are nucleic acids 3. The apparatus responsible for protein synthesis (the ribosome and related proteins) Since these targets are missing or changed in the cells of humans and other mammals, antibiotics typically do not damage our human cells and instead are targeted only against bacteria (Lade and Kim 2021).

4

Emergence of Antibiotic Resistance

The formation of resistance in our microbiota is the particular phenomenon connected to antibiotics. The phrase “gut microbiota” denotes to the collection of all bugs that squat the gastrointestinal system, including bacteria, viruses, and

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Fig. 14.1 The primary targets of antibiotics in Staphylococcus aureus. (Figure adapted from reference (Lade and Kim 2021))

eukaryotes. More than 3–5 million distinct genes are thought to be present in the microbiome, the collective genome of the gut bacteria, which is more than a hundred times larger than the genome of the human body (Cantón and Morosini 2011). Even when administered appropriately, antibiotics can cause the gut microbiota to shift into a condition known as dysbiosis, which is characterised by a number of variables such as a loss of diversity, changes in metabolic capacity, and lower colonisation resistance against invading pathogens (Hasan and Yang 2019). The impact of excessive and inappropriate use, such as the use of wide-spectrum antibiotics, is stronger on dysbiosis, which encourages the horizontal transfer of resistance genes and supports the advent of antibiotic-resistant germs. Pumping the antibiotic out of the bacterial cell and generating chemicals that can degrade the antibiotic are two frequent strategies to build antibiotic resistance (Munita and Arias 2016). Healthcare expenses, hospital stay times, morbidity, and mortality are all on the rise as a result of AMR, in both developed and developing nations. Only resistant, or non-susceptible, bacteria will survive or at the very least proliferate more quickly than susceptible bacteria and grow in quantity when the antibiotic is present (Munita and Arias 2016). Clinical resistance is the ability of a bacterium to grow in the body at the antibiotic doses attained during treatment, most frequently resulting in treatment failure. There are two more ways for bacteria to develop all sorts of resistance:

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• Resistance might happen by coincidence due to alterations in the bacterial DNA. • As an alternative, they can get resistance genes from adjacent bacteria. The term “horizontal gene transfer” refers to this action. The current resistance environment necessitates a quick and collaborative approach by the scientific community, doctors, pharmacologists, legislators, farmers, and, not less importantly, the culture. Additionally, by improving communication strategies regarding the prevalence and dangers of antimicrobial resistance (AMR), the mechanisms underlying the transfer and spread of multidrug-resistant (MDR) pathogens, and the proper and deliberate use of antibiotics, we can further raise civic cognizance of the importance of antibiotics. While it is necessary to consider all such measures and halting the spread of AMR is a critical challenge for the now and the forthcoming, it is insufficient to stop the looming antibiotic shortage. In order to combat such a scenario on a worldwide scale, new antimicrobial techniques—including novel antibiotics as well as alternative measures like antivirulence drugs, vaccines, etc.—must be researched and developed. In face of the fact that governmental and public sentience of AMR is expanding and that various studies have been initiated to explore innovative techniques to tackle AMR, the activities of gigantic therapeutic firms clearly contradict this inclination. This is correspondingly proven by the datum that fewer innovative and ground-breaking antimicrobial drugs are being presented to the market, and beholding at the existing pipelines for antibiotic R&D, it is evident that the “golden era of antibiotic discovery” has long ago conceded. Shoddier, there is a danger that the antibiotic supply will run out (Wright 2007). Despite Big Pharma’s progressive loss in interest in antibiotic R&D, contemporary research exertions in academia and the commercial segment show that there are still new lead combinations with remarkable antibacterial characteristics to uncover. For example, a lot of antibacterial medications now in medical trials fulfil wholly conditions for innovation, such as having a distinct chemical class, aim, and mechanism of feat, in addition to no cross-resistance (Wright 2007). Enterococcus spp., S. aureus, K. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. have all become more and more engaged in infectious disorders in humans (Kapoor et al. 2017). The WHO recently designated them as precedence antibiotic-resistant bacteria to escort research, discovery, and expansion of novel medicines, giving them the collective moniker ESKAPE and drawing even greater worldwide attention to them.

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Drivers for Resistance

Antimicrobial resistance is prevalent, has evolved against all classes of antimicrobial medicine, and appears to be extending into new therapeutic niches around the turn of the century. The following factors are likely to have an impact on the epidemiology and health effects of future antibiotic-resistant infections: antibiotic overuse and misuse, which hastens the advent of drug-resistant strains; meagre infection control

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procedures; unsanitary settings; indecorous food management; poverty; the absence or inadequacy of diagnostic tests; antibiotic overuse and misuse in agriculture and the environment; and travel. Different classes of antibiotics have various modes of action and forms of resistance (Table 14.1) (Wright 2007).

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Clinical and Economic Consequences of Antibiotic Resistance

Drug-resistant infections were previously associated with hospital settings, but in the last 10 years, resistant infections have been identified in the general community, including long-term care facilities for the elderly (Ventola 2015). According to studies, ESKAPE bacteria that are resistant have been linked to high hospital charges and higher healthcare costs (Founou et al. 2017). Coronavirus disease 2019 (COVID-19) may be a factor in the rise in AMR rates for specific individuals, as well as at institutional and regional levels (Clancy and Nguyen 2020). We risk losing the enormous progress we have made over the past century as a result of rising resistance. This includes: • Our battle against infectious diseases that might be fatal, like pneumonia. • In the fight against diseases like cancer, where antibiotics are essential for preventing and combating infection in chemotherapy patients. • We have achieved great advances in surgical operations such as organ transplants and caesarean deliveries as a result of our capacity to successfully prevent or cure acute infections using antibiotics (Table 14.2).

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Stewardship a Key to Combat the Antibiotic Resistance

The misuse of antimicrobials in human medicine, both in the community and in the clinical context, as well as its overuse in animals, is known as antimicrobial resistance (AMR). However, a comprehensive strategy necessitates the development of novel diagnostic and treatment approaches, as well as the improvement of surveillance and infection control procedures. Antimicrobial stewardship programmes (ASPs) are among the most strongly advised ways to stop the spread of AMR in this situation (Martínez et al. 2020). A more clinical description that may be beneficial and interesting for clinicians is the appropriate antibiotic for the right patient, at the right time, with the right dose, and with the least amount of risk to the patient and future patients (Probst et al. 2021). Antibiotic stewardship programmes (ASPs) are made to enhance patient outcomes, optimise antibiotic treatment, and reduce adverse effects (Doron and Davidson 2011). The idea of antimicrobial stewardship (AMS) is not new. Dr. Dale Gerding coined the term “antibiotic stewardship” in the 1980s to convey the idea of reasonable antibiotic prescribing (Probst et al. 2021). It can be summed up as the ideal antimicrobial choice, dosage, and duration for treating or

Table 14.1 Antibiotic resistance mechanisms that are commonplace. (Table adapted from reference (Kapoor et al. 2017))

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Country Colombia India Senegal Thailand Thailand Thailand Thailand Turkey

WHO region Americas South East Asia Africa South East Asia South East Asia South East Asia South East Asia Europe

World Bank classification Upper middle income Upper middle income Low income Upper middle income Upper middle income Upper middle income Upper middle income Upper middle income

Settings Tertiary hospital Tertiary hospital Hospital University hospital University hospital University hospital University hospital University hospital

Follow-up period 30 days NR NR 34 days 43 days NR NR 28 days

Overall healthcare costs Case group Control group 11,822 USD 7178 USD 1478 USD 790 USD 228 USD 122 USD 935 USD 122 USD 615 USD 214 USD 2731 USD 1199 USD 11,773 USD 7797.9 USD 35,277 USD 26,333 USD

Table 14.2 Statistics summary on the price of healthcare due to resistant infections (MacDougall and Polk 2005) p-value