Drug Discovery Targeting Drug-Resistant Bacteria [1 ed.] 0128184809, 9780128184806

Table of contents :
Chapter 1 - Antibiotics: past, present, and future
1 - Introduction
1.1 - History and discovery of antimicrobials
2 - Current status
2.1 - Dwindling drug discovery pipeline: no new class of antibiotics from the last three decades
2.2 - Pharmaceutical companies dropped out the research and development of antibiotics
3 - Solution to the problem: what can be done?
3.1 - A surveillance system to monitor the escalating antimicrobial resistance
3.2 - One Health approach
3.3 - Introducing new chemical entities and managing current antibiotics
References
CHAPTER 2 - Mechanisms of antibacterial drug resistance and approaches to overcome
1. Introduction
2. Mechanisms of antibacterial drug action
2.1 Interference with the cell-wall synthesis
2.2 Inhibition of protein synthesis
2.3 Interference with nucleic acid synthesis
2.3.1 Inhibit DNA synthesis
2.3.2 Inhibit RNA synthesis
2.4 Inhibition of metabolic pathways of essential metabolites such as the folic acid
2.5 Disruption and increased permeability of cytoplasmic membrane structure
3. Mechanisms of developing antibacterial resistance
3.1 Modification of the antibiotic molecule
3.2 Destruction of the antibiotic molecule
3.3 Decreased antibiotic penetration and efflux
3.3.1 Decreased permeability
3.3.2 Efflux pumps
3.4 Changes in target sites
3.4.1 Target protection
3.4.2 Modification of the target site
3.4.2.1 Mutations lead to alteration of the target site
3.4.2.2 Alteration of the target site by enzymes
3.4.2.3 Change or bypass of the target site
3.5 Resistance by global cell adaptations
4. Mechanisms of the spread of antibacterial resistance
4.1 Genetic basis and mechanisms of spreading antibacterial resistance
4.2 Emergence of antibacterial resistance
5. The most harmful multidrug-resistant strains
6. New antibacterial drug targets and novel approaches to drug development
7. Conclusion
Acknowledgments
References
Chapter 3 - Antibiotics targeting Gram-negative bacteria
1 - Gram-negative infections: the unmet need
1.1 - Mechanisms of resistance and their epidemiology
1.1.1 - Resistance to carbapenems
1.1.2 - Resistance to polymyxins
2 - Combating multidrug resistance with new drugs
2.1 - Compounds with novel targets
2.2 - Combinations of approved antibiotics with partners that enhance the activity of the antibiotic
2.2.1 - SPR741 combination
2.2.2 - β-Lactam and β-lactamase inhibitors
2.2.3 - Imipenem/cilastin + relebactam
2.2.4 - Sulbactam + ETX2514
2.2.5 - Meropenem + nacubactam
2.2.6 - Cefepime + zidebactam (WCK-5222)
2.2.7 - ETX0282 cefpodoxime proxetil
2.2.8 - Cefepime + VNRX-5133
2.2.9 - Cefepime + AAI101
2.3 - Analogs of existing classes of antibiotics (cefiderocol, tetracycline, aminoglycoside, and polymyxin)
2.3.1 - Compounds in clinical development
3 - What the future holds
References
Chapter 4 - Recent development of antibacterial agents to combat drug-resistant Gram-positive bacteria
1 - Introduction
2 - Antibacterial drugs under clinical development
3 - Nature-derived antibiotics
3.1 - Antibiotics discovered from the soil microbiome
3.1.1 - Teixobactin
3.1.2 - Malacidins
3.1.3 - Lysocins
3.2 - Antibiotics discovered from human microbiome
4 - Semisynthetic antibiotics
4.1 - Semisynthetic β-lactams
4.2 - Semisynthetic tetracyclines
4.3 - Semisynthetic macrolides
4.4 - Semisynthetic glycopeptides
5 - Synthetic antibacterial agents
5.1 - Synthetic antibacterial agents derived from screening process
5.1.1 - Retinoids
5.1.2 - Oxadiazoles
5.2 - Synthetic antibacterial agents inspired from antibacterial peptides
5.2.1 - Small molecular antibacterial peptide mimics
5.2.2 - Macromolecular antibacterial peptide mimics
6 - Conclusion and future prospects
References
Chapter 5 - Repurposing nonantibiotic drugs as antibacterials
1 - Introduction
2 - Ibuprofen
3 - Ethyl bromopyruvate
4 - Disulfiram
5 - Diphenyleneiodonium chloride
6 - Ivacaftor
7 - Ebselen
8 - Niclosamide
9 - Loperamide
10 - Antidepressants
11 - Sertraline
12 - Amitriptyline
13 - Anti-virulence agents
14 - Pentetic acid
15 - Diflunisal
16 - Metformin
17 - Auranofin
18 - Floxuridine
19 - Gemcitabine
20 - Streptozotocin
21 - Statins
22 - Nicotinamide
23 - Dexamethasone
24 - Celecoxib
25 - Pentamidine
26 - Zidovudine
27 - Ciclopirox
28 - Gallium compounds
29 - 5-Fluorouracil
30 - Conclusion
Acknowledgments
References
Further readings
Chapter 6 - Drugs against Mycobacterium tuberculosis
List of abbreviations
1 - Introduction
2 - Tuberculosis drug discovery and development of treatment regimens
3 - Intrinsic and acquired drug resistance in Mycobacterium tuberculosis
3.1 - Mechanisms of resistance against first-line drugs
3.2 - Mechanism of resistance against second-line drugs
4 - Drug susceptibility testing and its importance
5 - Drugs currently under development for tuberculosis
6 - Host-directed therapies for treating tuberculosis
7 - Conclusion
References
Chapter 7 - Combating biothreat pathogens: ongoing efforts for countermeasure development and unique challenges
Abbreviations
1 - Introduction
2 - Biothreat agents
2.1 - Bacterial biothreat agents
2.2 - Viral biothreat agents
3 - Screening strategies to identify therapeutics against biothreat agents
3.1 - High-throughput screening approaches to identify therapeutics against bacterial agents
3.2 - Screening platforms for biothreat viral agents
3.3 - Identification of host factors required for pathogen replication through knowledge-based or multiomics screening
4 - Development of countermeasures to biothreat agents
4.1 - Host-directed therapy
4.2 - Antibody therapy
4.3 - Antiviral medical countermeasures
4.4 - Combination therapies
5 - Unique preclinical challenges
6 - Clinical trials and the animal rule
7 - Summary and conclusion
References
Chapter 8 - New approaches to antibacterial drug discovery
1 - Introduction
2 - Natural products and drug discovery
2.1 - Innovative drug discovery methods from natural products
2.2 - Natural products diversification for drug discovery
2.2.1 - Chemical investigation of unexplored natural sources
2.2.1.1 - Activation of biosynthetic gene clusters
2.2.1.2 - Light-driven structure diversification
2.2.2 - Biological approaches
2.2.3 - Synthetic chemistry approaches
3 - Target-based drug discovery
3.1 - Peptide-based drug discovery: reverse pharmacology approach and phage display libraries
3.2 - Antibody-based drug discovery
3.3 - Fragment-based drug discovery
4 - Quorum sensing and drug discovery
4.1 - Quorum sensing inhibition mechanisms
4.2 - The antipathogenic effect of quorum sensing inhibitors
5 - Automation in molecule design
5.1 - Automated de novo design
5.2 - Molecular docking
5.3 - Microfluids-based synthesis
6 - Biosensors role in drug discovery
7 - Stem cells role in drug discovery
8 - Conclusion
References
Chapter 9 - New strategies and targets for antibacterial discovery
1 - Antimicrobial discovery strategies: a historical perspective
2 - New approaches to mining novel natural compounds
2.1 - Culturing the unculturable
2.2 - Awakening silent or cryptic secondary metabolic pathways
2.2.1 - Genome mining
2.2.2 - Metagenomics-guided antimicrobial discovery
2.2.3 - Ribosome engineering
2.2.4 - One strain-many compounds approach
2.2.5 - Modifying regulatory elements or promoter regions
2.2.6 - Heterologous expression in engineered hosts
2.2.7 - Combined-culture methods
3 - Beyond traditional antibiotics
3.1 - Antimicrobial peptides
3.2 - Bacteriophages
3.3 - Nanoparticles
4 - Antimicrobial targets
4.1 - New target identification strategies
4.2 - Novel antimicrobial targets
4.2.1 - Targeting virulence
4.2.2 - Preventing microbial communication
4.2.3 - Targeting two-component signal transduction systems
4.2.4 - Efflux pumps
4.2.5 - Fatty acid biosynthesis pathways as antimicrobial targets
4.2.6 - Aminoacyl-transfer RNA synthases
5 - Summary
References
Chapter 10 - Importance of efflux pumps in subjugating antibiotic resistance
List of abbreviations
1 - Introduction
1.1 - Mechanism of antibiotic resistance
1.2 - Development of antibiotic resistance
2 - Bacterial efflux system
2.1 - Types of resistance
3 - Efflux pump-mediated multidrug resistance
3.1 - β-Lactams (extended spectrum β-lactamase)
3.2 - Metallo β-lactamase
3.3 - AmpC β-lactamase
4 - Integrons
5 - Other antibiotic resistance genes
6 - Resistance mechanisms in Escherichia coli
6.1 β-Lactams
6.2 Aminoglycosides
6.3 Trimethoprim
6.4 Fluoroquinolones
6.5 Sulfonamides
7 - Families of efflux pumps
8 - Types of efflux pump inhibitors based on their mechanism of action
8.1 Energy dissipation
8.2 Inhibition by direct binding
9 - Types of efflux pump inhibitors based on their origin
9.1 - Plant-derived efflux pump inhibitors
9.2 - Efflux pump inhibitors of synthetic origin
9.3 - Efflux pump inhibitors derived from microbes
10 - Overcoming efflux-mediated antimicrobial resistance
11 - Efflux pumps of important bacteria
11.1 Staphylococcus aureus
11.2 Escherichia coli
11.3 Pseudomonas aeruginosa
12 - Regulation of multidrug efflux pumps
12.1 Significance of inhibitors as novel therapeutic agents
12.2 Current challenges for efflux pump inhibitors as therapeutic agents
13 - Future studies
14 - Conclusion
References
Chapter 11 - Phage therapy—bacteriophage and phage-derived products as anti-infective drugs
1 - Introduction
2 - Lysins—an overview
3 - Development path for lysin drugs
3.1 - Stage 1: identification of lysins
3.1.1 - Design, construction, and characterization of chimeric lysin P128
3.1.1.1 - Identification of ectolysin ORF56 from phage K
3.1.1.2 - Construction of a potent antistaphylococcal chimeric ectolysin
3.1.1.3 - Mechanism of action of P128
3.2 - Stage 2: lysin characterization
3.2.1 - Assessment, spectrum, and preclinical testing of P128
3.2.1.1 - Assessment of the antimicrobial activity of P128
3.2.1.2 - Spectrum of P128 activity
3.2.1.3 - Preclinical efficacy and safety testing of P128
3.3 - Stage 3: lysin manufacture
3.3.1 - The chemistry, manufacturing, and controls for lysin P128
3.3.1.1 - Overview
3.3.1.2 - Manufacturing process development
3.3.1.3 - Purification process
3.3.1.4 - Analytical methods and specifications
3.4 - Stage 4: clinical development
3.4.1 - Preclinical studies: pharmacology/toxicology
3.4.2 - Clinical development
3.4.2.1 - Defining target medical condition
3.4.2.2 - Target product profile
3.4.2.3 - Target product profile for the systemic use of P128
4 - Conclusion
5 - Summary
Acknowledgments
References
Chapter 12 - Drug discovery targeting drug-resistant nontuberculous mycobacteria
1 - The need for drug discovery targeting drug-resistant nontuberculous mycobacteria
1.1 - Innate resistance of nontuberculous mycobacteria to antimicrobials
1.2 - Challenges of nontuberculous mycobacteria drug development
1.3 - New compounds tackling drug resistance
1.4 - New targets for compound development
1.5 - Existing antimicrobials being repurposed for nontuberculous mycobacteria treatment
1.6 - Therapeutic vaccine approaches
References
Chapter 13 - New strategies to combat drug resistance in bacteria
Appendices and Nomenclatures
1 - Introduction
1.1 - Development of antimicrobial drugs
1.2 - Spread of antibiotic resistance
1.3 - Types of antibiotic resistance
1.4 - Mechanisms of multidrug resistance
1.5 - Antibiotics in the management of infections due to Gram-negative bacteria
2 - Combating drug resistance
2.1 - Antibiotic research in the age of omics
2.1.1 - Transcriptomics
2.1.2 - Proteomics
2.1.3 - Genomics
2.2 - Bioinformatics and high-throughput screening for antibiotics discovery
2.2.1 - Genomics-based applications
2.3 - Synthesis of nanomaterials
2.4 - Phage therapy
2.5 - Molecular targets and strategies to combat drug resistance in bacteria
2.5.1 - Targeting essential processes
2.5.2 - Targeting nonessential processes
3 - Conclusion and future perspectives
References

Citation preview

DRUG DISCOVERY TARGETING DRUGRESISTANT BACTERIA Edited by

PRASHANT KESHARWANI Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India

SIDHARTH CHOPRA Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India

ARUNAVA DASGUPTA Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2020 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www. elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-818480-6 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Andre Gerhard Wolff Acquisitions Editor: Erin Hill-Parks Editorial Project Manager: Timothy Bennett Production Project Manager: Swapna Srinivasan Designer: Victoria Pearson Typeset by Thomson Digital

This book is dedicated to the unsung heroes of the entire antibacterial drug discovery and development community, who have toiled far too long in the shadows to bring life-saving drugs to the market in spite of multiple hindrances.Without these drugs, most of us including the editors and readers would not have been alive today. May the force be with you! Prashant Kesharwani, Sidharth Chopra, and Arunava Dasgupta

Contributors

Yash Acharya Antimicrobial Research Laboratory, New Chemistry Unit and School of Advanced Materials, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, Karnataka, India Tanjore Balganesh GangaGen Biotechnologies Pvt. Ltd., Bangalore, India Swagatam Barman Antimicrobial Research Laboratory, New Chemistry Unit and School of Advanced Materials, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, Karnataka, India Edward D. Chan Department of Medicine, Rocky Mountain Regional Veterans Affairs Medical Center, Denver, CO, United States; Departments of Medicine and Academic Affairs, National Jewish Health, Denver, CO, United States; Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Anschutz Medical Campus, Denver, CO, United States Michael M. Chan Department of Medicine, Rocky Mountain Regional Veterans Affairs Medical Center, Denver, CO, United States; Departments of Medicine and Academic Affairs, National Jewish Health, Denver, CO, United States; Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Anschutz Medical Campus, Denver, CO, United States Chih-Yuan Chiang Countermeasures Division, US Army Medical Research Institute of Infectious Diseases, Frederick, MD, United States; Cherokee Nation Assurance, Frederick, MD, United States Sidharth Chopra Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India Sajad Ahmad Dar Department of Microbiology, University College of Medical Sciences (University of Delhi) and GTB Hospital, Delhi, India; Research and Scientific Studies Unit, College of Nursing and Allied Health Sciences, Jazan University, Jazan, Saudi Arabia Shukla Das Department of Microbiology, University College of Medical Sciences (University of Delhi) and GTB Hospital, Delhi, India Arunava Dasgupta Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India Kathakali De Antimicrobial Research Laboratory, New Chemistry Unit and School of Advanced Materials, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, Karnataka, India Allen J. Duplantier Countermeasures Division, US Army Medical Research Institute of Infectious Diseases, Frederick, MD, United States; Cherokee Nation Assurance, Frederick, MD, United States xv

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Contributors

Jayanta Haldar Antimicrobial Research Laboratory, New Chemistry Unit and School of Advanced Materials, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, Karnataka, India Shafiul Haque Research and Scientific Studies Unit, College of Nursing and Allied Health Sciences, Jazan University, Jazan, Saudi Arabia Sukumar Hariharan GangaGen Biotechnologies Pvt. Ltd., Bangalore, India Sven Hoffner Department of Public Health Sciences, Karolinska Institutet, Stockholm, Sweden Grace Kaul Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh India Shashimohan Keelara GangaGen Biotechnologies Pvt. Ltd., Bangalore, India Prashant Kesharwani Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India Mohini Mohan Konai Antimicrobial Research Laboratory, New Chemistry Unit and School of Advanced Materials, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, Karnataka, India Pradeep Kumar Division of Infectious Disease, Department of Medicine and the Ruy V. Lourenço Center for the Study of Emerging and Reemerging Pathogens, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ, United States Cheryl Miller Countermeasures Division, US Army Medical Research Institute of Infectious Diseases, Frederick, MD, United States; National Research Council (NRC) Research Associateship Program at USAMRIID, Washington, DC, United States Diane Ordway Mycobacteria Research Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, United States Rekha G. Panchal Countermeasures Division, US Army Medical Research Institute of Infectious Diseases, Frederick, MD, United States Vivek Daniel Paul GangaGen Biotechnologies Pvt. Ltd., Bangalore, India Robert Penchovsky Department of Genetics, Faculty of Biology, Sofia University “St. Kliment Ohridski”, Sofia, Bulgaria Radha Rangarajan Vitas Pharma Research Private Limited, University of Hyderabad, Hyderabad, Telangana, India

Contributors

Manjulika Shukla Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India Amy C. Shurtleff Countermeasures Division, US Army Medical Research Institute of Infectious Diseases, Frederick, MD, United States; The Geneva Foundation, Tacoma, WA, United States Ramandeep Singh Tuberculosis Research Laboratory, Translational Health Science and Technology Institute, Faridabad, Haryana, India Taru Singh Department of Microbiology, University College of Medical Sciences (University of Delhi) and GTB Hospital, Delhi, India Nanduri Srinivas Department of Medicinal Chemistry, NIPER-HYD, Hyderabad, India Bharathi Sriram GangaGen Biotechnologies Pvt. Ltd., Bangalore, India Melek Sunay Countermeasures Division, US Army Medical Research Institute of Infectious Diseases, Frederick, MD, United States; Oak Ridge Institute for Science and Education (ORISE) Fellowship Program at USAMRIID, Oak Ridge, TN, United States Kapil Tahlan Actinobacterial Research Laboratory, Department of Biology, Memorial University of Newfoundland, St. John’s, NL, Canada Ritesh Thakare Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India Martina Traykovska Department of Genetics, Faculty of Biology, Sofia University “St. Kliment Ohridski”, Sofia, Bulgaria Nouha Bakaraki Turan Environmental Engineering Department, Civil Engineering Faculty,Yildiz Technical University, Esenler, İstanbul, Turkey Meera Unnikrishnan Warwick Medical School, University of Warwick, Coventry, United Kingdom Aikaterini Valsamatzi-Panagiotou Department of Genetics, Faculty of Biology, Sofia University “St. Kliment Ohridski”, Sofia, Bulgaria Rasika Venkataraman Fred Hutchinson Cancer Research Center, Seattle, WA, United States Aradhana Amin Vipra GangaGen Biotechnologies Pvt. Ltd., Bangalore, India Kate Watkins Warwick Medical School, University of Warwick, Coventry, United Kingdom

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Foreword 1

Our history with antibiotics is remarkably short. In 1906, Paul Ehrlich developed Compound 606 or Arsphenamine, the first chemotherapeutic agent that was able to selectively attack a bacterial organism (spirochetes, which causes syphilis). However, the modern age of antibiotics that were safe to humans but able to kill bacteria at the site of infection goes back only to 1928 with the discovery of penicillin. In fact, the first person to be treated with an antibiotic was as recent as 1942, within current living memory. Nevertheless, resistance has arisen to most antibiotics soon after they were introduced into clinical practice. But that is not unexpected. As many antibiotics are derived from naturally occurring compounds that are derived from fungi, it is but natural that the genetic basis for resistance is present in a small proportion of bacteria.The massive selection pressure we have applied on these “resistant” strains of bacteria through the use of millions of tons of antibiotics, in humans, animals, and the environment, has ensured that resistance is no longer a rare phenomenon. Indeed, in the case of many bacteria pathogens, a significant proportion of bacteria causing infections no longer respond to antibiotics. Antimicrobial resistance (AMR) has been compared to climate change. Resistance can emerge in any part of the world because of antibiotic overuse or misuse and the rapid dissemination of resistant pathogens globally. There are 10 million people on an airplane on any given day and bacteria do not need passports or visas to move around the world. Like with climate change, individuals and countries are not fully incentivized to tackle the AMR problem on their own. However, there is one important respect in which AMR is not like climate change. The potential for drug resistance was known even before penicillin was used on a single patient, unlike with climate change, where the impact on climate was not understood at the dawn of the fossil fuel age. Alexander Fleming warned of drug resistance in his Nobel Lecture in 1945, “It is not difficult to make microbes resistant to penicillin in the laboratory by exposing them to concentrations not sufficient to kill them, and the same thing has occasionally happened in the body. The time may come when penicillin can be bought by anyone in the shops. Then there is the danger that the ignorant man may easily underdose himself and by exposing his microbes to non-lethal quantities of the drug make them resistant.” Despite these early warnings, we have failed to ensure that penicillin and other antibiotics are not bought by anyone without medical knowledge. And we have seen the consequences. In this book “Antibiotics: Past, present and future,” the editors Prashant Kesharwani, Arunava Dasgupta, and Sidharth Chopra have put together a remarkable compendium of papers addressing various aspects of the AMR challenge. AMR is global, and it is but natural that science to address the problem of resistance including on discovery of new

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Foreword

antimicrobial agents should also be global. India, as the world’s large consumer of antibiotics for use in humans, and as a country with a challenged public health and sanitation system, is bearing the brunt of the AMR burden. It is timely that researchers in India have enthusiastically taken up the scientific challenge of tackling AMR. There may come a time when we are no longer dependent on antibiotics because of advances in science that we cannot yet see. Perhaps phage therapies, vaccines, monoclonal antibodies, and probiotics would have advanced to a stage where our reliance on antibiotics is much diminished. But until that time, we owe it to future generations to ensure that they do not enter a world where antibiotics no longer work, for no fault of their own. Ramanan Laxminarayan

Foreword 2

Antimicrobial Resistance (AMR) is one of the biggest challenges the world faces today. The issue is as complex as global warming and global poverty, requiring coordinated efforts of all stakeholders, including the medical community, scientists, policymakers, pharmaceutical industry, politicians, and the public. One of the basic reasons for the current AMR scenario is the dry anti-infective pipeline. The reasons behind this dry pipeline are well known. The book “Drug Discovery Targeting Drug-Resistant Bacteria” is very comprehensive, with an introductory chapter on why there is an urgent need for newer antiinfectives, and multiple well-written sections covering very important aspects such as mechanisms of drug resistance, drugs targeting various groups of bacteria, newer approaches to drug discovery including peptides and a detailed discussion on the role of phages. The monogram is an excellent resource that helps us to delve deep into the intricacies of the current AMR scenario and a souvenir on the light at the far end of the tunnel. I congratulate Dr. Sidharth Chopra and team for their sincere effort on a topic of high public health significance.  Abdul Ghafur

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Acknowledgments We wish to sincerely thank the authors for offering to write comprehensive chapters on a tight schedule. This is generally an added responsibility in the hectic work schedules of researchers. We express our earnest gratitude to the reviewers, who provided their critical views for the improvement of the book chapters. We would also like to thank reviewers of our book proposal for their suggestions in the framing of the chapters. We also thank Timothy Bennett (Editorial Project Manager, Elsevier), whose efforts during the preparation of this book were very useful. Editors

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CHAPTER 1

Antibiotics: past, present, and future Ritesh Thakarea, Prashant Kesharwanib, Arunava Dasguptaa, Nanduri Srinivasc, Sidharth Chopraa Division of Microbiology, CSIR-Central Drug Research Institute, Lucknow, Uttar Pradesh, India Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India c Department of Medicinal Chemistry, NIPER-HYD, Hyderabad, India a

b

1 Introduction 1.1  History and discovery of antimicrobials Selman Waksman, one of the very first investigators in the field of antimicrobial chemotherapy and bacteriology, coined the term “antibiotic” [1]. The term was singularly used to refer to a molecule that was used against bacteria and exhibited bacteriostatic or bactericidal activity. However today, the term antibiotics or antimicrobials is often used interchangeably for compounds used in the treatment of bacterial, fungal, protozoan, or other microbial infections. As has been well documented, infectious diseases have a significant impact on human morbidity and mortality and have shaped human evolution and history. The discovery of antimicrobials is one of the most significant breakthroughs that revolutionized human medical sciences as antibiotics have saved millions of lives and increased life expectancy all across the globe. Paul Ehrlich started the never-ending quest of discovery of antimicrobials along with Sahachiro Hata. They identified a compound, marketed as Salvarsan, that showed antibacterial activity against Treponema pallidum, the causative agent of syphilis that had a major impact on health care in 19th century Europe.The discovery of salvarsan gave the concept of magic bullet, a compound that specifically acts on the disease-causing agent but does not damage the host. The discovery of salvarsan was followed by the discovery of sulfa drugs by Gerhard Domagk in the 1930s [2]. Soon, penicillin was discovered by Sir Alexander Fleming from Penicillium notatum in 1928 [3]. The discovery of penicillin was a real game-changer because in the past we were dependent on synthetic antimicrobial discovery, but this opened up the avenue of natural products as antimicrobials. Penicillin came into clinical use in the 1940s and was a miracle drug. Suddenly, one’s untreatable infections that had been death sentence before now were cured in days. The finding led to the understanding that a microorganism can produce substances that could inhibit the growth of other microorganisms.The best source of new antibiotics was from a naturally occurring microorganism, and every effort was made to reach all the parts of the world to isolate the antibiotic-producing organism [4]. After the successful introduction of penicillin, there was a huge expansion in the arsenal against microbial infections through Drug Discovery Targeting Drug-Resistant Bacteria. http://dx.doi.org/10.1016/B978-0-12-818480-6.00001-1 Copyright © 2020 Elsevier Inc. All rights reserved.

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Drug Discovery Targeting Drug-Resistant Bacteria

the discovery of new molecules. Waksman and Schatz discovered streptomycin from Streptomyces sp. that was active against both Gram-positive and Gram-negative bacteria, including tuberculosis caused due to Mycobacterium tuberculosis [5]. In subsequent years, many new antibiotics such as chloramphenicol, tetracycline, macrolides, quinolones, etc. were deployed in clinical settings. In fact, so many molecules were discovered in between 1950s and 1970s that is considered to be the “golden era of antibiotics.” Most of the classes of antibiotics were discovered and introduced in the health-care system but since then almost no new class of antibiotics has been discovered [6]. In the current economic scenario, India and China mass produce antibiotics and sell them globally. In the United States in 2011, total antibiotic use in human medicine was 3290 t and the use of medically important antibiotics in food animals was almost three times higher than human use [7]. According to a recent study in China, the annual consumption of antibiotics is 138 g per person which is 10 times more in comparison to that in the United States. Interestingly, an average growing pig excretes 175 mg of antibiotics per day [8]. According to Wang et al. [9], 21 different types of antibiotics were reported in the urine of Chinese primary students who have not been treated by antibiotics for years. Adding to the perfect storm, the Indian subcontinent is proving to be perfect superbug petri dish mainly due to substandard antibiotic policies, lack of strict regulation from authorities, poor sanitation, and overcrowding [10,11].The global consumption of antibiotics has increased by 40% in the last decades without any exception [12]. Apart from their central role in treating infections, antibiotics are used to prevent infections in surgical patients, in organ transplant, as adjunct to chemotherapy as well as to stave of infections in critically ill patients in ICUs. Taken together, most of the advances in human medicine would not have been possible without antibiotics. In time, a perception was generated that antibiotics could cure anything, so they were used extensively, many times unnecessarily and inappropriately. Inappropriate use of antibiotics all over the world has led to antimicrobial resistance (AMR). AMR is defined as the ability of microbes to grow in the presence of antimicrobial that would usually kill them [13]. It is not a new phenomenon. In fact, in 1940, Abraham and Chain [14] demonstrated that Escherichia coli cell extract could destroy the antimicrobial activity of penicillin by enzymatic action. Incidentally, Sir Alexander Fleming also warned about the development of AMR in his Nobel lecture. He mentioned that it is not difficult to make microbes resistant to penicillin in the laboratory and the same thing can occasionally happen in the body “The ignorant man may easily under dose himself and by exposing his microbes to non-lethal quantities of the drug and make them resistant” [15]. Unfortunately, his words turned out to be true. Extensive use of antimicrobials causes a spread of resistant phenotypes and the emergence of multidrug-resistant (MDR) pathogens across the globe. Over the years, the microbes have adapted and evolved in a way that even our last precious antibiotics become potently useless against these superbugs. Pathogens are acquiring resistance to multiple antibiotics and these resistances are being disseminated rapidly. Resistance in

Antibiotics: past, present, and future

microorganisms is happening due to mutation in preexisting DNA or by the acquisition of foreign DNA containing the antibiotic resistance gene.These genes confer a variety of resistance mechanisms to bacteria. According to Infectious Diseases Society of America, the emergences of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus, multidrug resistance in Gram-negatives especially ESKAPE pathogen and in M. tuberculosis have led to the many challenges during their treatments [16]. Furthermore, AMR is a global problem and is not restricted to a particular part of the world. Although there are considerable variations in the pattern of AMR, it affects all the parts of the world and all classes of people. Even in a modern, well-funded health-care system where the patient gets easy access to the second- and third-line therapies, these are often associated with higher mortality rates in infections caused due to drug-resistant bacteria. According to Centers for Disease Control and Prevention, each year in the United States, at least 2 million people get an antibiotic-resistant infection and out of those, 23,000 people die. Antimicrobial-resistant infections claimed 50,000 lives each year in Europe and the United States [17]. However, lower middle-class countries suffer from emerging resistance to treatment of tuberculosis, malaria, and HIV in addition to lack of access to medicines. The drug-resistant strains of tuberculosis are prevalent in many parts of the world. Majority of these cases are often left untreated due to late or no diagnosis, inaccessibility to second- or third-line therapy and associated cost of treatment [18]. It is estimated that superbugs are on track to kill 10 million people by the year 2050 [17].The rapid dissemination of AMR and the emergence of MDR pathogens have created a pressing need for new classes of antimicrobial agents. Currently, the complexity and speed of international global travel create new opportunities for the drug-resistant pathogen to disseminate globally.Therefore, no country in isolation can tackle AMR and needs a coordinated, dedicated international multidisciplinary effort.

2  Current status 2.1  Dwindling drug discovery pipeline: no new class of antibiotics from the last three decades A new report from the World Health Organization (WHO) mentioned that the antibiotics currently in clinical development are not sufficient to counter rising AMR, especially against those pathogens that posed the greatest threat to human health. Currently, only a few molecules in clinical trials can potentially counter the multidrug resistance in Gram-negative bacteria. Many of these drugs are modifications of existing antibiotics classes and add little to the already existing arsenal of resistance mechanisms. Potential treatment options are lacking for most critical resistant bacteria, especially for multidrug and extensively drug-resistant Gram-negative pathogens. As per the report in May 2017, antibacterials under clinical development include 51 antibiotics (including combinations) and 11 biologicals that have the potential to treat serious bacterial infections and were in phase 1–3 clinical trials.

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Recently, WHO also published the list of bacteria for which new antibiotics are urgently needed [19]. The list contains priority pathogens that are categorized in critical, medium, and high, depending upon the urgency of treatment and increasing drug resistance to existing antibiotics. The critical category contains three Gram-negative bacteria that are resistant to multiple antibiotics carbapenem-resistant Pseudomonas aeruginosa, carbapenem-resistant Acinetobacter baumannii, and carbapenem-resistant, extended-spectrum beta-lactamase-producing Enterobacteriaceae. The WHO listed nine bacteria, including S. aureus, Salmonella, Neisseria gonorrhoeae, and Shigella as high-priority pathogens. In treatment options, only 12/33 antibiotics are active against these pathogens that are in the current drug pipeline and only two agents, GSK-3342830 (phase 1) and cefiderocol (phase 3), are expected to be active against all three critical priority pathogens. Cefiderocol is siderophore cephalosporin with a novel mechanism to penetrate the outer membrane of Gram-negative pathogens. None of these targets vancomycin-resistant Enterococci or fluoroquinolone-resistant Salmonella, both listed as high-priority pathogens. On the other hand, drug pipeline for other priority pathogens is robust with most of 16 new antibiotics (which include 2 new antibiotic classes and 7 biological agents) specifically targeting MRSA. Modifications in existing antibiotic classes are a valuable short-term approach, but innovative approaches to antibacterial treatment are required to overcome resistance sustainably. Collectively, only 8 of the 51 new antibiotics act with a novel mechanism of action. In the past decade, only six new agents, namely, telithromycin, gemifloxacin, daptomycin, tigecycline, linezolid, and quinupristin–dalfopristin have been approved for clinical use by the US FDA. Majority of the newly approved drugs exhibit a narrow spectrum and are active against Gram-positive pathogens only. Only tigecycline is active against Gram-negative and Gram-positive human pathogens.

2.2  Pharmaceutical companies dropped out the research and development of antibiotics Most pharmaceutical companies have dropped their programs on new antibiotic discovery and development (Fig. 1.1). Unfortunately, recent hunts for novel targets by genomic and proteomics-based approaches have also produced limited success [20,21].The reason for this drop was because of lack of return on investment on antiinfectives amongst a multitude of factors. The antibiotic market is no longer attractive to the pharmaceutical investors due to developmental cost and stringent policies of regulatory authorities. If the drug molecules passed these hurdles and gained approval, then it was held in reserve and only prescribed for the resistant infection, thus further lowering the return on investment. Furthermore, as compared to other drugs for chronic diseases, antibiotics are administered for a very short duration and include inherent risk of resistance. So in the long run, investors find antibiotic drug development less profitable than other drugs. Given the upsetting situation, the search for new antimicrobial agents against the drugresistant pathogen is an urgent and unmet medical need.

Antibiotics: past, present, and future

Figure 1.1  Timeline of antibiotic discovery and deployment and emergence of AMR. AMR, antimicrobial resistance.

3  Solution to the problem: what can be done? 3.1  A surveillance system to monitor the escalating antimicrobial resistance According to a report by the Food and Agriculture Organization of the United Nations, World Organisation for Animal Health (OIE) and the WHO, 100 countries now have national action plans for AMR and 51 countries have plans under development. The report includes surveillance, education, monitoring, and regulating consumption and use of antimicrobials in human, in animal health and production, as well as plants and the environment as recommended in the Global Action Plan published in 2015. Progress in implementing plans is greater in high- than low-income countries [22]. The Government of India has recognized AMR as a key priority and in April 2017 and finalized India’s National Action Plan on Antimicrobial Resistance [23]. Surveillance findings are playing an important role to inform clinical therapy decisions, guiding policy recommendations, and assessing the impact of resistance globally. WHO has launched Global Antimicrobial Resistance Surveillance System to support a standardized approach to the collection, analysis, and sharing of data on AMR at a global level [24]. Surveillance and epidemiological data are essential to keep an eye on increasing global resistance pattern in pathogens. Surveillance data help one to track the changes in microbial populations and permit the early detection of resistant strains. Further, the

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European Antimicrobial Resistance Surveillance System and the European Surveillance of Antimicrobial Consumption program have also joined the venture.

3.2  One Health approach One Health is defined as a collaborative, multisectoral, and transdisciplinary approach to work at the local, regional, national, and global levels to achieve optimal health outcomes recognizing the interconnection between people, animals, plants, and their shared environment (Fig. 1.2) [25]. It encourages collaborative efforts of many experts such as physicians, veterinarians, and ecologists working together to monitor, control, and learn about public health threats, the spread of diseases among the animal, human, and environment.The health of the people is strongly related to the health of the animal and environment. Many human diseases are shared between animals and people and hence they can serve as an early warning sign for the potential human illness in the future. The diseases include zoonotic diseases such as rabies, Salmonella infection, West Nile virus fever, and Q fever. The One Health concept is not new but now it is extremely important in the context of a current situation where many factors have changed the interaction in between

Figure 1.2  One Health. (Adapted from CDC).

Antibiotics: past, present, and future

the human, animal and their shared environment. Intriguingly, the problem of misuse of antibiotics originates from its utilization in livestock where ∼80% of all the antibiotics produced are used as growth promoters and prophylactic agents in animals (nontherapeutic uses) worldwide [26]. These animals are often grown at places where they prone to infection due to unsanitary, filthy, and stressful conditions. The economic cost of maintaining a sanitary environment is a lot greater than that of addition of antibiotics as growth promoters. So addition of antibiotics to food and water for livestock makes them grow faster and prevent infections. This situation leads to incredible increase in AMR, and often these animals are home to multiple antibiotic-resistant organisms.

3.3  Introducing new chemical entities and managing current antibiotics New chemical entities should be continuously introduced with a novel mechanism of action by incentivizing the research and development in drug discovery. But the introduction of new antibiotics is not the only solution to the problem. The solution to the problem is relying on our ability to manage current antimicrobial agents effectively. The problem is in the broken system, which we have developed over the years. We introduce our precious drugs into the broken system that gives millions of chances to bacteria to develop a resistance. Instead, antibiotics are the societal drugs and every person should bear the responsibility of using it appropriately [27]. Antibiotics are not the same as other drugs that we used to treat chronic diseases such as diabetes or cancer. For example, if a person takes paracetamol and accidentally gets overdosed, it can lead to the liver damage and kidney failure. But that does not change or affect anybody else’s ability to take paracetamol. If the same person is going to misuse antibiotics, it creates drug-resistant bacteria that spread to the other people in society and prevent them from being treated with that same antibiotic. We should embrace the antibiotics like a precious treasure and save them for future generation.

References [1] H.B. Woodruff, S.A. Waksman, Winner of the 1952 Nobel Prize for physiology or medicine, Appl Environ Microbiol 80 (2014) 2–8. [2] M.E. Török, F.J. Cooke, E. Moran, Oxford handbook of infectious diseases and microbiology, 2nd ed., Oxford University Press, United Kingdom, (2016). [3] S.Y.Tan,Y.Tatsumura, Alexander Fleming (1881–1955): discoverer of penicillin, Singapore Med J 56 (7) (2015) 366–367. [4] T. Saga, K.Yamaguchi, History of antimicrobial agents and resistant bacteria, JMAJ 52 (2009) 103–108. [5] A. Schatz, E. Bugle, S.A. Waksman, Streptomycin, a substance exhibiting antibiotic activity against Gram-positive and Gram-negative bacteria, Exp Biol Med 55 (1944) 66–69. [6] R.I. Aminov, A brief history of the antibiotic era: lessons learned and challenges for the future, Front Microbiol 1 (2010) 134. [7] Food and Drug Administration: antibacterial drug usage analysis. Available from: https://www.fda.gov/ downloads/Drugs/DrugSafety/InformationbyDrugClass/UCM319435.pdf. [Accessed May 31, 2019]. [8] D. Cui, X. Liu, P. Hawkey, H. Li, Q. Wang, Z. Mao, et al. Use of and microbial resistance to antibiotics in China: a path to reducing antimicrobial resistance, J Int Med Res 45 (2017) 1768–1778.

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[9] H. Wang, N. Wang, B. Wang, H. Fang, C. Fu, C. Tang, et al. Antibiotics detected in urines and adipogenesis in school children, Environ Int 89–90 (2016) 204–211. [10] S.G. Kumar, C. Adithan, B.N. Harish, S. Sujatha, G. Roy, A. Malini, Antimicrobial resistance in India: a review, J Nat Sci Biol Med 4 (2) (2013) 286–291. [11] J.A. Ayukekbong, M. Ntemgwa, A.N. Atabe, The threat of antimicrobial resistance in developing countries: causes and control strategies, Antimicrob Resist Infect Control 6 (2017) 47. [12] T.P. Van Boeckel, S. Gandra, A. Ashok, Q. Caudron, B.T. Grenfell, S.A. Levin, et al. Global antibiotic consumption 2000 to 2010: an analysis of national pharmaceutical sales data, Lancet Infect Dis 14 (2014) 742–750. [13] H.D. Marston, D.M. Dixon, J.M. Knisely, T.N. Palmore, A.S. Fauci, Antimicrobial resistance, JAMA 316 (2016) 1193. [14] E.P. Abraham, E. Chain, An enzyme from bacteria able to destroy penicillin, Nature 146 (1940) 837. [15] F. Alexander, Penicillin Nobel lecture, 1945, Available from: https://www.nobelprize.org/nobel_prizes/medicine/laureates/1945/fleming-lecture.pdf. Accessed May 31, 2019. [16] H.W. Boucher, H.T. George, J.H. Bradley, J.E. Edwards, D. Gilbert, L.B. Rice, et al. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America, Clin Infect Dis 48 (1) (2009) 1–12. [17] J. O'Neill, 2014. Antimicrobial resistance: tackling a crisis for the health and wealth of nations, Available from: https://amr-review.org/sites/default/files/AMR Review Paper-Tackling a crisis for the health and wealth of nations_1.pdf. [Accessed May 31, 2019]. [18] World Health Organization: global tuberculosis report 2014. Available from: http://www.who.int/tb/ publications/global_report/gtbr14_executive_summary.pdf. [Accessed May 10, 2019]. [19] World Health Organization: antibacterial agents in clinical development. Available from: https:// www.who.int/medicines/news/2017/IAU_AntibacterialAgentsClinicalDevelopment_webfinal_2017_09_19.pdf. [Accessed May 10, 2019]. [20] H. Brötz-Oesterhelt, P. Sass, Postgenomic strategies in antibacterial drug discovery, Future Microbiol 5 (2010) 1553–1579. [21] S. Donadio, S. Maffioli, P. Monciardini, M. Sosio, D. Jabes, Antibiotic discovery in the twenty-first century: current trends and future perspectives, J Antibiot (Tokyo) 63 (2010) 423–430. [22] World Health Organization: countries step up to tackle antimicrobial resistance. Available from: https:// www.who.int/news-room/detail/18-07-2018-countries-step-up-to-tackle-antimicrobial-resistance. [Accessed May 31, 2019]. [23] www.searo.who.int/india/topics/antimicrobial_resistance/nap_amr.pdf. [Accessed May 31, 2019]. [24] World Health Organization: global antimicrobial resistance surveillance system report 2017. Available from: https://www.who.int/glass/resources/publications/early-implementation-report/en/[Accessed May 10, 2019]. [25] Centers for Disease Control and Prevention (CDC), 2018. CDC-One Health. Available from: https:// www.cdc.gov/onehealth/basics/index.html. [Accessed May 31, 2019]. [26] A. Hollis, Z. Ahmed, Preserving antibiotics, rationally, N Engl J Med 369 (2013) 2474–2476. [27] P. Sarkar, I.M. Gould, Antimicrobial agents are societal drugs, Drugs 66 (7) (2006) 893–901.

CHAPTER 2

Mechanisms of antibacterial drug resistance and approaches to overcome Aikaterini Valsamatzi-Panagiotou, Martina Traykovska, Robert Penchovsky Department of Genetics, Faculty of Biology, Sofia University “St. Kliment Ohridski”, Sofia, Bulgaria

1. Introduction It is universally known that antibiotics are one of the most important drugs against infectious diseases over the past 100 years. The father of modern chemotherapy is believed to be Paul Ehrlich who discovered the first antibiotic named Salvarsan in 1909. It was the first drug to be used for the treatment of syphilis, a sexually transmitted disease caused by Treponema pallidum. His idea about the production of an agent that would be harmful against bacteria but would not affect the host was characterized as a “magic bullet.” Gerhard Domagk discovered the sulfa drugs in the 1930s. Penicillin discovery made by Alexander Fleming followed in the 1940s. Fleming predicted that the overuse of penicillin may lead to resistance development and he noted that it should be used with caution [1,2]. The antibiotic discovery is considered to be the main reason that led to the eradication of many infectious diseases that were considered to be fatal until then. Some physicians believed that many infections after some years would belong to the past. Although the rate of morbidity and mortality decreased after the introduction of the antibiotics in comparison with the preantibiotic era, every discovery of a drug was followed eventually by the emergence of antibiotic-resistant bacterial strains [3]. The existence of resistance against antibacterial agents is as ancient as the bacteria. Indeed, from the period of penicillin discovery and before the beginning of its massive use, it was found out that the penicillin could be destroyed by some bacteria through enzymatic degradation [1]. According to the World Health Organization (WHO), antibiotic resistance (AR) and its spread constitutes one of the three biggest threats in the 21st century worldwide [4]. It is of great importance to be aware of the factors that are contributing to the development of AR. It is widely accepted that AR is the consequence of the inappropriate and heavy use of antibiotics against diseases caused by some bacteria or even by nonbacterial agents. The overuse of antibiotics in human and in veterinary medicine in combination with the absence of vaccination, which is extremely important for the prevention of many infections, is considered to be some of the most significant reasons that led to the development of resistance in bacteria. Indeed, it is accepted that antibiotics are drugs lifespan of utility of which is limited. Nowadays, a growing number of bacterial strains Drug Discovery Targeting Drug-Resistant Bacteria. http://dx.doi.org/10.1016/B978-0-12-818480-6.00002-3 Copyright © 2020 Elsevier Inc. All rights reserved.

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are becoming multidrug-resistant (MDR). MDR bacterial infections are worrisome and related to longer hospitalization of patients, prolonged treatments with broad-spectrum antibiotics, therefore, increase health-care costs in combination with an increase in disability and mortality rates [3,5-7]. The mechanisms by which bacteria manage to develop resistance are related to the mechanisms of antibiotic action. During the last decades the understanding of the mechanisms of emergence of AR has been a subject of vigorous research. Some factors that seem to contribute to the rapid evolution of AR are the huge bacteria populations, as well as, the elevation of mutation rates that is facilitated by stress and spatial heterogeneity. Several genes blamed for developing AR have the capability of spreading rapidly through horizontal transfer of genetic information among bacteria. It is critically important to understand thoroughly the genetics likewise the biochemical basis of AR in order to find out ways to fight against it. New drugs and therapeutic approaches, which will be potent against MDR bacteria, should be developed based on novel strategies [4,8-10].

2. Mechanisms of antibacterial drug action To understand the mechanisms of AR, at first, we should be familiar with the mechanisms of antibacterial drug action. In general, antibiotics can be classified based on a variety of criteria. We can divide them into two categories by their way of action against bacteria. If they inhibit the growth of bacteria, they are called “bacteriostatic,” while if they manage to kill bacteria, they are called “bactericidal” [11]. Taking into consideration their capability of affecting various bacteria, “narrow-” or “broad-” spectrum antibiotics exist.

2.1 Interference with the cell-wall synthesis In microbiology, bacteria are classified into Gram-positive and Gram-negative due to their response to a Gram stain that is a crystal violet dye [5]. During the observation of the bacteria in the microscope, Gram-positive bacteria look purple in color, while Gram-negative bacteria are decolorized and look red. In general, bacteria are surrounded by the cell wall that is responsible for the preservation of their shape and size (Fig. 2.1). The strength of their cell wall is due to a layer known as peptidoglycan (PG), murein, or mucopeptide. PG is a substance that consists of three parts (1) a backbone, (2) tetrapeptide side chains, and (3) peptide cross-bridges. Any disruption of the PG layer leads to an impairment of the cell wall that results in lysis of the cell itself [12]. The structure of the bacterial cell wall plays an important role in antibacterial drug delivery. The anatomy of the cell wall between Gram-positive and Gram-negative bacteria differs significantly. Gram-positive bacteria are surrounded from inside out, by the plasma membrane and the cell wall, which are composed of PG (Fig. 2.1).Their cell wall consists of some special components that are teichoic and teichuronic acids, as well as,

Mechanisms of antibacterial drug resistance and approaches to overcome

Figure 2.1  Molecular antibacterial drug targets.

polysaccharides.The layer of PG in Gram-positive bacteria is thicker in comparison with Gram-negative bacteria. Gram-negative bacteria are also surrounded by a plasma membrane, although they differ from Gram-positive in the existence of a periplasmic space that can be found between the inner and an extra outer membrane. The components of periplasmic space are built of PG and a solution of proteins. The outer membrane is bilayered and is composed of lipopolysaccharides and is responsible for the prevention of hydrophobic molecules entering the bacteria. However, low-molecular-hydrophilic molecules are capable of entering through special channels, a process completed with the help of some protein molecules called porins. Low-molecular-hydrophilic molecules enter the cell through passive diffusion [12]. An ideal antimicrobial agent has the particularity of the selective toxicity, which means that it can cause damage only against a pathogen without affecting the host organism. In general, toxicity is not absolute but relative, which means that the antibacterial agent is given in a concentration that is adjusted in order to be safe for the host and at the same time harmful against the pathogen [12]. Antibacterial drugs act by interfering with the physiological processes of bacterial growth and division [11,13,14].

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As mentioned previously, PG is a component of the cell wall found in both Grampositive and Gram-negative bacteria. It is considered a cross-linked polymer matrix that is responsible for the mechanical strength that bacteria can afford in order to survive. Thus it is an important component for the structure of the bacteria and in the case of any disruption of the PG layer, the osmotic pressure is increased that results in lysis of the cell. The structural integrity of the cell is closely related to PG cross-linking. Transglycosylase and transpeptidase are two enzymes responsible for maintenance of the PG layer. Their role is to expand the glycan strands of the PG molecules. They manage to implement this procedure, by adding disaccharide pentapeptides, while at the same time they are capable of cross-linking close strands of peptides in which the PG units are not mature enough. The B-lactam antibiotics and glycopeptides interfere with the cell-wall synthesis by inhibiting PG biosynthesis [11,15-17]. The B-lactam antibiotics include several groups such as penicillins, cephalosporins, carbapenems, and monobactams. Their mechanism of action is common and relates to binding to special cell receptors known as penicillin-binding proteins (PBPs). By creating a bond with one or more receptors, the drug manages to inhibit the reaction of transpeptidation (carried out by transpeptidase enzymes) leading in that way to an impairment of PG synthesis (Fig. 2.1). The driving scenario reported for this reaction is the structural similarity of (-lactams to acyl-d-alanyl-d-alanine. As a result, this group of drugs becomes capable of inhibiting the process of transpeptidation that in its turn impairs the conversion of immature to mature PG. Consequently, PBPs are considered the main target of (-lactams. After the interaction between the ring of (-lactams and PBPs, synthesis of PG is impaired, the layer of PG is disrupted, and bacterial cell lysis is the result. The mechanism of action of (-lactams is thought to have slight differences between Gram-negative and Gram-positive bacteria.This could be explained by the difference of the cell wall structure between these two different groups of bacteria [18-21]. The most common glycopeptides are vancomycin, teicoplanin, and oritavancin. They have a similar mechanism of action as (-lactams that involves the inhibition of the cell-wall synthesis. Each drug binds with a special part of the PG chain, the d-alanyl-dalanine part. Vancomycin is the drug of glycopeptide category, which is mostly used. It acts by impeding d-alanyl subunit to bind with PBPs and, thus, manages to inhibit the synthesis of the cell wall. Glycopeptides are used mostly against Gram-positive bacteria such as Staphylococcus aureus and the strains of S. aureus, which are resistant to methicillin (MRSA), while (-lactams are used both against Gram-positive and Gram-negative bacteria [19,22].

2.2 Inhibition of protein synthesis Two main processes are involved in the synthesis of proteins: transcription and translation. In the beginning, through the process of transcription, RNA is synthesized using the bacterial DNA as a template (Fig. 2.1). Then translation follows that is the synthesis

Mechanisms of antibacterial drug resistance and approaches to overcome

of proteins, during which messenger (m)RNA is used as a template. The process is catalyzed by ribosomes and other factors of the cytoplasm. The process of translation is divided into three phases, (1) initiation, (2) elongation, and (3) termination [11]. The ribosome in bacteria is composed of two subunits: the small subunit (the 30S) and the large subunit (the 50S). The ribosome is composed of three transfer transfer-RNA [(t) RNA] sites, the donor P-site, the acceptor A-site, and the exit E-site. In the middle is located the peptidyl transferase center. Aminoacyl-tRNA (aa-tRNA) is bound with the A site, a process that depends on the presence of GTP (guanosine triphosphate), while peptidyl-tRNA is bound with the P-site. Antibiotics can block protein synthesis at different steps by impairing ribosome’s function or the function of cytoplasm factors. More specifically, the 30S is responsible for codon-anticodon interaction so the 30S inhibitors impair the process of initiation or mRNA codon to aa-tRNA anticodon pairing. The large subunit of the ribosome, the 50S, is responsible for the formation of a peptide bond between aa-tRNA and peptidyl-tRNA sites; therefore the 50S inhibitors disrupt the process of elongation (or peptidyl-transferase reaction) [19,23-25]. The drugs that affect the 30S subunit are aminoglycosides and tetracyclines. Aminoglycosides alter the formation of the complex between mRNA and aa-tRNA that leads to the improper matching of tRNA. This causes misreading of the genetic code and finally results in the inhibition of protein synthesis.The tetracyclines also act on 16S ribosomal RNA (rRNA) components of the 30S subunit by the impedance of tRNA binding with the acceptor A-site of the ribosome [19,26-28]. Several drugs such as macrolides, chloramphenicol, clindamycin, quinupristin, dalfopristin, linezolid, and lincomycin act on the 50S subunit of the ribosome. More specifically, after binding with the 50S subunit, they affect either the translocation or peptidyltransferase reaction. Indeed, in some cases, they can affect both the processes simultaneously, which finally leads to detachment of incomplete chains. Chloramphenicol acts through binding to the peptidyl-transferase enzyme on the peptidyl-transfer center of the 50S subunit, which results in impairment of formation of the peptide bonds [19,23].

2.3 Interference with nucleic acid synthesis 2.3.1 Inhibit DNA synthesis DNA topoisomerase enzymes are important for DNA synthesis, mRNA transcription, as well as cell division [29-31].This is attributed in their capability of modulating chromosomal supercoiling by catalyzing the breakage and rejoin reactions of DNA strands [11]. There are two types of topoisomerases, I and II. Type I topoisomerase can break only one strand of DNA, while type II is capable of breaking both DNA strands [24,32,33]. The topological state of DNA is controlled by those enzymes [33,34]. Topoisomerase II is also known as a DNA gyrase.The gyrase is made of two GyrA and two GyrB subunits (Fig. 2.1). The domain responsible for DNA cleavage and the quinolone binding site is located in GyrA subunit. In the presence of ATP, DNA gyrase supercoils negatively

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DNA while in the absence of ATP relaxes it. DNA gyrase also takes part in catenation and decatenation reaction of the circled DNA and is responsible for resolving knots that may be present in DNA. Thus DNA gyrase is vital for the processes of replication, recombination, and transcription. It constitutes an ideal drug target because it is found only in prokaryotes and is considered to be important for their survival [33,35]. A class of antimicrobial drugs, which act by interference with nucleic acid synthesis, are quinolones. They were discovered by accident during the synthesis of chloroquine, introduced in the 1960s. Quinolones were used mostly for the treatment of urinary tract infections [11,33]. Their target is DNA gyrase and topoisomerase IV enzymes. They get trapped in complexes made by the drug and cleaved DNA and thus, the rejoining of the DNA strand is prevented [36-38]. More specifically, DNA gyrase interacts with the subunit A (GyrA); therefore it manages to affect the process of breakage and reunion [39]. Although the antimicrobial activity of first-generation quinolones such as nalidixic acid is weak, the next generations were proved to be potent antibiotics [33]. Except topoisomerase II that already constitutes a drug target, topoisomerase I can be a promising target for the investigation of new antibacterial agents [34]. 2.3.2 Inhibit RNA synthesis Rifamycins are broad-spectrum antibiotics and the first-line of drugs, which are used for the treatment of infections with Mycobacterium tuberculosis and in combinational therapies with other drugs. This is because they are capable of inducing cell death in mycobacteria species, although resistance can rapidly be developed. Except for their use against infections with M. tuberculosis, rifamycins can also be used against chronic staphylococcal infections [40-42]. The RNA polymerase is an enzyme responsible for DNA transcription [41]. Rifamycins bind with high-affinity to (-subunit of a DNA-bound of actively transcribing RNA polymerase and in this way they manage to inhibit DNA-dependent transcription [43-45].

2.4 Inhibition of metabolic pathways of essential metabolites such as the folic acid The result of folic acid metabolism is the synthesis of thymine, which is a necessary component for DNA synthesis (Fig. 2.1). Sulfonamides considered to be the antibacterial agents that have been in long use. Sulfonamides and trimethoprim interfere with bacterial folic acid metabolism. They achieve this by blocking the synthesis of tetrahydrofolate, which is an important substance for DNA, RNA, and the synthesis of bacterial cell wall proteins. Bacteria are not able to obtain folic acid from exogenous resources so they should synthesize it by their one. Sulfonamides and trimethoprim affect the formation of tetrahydrofolate. Each of them interferes with a different step of this process. Dihydropteroate synthase catalyzes the formation of dihydropteroic acid from pteridine and p-aminobenzoic acid (PABA). Due to their greater affinity than PABA for pteridine

Mechanisms of antibacterial drug resistance and approaches to overcome

synthetase, sulfonamides inhibit competitively dihydropteroate synthase and, thus, affect an early stage in folic acid synthesis. On the other side, trimethoprim affects a later stage by the inhibition of dihydrofolate reductase (DHFR) enzyme that is responsible for the formation of tetrahydrofolic acid from dihydrofolic acid [19,46,47].

2.5 Disruption and increased permeability of cytoplasmic membrane structure The cytoplasmic membrane is composed of lipids, proteins, lipoproteins, and other biological membranes and surrounds the cytoplasm. It controls the internal composition of the cell by acting as a diffusion barrier (Fig. 2.1). Any impairment of its integrity results in leakage of ions and macromolecules and, therefore, cell damage. The antimicrobial agents capable of disorganizing the cytoplasmic membrane are divided into cationic, anionic, and neutral. Polymyxin B and polymyxin E (colistimethate) are some common examples of drugs in this category [24]. Polymyxins act selectively by damaging membranes, which contain phosphatidylethanolamine.They have this capability because they consist of detergent-like cyclic peptides. Polymyxin B is a cationic antibacterial agent that acts by increasing the outer membrane’s permeability to lysozyme and hydrophobic compounds. Although it has a variety of damaging properties, initially it manages to disrupt the cytoplasmic membrane, which allows some substances to enter the cell, and at the same time, it impairs some metabolic processes. Daptomycin is another antimicrobial agent that uses a calcium-dependent interaction to bind to the cell membrane and causes depolarization that followed by potassium release inside the cell. Several other drugs as amphotericin B, colistin, imidazoles, and triazoles act by inhibiting cytoplasmic membrane function [24,48,49]. Note that the compromising of the cell membrane bacteria leads to block its ability to produce energy.

3. Mechanisms of developing antibacterial resistance The appearance of AR was observed even from the discovery of the first antibiotics [5,50]. Antibiotics are considered one of the most successful categories of drugs. However, their misuse in combination with the inability to produce new antibiotics capable of coping with MDR pathogens constitutes a serious public health-care problem [5,7]. It is of great importance to understand the sophisticated mechanisms, through which bacteria develop AR during the years. To classify the mechanisms of antibacterial drug resistance properly, multiple biochemical routes are used. The basic categories of biochemical routes for developing AR are discussed in details next [4,13,51,52].

3.1 Modification of the antibiotic molecule Bacteria produce specific enzymes that may either inactivate the drug through some chemical changes or destroy it. That is the way to cope with antibiotics and make them

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Figure 2.2  Mechanisms for developing antibacterial drug resistance.

unable to act properly (Fig. 2.2). The antibiotics, which belong to this category, are aminoglycosides, carbapenems, cephalosporins, clindamycin, fluoroquinolones, isoniazid, penicillin, rifampin, streptomycin, and tetracyclines [4,5,7]. Enzymes able to alter chemically an antibiotic molecule are found in Gram-negative and in Gram-positive bacteria. They are able to modify the antibiotics, catalyzing the following reactions: (1) adenylation, observed in aminoglycosides and lincosamides, (2) phosphorylation that concerns aminoglycosides and chloramphenicol, and (3) acetylation, noticed in aminoglycosides and streptogramins (Fig. 2.2). The two basic examples of antibiotics in this category are aminoglycosides and chloramphenicol. Aminoglycosides are mostly bactericidal. They are given as therapeutic agents against infections occurred in hospitals and more specifically, infections caused either by Gram-negative or by aerobic bacteria. The modification by aminoglycoside-modifying enzymes (AMEs) is one of the most important mechanisms by which emergences resistance against aminoglycosides [53]. AMEs act through alteration of the amino or hydroxyl group of the antibacterial

Mechanisms of antibacterial drug resistance and approaches to overcome

molecule, and thus, manage to render the drug unable to function by its physiological mechanism and as a result, AR occurs [54]. The genes that are responsible to encode AMEs are usually found in plasmids, although they can also be found in integrons and transposons [55,56]. There is a great variety of AMEs. Some of them are aminoglycoside nucleotidyltransferases, aminoglycoside phosphotransferases (APHs), and aminoglycoside acetyltransferases (AACs) [57]. AMEs are classified by a nomenclature that takes into consideration their biochemical activity and the position of the drug, which they alternate [58]. Note that some enzymes may have numerous biochemical activities. One example is the AAC(6′)APH(2″) enzyme that is bifunctional. It can have both acetylation and phosphotransferase activities [4]. Another drug that is modified by enzymatic alteration is chloramphenicol. Its mechanism of action against bacteria involves inhibition of protein synthesis. There are two types of enzymes based on the level of AR that they evoke: high resistance (type A) or low resistance (type B). They are responsible for chemical modification of chloramphenicol and they are called chloramphenicol acetyltransferases (CATs). CATs develop AR via enzymatic inactivation by acetylation of chloramphenicol [59,60].

3.2 Destruction of the antibiotic molecule Enzymes may destruct the antibiotic molecule to induce A. One common example in this category is (-lactam resistance. It usually occurs in Gram-negative bacteria (Fig. 2.2). The enzymes responsible for the protection of bacteria against (-lactams such as cephalosporins, penicillins, and other compounds are (-lactamases. They are encoded by bla genes. (-Lactamases act by destroying the amide bond of the (-lactam ring. There are two systems of classification of (-lactamases: Ambler and Bush-Jacoby. The Ambler classification includes four groups of (-lactamases (A, B, C, and D) that are identified and separated by an amino acid sequence. The Bush-Jacoby classification is based on the biochemical function of (-lactamases [61,62]. The mechanism responsible for penicillin-resistant S. aureus is thought to be a plasmid-encoded penicillinase. New antibiotic molecules with lesser susceptibility to penicillinase and a wider spectrum of action were investigated in an effort to overcome this AR such as ampicillin. Although during the 1960s it came up that it could be hydrolyzed by a plasmid-encoded lactamase, since that time, the discovery of each new drug was followed by the development of enzymes capable of destroying it [63-65].

3.3 Decreased antibiotic penetration and efflux 3.3.1 Decreased permeability This mechanism of AR concerns antibiotics whose bacterial target is either intracellular or inner cytoplasmic membrane. Therefore to express its antimicrobial properties, the antibiotic has to penetrate the outer and/or the inner membrane of the cell. Especially,

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in Gram-negative bacteria antibiotics should reach the inner membrane to exert their antimicrobial function so they are considered to be not as permeable in comparison with Gram-positive bacteria [66].The outer membrane acts as a gatekeeper and protects the bacterial cell [67]. The antibiotics that belong to this category are aminoglycosides, methicillin, erythromycin, tetracycline, vancomycin, linezolid, macrolides, penicillin, ciprofloxacin, cephalosporins, carbapenems, clindamycin, isoniazid, rifampin, streptomycin, and tetracycline [5]. Fluoroquinolones, tetracycline, and (-lactams have hydrophilic properties. Antibiotics that have hydrophilic properties to penetrate diffuse through porin proteins, which are located on the outer membrane. These categories of antibiotics develop resistance by the mechanism of decreased permeability. If the permeability of membrane decreases, antibiotics will not manage to cross the barrier, which is normally achieved by the use of porins (water-filled diffusion channels). One common antibiotic in this category is vancomycin that renders unable to penetrate the outer membrane of the cell; therefore its antibacterial drug action against Gram-negative bacteria is greatly decreased (Fig. 2.2).The number of porins varies. They can be classified according to three features: (1) structure, (2) selectivity, and (3) regulation of their expression. If there is a change in porins (either in their level of expression, the type of porins expressed or inability to function), this can lead to a low level of AR and it is often accompanied by overexpression of the efflux pump [67]. Two examples that use the porin-mediated mechanism in order to emergence resistance are Pseudomonas aeruginosa and Klebsiella pneumoniae. P. aeruginosa is mostly involved in respiratory infections (pneumonia syndromes), urinary tract infections, skin and soft tissue infections, as well as, bacteremia [68-71]. In the case of P. aeruginosa, the AR is caused by a mutation in the oprD genes. Normally, oprD (D2 protein) is a specific porin that is produced helps in the uptake of imipenem, as well as, amino acids. After the mutation the uptake of imipenem and amino acids is impaired.This mechanism of AR can be used alone or in combination with overexpression of an efflux pump [72-75]. K. pneumoniae belongs to Gram-negative bacteria and is responsible for many infections in the hospital care unit, community-acquired pneumonia, and for complications such as meningitis and endophthalmitis. K. pneumoniae has two major porins on its outer membrane: the OmpK35 and the OmpK36. Porin expression is alternated from OmpK35 to OmpK36; thus there is less susceptibility to (-lactams because the porin channel size of OmpK36 is smaller. Note that extended-spectrum (-lactamases (ESBLs) are produced by K. pneumoniae and are blamed for the increment of AR. This is due to the fact that ESBL strains usually express only OmpK36 and it is believed that one of the contributing factors of the AR is the absence of OmpK35 [76,77]. 3.3.2 Efflux pumps AR can be achieved either through the extraction of the antibacterial agent out of the cell, a process done by complex bacterial mechanisms, or by overexpression of efflux

Mechanisms of antibacterial drug resistance and approaches to overcome

pumps mediated by mutations [9,78] (Fig. 2.2). Each efflux pump is formed by three components: (1) inner membrane transporter, (2) outer membrane transporter, and (3) periplasmic lipoprotein [79]. The substrate specificity of the efflux pumps varies from narrow range (e.g., tetracycline pumps) to wide range as some of them can transport multidrug resistance efflux pumps (Table 2.1). The efflux pumps can be classified into five families on the basis of their structure, their existence in different types of bacteria, their capability of extruding substrates out of the cell, and their source of energy (Table 2.1). The five families are as follows: (1) major facilitator superfamily (MFS), (2) small multidrug resistance family, (3) resistance-nodulation-cell-division family, (4) ATP-binding cassette family, and (5) multidrug and toxic compound extrusion family [9,78,80]. Tetracyclines, aminoglycosides, clindamycin, carbapenems, cephalosporins, fluoroquinolones, penicillin, linezolid, erythromycin, and methicillin are the antibiotics that belong in this category [5]. Two common examples of antibacterial agents are tetracyclines and macrolides. Tetracycline efflux pump (Tet efflux pump) is responsible for tetracycline extraction and belongs to the MFS family.The source of energy that uses in order to achieve tetracycline extraction is the proton pump exchange.Tet genes are mostly met in Gram-negative bacteria with some exceptions as Tet(K) and Tet(L) genes that can be found in Gram-positive bacteria. Macrolides also use the mechanism of efflux pumps in order to emergence resistance. They are mainly used against Gram-positive bacteria and anaerobic microorganisms.The first antibiotic of the category, which was identified in 1952, was erythromycin.

Table 2.1  Families of the efflux pump in Gram-negative and Gram-positive bacteria and drugs that are extracted through these efflux pumps. Gram-positive bacteria The family of efflux pump

Drug extruded by this family

ABC superfamily MFS family

Numerous drugs Acriflavine, benzalkonium, cetrimide, chlorhexidine, pentamidine Aminoglycosides, fluoroquinolones, cationic drugs Acriflavine, benzalkonium, cetrimide

MATE family SMR family Gram-negative bacteria The family of efflux pump RND family ABC superfamily MFS family

Drug which extracted Numerous drugs Macrolides Nalidixic acid, novobiocin

ABC, ATP-binding cassette; MATE, multidrug and toxic compound extrusion; MFS, major facilitator superfamily; RND, resistance-nodulation-cell-division; SMR, small multidrug resistance.

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The mef genes (A and E) encode the efflux pumps that extrude erythromycin out of the cell. Streptococci (pyogenes, pneumonia), as well as Gram-positive organisms, are the main bacteria in which mef pumps are found [4,81-83]. S. aureus, P. aeruginosa, and Enterobacteriaceae use the mechanism of overexpression of the efflux pumps [9].

3.4 Changes in target sites A common technique that bacteria use in order to develop AR is the alteration of the target site. This can be achieved in several ways. Some of them follow the mutation of the target site, modification of the target site, and bypass of the target site.These processes render the antibiotic unable to bind with the target site and, thus, unable to act against bacteria [4,8,9]. 3.4.1 Target protection This mechanism is used by numerous important antibiotics. Some of them are tetracyclines, fluoroquinolones, and fusidic acid [9]. Mobile genetic elements (MGEs) play an important role because they are responsible for transferring genes that encode proteins used in target protection (Fig. 2.2).These genes may also be found in the bacterial chromosome.The most important drugs, which belong to this category, are tetracyclines and quinolones. The mechanism by which tetracyclines develop AR involves basically two ribosomal protection proteins that are called Tet(M) and Tet(O). They are capable of dislodging tetracycline from the ribosome and this process depends on the presence of GTP. As a result, tetracycline is released and the antibiotic is unable to bind again at the binding site and, therefore, unable to act [26,84,85]. The mechanism of quinolones action is the inhibition of DNA synthesis through their binding with the enzymes DNA gyrase and topoisomerase IV. The QNR genes discovered first in clinical isolates of K. pneumoniae encode the QNR proteins. They are transmitted by plasmids and considered responsible for the development of AR. Plasmidmediated resistance in quinolones can be transmitted either horizontally or vertically. Quinolone resistance can be achieved with the help of QNR proteins. QNR’s way of action is to decrease the ability of DNA gyrase and topoisomerase IV to bind to DNA; therefore they manage to decrease the number of enzyme targets on the chromosome. As a result, the cell is protected from quinolones action. Another mechanism that QNR proteins employ involves their binding to DNA gyrase and topoisomerase IV instead of quinolones. By this way, quinolones render unable to enter the cleavage complexes that enzymes form [38,86-90]. 3.4.2 Modification of the target site The main processes involved in the alteration of the target site are mutations of the genes that are responsible to encode the target, alteration of the target site by enzymes, and change or bypass of the target site [4,8,9].

Mechanisms of antibacterial drug resistance and approaches to overcome

3.4.2.1 Mutations lead to alteration of the target site The most common drugs, which belong to this category, are rifampin, fluoroquinolones, and oxazolidinones (linezolid and tedizolid). The rpoB gene found in Escherichia coli is responsible for encoding the (-subunit of RNA polymerase. In the case of rifampin a point mutation in the rpoB gene leads to a high level of AR (Fig. 2.2). This is due to some genetic changes in the rpoB gene [91,92]. Fluoroquinolones act by inhibiting DNA gyrase and topoisomerase IV enzymes which are necessary for replication of the DNA so they develop AR through mutations in the genes that encode these enzymes [93,94]. Linezolid and tedizolid belong to the class of oxazolidinones. Linezolid is used mostly against Gram-positive bacteria and MDR pathogens such as MRSA. AR against linezolid is developed by mutations in genes of the 50S ribosomal subunit; thus the affinity of the drug for the target located in this region of the ribosome is decreased [95-97]. 3.4.2.2 Alteration of the target site by enzymes Macrolides and linezolid are the most common drugs in this category. In macrolides the so-called erm (erythromycin ribosomal methylation) genes encode an enzyme, which causes methylation of the ribosome, and therefore, macrolide resistance develops. Numerous erm genes have been described but the most commonly found are erm(A), erm(C) in staphylococci and erm(B) in Pneumococci, and Enterococci [98,99]. Linezolid uses an enzyme called CFR enzyme that is encoded by CFR gene to develop AR against phenolics, streptogramin A, lincosamides, and pleuromutilins, the so-called CFRmediated linezolid resistance [100,101]. 3.4.2.3 Change or bypass of the target site In this case the old target is replaced by a new one that has almost the same biochemical function, although the antibacterial agents are incapable of inhibiting it. In the case of MRSA the PBP2a that is encoded by the mecA gene plays an important role in the development of AR. It renders the bacteria able to sustain their cell-wall synthesis, while the (-lactam antibiotics are capable of inhibiting other PBPs [63,102-106].Vancomycin resistance is common among Enterococci, although it was also described a high-level AR against it in S. aureus. In Enterococci, the so-called van gene clusters are acquired. These genes affect the synthesis of PG [4,107,108]. The second strategy, which bacteria use in this category, is to bypass the target site. They achieve this through overproduction of antibiotic target, which results in decreased concentration of antibiotic in comparison with an increment concentration of the targeted molecules. For instance, trimethoprimsulfamethoxazole (TMP-SMX) acts by changing the bacterial production of folate and, thus, the bacteria are unable to synthesize purines, as well as, other useful amino acids. Two enzymes are involved in the synthesis of folate called dihydropteroic acid synthase (DHPS) and DHFR. These enzymes take part in two processes. The first process is the formation of dihydrofolate with the action of DHPS from PABA. In the second process,

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dihydrofolate is transformed in tetrahydrofolate through DHFR. TMP-SMX resistance is achieved by the inhibition of PABA from SMX and by the inhibition of dihydrofolate (Fig. 2.2). However, there is another mechanism of resistance that involves increased production of DHFR and DHPS, which impairs the inhibition of folate production by TMP-SMX. Therefore bacteria manage to survive [109-111].

3.5 Resistance by global cell adaptations Human body is one of the most hostile environments for bacteria. After their entrance in a human’s body to avoid immune’s system attack and gain sufficient nutrients to survive, bacteria developed complex mechanisms.Through these mechanisms, they manage to retain the process of cell-wall synthesis and membrane homeostasis, which are vital for their survival. Daptomycin and vancomycin are two drugs that belong to this category. Daptomycin is a lipopeptide that acts as an antibacterial agent against Enterococci by alternating the homeostasis of the cell envelope [112]. Cationic antimicrobial peptides (CAMPs), produced by the innate immune system, play an important role in daptomycin’s action. Therefore if CAMPs action is inhibited and the cell envelope is protected, AR can occur. In Enterococci (Enterococcus faecalis) the mechanism of daptomycin resistance involves mutations in LiaFSR that is a system found in Gram-positive bacteria composed of three components responsible for regulating the response to stress on the cell envelope [113-115]. However, the direct mechanism by which LiaFSR develops AR and the mechanism by which regulates cell envelope’s stress response are not fully understood. The cell synthesis and homeostasis is also regulated by another system called YycFG (WalKR). This regulatory system is important not only for Enterococci but also for S. aureus [115].

4. Mechanisms of the spread of antibacterial resistance 4.1 Genetic basis and mechanisms of spreading antibacterial resistance AR is classified into two types, “intrinsic” or “inherited” and “acquired” (Table 2.2). Intrinsic AR is defined as the type of resistance in which a specific characteristic of an organism is inherited (Table 2.2). This happens through the processes of horizontal or vertical transfer of genetic information [4,8,13,116]. The second type of AR is acquired which means that a previously effective drug became ineffective, either through a mutation in a chromosomal gene or through the acquisition of a foreign gene(s), that develops AR by horizontal gene transfer (HGT) [4,8,9,51,117]. HGT is considered to play an important role in the development of AR [118]. The transmission of AR can be done either in a horizontal or vertical transfer of genetic material [5]. Bacteria are capable of rapidly spreading the antibacterial resistant mutations. Resistant strains of bacteria may transmit new genetic information to susceptible bacteria by HGT. This can be done through three mechanisms such as transformation, transduction,

Mechanisms of antibacterial drug resistance and approaches to overcome

Table 2.2  Subtypes of the intrinsic and acquired type of resistance. Intrinsic resistance

Acquired resistance

(1) The target is absent (2) The target site has a species-specific structure (3) Transfer of the gene (4) Low drug delivery (5) Cell-cycle effects (6) The detoxication capacity is high (7) Response to stress

(1) The selection is natural (2) Adaptive changes (3) Transfer of the gene (4) Amplification of the gene (5) Response to stress

and conjugation. Transformation is the process by which bacteria incorporate DNA. Pure DNA known also as naked DNA has the ability to pass from the donor to the recipient cell and finally be incorporated into recipients cell DNA [5,13,119]. In the process of transduction the transmission of genetic information between two relative bacteria is performed by bacteriophages that are viruses that infect bacteria. At first, a bacterial cell is infected by a phage, after that the phage is released and gets attached to the recipient cell. When the phage is inserted into the recipient cell, its DNA gets incorporated in the bacterial genome. Plasmids that are circular DNA play an important role in conjugation which is the most common mechanism used for genetic transmission in bacteria. The plasmid replication is not dependent on the replication of the bacterial genome. To transmit between two bacterial cells, plasmids use a temporal connection between the donor and the recipient cell, which is called pilus. They may carry one or more genes responsible for AR. Gram-negative bacteria often use plasmids in order to transmit genes blamed for developing AR [4,5,13,51].Transposons are MGEs that also take part in the transmission of AR genes. For the transmission of tetracyclineresistant genes, is used the transposon Tn10 [5]. The way by which bacteria may manifest AR varies [13]. It is important to understand the genetic basis of resistance to find ways to overcome it and develop new therapeutic approaches [9].

4.2 Emergence of antibacterial resistance Nowadays, there is no doubt that every discovery of a new antibiotic sooner or later will be followed by the emergence of AR [120]. However, when the first antibiotics started to be used against infections which until that time were fatal, it was believed that all infectious diseases will be eradicated. It is well known that antibiotics are used in human medicine, veterinary medicine, and agriculture and are considered to be one of the most important drugs [3,121] (Fig. 2.3). Many of the mechanisms that lead to AR are dated million years ago [122]. There is a conviction that many factors are involved in the emergence of AR. The overuse and

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Figure 2.3  Pathway of spreading antibiotic resistance from domestic animals (A) to humans (along with the food chain) and between humans (B).

misuse of antibiotics as well as, the decreased use of vaccines are some of the basic factors that played an important role in the increase of selection of antibiotic-resistant pathogens [5,121]. Note that many antibiotics are prescribed without an obvious reason, sometimes only as prophylaxis. This happens in up to 50% of the cases. Bacteria should survive in different environments to be able to have offspring. That is why they have developed adaptation mechanisms. Some of the bacteria produce spores that are able to remain in the environment for many years and infect either animals or humans. Bacteria reproduce extremely fast under favorable conditions (every 20-30 min); thus they achieve to create huge populations. They have this ability because they are unicellular organisms and they use cell division for their multiplication [5]. The chain of the spread of the AR involves domestic animals, human population, and health-care facilities. At first, domestic animals are treated with antibiotics to prevent and control diseases; therefore drug-resistant bacteria develop in their gastrointestinal (GI) tracts and may remain either in the meat, milk, eggs, and other products of the animals that will be sold in the markets or spread through water contaminated with animal feces (Fig. 2.3). Animal feces contain drug-resistant bacteria, which will end up in crops and fruits by watering with contaminated water, so humans who will consume the crops and fruits are going to get into their GI tract-resistant bacteria. Another way of spreading AR bacteria occurs in health-care units when patients are treated with antibiotics and develop by their one into their GI tract-resistant bacteria or get infected by the environment in the hospital. In fact, some patients even before their admission in the hospital already carry resistant bacteria that are going to spread during their hospitalization in other, hospitalized patients. The way that bacteria can spread is either directly from human to human or indirectly from surfaces, unclean hands, or contaminated equipment

Mechanisms of antibacterial drug resistance and approaches to overcome

Figure 2.4  Timeline of antibiotics development and the emergence of AR against them. AR, antibiotic resistance.

to humans (Fig. 2.4). By this way, resistant bacteria end up in the general community when a patient is discharged from health-care units and returns back home (Fig. 2.3) [5].

5. The most harmful multidrug-resistant strains Nowadays, there is a growing need of antibacterial agents, while at the same time new bacterial strains, single drug-resistant, or MDR develop and spread extremely fast. The emergence of AR strains has been characterized by some health organizations as a “crisis.” The Centers for Disease and Control Prevention (CDC) in 2013 has recorded 18

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Table 2.3  The most harmful pathogens and response to antibiotics (according to a CDC 2013 report). Threats

Species

Resistance against drugs

Urgent threats

Clostridium difficile Escherichia coli Klebsiella spp. Neisseria gonorrhoeae

Serious threats

Acinetobacter Campylobacter Candida spp.

Fluoroquinolone resistance (2000 year strain) Carbapenem-resistance Carbapenem-resistance Cefixime, ceftriaxone, azithromycin, tetracycline resistance Multidrug-resistant Ciprofloxacin, azithromycin resistance Fluconazole resistance (azole and echinocandin resistant strains) Penicillin, cephalosporin resistance Penicillin, cephalosporin resistance Vancomycin resistance Vancomycin resistance Vancomycin resistance

ESBL E. coli ESBL Klebsiella spp. Enterococcus faecium Enterococcus faecalis Enterococcus (not determined species) Pseudomonas aeruginosa

Shigella MRSA Streptococcus pneumoniae Mycobacterium tuberculosis S. aureus

Multidrug-resistant (aminoglycosides, cephalosporins, fluoroquinolones, and carbapenems) Ceftriaxone, ciprofloxacin, multiple drug resistant Ceftriaxone, azithromycin, ciprofloxacin resistant (very common) Azithromycin, ciprofloxacin resistance Methicillin, nafcillin, oxacillin resistance Resistant to penicillin and erythromycin groups Resistance to isoniazid, multidrug-resistant, multidrug-resistant plus Vancomycin resistance

Group A Streptococcus Group B Streptococcus

Erythromycin resistance Clindamycin resistance

Nontyphoidal salmonella Salmonella serotype typhi

Concerning threats

CDC, Centers for Disease and Control Prevention; ESBL, extended-spectrum (-lactamase; MRSA, methicillin-resistant Staphylococcus aureus.

resistant strains that were categorized in three classes: (1) urgent, (2) serious, and (3) concerning (Table 2.3). Seven factors were taken into consideration in order to assess the threat and classify them: the clinical impact, the economic impact, the incidence, a 10-year incidence projection, the transmissibility, the availability of effective antibiotics, and the prevention barriers [123,124].

Mechanisms of antibacterial drug resistance and approaches to overcome

Resistant strains emerged both in Gram-positive and Gram-negative species. Some Gram-negative bacteria have developed MDR, considered to be a real threat to public health [125]. Carbapenem-resistant Enterobacteria (Klebsiella spp., E. coli), Clostridium difficile, and Neisseria gonorrhoeae are at the top of the CDC list from 2013 as the most threatening groups (Table 2.3) of bacteria [124]. One of the most common carbapenemresistant Enterobacteria is K. pneumonia (CRKP), a Gram-negative bacterium that constitutes one of the most common pathogens met in health-care units.The last lines of antibiotics used against MDR bacteria are carbapenems; thus the prognosis of the treatment of bacteria that are resistant to carbapenems as CRKP is poor and the mortality rate is high particularly among patients with compromised immune systems [126]. C. difficile is a Gram-negative spore-forming bacterium, found mostly in hospitalized patients and blamed for life-threatening diarrhea associated with antibiotic use.The treatment of infections with C. difficile is difficult due to the development of more toxic strains. Particular strains are related to the increased probability of colitis development. Indeed, a hypervirulent strain (polymerase chain reaction ribotype 027) is associated with fulminant pseudomembranous colitis and causes higher mortality [127]. N. gonorrhoeae is a Gram-negative diplococcus that causes the sexually transmitted infection gonorrhea. In the past, penicillin was the drug preferred for the treatment of infections with N. gonorrhoeae, although during the years new MDR strains developed and major classes of antibiotics became ineffective (Table 2.4). Drug-resistant M. tuberculosis and Table 2.4  The antibiotics are needed urgently for most threatening pathogenic bacteria according to a WHO 2017 report. Urgency

Bacteria

Resistance

Critical-priority 1

Acinetobacter baumannii Pseudomonas aeruginosa Enterobacteriaceae (Klebsiella, E. coli, Serratia, and Proteus) Enterococcus faecium Staphylococcus aureus

Carbapenem-resistant Carbapenem-resistant Carbapenem-resistant and ESBLproducing Vancomycin-resistant Methicillin-resistant Vancomycin intermediate and resistant Clarithromycin-resistant Fluroquinolone-resistant Fluroquinolone-resistant Cephalosporin-resistant Fluroquinolone-resistant Penicillin-non susceptible Ampicillin-resistant Fluroquinolone-resistant

High-priority 2

Helicobacter pylori Campylobacter spp. Salmonellae Neisseria gonorrhoeae Medium-priority 3

Streptococcus pneumoniae Haemophilus influenzae Shigella spp.

ESBL, extended-spectrum (-lactamase; WHO, World Health Organization.

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MDR P. aeruginosa should also be mentioned here because they constitute serious threats for public health and cause a great number of deaths worldwide [5,128]. The WHO published its first list that includes the bacteria for which new antibiotics are needed urgently in 2017 (Table 2.4). The list is constituted of 12 families of bacteria and divides them into three categories: (1) critical, (2) high, and (3) medium. The list is based on the need of antibiotics against these families due to the existence of AR (Table 2.4). The most threatening bacteria are MDR Gram-negative bacteria that are able to adapt and acquire or develop AR against many drugs, while at the same time they can transfer their genetic material to other bacteria, thus rendering them resistant. The following criteria were used for the creation of the list: the mortality of infections, the days of hospitalization, and the ways of the spread of the bacteria, and the existence or development of antibiotics able to treat infections caused by MDR bacteria. The group of critical bacteria is placed at the top of the list and includes pathogens that can cause serious and life-threatening infections such as pneumonia and bloodstream infections, while those pathogens, which are included in high and medium groups, can cause infections such as gonorrhea and food poisoning. The basic aim for the creation of the list was the promotion of research of development of new antibiotics capable of fighting MDR pathogens [129]. The WHO produces a list since 2005 that includes the most important antibiotics. The last meeting of Advisory Group of Integrated Surveillance of Antimicrobial Resistance in which the list was updated was held in the United States, in 2016 (Table 2.5). The purpose of this list is to highlight the important categories of antibiotics to prevent their unnecessary use in veterinary and human medicine, to preserve their effectiveness and to prevent the development of AR against them (Fig. 2.4). The antibiotics are divided into three categories based on their importance as follows: (1) critically important, (2) highly important, and (3) important.Two criteria and three prioritization criteria are used in the classification (Table 2.5) [130].

6. New antibacterial drug targets and novel approaches to drug development It is important to realize that developing new antibiotics may be more productive if novel molecular targets are applied. Novel antibacterial drug targets present the opportunity to develop new antibiotics against which bacteria have not still developed AR. Therefore we have to analyze bacterial genomes and many essential biochemical pathways in bacteria to figure out such new molecular targets. The suitable molecular targets should be specific to bacteria and should not be found in humans. However, this assumption can be ignored if the antibacterial drug can be delivered to the bacterial cell only. For instance, there are cell penetrating peptides (CPPs) that enter bacteria only and do not penetrate eukaryotic cells. If such CPPs are attached to antibiotics, they can

Mechanisms of antibacterial drug resistance and approaches to overcome

Table 2.5  Classification of antimicrobial drugs based on their importance according to a WHO 2016 report. Importance

Antimicrobial class

Critically important–highest priority

Cephalosporins (3rd, 4th, and 5th generation) Glycopeptides Macrolides and ketolides Polymyxins Quinolones Aminoglycosides Ansamycins Carbapenems and other penems Glycylcyclines Lipopeptides Monobactams Oxazolidinones Penicillins (natural, aminopenicillins, and antipseudomonal) Phosphonic acid derivatives Drugs used solely to treat tuberculosis or other mycobacterial diseases Aminopenicillins Amphenicols Cephalosporins (1st and 2nd generation) and cephamycins Lincosamides Penicillins (antistaphylococcal) Pseudomonic acids Riminophenazines Steroid antibacterials Streptogramins Sulfonamides, dihydrofolate reductase inhibitors, and combinations Sulfones Tetracyclines Aminocyclitols Cyclic polypeptides Nitrofurantoins Nitroimidazoles Pleuromutilins

Critically important–high priority

Highly important

Important

WHO, World Health Organization.

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specifically deliver the drugs into bacterial cells only. In fact, some CPPs possess antibacterial properties by themselves. Different molecular targets can be selected to develop different types of antibiotics. For instance, the inhibition of a particular target in bacteria can lead to either bacteriostatic or bactericide effects. In addition, if our molecular target is found in various bacterial species, we can develop broad-spectrum antibiotics against it. In contrast, if the molecular target is specific to a limited number of bacterial species, we can develop narrow-spectrum antibiotics against it. In general, it is preferable to apply narrow-spectrum antibiotics when possible because they can induce AR in a limited number of bacterial species in contrast to broad-spectrum antibiotics. In general, it is believed that bacteriostatic drugs may induce less frequently AR than that of bactericidal antibiotics due to smaller selection pressure they imposed on bacteria to evolve and develop AR. Therefore it is preferable to apply bacteriostatic and narrow-spectrum antibiotics when possible. In fact, there are some infections caused by toxin-producing strains of pathogenic bacteria that should be treated with bacteriostatic antibiotics because the simultaneous death of many bacteria can release a lethal dose of toxins in the human body leading to human death. In general, we need to develop all types of antibiotics and therefore we have to apply different types of molecular targets. However, independently of the target type, we can target the highly conservative parts of target molecules that are difficult to mutate without losing their essential functions. In recent years, there are several types of RNAs that are becoming promising targets for antibacterial drug discovery. rRNAs were usually used for many years as targets for antibacterial drug discovery. However, after the determination of the 3D structure of the bacterial ribosome, there are new opportunities to design rationally new antibiotics that target bacterial ribosomes. It is important to note that developing new antibiotics may be more productive not only when novel molecular targets are applied but also when novel approaches to drug developing are used. The combination of new drug targets and novel mechanisms of drug actions can be very productive for the development of new effective antibiotics that can tackle the challenges of the MDR pathogenic bacteria. For example, we can use antisense oligonucleotide technology to promptly design novel antibacterial drugs [4].

7. Conclusion We cannot eradicate pathogenic bacteria from the Earth. We have been coexisting with pathogenic bacteria since our existence. Some strains of pathogenic bacteria such as Yersinia pestis caused millions of deaths throughout human history when no antibiotics were available. Applying various antibiotics, we have successfully tackled the bacterial infection diseases over the last 80 years. However, the currently available antibiotics are becoming not effective against many MDR pathogenic bacteria (Fig. 2.5). At the same

Mechanisms of antibacterial drug resistance and approaches to overcome

Figure 2.5  The pipeline of antibacterial drug production (A) and the frequency of emergence of AR over the years (B). AR, antibiotic resistance.

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time, the pipeline of producing new antibacterial drugs is slowing down over the last decade. Therefore we have to promptly develop new antibiotics using new molecular targets and new mechanisms of drug action to address the threat imposed by the global spread of MDR pathogenic bacteria.

Acknowledgments The current research in Robert Penchovsky laboratory is funded by a grant no. DN13/14/20.12.2017 awarded by the Bulgarian National Science Fund (BNSF).

References [1] R.I. Aminov, A brief history of the antibiotic era: lessons learned and challenges for the future, Front Microbiol 1 (2010) 134. [2] K.J. Williams, The introduction of ‘chemotherapy’ using arsphenamine-the first magic bullet, J R Soc Med 102 (8) (2009) 343–348. [3] S. Rodriguez-Mozaz, S. Chamorro, E. Marti, B. Huerta, M. Gros, A. Sanchez-Melsio, et al. Occurrence of antibiotics and antibiotic resistance genes in hospital and urban wastewaters and their impact on the receiving river, Water Res 69 (2015) 234–242. [4] J.M. Munita, C.A. Arias, Mechanisms of antibiotic resistance, Microbiol Spectr 4 (2) (2016) 1–37. [5] R. Penchovsky, M. Traykovska, Designing drugs that overcome antibacterial resistance: where do we stand and what should we do?, Expert Opin Drug Discov 10 (6) (2015) 631–650. [6] C.M. Kunin, Antibiotic armageddon, Clin Infect Dis 25 (2) (1997) 240–241. [7] G.D. Wright, Molecular mechanisms of antibiotic resistance, Chem Commun (Camb) 47 (14) (2011) 4055–4061. [8] J.D. Hayes, C.R. Wolf, Molecular mechanisms of drug resistance, Biochem J 272 (2) (1990) 281–295. [9] J.M. Blair, M.A. Webber, A.J. Baylay, D.O. Ogbolu, L.J. Piddock, Molecular mechanisms of antibiotic resistance, Nat Rev Microbiol 13 (1) (2015) 42–51. [10] R. Hermsen, J.B. Deris, T. Hwa, On the rapidity of antibiotic resistance evolution facilitated by a concentration gradient, Proc Natl Acad Sci U S A 109 (27) (2012) 10775–10780. [11] M.A. Kohanski, D.J. Dwyer, J.J. Collins, How antibiotics kill bacteria: from targets to networks, Nat Rev Microbiol 8 (6) (2010) 423–435. [12] MA Jawetz, Medical microbiology, 26th ed., Lange, Saint Louis, MO, USA, 2013, pp. 11–31. [13] F.C. Tenover, Mechanisms of antimicrobial resistance in bacteria, Am J Med 119 (6 Suppl 1) (2006) S3–S10 discussion S62-70. [14] S.B. Levy, B. Marshall, Antibacterial resistance worldwide: causes, challenges, and responses, Nat Med 10 (12 Suppl) (2004) S122–S129. [15] T.D. Bugg, C.T. Walsh, Intracellular steps of bacterial cell wall peptidoglycan biosynthesis: enzymology, antibiotics, and antibiotic resistance, Nat Prod Rep 9 (3) (1992) 199–215. [16] J.V. Holtje, Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli, Microbiol Mol Biol Rev 62 (1) (1998) 181–203. [17] J.T. Park,T. Uehara, How bacteria consume their own exoskeletons (turnover and recycling of cell wall peptidoglycan), Microbiol Mol Biol Rev 72 (2) (2008) 211–227 table of contents. [18] L.P. Kotra, S. Mobashery, Mechanistic and clinical aspects of beta-lactam antibiotics and beta-lactamases, Arch Immunol Ther Exp (Warsz) 47 (4) (1999) 211–216. [19] G. Kapoor, S. Saigal, A. Elongavan, Action and resistance mechanisms of antibiotics: a guide for clinicians, J Anaesthesiol Clin Pharmacol 33 (3) (2017) 300–305. [20] P.E. Reynolds, Structure, biochemistry and mechanism of action of glycopeptide antibiotics, Eur J Clin Microbiol Infect Dis 8 (11) (1989) 943–950. [21] M.S. Wilke, A.L. Lovering, N.C. Strynadka, Beta-lactam antibiotic resistance: a current structural perspective, Curr Opin Microbiol 8 (5) (2005) 525–533.

Mechanisms of antibacterial drug resistance and approaches to overcome

[22] R. Nagarajan, Antibacterial activities and modes of action of vancomycin and related glycopeptides, Antimicrob Agents Chemother 35 (4) (1991) 605–609. [23] P. Vannuffel, C. Cocito, Mechanism of action of streptogramins and macrolides, Drugs 51 (Suppl 1) (1996) 20–30. [24] H. Jaka, A. Liwa, Antimicrobial resistance: mechanisms of action of antimicrobial agents, The Battle Against Microbial Pathogens: Basic Science, Technological Advances and Educational Programs, 2015. [25] T. Pape, W. Wintermeyer, M.V. Rodnina, Conformational switch in the decoding region of 16S rRNA during aminoacyl-tRNA selection on the ribosome, Nat Struct Biol 7 (2) (2000) 104–107. [26] I. Chopra, M. Roberts, Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance, Microbiol Mol Biol Rev 65 (2) (2001) 232–260 Second page, table of contents. [27] I. Chopra, P.M. Hawkey, M. Hinton, Tetracyclines, molecular and clinical aspects, J Antimicrob Chemother 29 (3) (1992) 245–277. [28] R. Schroeder, C. Waldsich, H. Wank, Modulation of RNA function by aminoglycoside antibiotics, EMBO J 19 (1) (2000) 1–9. [29] O. Espeli, K.J. Marians, Untangling intracellular DNA topology, Mol Microbiol 52 (4) (2004) 925–931. [30] K. Drlica, M. Snyder, Superhelical Escherichia coli DNA: relaxation by coumermycin, J Mol Biol 120 (2) (1978) 145–154. [31] M. Gellert, K. Mizuuchi, M.H. O’Dea, H.A. Nash, DNA gyrase: an enzyme that introduces superhelical turns into DNA, Proc Natl Acad Sci U S A 73 (11) (1976) 3872–3876. [32] L.F. Liu, C.C. Liu, B.M. Alberts,Type II DNA topoisomerases: enzymes that can unknot a topologically knotted DNA molecule via a reversible double-strand break, Cell 19 (3) (1980) 697–707. [33] F. Collin, S. Karkare, A. Maxwell, Exploiting bacterial DNA gyrase as a drug target: current state and perspectives, Appl Microbiol Biotechnol 92 (3) (2011) 479–497. [34] D.E. Ehmann, S.D. Lahiri, Novel compounds targeting bacterial DNA topoisomerase/DNA gyrase, Curr Opin Pharmacol 18 (2014) 76–83. [35] M. Chatterji, S. Unniraman, S. Mahadevan,V. Nagaraja, Effect of different classes of inhibitors on DNA gyrase from Mycobacterium smegmatis, J Antimicrob Chemother 48 (4) (2001) 479–485. [36] K. Drlica, Mechanism of fluoroquinolone action, Curr Opin Microbiol 2 (5) (1999) 504–508. [37] C.R. Chen, M. Malik, M. Snyder, K. Drlica, DNA gyrase and topoisomerase IV on the bacterial chromosome: quinolone-induced DNA cleavage, J Mol Biol 258 (4) (1996) 627–637. [38] K. Drlica, X. Zhao, DNA gyrase topoisomerase IV, and the 4-quinolones, Microbiol Mol Biol Rev 61 (3) (1997) 377–392. [39] A.Y. Chen, L.F. Liu, DNA topoisomerases: essential enzymes and lethal targets, Annu Rev Pharmacol Toxicol 34 (1994) 191–218. [40] W.J. Burman, K. Gallicano, C. Peloquin, Comparative pharmacokinetics and pharmacodynamics of the rifamycin antibacterials, Clin Pharmacokinet 40 (5) (2001) 327–341. [41] W. Wehrli, Rifampin: mechanisms of action and resistance, Rev Infect Dis 3 (5 Suppl) (1983) S407–S411. [42] G.L. Hobby, T.F. Lenert, The action of rifampin alone and in combination with other antituberculous drugs, Am Rev Respir Dis 102 (3) (1970) 462–465. [43] G. Hartmann, K.O. Honikel, F. Knusel, J. Nuesch, The specific inhibition of the DNA-directed RNA synthesis by rifamycin, Biochim Biophys Acta 145 (3) (1967) 843–844. [44] E.A. Campbell, N. Korzheva, A. Mustaev, K. Murakami, S. Nair, A. Goldfarb, et al. Structural mechanism for rifampicin inhibition of bacterial RNA polymerase, Cell 104 (6) (2001) 901–912. [45] T. Naryshkina, A. Mustaev, S.A. Darst, K. Severinov, The β′ subunit of Escherichia coli RNA polymerase is not required for interaction with initiating nucleotide but is necessary for interaction with rifampicin, J Biol Chem 276 (16) (2001) 13308–13313. [46] H.Yoneyama, R. Katsumata, Antibiotic resistance in bacteria and its future for novel antibiotic development, Biosci Biotechnol Biochem 70 (5) (2006) 1060–1075. [47] C. Walsh, Where will new antibiotics come from?, Nat Rev Microbiol 1 (1) (2003) 65–70. [48] MA Jawetz, Medical microbiology, 26th ed., Lange, Saint Louis, MO, USA, (2013) 373. [49] M. Krupovic, R. Daugelavicius, D.H. Bamford, Polymyxin B induces lysis of marine pseudoalteromonads, Antimicrob Agents Chemother 51 (11) (2007) 3908–3914.

33

34

Drug Discovery Targeting Drug-Resistant Bacteria

[50] L.L.D. Mayers, J. Sobel, M. Ouellette, K. Kaye, D. Marchaim, , Antimicrobial drug resistance: mechanisms of drug resistance,Volume 12017. [51] N.A. Lerminiaux, A.D.S. Cameron, Horizontal transfer of antibiotic resistance genes in clinical environments, Can J Microbiol 65 (1) (2019) 34–44. [52] D.N. Wilson, Ribosome-targeting antibiotics and mechanisms of bacterial resistance, Nat Rev Microbiol 12 (1) (2014) 35–48. [53] K.J. Shaw, P.N. Rather, R.S. Hare, G.H. Miller, Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes, Microbiol Rev 57 (1) (1993) 138–163. [54] L.P. Kotra, J. Haddad, S. Mobashery, Aminoglycosides: perspectives on mechanisms of action and resistance and strategies to counter resistance, Antimicrob Agents Chemother 44 (12) (2000) 3249–3256. [55] S. Shakil, R. Khan, R. Zarrilli, A.U. Khan, Aminoglycosides versus bacteria-a description of the action, resistance mechanism, and nosocomial battleground, J Biomed Sci 15 (1) (2008) 5–14. [56] M.P. Mingeot-Leclercq,Y. Glupczynski, P.M. Tulkens, Aminoglycosides: activity and resistance, Antimicrob Agents Chemother 43 (4) (1999) 727–737. [57] K. Poole, Aminoglycoside resistance in Pseudomonas aeruginosa, Antimicrob Agents Chemother 49 (2) (2005) 479–487. [58] M.S. Ramirez, M.E. Tolmasky, Aminoglycoside modifying enzymes, Drug Resist Updat 13 (6) (2010) 151–171. [59] I.A. Murray, W.V. Shaw, O-Acetyltransferases for chloramphenicol and other natural products, Antimicrob Agents Chemother 41 (1) (1997) 1–6. [60] S. Schwarz, C. Kehrenberg, B. Doublet, A. Cloeckaert, Molecular basis of bacterial resistance to chloramphenicol and florfenicol, FEMS Microbiol Rev 28 (5) (2004) 519–542. [61] R.P. Ambler, A.F. Coulson, J.M. Frere, J.M. Ghuysen, B. Joris, M. Forsman, et al. A standard numbering scheme for the class A beta-lactamases, Biochem J 276 (Pt 1) (1991) 269–270. [62] K. Bush, G.A. Jacoby, A.A. Medeiros, A functional classification scheme for beta-lactamases and its correlation with molecular structure, Antimicrob Agents Chemother 39 (6) (1995) 1211–1233. [63] B. Blazquez, L.I. Llarrull, J.R. Luque-Ortega, C. Alfonso, B. Boggess, S. Mobashery, Regulation of the expression of the beta-lactam antibiotic-resistance determinants in methicillin-resistant Staphylococcus aureus (MRSA), Biochemistry 53 (10) (2014) 1548–1550. [64] D.L. Paterson, R.A. Bonomo, Extended-spectrum beta-lactamases: a clinical update, Clin Microbiol Rev 18 (4) (2005) 657–686. [65] J.F. Fisher, S. Mobashery, β-Lactam resistance mechanisms: Gram-positive bacteria and Mycobacterium tuberculosis, Cold Spring Harb Perspect Med 6 (5) (2016). [66] H. Nikaido, M.Vaara, Molecular basis of bacterial outer membrane permeability, Microbiol Rev 49 (1) (1985) 1–32. [67] J.M. Pages, C.E. James, M. Winterhalter, The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria, Nat Rev Microbiol 6 (12) (2008) 893–903. [68] M.A. Gharabaghi, S.M. Abdollahi, E. Safavi, S.H. Abtahi, Community acquired Pseudomonas pneumonia in an immune competent host, BMJ Case Rep (2012) 2012. [69] S.M. Bagshaw, K.B. Laupland, Epidemiology of intensive care unit-acquired urinary tract infections, Curr Opin Infect Dis 19 (1) (2006) 67–71. [70] X. Ji, P. Jin,Y. Chu, S. Feng, P. Wang, Clinical characteristics and risk factors of diabetic foot ulcer with multidrug-resistant organism infection, Int J Low Extrem Wounds 13 (1) (2014) 64–71. [71] V.H. Tam, C.A. Rogers, K.T. Chang, J.S. Weston, J.P. Caeiro, K.W. Garey, Impact of multidrug-resistant Pseudomonas aeruginosa bacteremia on patient outcomes, Antimicrob Agents Chemother 54 (9) (2010) 3717–3722. [72] R.E. Hancock, F.S. Brinkman, Function of pseudomonas porins in uptake and efflux, Annu Rev Microbiol 56 (2002) 17–38. [73] J.P. Quinn, E.J. Dudek, C.A. DiVincenzo, D.A. Lucks, S.A. Lerner, Emergence of resistance to imipenem during therapy for Pseudomonas aeruginosa infections, J Infect Dis 154 (2) (1986) 289–294. [74] H. Huang, R.E. Hancock, The role of specific surface loop regions in determining the function of the imipenem-specific pore protein OprD of Pseudomonas aeruginosa, J Bacteriol 178 (11) (1996) 3085–3090.

Mechanisms of antibacterial drug resistance and approaches to overcome

[75] J. Trias, H. Nikaido, Outer membrane protein D2 catalyzes facilitated diffusion of carbapenems and penems through the outer membrane of Pseudomonas aeruginosa, Antimicrob Agents Chemother 34 (1) (1990) 52–57. [76] U.O. Hasdemir, J. Chevalier, P. Nordmann, J.M. Pages, Detection and prevalence of active drug efflux mechanism in various multidrug-resistant Klebsiella pneumoniae strains from Turkey, J Clin Microbiol 42 (6) (2004) 2701–2706. [77] Y.K. Tsai, C.P. Fung, J.C. Lin, J.H. Chen, F.Y. Chang, T.L. Chen, et al. Klebsiella pneumoniae outer membrane porins OmpK35 and OmpK36 play roles in both antimicrobial resistance and virulence, Antimicrob Agents Chemother 55 (4) (2011) 1485–1493. [78] L.J. Piddock, Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria, Clin Microbiol Rev 19 (2) (2006) 382–402. [79] H.I. Zgurskaya, H. Nikaido, Multidrug resistance mechanisms: drug efflux across two membranes, Mol Microbiol 37 (2) (2000) 219–225. [80] X.Z. Li, H. Nikaido, Efflux-mediated drug resistance in bacteria: an update, Drugs 69 (12) (2009) 1555–1623. [81] K. Poole, Efflux-mediated antimicrobial resistance, J Antimicrob Chemother 56 (1) (2005) 20–51. [82] M.C. Roberts, Update on acquired tetracycline resistance genes, FEMS Microbiol Lett 245 (2) (2005) 195–203. [83] T. Golkar, M. Zielinski, A.M. Berghuis, Look and outlook on enzyme-mediated macrolide resistance, Front Microbiol 9 (2018) 1942. [84] S.R. Connell, D.M.Tracz, K.H. Nierhaus, D.E.Taylor, Ribosomal protection proteins and their mechanism of tetracycline resistance, Antimicrob Agents Chemother 47 (12) (2003) 3675–3681. [85] A. Donhofer, S. Franckenberg, S.Wickles, O. Berninghausen, R. Beckmann, D.N.Wilson, Structural basis for TetM-mediated tetracycline resistance, Proc Natl Acad Sci U S A 109 (42) (2012) 16900–16905. [86] D.C. Hooper, Mechanisms of fluoroquinolone resistance, Drug Resist Updat 2 (1) (1999) 38–55. [87] D.C. Hooper, G.A. Jacoby, Mechanisms of drug resistance: quinolone resistance, Ann N Y Acad Sci 1354 (2015) 12–31. [88] L. Martinez-Martinez, A. Pascual, G.A. Jacoby, Quinolone resistance from a transferable plasmid, Lancet 351 (9105) (1998) 797–799. [89] J.M. Rodriguez-Martinez, M.E. Cano, C.Velasco, L. Martinez-Martinez, A. Pascual, Plasmid-mediated quinolone resistance: an update, J Infect Chemother 17 (2) (2011) 149–182. [90] K.J. Aldred, R.J. Kerns, N. Osheroff, Mechanism of quinolone action and resistance, Biochemistry 53 (10) (2014) 1565–1574. [91] H.G. Floss, T.W.Yu, Rifamycin-mode of action, resistance, and biosynthesis, Chem Rev 105 (2) (2005) 621–632. [92] D.J. Jin, C.A. Gross, Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance, J Mol Biol 202 (1) (1988) 45–58. [93] D.C. Hooper, Fluoroquinolone resistance among Gram-positive cocci, Lancet Infect Dis 2 (9) (2002) 530–538. [94] C.J. Willmott, A. Maxwell, A single point mutation in the DNA gyrase A protein greatly reduces binding of fluoroquinolones to the gyrase-DNA complex, Antimicrob Agents Chemother 37 (1) (1993) 126–127. [95] R.E. Mendes, L.M. Deshpande, R.N. Jones, Linezolid update: stable in vitro activity following more than a decade of clinical use and summary of associated resistance mechanisms, Drug Resist Updat 17 (1-2) (2014) 1–12. [96] R.E. Mendes, L. Deshpande, J.M. Streit, H.S. Sader, M. Castanheira, P.A. Hogan, et al. ZAAPS programme results for 2016: an activity and spectrum analysis of linezolid using clinical isolates from medical centres in 42 countries, J Antimicrob Chemother (2018) 1880–1887. [97] H. Chen, W. Wu, M. Ni, Y. Liu, J. Zhang, F. Xia, et al. Linezolid-resistant clinical isolates of Enterococci and Staphylococcus cohnii from a multicentre study in China: molecular epidemiology and resistance mechanisms, Int J Antimicrob Agents 42 (4) (2013) 317–321. [98] R. Leclercq, Mechanisms of resistance to macrolides and lincosamides: nature of the resistance elements and their clinical implications, Clin Infect Dis 34 (4) (2002) 482–492. [99] B.Weisblum, Erythromycin resistance by ribosome modification, Antimicrob Agents Chemother 39 (3) (1995) 577–585.

35

36

Drug Discovery Targeting Drug-Resistant Bacteria

[100] S.M. Toh, L. Xiong, C.A. Arias, M.V.Villegas, K. Lolans, J. Quinn, et al. Acquisition of a natural resistance gene renders a clinical strain of methicillin-resistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid, Mol Microbiol 64 (6) (2007) 1506–1514. [101] S. Schwarz, C. Werckenthin, C. Kehrenberg, Identification of a plasmid-borne chloramphenicol-florfenicol resistance gene in Staphylococcus sciuri, Antimicrob Agents Chemother 44 (9) (2000) 2530–2533. [102] J. Fishovitz, J.A. Hermoso, M. Chang, S. Mobashery, Penicillin-binding protein 2a of methicillinresistant Staphylococcus aureus, IUBMB Life 66 (8) (2014) 572–577. [103] J. Cha, S.B.Vakulenko, S. Mobashery, Characterization of the beta-lactam antibiotic sensor domain of the MecR1 signal sensor/transducer protein from methicillin-resistant Staphylococcus aureus, Biochemistry 46 (26) (2007) 7822–7831. [104] T.K. McKinney,V.K. Sharma, W.A. Craig, G.L. Archer, Transcription of the gene mediating methicillin resistance in Staphylococcus aureus (mecA) is corepressed but not coinduced by cognate mecA and beta-lactamase regulators, J Bacteriol 183 (23) (2001) 6862–6868. [105] A.E. Rosato, B.N. Kreiswirth, W.A. Craig, W. Eisner, M.W. Climo, G.L. Archer, mecA-blaZ corepressors in clinical Staphylococcus aureus isolates, Antimicrob Agents Chemother 47 (4) (2003) 1460–1463. [106] C. Ryffel, F.H. Kayser, B. Berger-Bachi, Correlation between regulation of mecA transcription and expression of methicillin resistance in staphylococci, Antimicrob Agents Chemother 36 (1) (1992) 25–31. [107] D. Billot-Klein, L. Gutmann, E. Collatz, J. van Heijenoort, Analysis of peptidoglycan precursors in vancomycin-resistant Enterococci, Antimicrob Agents Chemother 36 (7) (1992) 1487–1490. [108] M. Baptista, F. Depardieu, P. Reynolds, P. Courvalin, M. Arthur, Mutations leading to increased levels of resistance to glycopeptide antibiotics in VanB-type Enterococci, Mol Microbiol 25 (1) (1997) 93–105. [109] P. Huovinen, L. Sundstrom, G. Swedberg, O. Skold,Trimethoprim and sulfonamide resistance, Antimicrob Agents Chemother 39 (2) (1995) 279–289. [110] P. Huovinen, Resistance to trimethoprim-sulfamethoxazole, Clin Infect Dis 32 (11) (2001) 1608–1614. [111] M.K.Yun, Y. Wu, Z. Li, Y. Zhao, M.B. Waddell, A.M. Ferreira, et al. Catalysis and sulfa drug resistance in dihydropteroate synthase, Science 335 (6072) (2012) 1110–1114. [112] C.A. Arias, D. Panesso, D.M. McGrath, X. Qin, M.F. Mojica, C. Miller, et al. Genetic basis for in vivo daptomycin resistance in Enterococci, N Engl J Med 365 (10) (2011) 892–900. [113] J.M. Munita, T.T. Tran, L. Diaz, D. Panesso, J. Reyes, B.E. Murray, et al. A liaF codon deletion abolishes daptomycin bactericidal activity against vancomycin-resistant Enterococcus faecalis, Antimicrob Agents Chemother 57 (6) (2013) 2831–2833. [114] S. Jordan, A. Junker, J.D. Helmann,T. Mascher, Regulation of LiaRS-dependent gene expression in Bacillus subtilis: identification of inhibitor proteins, regulator binding sites, and target genes of a conserved cell envelope stress-sensing two-component system, J Bacteriol 188 (14) (2006) 5153–5166. [115] L. Diaz, T.T. Tran, J.M. Munita, W.R. Miller, S. Rincon, L.P. Carvajal, et al. Whole-genome analyses of Enterococcus faecium isolates with diverse daptomycin MICs, Antimicrob Agents Chemother 58 (8) (2014) 4527–4534. [116] D.I. Andersson, D. Hughes, J.Z. Kubicek-Sutherland, Mechanisms and consequences of bacterial resistance to antimicrobial peptides, Drug Resist Updat 26 (2016) 43–57. [117] P.A. Smith, F.E. Romesberg, Combating bacteria and drug resistance by inhibiting mechanisms of persistence and adaptation, Nat Chem Biol 3 (9) (2007) 549–556. [118] K.S. Baker, T.J. Dallman, N. Field, T. Childs, H. Mitchell, M. Day, et al. Horizontal antimicrobial resistance transfer drives epidemics of multiple Shigella species, Nat Commun 9 (1) (2018) 1462. [119] S. Jiang, J. Zeng, X. Zhou, Y. Li, Drug resistance and gene transfer mechanisms in respiratory/oral bacteria, J Dent Res 97 (10) (2018) 1092–1099. [120] J. Davies, D. Davies, Origins and evolution of antibiotic resistance, Microbiol Mol Biol Rev 74 (3) (2010) 417–433. [121] J.L. Martinez, F. Baquero, Emergence and spread of antibiotic resistance: setting a parameter space, Ups J Med Sci 119 (2) (2014) 68–77. [122] I.M. Gould, A.M. Bal, New antibiotic agents in the pipeline and how they can help overcome microbial resistance,Virulence 4 (2) (2013) 185–191. [123] C.L.Ventola, The antibiotic resistance crisis: Part 1: Causes and threats, P T 40 (4) (2015) 277–283.

Mechanisms of antibacterial drug resistance and approaches to overcome

[124] T. Friedan, Antibiotic resistance threats in the United States. Report. United States: 2013 23. April Report No., 2013, 1-114. [125] G.M. Rossolini, F. Arena, P. Pecile, S. Pollini, Update on the antibiotic resistance crisis, Curr Opin Pharmacol 18 (2014) 56–60. [126] B. Zheng, Y. Dai, Y. Liu, W. Shi, E. Dai, Y. Han, et al. Molecular epidemiology and risk factors of carbapenem-resistant Klebsiella pneumoniae infections in Eastern China, Front Microbiol 8 (2017) 1061. [127] S. Nishimura, T. Kou, H. Kato, M. Watanabe, S. Uno, M. Senoh, et al. Fulminant pseudomembranous colitis caused by Clostridium difficile PCR ribotype 027 in a healthy young woman in Japan, J Infect Chemother 20 (11) (2014) 729–731. [128] L. Sanchez-Buso, S.R. Harris, Using genomics to understand antimicrobial resistance and transmission in Neisseria gonorrhoeae, Microb Genom (2019). [129] Organization WH. Global priority list of antibiotic-resistant bacteria 2017. Available from: https:// www.who.int/news-room/detail/27-02-2017-who-publishes-list-of-bacteria-for-whichnew-antibiotics-are-urgently-needed. [130] WHO Advisory Group on Integrated Surveillance of Antimicrobial Resistance (AGISAR)Critically important antimicrobials for human medicine, 5th revision, World health organization, Geneva, (2017).

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Antibiotics targeting Gram-negative bacteria Radha Rangarajana, Rasika Venkataramanb

Vitas Pharma Research Private Limited, University of Hyderabad, Hyderabad, Telangana, India Fred Hutchinson Cancer Research Center, Seattle, WA, United States

a

b

The discovery of penicillin in the 1940s was a watershed moment in human history; not only did it contribute to reducing mortality and morbidity but also laid the foundations for the science of drug discovery. Following the introduction of penicillin, a number of other antibiotics were discovered and brought into clinical use in quick succession. Analogs followed, as synthetic chemistry processes evolved.This period is often known as the golden period of antibiotic drug discovery. It was followed by a brief lull in the 1980s, but activity picked up again in the 1990s, with the advent of the Genomics era. Cellular targets became the focal point of discovery campaigns. To efficiently identify chemical hits targeting these proteins, pharmaceutical companies employed high-throughput screening to rapidly evaluate the activity of millions of compounds from in-house libraries. Despite extensive efforts, dedicated manpower and generous outlays of funding, this approach failed to deliver new drugs. Not surprisingly, this led to an exodus of pharmaceutical and biotech companies marking the end of the phase. Paradoxically, this largescale exit coincided with rising morbidity and mortality due to infections. Responding to this unmet need now, is a clutch of small pharmaceutical and biotech companies, marking perhaps the third phase in the history of antibiotics. What clinical unmet needs are driving the efforts of these companies? What do compounds in development promise to deliver? What are the gaps? This chapter addresses these questions in the context of Gram-negative infections.

1  Gram-negative infections: the unmet need Gram-negative bacteria are associated with serious, life-threatening infections, such as bloodstream, surgical site, complicated urinary tract, and lung.The most commonly used antibiotics for such infections are cephalosporins (ceftriaxone–cefotaxime, ceftazidime, and others), fluoroquinolones (ciprofloxacin, levofloxacin), tetracyclines, aminoglycosides (gentamicin, amikacin), carbapenems, broad-spectrum penicillins with or without β-lactamase inhibitors (BLIs) (amoxicillin–clavulanic acid, piperacillin–tazobactam), Drug Discovery Targeting Drug-Resistant Bacteria. http://dx.doi.org/10.1016/B978-0-12-818480-6.00003-5 Copyright © 2020 Elsevier Inc. All rights reserved.

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fosfomycin, and trimethoprim–sulfamethoxazole. Despite the availability of these classes of antibiotics, Gram-negative infections are associated with high morbidity and mortality. Multidrug resistance (MDR) is a key factor. MDR limits therapeutic choices, leading to suboptimal outcomes for patients. The World Health Organization has identified priority pathogens, responsible for the most tough-to-treat infections [1]. The Gram-negative species of this set include Escherichia coli, Klebsiella, Acinetobacter, and Pseudomonas spp., which define the focus of this chapter.

1.1  Mechanisms of resistance and their epidemiology MDR in Gram-negatives is mostly characterized by increased resistance to third- and fourth-generation cephalosporins, resistance to carbapenems, combined resistance to fluoroquinolones, aminoglycosides, in addition to carbapenems and resistance to polymyxins. Of particular concern, is resistance to last-line antibiotics such as carbapenems and polymyxins, which is driving current discovery and development efforts. Carbapenem resistance is driven by genes encoding carbapenem-hydrolyzing enzymes, carbapenemases (classes A, B, C, and D), upregulation of efflux pumps, and porin loss (Fig. 3.1) [2]. Resistances to polymyxins B and E are caused by modifications of the lipopolysaccharide layer through genes altering lipid A [mcr (1–8), mgrB, phoP/phoQ, pmrA, and others], and upregulation of efflux pumps (Fig. 3.1) [3]. A brief summary of global epidemiological data and the diversity of resistance mechanisms is provided here. 1.1.1  Resistance to carbapenems Carbapenem-resistant Enterobacteriaceae (CRE) are an important and rapidly increasing threat to global health.Their rapid and wide dissemination is contributed by both clonal transmission and plasmid-mediated horizontal transmission of resistance genes in bacteria. Data from large surveillance networks, such as International Nosocomial Infection Control Consortium, SENTRY Antimicrobial Surveillance Program, and European Antimicrobial Resistance Surveillance Network (EARS-NET), report a continuing dissemination of carbapenem-resistant hospital-acquired infections in intensive care units (ICUs) across Europe, Asia, and the United States, with an overall prevalence of up to 4% in E. coli, 7% in Klebsiella pneumoniae, 45% in Pseudomonas aeruginosa, and up to 63% in Acinetobacter baumannii [4]. A multicenter study in 2015, which covered 25 hospitals across 14 provinces in China, reported an overall CRE infection incidence of 4.0 per 10,000 discharges [5]. In comparison, overall CRE infection incidences were 2.93 per 100,000 population and 1.3 per 10,000 hospital admissions in the United States and European countries, respectively [5].

Antibiotics targeting Gram-negative bacteria

Figure 3.1  Mechanisms of resistance for (A) carbapenems and (B) polymyxins. (A) Resistance to carbapenems is mediated by plasmid or chromosome encoded genes expressing carbapenemases, which enzymatically degrade the antibiotic and render it inactive, reduction of outer membrane permeability by loss of or mutation in OprD porin channels, and upregulation of efflux pump systems. (B) Resistance to polymyxins is caused by modifications to the lipopolysaccharide, which include addition of positively charged residues of 4-amino-deoxy-l-arabinose (l-Ara4-N) and/or phosphoethanolamine (pEtN) to Lipid A moiety. These modifications to the negative charge of the outer membrane prevent binding of positively charged polymyxin, thus conferring resistance to polymyxins. Polymyxin resistance is regulated by genes such as mgrB, phoP/phoQ, pmrA-D, and mcr1, and by the upregulation of efflux pumps.

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Class A Klebsiella pneumoniae carbapenemase (KPC)-producing CRE are most prevalent in the United States, where the prevalence of blaKPC within K. pneumoniae was 13% in 2013–2014 [8]. In Europe, Italy and Greece reported to have an “endemic situation” for blaKPC in 2014–2015, where a single-center study from Italy reported 99% of carbapenemase-producing strains were blaKPC positive between 2012 and 2014 [8]. Carbapenem resistance in K. pneumoniae has become one of the most important epidemiologic and therapeutic challenges. EARS-NET 2013 indicated the highest rates of carbapenem resistance in K. pneumoniae in Greece (59.4%) and Italy (34.3%) [6]. An epidemiological study in the United States in 2015 identified 599 CRE isolates in 481 patients of which 58.6% were K. pneumoniae and positive for KPC [6]. A 2011 study from China reported 71% of 109 ertapenem-resistant K. pneumonia isolates were blaKPC-2 positive, some in combination with CTX-M-type extended-spectrum β-lactamase (ESBL) enzymes [8]. In contrast, blaKPC has not been found frequently in Enterobacteriaceae from patients in India [8]. Class B metallo-β-lactamase (MBL)-producing CRE have been most commonly associated with the Indian subcontinent as well as specific countries in Europe, including Romania, Poland, and Denmark, with an interregional spread of resistance [8]. In India a molecular characterization study of carbapenem-resistant Enterobacteriaceae in a tertiary care hospital in Mumbai, West India, revealed New Delhi metallo-β-lactamase-1 (NDM-1) to be the most prevalent carbapenemase that accounted for 75.22% of the isolates and that an alarming 18.5% of the clinical isolates possessed dual carbapenemase genes [7]. Although MBL has been an uncommon etiology for carbapenem resistance in the United States, 157 NDM-producing Enterobacteriaceae from 25 states were reported to the Centers for Disease Control (CDC), in 2016 [8]. Verona integron– mediated (VIM) and Imipenemase (IMP)-producing isolates are far less common in the United States, with only 17 and 10 isolates reported, respectively, as per the CDC in 2016 [8]. Class C β-lactamases confer resistance to carbapenems and third-generation cephalosporinases by the hyperproduction of chromosomally encoded AmpC in combination with other mechanisms of resistance such as porin loss or upregulated efflux activity primarily found in Enterobacteriaceae and P. aeruginosa [9]. Class D OXA-48-like-producing K. pneumoniae clones are the leading cause of nosocomial infections in Turkey, which was reported to have the highest epidemiological level of these strains in 2014–2015 [8]. The underestimation of the incidence of OXA-48-like resistance is of notable importance, since most clinical microbiology laboratories may not test for the presence of OXA-48-like enzymes, given the low level of carbapenem resistance in some strains [8]. Carbapenem-resistant A. baumannii (CRAB) and MDR A. baumannii have been reported globally with resistance mainly driven by MBLs such as VIM and IMP, and three unrelated groups of clavulanic acid–resistant β-lactamases, OXA-23, OXA-24, and OXA-58, which are encoded chromosomally or via plasmid [10].

Antibiotics targeting Gram-negative bacteria

In Europe, reports of interregional and endemic spread of A. baumannii resistant to carbapenems have been on the rise over the past few years. A regional analysis of invasive carbapenem-resistant Acinetobacter isolated in Switzerland between 2005 and 2016 reported a resistance rate of 9.2% and highlighted the existence of a diverse pool of A. baumannii in hospital settings in Switzerland, as also observed in France and Germany in other studies [11]. In the Middle East a 2011–2013 study elucidating the molecular epidemiology and mechanisms of resistance of CRAB in the Arab states of the Gulf reported the presence of blaOXA-23-like gene in 91% and blaOXA-24-type in 4.3% of 117 isolates. Around 84% of the isolates were associated with health-care exposure [12]. In the United States, CDC reported MDR Acinetobacter as the causative agent of ∼7000 infections and ∼500 deaths in 2013 [13]. A study in the United States from hospital clinical microbiology laboratories reported an increase in CRAB isolates from 21% in 2003–2005 to 48% in 2009–2012 [13]. Further, laboratory- and population-based surveillance in select metropolitan areas in the United States between 2012 and 2015 identified 621 cases of CRAB cases from 537 unique patients and reported a crude annual incidence rate of 1.2 cases per 100,000 persons [14]. Most of these patients were reported to have had an exposure to a health-care facility in the previous year [14]. A 2016 review reported CRAB as the major cause of health care–associated infections in large hospitals throughout South and Southeast Asia, with >50% carbapenem resistance rates in the ICU settings [15]. The OXA-23 carbapenemase enzyme was predominant in CRAB in India and Pakistan, with carbapenem resistance rates greater than 40% in India and between 62% and 100% in Pakistan [15]. In 2011–2013, in Kathmandu, Nepal, carbapenem resistance among A. baumannii isolates ranged from 17% to 98% in hospitals with the presence of blaOXA-23 in all isolates, 25% of which also tested positive for the blaNDM-1 [15]. In Southeast Asia, two outbreaks in Singaporean public sector hospitals marked a rise in carbapenem resistance from 46% in 2006 to 62% by 2010 [15]. In 2014 a national surveillance report for antimicrobial resistance in Malaysia highlighted that 57.3% of 15,533 A. baumannii isolates from 39 microbiology laboratories were resistant to meropenem [15]. In 2010 a study, including 577 A. baumannii isolates from Northern Taiwan hospitals, identified blaOXA-23-like genes in 67.8% of the isolates and blaOXA-24-like genes in 2.2% of the isolates [16]. Carbapenem resistance in Pseudomonas is mainly driven by chromosomal mutations that alter porins or modify efflux pump activity. In addition, carbapenemase genes such as VIM MBLs, which are commonly carried on mobile genetic elements, have led to the rapid dissemination of carbapenem-resistant P. aeruginosa (CRPA), with VIM-2 and VIM-4 being the most common carbapenemases. In 2009 the growing incidence of meropenem-resistant P. aeruginosa due to overexpression of both extended-spectrum cephalosporinases and efflux pump was reported from a hospital in France, where 87.5% of the cases showed this combination of resistance [17]. Fourteen countries in Europe

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reported an alarming increase in the prevalence of MBL-producing P. aeruginosa from 12.3% in 2010 to 30.6% in 2011 [18]. High prevalence of CRPA has been specifically reported in Greece, with 56.4% resistance in ICU patients at a national level between 2010 and 2013 [19]. In 2015 a hospital in Algeria reported a 18.75% carbapenem resistance rate in P. aeruginosa isolates [20]. Another surveillance study in 2015 for CRPA from five metropolitan cities in the United States identified 384 cases of CRPA from nine clinical laboratories, where carbapenem resistance ranged from 4.6% at Oregon in the west coast to 12% at Georgia in the east coast of the United States. Of this, only 2% of CRPA isolates tested positive for a carbapenemase gene suggesting other mechanisms of resistance at these surveillance sites [18]. Intrahospital dissemination of MDR/ Extensive Drug-Resistant (XDR) strains of P. aeruginosa has led to the rise of epidemic strains at an international level, such as ST111, ST175, and ST235 [21]. A recent study from a hospital in Spain reported most isolates tested belonged to ST175, consistent with national level data [21]. 1.1.2  Resistance to polymyxins The widespread use of polymyxins—polymyxin B and colistin (polymyxin E)—to treat MDR Gram-negative bacterial infections, particularly infections caused by carbapenemase-producing Enterobacteriaceae, has led to the increase in polymyxin-resistant K. pneumoniae (PRKP) worldwide. Apart from clinical medicine, the heavy use of polymyxin in veterinary medicine for animal husbandry has resulted in polymyxin-resistant bacteria that can potentially transmit from animals to humans [22]. There has been a widespread and rapid dissemination of resistance against colistin worldwide, especially in settings of high KPC-producing K. pneumoniae endemicity such as Italy, Brazil, Greece, and Serbia. A study in Brazil between 2007 and 2013 identified 10 polymyxin B–resistant K. pneumoniae isolates from rectal swabs, with mutations in the mgrB gene mediating polymyxin resistance [23]. Most of these PRKP isolates harbored blaKPC-2 and belonged to the clonal complex 258. Data from SENTRY antimicrobial surveillance program reported a 3.2% increase of PRKP isolates in Brazil between 2008 and 2010, which further increased in 2011 to 6.6% for ESBL-producing K. pneumoniae in Latin America and to 9.7% for KPC-2-producing K. pneumoniae from Brazil in 2010 [23]. A 2014 multicenter survey from Italy across 21 hospital laboratories reported colistin resistance in 43% of KPC-producing K. pneumoniae isolates [24]. A 2017 study reported a country-wide colistin resistance in Serbia between 2013 and 2016, where 27 colistin- and carbapenem-resistant K. pneumoniae isolates were identified in tertiary care hospitals from adults with no prior evidence of colistin treatment [25]. Most of the isolates were blaOXA-48 positive, a few were blaNDM-1 positive, and all were blaCTX-M-15 positive.This clear association between polymyxin resistance and other acquired mechanisms of resistance is worrisome, especially where polymyxin is used as a last-resort antibiotic against carbapenemase-producing K. pneumoniae isolates endemic to a region [25].

Antibiotics targeting Gram-negative bacteria

In the United States, between 2013 and 2014, multicenter surveys reported a colistin resistance rate of up to 18% among carbapenem-resistant E. coli and K. pneumoniae isolates and 6.7% in Canada [26]. Most of the outbreaks with colistin-resistant KPCproducing K. pneumoniae isolates have been attributed to the international epidemic clone-type ST258 in the United States, the Netherlands, Italy, and Greece [26]. Several outbreaks of KPC-producing, colistin-resistant K. pneumoniae isolates in Greece and Spain have marked the rapid increase in colistin resistance in these areas from 20% after 2010 in Greece, and from 13.5% to 31.7% in Spain, where colistin-resistant VIM-1-producing K. pneumoniae isolate was reported [26]. France, however, had a low rate of colistin resistance (6.2%), although an outbreak of OXA-48-producing colistin-resistant isolate was reported [26]. In contrast to the United States and Europe, low rates of colistin resistance have been reported in Africa and the Middle East—2.7% and 4.5% in Turkey and Israel, respectively [26]. In Asia, moderate levels of colistin resistance (4.4%–11%) have been reported in China, South Korea, and Taiwan between 2009 and 2012 [26]. However, the emergence of the transferable gene mcr-1 that causes colistin resistance is being increasingly reported across countries in Asia. In 2014 Tata Medical Center in Kolkata, India, reported a series of 24 cases of colistin-resistant Klebsiella infection [27]. A high prevalence of ESBL (70%) and carbapenemase (39%) producers in this area resulted in the high first-line use of meropenem and colistin in this hospital [27]. A multicenter hospital study in China by Zhang et al. also reported the emergence of colistin resistance in three CRE isolates, of which one was identified to cocarry blaNDM-5 and the colistin resistance gene mcr-1 [5]. Another surveillance study of CRE in a hospital in Shanghai, China reported an independent emergence of colistin-resistant Enterobacteriaceae in 4 out of 82 clinical isolates, without previous colistin treatment [28]. Colistin resistance in A. baumannii, although rare, is rapidly emerging, particularly in patients in ICU settings. A study between 2007 and 2014 by Qureshi et al. reported 20 patients with colistin-resistant A. baumannii infections from ventilator-associated pneumonia in a hospital center in Pennsylvania, United States [29]. Lipid A modification accounted for colistin resistance in all isolates and most patients had previously received colistin methanesulfonate for the treatment of carbapenem-resistant, colistin-susceptible A. baumannii infection [29].

2  Combating multidrug resistance with new drugs The prevalence of a diverse range of resistance mechanisms brings into focus the complexity of developing novel antibacterial drugs. New compounds must not only have activity against specific species but also target the associated mechanisms of resistance, while taking into account clinical usage (indications and types of infection) and geographic distribution.

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The Pew’s Trust provides a summary of compounds in development for antibacterial applications [30]. As per its March 2019 update, there are 42 compounds in clinical development but only 18 address serious, life-threatening infections caused by MDR Gram-negative priority organisms [30]. The following section reviews preclinical and clinical data for novel, small molecules that are in clinical development (or have been recently approved). The compounds have been classified into the following categories based on their mechanism: (1) compounds with novel cellular targets, (b) combinations of approved antibiotics with partners that enhance the activity of the antibiotics, and (c) analogs of existing chemical classes.

2.1  Compounds with novel targets This category refers to antibacterial compounds that target novel cellular proteins. The chemical scaffolds theoretically overcome all existing mechanisms of resistance and offer wholly new approaches for treating infections. Such compounds are also more difficult to develop and carry a higher risk of failure. Only one compound in the current pipeline falls into this group. Murepavadin (POL7080) is a 14-amino-acid macrocyclic peptide, which binds to the lipopolysaccharide transport protein D, an outer membrane protein in Gram-negative bacteria [31]. Disruption of the lipopolysaccharide transport causes alterations in the outer membrane of the bacterial cell and, ultimately, death [31].The compound is in Phase 3 of clinical development as a parenteral antibiotic for the treatment of hospitalacquired bacterial pneumonia (HABP) and ventilator-associated bacterial pneumonia (VABP), caused by Pseudomonas. The compound has been tested against 1219 P. aeruginosa isolates from 112 medical centers. Murepavadin demonstrated a minimum inhibitory concentration (MIC)50/90 of 0.12/0.12 mg/L and inhibited 99.1% of isolates at ≤0.5 mg/L, with no differences in activity against isolates from Europe, United States, or China [32]. In vivo efficacy for POL7080 has been tested in the mouse septicemia model using P. aeruginosa ATCC 9027 or ATCC 27853 at doses of 10, 3, 1, 0.3, and 0.1 mg/kg, administered subcutaneously at 1 and 5 h. POL7080 demonstrated substantial activity against both strains with an Effective Dose-50 (ED)50 in the range from 0.25 to 0.55 mg/kg [33]. A Pharmacokinetics-Pharmacodynamics (PK–PD) model was undertaken in CD1 neutropenic mice at different dosing frequencies for 24 h, and the number of CFU/lung was determined. The mean area under curve (AUC) required to provide a static effect was 36.83 mg h/L (fAUC = 8.25 mg h/L), and that to provide a 1-log reduction was 44.0 mg h/L (fAUC = 9.86 mg h/L). The mean static fAUC/MIC was determined to be 27.78, and that for a 1-log reduction was 39.85 [34]. Murepavadin has completed six Phase 1 and two Phase 2 trials [35]. The drug was well tolerated; only mild, transient adverse events were reported [36]. A Phase 2 openlabel study in VABP enrolled 25 VABP patients who received murepavadin; 12 had a

Antibiotics targeting Gram-negative bacteria

microbiologically documented infection due to P. aeruginosa (5 with MDR isolates).The clinical cure rate was 91% and the 28-day all-cause mortality rate of 9%, below the expected mortality rate for such infections [37]. No development of treatment-emergent resistance to murepavadin was detected. Currently, a Phase 3 clinical study of murepavadin in HABP/VABP is underway [31].

2.2  Combinations of approved antibiotics with partners that enhance the activity of the antibiotic These are novel compounds that by themselves do not have significant antibacterial activity but enhance or potentiate the activity of a known antibiotic through improved cell penetration or inhibition of the enzyme responsible for resistance. 2.2.1  SPR741 combination SPR741 is a polymyxin-derived novel compound with no significant direct antibacterial activity of its own but its interactions with the bacterium’s outer membrane allow partner antibiotics, particularly hydrophobic ones, to enter the Gram-negative cell more efficiently. Unlike polymyxin B, SPR741 has lower nephrotoxicity due to a reduced positive charge and the absence of highly lipophilic fatty-acid side chains [38]. SPR741 is currently in Phase 1 of clinical development as a parenteral antibiotic. The indications it will be developed for will depend on the partner antibiotic chosen. In combinations with SPR741, the MICs of 13 antibiotics (azithromycin, clarithromycin, erythromycin, fidaxomicin, fosfomycin, fusidic acid, mupirocin, novobiocin, quinupristin–dalfopristin, ramoplanin, retapamulin, rifampin, and telithromycin) against E. coli ATCC 25922 were reduced at least 32-fold, with substantial reductions in the MIC for rifampin (>8000-fold to 0.002 µg/mL) in the presence of 8 µg/mL SPR741 [38]. Against K. pneumoniae, the potentiation of activity was lower than for E. coli. The MICs of 10 antibiotics (azithromycin, clarithromycin, erythromycin, fusidic acid, mupirocin, novobiocin, retapamulin, rifampin, telithromycin, and vancomycin) were reduced at least 32-fold in combination with SPR741, with a maximum reduction of 128-fold for rifampin, clarithromycin, and retapamulin [38]. For A. baumannii NCTC 12156 the MICs of eight antibiotics (clarithromycin, erythromycin, fusidic acid, quinupristin–dalfopristin, ramoplanin, retapamulin, rifampin, and teicoplanin) were reduced ≥32-fold against this strain. Of these, MIC reductions of 128-fold were observed for clarithromycin, erythromycin, fusidic acid, and rifampin [38]. In a study exclusively focused on A. baumannii, Zurawski et al. showed that among the 28 extensively drug-resistant strains, 27 strains were susceptible to the combination of SPR741 (4 µg/mL) and rifampin (1 µg/mL), with a minimum fourfold reduction of most MICs [39]. In the K. pneumoniae murine model of urinary tract infection (UTI), a combination of SPR741 (10 mg/kg, every 8 h) and rifampin (4 mg/kg, every 8 h) was very effective in decreasing the bacterial load in the kidneys, compared to single agent [40]. Other

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combinations such as SPR741 (10 mg/kg, every 8 h, subcutaneously) and SPR720 (gyrB inhibitor, 60 mg/L oral) were effective in decreasing the kidney bacterial load of carbapenem-resistant K. pneumoniae compared to single agent [40]. In the A. baumannii murine lung model, the combination of SPR741 (10 mg/kg, every 8 h) and clarithromycin (100 mg/kg once daily) decreased the bacterial load by 1.15 log10 compared to pretreatment [40]. In an A. baumanni pneumonia model, SPR741 (60 mg/kg every 12 h) and rifampin (5 mg/kg every 12 h), the combination decreased bacterial burden by 6.0 log10 CFU/g; this was a 2.0-log10 greater reduction in bacterial burden compared to rifampin alone [39]. In Phase 1 clinical trials, SPR741 was well tolerated in single doses of up to (and including) 800 mg and at doses up to and including 600 mg every 8 h for 14 days [41]. 2.2.2  β-Lactam and β-lactamase inhibitors This group of compounds includes combinations of β-lactam antibiotics with inhibitors of β-lactamases. The BLIs belong to three chemical classes, diazabicyclooctanes (DBOs), cyclic boronic acids, and penicillanic acid sulfones. They inhibit the different types of β-lactamases to varying degrees, leading to differences in the overall spectrum of activity for each combination.Two recently approved drugs that belong to this group are ceftazidime/avibactam and meropenem/vaborbactam [42,43]. Tables 3.1 and 3.2 show the indications, spectrum of microbiological activity and mechanisms of resistance covered by approved BLIs and BLIs in clinical development, respectively. 2.2.3  Imipenem/cilastin + relebactam Relebactam (REL) (MK-7655) is a DBO-type BLI with activity against class A (including KPC-type carbapenemases) and C β-lactamases [44]. REL is structurally related to avibactam (Fig. 3.2), with its conserved DBO core, but unlike avibactam, REL does not

Table 3.1  Recently approved BLIs. Microbiological spectrum (Gram-negative)

Mechanisms of resistance covered

Classes A (including KPC) and C, some D Classes A and C, including KPC

Drug

Chemical class of BLI

Ceftazidime/ Avibactam (IV)

Diazabicyclooctane (DBO)

IAI, UTI, HABP,VABP

Enterobacteriaceae, Pseudomonas

Meropenem/ Vaborbactam (IV)

Cyclic boronic acid

cUTI

Enterobacteriaceae

Indication

BLI, β-lactamase inhibitor; cUTI, complicated urinary tract infection; DBO, diazabicyclooctane; HABP, hospital-acquired bacterial pneumonia; IAI, intraabdominal infections; IV, intravenous; UTI, urinary tract infections; VABP, ventilatorassociated bacterial pneumonia.

Antibiotics targeting Gram-negative bacteria

Table 3.2  BLIs in clinical development. Name of compound

Chemical class of BLI

Phase of develop­ ment

Indication

Microbiological spectrum

Mechanisms of resistance covered

Imipenem/ Cilastatin +  relebactam (IV) Sulbactam +   ETX2514 (IV) Meropenem +  nacubactam (IV) Cefepime +  zidebactam (IV)

DBO

Phase 3

HABP,VABP, cIAI, and cUTI

Enterobacteriaceae, P. aeruginosa

Class A and C βlactamases

DBO

Phase 3

cUTI, HABP, VABP

Acinetobacter spp.

Classes A, C, and D

DBO

Phase 1

cUTI, HABP, VABP, cIAI

Enterobacteriaceae

Classes A, C, and D

DBO

Phase 1

cUTI, HABP, VABP

Classes A, C, and D

Phase 1

UTI

Enterobacteriaceae, Acinetobacter, P. aeruginosa Enterobacteriaceae

Phase 1

cUTI, cIAI

Classes A, B, C, and D

Phase 3

cUTI, cIAI, HABP, VABP

Enterobacteriaceae, P. aeruginosa Enterobacteriaceae

Cefpodoxime +  DBO; proETX0282 drug of (oral) ETX1317 Cefepime +  Cyclic VNRX-5133 boronic (IV) acid Cefepime +  PenicilAAI101 (IV) lanic acid sulfone

Classes A, C, and D

Classes A, C, and some D

BLI, β-lactamase inhibitor; cIAI, complicated intraabdominal infection; cUTI, complicated urinary tract infection; DBO, diazabicyclooctane; HABP, hospital-acquired bacterial pneumonia; IV, intravenous; UTI, urinary tract infections; VABP, ventilator-associated bacterial pneumonia.

Figure 3.2  Structures of avibactam and relebactam.

inhibit class D carbapenemases (e.g., OXA-48-like) [44]. Mechanistically, avibactam has a two-part process of exerting its effects. First there is a noncovalent binding with the β-lactamase, followed by a covalent acylation at the β-lactamase serine residue [45]. The same mechanism of action is predicted for REL [45].

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The structures show a conserved DBO core with differences in the side chains. Avibactam has a carboxymide, while REL has a piperidine ring. The combination possesses inhibitory activity against clinical isolates of K. pneumoniae carrying ESBLs, variant KPC-3 enzymes that are resistant to ceftazidime–avibactam [46,47] and against P. aeruginosa isolates that are carbapenem resistant due to porin loss and AmpC expression [47]. Imipenem (IMI)–REL is inactive against MBL-producing Gram-negative bacilli [46,47]. The compound is in Phase 3 of clinical development for HABP, VABP, complicated intraabdominal infection (cIAI), and complicated UTI (cUTI) as a parenteral drug. The in vitro activity of this combination has been reported in several studies. The data from key large studies are summarized here. In a study of 5447 isolates of P. aeruginosa as part of the SMART global surveillance program (with contributions from 22 European countries) in 2015–2017, 69.4% and 92.4% of isolates were susceptible to IMI and IMI/REL, respectively. Among MDR IMI nonsusceptible isolates, REL restored sensitivity to IMI for 69.6% of the isolates [48]. In a study of clinical isolates of Enterobacteriaceae (n = 3419) and P. aeruginosa (n = 896) as part of the SMART surveillance program conducted in the United States in 2016, susceptibilities to IMI–REL for non-Proteeae Enterobacteriaceae (n = 3143) and P. aeruginosa were 99.1% and 95.9% and for IMI alone was 94.4% and 74.7%, respectively. REL was used at a fixed concentration of 4 µg/mL. Among the resistant subtypes, REL restored IMI susceptibility to 78.5% of IMI-nonsusceptible non-Proteeae Enterobacteriaceae and to 78.0% of IMI-nonsusceptible P. aeruginosa isolates. For MDR non-Proteeae Enterobacteriaceae and MDR P. aeruginosa, susceptibility to IMI–REL was 98.2% (444/452) and 82.2% (217/264), respectively [49]. In a study of 314 non-MBL carbapenemase-producing K. pneumoniae clinical strains from Greece, IMI–REL inhibited 98.0% of the KPC-producing isolates with a MIC50 and MIC90 of 0.25 and 1 mg/L compared to IMI alone which had a MIC50 and MIC90 of 32 and >64 mg/L, respectively. Reduced activity of IMI–REL was detected in 2% of the isolates and was associated with ompK35 disruption and/or mutated ompK36. REL did not improve IMI’s activity against K. pneumoniae with class D OXA-48-like enzymes [50]. Two Phase 1 and two Phase 2 studies have been conducted [51–53]. A Phase 3 randomized, active comparator–controlled, double-blind study (RESTORE-IMI 1) was conducted to evaluate the efficacy and safety of IMI/REL, compared with colistimethate sodium (CST) + IMI in patients with HABP/VABP, cIAI, or cUTI with causative Gram-negative pathogens nonsensitive to IMI but susceptible to both IMI/REL and CST [54,55] (Fig. 3.3). Pathogens expressed either class A or C β-lactamases. Patients received either IMI/REL (500 mg/250 mg) intravenously every 6 h (n = 31) or CST loading dose of 300 mg colistin base activity (CBA) ∼9 million IU, followed 12 h later by a CST maintenance dose (150 mg CBA) every 12 h, plus IMI (500 mg) every 6 h

Antibiotics targeting Gram-negative bacteria

Figure 3.3  RESTORE-IMI-1 study design.

(n = 16). The minimum duration of therapy was 5 days (cIAI, cUTI) or 7 days (HABP/ VABP) and the maximum duration was 21 days. Primary outcomes were mortality, overall clinical cure, and safety outcomes. As this was a multicentric study, the clinical response was analyzed by stratifying the patients using central laboratory data or local microbiology data (Fig. 3.3). Overall response in the patients stratified according to central laboratory data (microbiologic modified intent-to-treat population, primary efficacy population, n = 31) was 71.4% in the IMI/REL group (n = 21) compared with 70.0% in the CST + IMI group (n = 10). Overall response was defined as hospital-acquired pneumonia (HAP)/ventilator-associated pneumonia (VAP) survival through day 28, cIAI clinical response at day 28, and cUTI composite clinical and microbiological response 5–9 days after the end of therapy. At day 28, clinical response in this group was 71.4% in the IMI/REL group versus 40% in the CST + IMI group. All-cause mortality at day 28 was 9.5% in the IMI/REL group compared to 30.0% in the CST + IMI group. In the patient group stratified based on local microbiology data, the overall response was 75.0% in the IMI/REL group (n = 31) compared with 76.9% in the CST + IMI group (n = 16). The clinical response at day 28 was 75% in the IMI/REL group versus 53% in the CST + IMI group. All-cause mortality was 10.7% in the IMI/REL group compared to 23.1% in the CST + IMI group. While overall response was similar between groups, clinical response and mortality were more favorable for IMI/REL, regardless of how the patients were stratified [54,55]. This study enrolled patients from 16 countries. Even so, the number of patients remained small. While the current data establish proof of efficacy in the target population, additional studies with larger number of patients and diversity of molecular mechanisms, are necessary to assess the full potential of the drug.

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The primary efficacy population consisted of those patients who come under pathogen eligibility criteria (primarily susceptibility) in central laboratory testing. They were classified as microbiologic intent-to-treat population. 2.2.4  Sulbactam + ETX2514 ETX2514 is a DBO type of BLI, which inhibits class A and C β-lactamases and a broad range of class D β-lactamases differentiating itself from other BLIs [56] (Fig. 3.4). Sulbactam has intrinsic antibacterial activity against Acinetobacter spp., due to its inhibition of penicillin-binding proteins (PBP1 and PBP3) [57]. It is subject to hydrolysis by certain β-lactamases, such as TEM-1 and ADC-30 [58,59]. However, in combination with ETX2514, its activity is significantly improved, through complementary inhibition of PBP2 [60].The combination is in Phase 3 of clinical development as a parenteral antibiotic for cUTI, HABP, and VABP caused by Acinetobacter. The in vitro activity of sulbactam–ETX2514 was determined against 1131 A. baumannii clinical isolates from globally diverse settings in 2014 [60]. Sulbactam in combination with ETX2514 at 4 mg/mL had a MIC90 value of 4 mg/L in contrast with the MIC90 of sulbactam alone, which was 64 mg/L. This potency was unchanged in the subset of 731 meropenem-resistant, 56 colistin-resistant, and 778 MDR isolates [61]. In another study of 98 isolates of A. baumannii, 72 from the Walter Reed Army Medical Center (WRAMC) and 26 from Brooke Army Medical Center, University Hospitals of Cleveland, and Cleveland Clinic Foundation, the combination of ETX2514 and sulbactam was highly potent [56]. A majority of the WRAMC isolates encoded class C (blaADC) and D (blaOXA-69-like) genes. Furthermore, 40% of strains carried a class A blaTEM gene, and 11% and 12.5% of the isolates carried class D blaOXA-23-like and blaOXA-58-like genes, respectively. Around 47% of the isolates were resistant to sulbactam alone (MIC ≥ 16 mg/L) and 24% were resistant to meropenem (R ≥ 8 mg/L).The MIC50 and MIC90 for sulbac-

Figure 3.4  Structures of the DBO-based β-lactamase inhibitors. DBO, diazabicyclooctane.

Antibiotics targeting Gram-negative bacteria

tam with a fixed concentration of ETX2514 (4 mg/L) for the WRAMC collection were 1 and 2 mg/L, respectively. For the 26 carbapenem-resistant isolates (molecular determinants not described), the addition of 4 mg/L of ETX2514 resulted in MICs of ≤4 mg/L for sulbactam. Overall, the MIC90 against the 98 isolates was lowered from 32 mg/L for sulbactam alone to 2 mg/L in combination with 4 mg/L of ETX2514. Efficacy studies of sulbactam–ETX2514 in thigh and lung murine infection models showed a dose-dependent reduction in MDR A. baumannii bacterial counts when sulbactam and ETX2514 were used in a 4:1 ratio or when sulbactam was used at a fixed dose of 15 mg/kg. The strain used for the study was OXA-66+, OXA-72+, TEM-1+, and ADC-30+ [60]. The in vivo efficacy of the sulbactam–ETX2514 combination was also evaluated in a neutropenic mouse thigh infection model using A. baumannii ARC5955 carrying blaTEM-1, blaADC-82, blaOXA-23, and blaOXA-66 (OXA-51-like) genes. This strain had an in vitro MIC of 64 mg/L for sulbactam alone and an MIC of 4 mg/L in combination with ETX2514 at a concentration of 4 mg/L [62]. Sulbactam and ETX2514 were administered subcutaneously individually or in combination, every 3 h at doses of 75 and 50 mg/kg of body weight. Individual administration of the drugs resulted in >2 log10 CFU/g of growth at 24 h similar to vehicle controls. In combination, however, >1-log CFU/g of bactericidal activity was observed relative to the bacterial burden at the beginning of treatment. When ETX2514 was dosed at 200 mg/kg in combination with 50 mg/ kg sulbactam, there was a 1.5 log10 CFU/g reduction, similar to the efficacy of colistin (−1.36 log10 CFU/g), a comparator in the study [56]. In Phase 1 studies, sulbactam + ETX2514 was well tolerated in healthy subjects [63]. In a Phase 2 study of 80 patients with cUTI, the microbiological eradication rates were similar for the group treated with sulbatam + ETX2514 and IMI and those treated with placebo + IMI [64]. In the study, seven patients had a cUTI caused by IMI-nonsusceptible pathogens (three in the treatment arm and four in the placebo arm). Sulbactam + ETX2514 and IMI showed microbiological eradication in 3/3 patients compared to 3/4 in patients receiving placebo plus IMI. Notably, the study did not include any patients with Acinetobacter infections, making it difficult to make conclusions about efficacy. Overall, sulbactam + ETX2514 was well tolerated [64]. These data support progression to Phase 3 clinical studies. 2.2.5  Meropenem + nacubactam Nacubactam (OP-0595, RG6080) is a novel DBO with in vitro activity against class A, C, and some D β-lactamases (Fig. 3.4). In addition, the compound has intrinsic antibiotic effects against Enterobacteriaceae due to its binding affinity for PBP2 [65]. Activity for Acinetobacter and Pseudomonas is modest [66]. The combination is currently in Phase 1 and is being developed for the treatment of cUTI, HABP,VABP, and cIAI as an intravenous (IV) drug.

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In a study of 210 consecutive Enterobacteriaceae with NDM or VIM MBLs and 99 supplementary MBL-producing Enterobacteriaceae representing less prevalent phenotypes, species and enzymes, MICs of nacubactam alone clustered at 1–8 or >32 mg/L. More than 85% of E. coli and Enterobacter spp. fell into the low MIC cluster, whereas for Klebsiella spp., meropenem/nacubactam and cefepime/nacubactam inhibited 93.3% and 87.1% of MBL producers, respectively. Against the most resistant isolates (57 organisms with MICs of nacubactam >32 mg/L, cefepime ≥128 mg/L, and meropenem ≥128 mg/L), cefepime/nacubactam at 8 + 4 mg/L inhibited 63.2% and meropenem/ nacubactam at 8 + 4 mg/L inhibited 43.9% of the isolates [67]. In a study of 1553 Enterobacteriaceae isolates from United States and European hospitals, expressing β-lactamases belonging to classes A, B, C, and D, nacubactam alone had a MIC50 of 2 or 4 µg/mL against most isolate subgroups but showed lower activity against class D isolates. Meropenem combined with nacubactam at 4 µg/mL showed a MIC50/MIC90 of ≤0.004/0.015 µg/mL for class A expressing isolates, ≤0.004/64 µg/ mL for class B expressing isolates, ≤0.004/0.015  µg/mL for class C expressing isolates, and 0.12/4 µg/mL for class D expressing isolates. For KPC-producing isolates the MIC50/MIC90 was ≤0.004/0.25 µg/mL [68]. In a neutropenic murine cUTI model, meropenem–nacubactam was compared with meropenem or nacubactam alone against 10 K. pneumoniae, E. coli, and Enterobacter cloacae isolates with diverse genotypic and phenotypic profiles, including NDM, KPC, OXA, CTX-M, SHV, and TEM enzyme-producing isolates. Meropenem–nacubactam achieved a ≥3-log reduction from the 48-h control in 9 out of 10 isolates tested. Against ceftazidime–avibactam-resistant isolates, meropenem–nacubactam demonstrated antibacterial efficacy upward of 6 log10 CFU in comparison to the 48-h control [69]. The efficacy of meropenem and nacubactam against class A serine carbapenemaseproducing Enterobacteriaceae isolates has been tested in the neutropenic murine lung infection model against 12 meropenem-resistant K. pneumoniae, E. coli, and E. cloacae isolates harboring KPC- or IMI-type β-lactamases. Human-simulated epithelial lining fluid (ELF) exposures of each drug were established.The meropenem–nacubactam combination resulted in reductions of 1.50 ± 0.59 log10 CFU/lung, establishing efficacy for isolates harboring this particular molecular type of resistance [70]. In Phase 1 clinical studies, nacubactam alone or in combination with meropenem was well tolerated in healthy volunteers [30]. 2.2.6  Cefepime + zidebactam (WCK-5222) Zidebactam belongs to the DBO chemical class with dual modes of action. It has βlactamase inhibitory activity as well as intrinsic antibacterial activity due to selective, high-affinity binding for Gram-negative bacterial PBP2 protein [71,72] (Fig. 3.4). Zidebactam inhibits class A, C, and some D β-lactamases [73]. The combination is in Phase 1 clinical studies and is being developed for cUTI, HABP, and VABP as an IV formulation.

Antibiotics targeting Gram-negative bacteria

In a study of 132 carbapenem-resistant isolates consisting of Enterobacteriaceae producing A, B, and D classes of β-lactamases, ESBLs and AmpC, Acinetobacter expressing class A, B, and D β-lactamases and P. aeruginosa expressing class A and B β-lactamases as well as combinations of upregulated efflux, diminished or nonfunctional OprD porins, and AmpC overproduction, the MIC50/MIC90 for cefepime in combination with zidebactam (1:1 ratio) were 0.5/16 µg/mL [73]. Within the collection, for isolates expressing class A β-lactamases, MIC50/MIC90 for cefepime was 0.5/8 µg/mL, against class B it was 2/32 µg/mL, and against class D it was 2/16 µg/mL. For plasmid-mediated AmpC expressing Enterobacteriaceae, the cefepime MIC50/MIC90 was 0.12/2 µg/mL. For all P. aeruginosa isolates the MIC50/MIC90 of cefepime was 2/32 µg/mL. In a large study the in vitro activity of cefepime–zidebactam against 7876 contemporary (2015) clinical isolates of Enterobacteriaceae (n = 5946), P. aeruginosa (n = 1291), and Acinetobacter spp. (n = 639) from the United States (n = 2919), Europe (n = 3004), the Asia-Pacific (n = 1370), and Latin America (n = 583), the MIC50 and MIC90 of cefepime against Enterobacteriaceae was ≤0.03 and 0.12 µg/L, respectively, in a 1:1 combination with zidebactam and 0.06/0.25 µg/mL, in a 2:1 combination; 99.9% of isolates were inhibited at ≤4 (1:1) and ≤8 µg/mL (2:1) [72]. When analyzed for activity against carbapenem-resistant isolates, cefepime–zidebactam at a 1:1 ratio had a MIC50/90 or 1/4 µg/mL, with 99.3% of isolates being inhibited at ≤8 µg/mL. Against P. aeruginosa isolates, the MIC50/90 values for cefepime were 1/4 µg/mL and for the meropenem-resistant subset, the MIC50/90 values were 4/8 µg/mL. For Acinetobacter spp. the MIC50/90 values were 16/32 µg/mL. The in vivo efficacy of human-simulated WCK 5222 (cefepime–zidebactam) against cefepime-resistant A. baumannii strains (n = 13) was tested in the neutropenic murine lung infection model [74]. Twelve isolates were meropenem resistant, including isolates expressing OXA-23/24, oxacillinases commonly associated with carbapenem resistance in A. baumannii. In control animals and those that received cefepime or zidebactam alone, the mean bacterial growth at 24 h was >2 log10 CFU/lung compared with 0-h control (6.32 ± 0.33 log10 CFU/lung). WCK 5222 produced a decline in the bacterial burden for all isolates (mean reduction, 3.34 ± 0.85 log10 CFU/lung). In Phase 1 clinical studies the drug combination was well tolerated in healthy subjects with normal and impaired renal function [75]. 2.2.7  ETX0282 cefpodoxime proxetil ETX0282 is a prodrug BLI that is hydrolyzed to become ETX1317 in combination with cefpodoxime proxetil (CPDP), which is hydrolyzed in vivo to release CPD [76] (Fig. 3.4). The combination is in Phase 1 of clinical development as an oral agent for cystitis and pyelonephritis in outpatient settings or as oral step-down therapy in hospital settings. ETX1317 inhibits class A β-lactamases. CPD-ETX1317 was tested in a fixed 1:2 ratio against 910 Enterobacteriaceae (including E. coli, Klebsiella, Citrobacter, Proteus, and Enterobacter spp.) collected between 2013

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and 2015 from geographically diverse medical centers around the world [77]. The isolates were enriched for ESBL genotypes but also included KPC, OXA-48-like, and MBLs. The MIC90 of CPD alone was reduced from >32 to 0.5 mg/L when tested in a 1:2 ratio with ETX1317.The MIC50/90 for ESBL expressing isolates was 0.125/0.25 µg/ mL; for KPC-producing isolates, it was 0.25/2 µg/mL; for OXA-48-like-producing isolates, it was 0.5/2 µg/mL; for derepressed AmpC, it was 0.5/1 µg/mL; and for MBL expressing isolates, it was 0.5/8 µg/mL. The in vivo efficacy of ETX0282 was evaluated with and without CPDP, in a murine ascending UTI model against CTX-M14 E. coli and KPC-2 K. pneumoniae clinical isolates [78]. Bacterial titers for the E. coli were 6.53, 6.02, and 6.64 log10 CFU in kidneys, bladders, and urine, respectively, for the untreated controls on day 7. An approximately 1-log CFU decrease in E. coli titers in kidneys and urine was observed for each drug administered singly. For the combination (CPDP, 50 mg/kg + ETX0282, 5–50 mg/kg), titers came down to 2.62–2.93 log10 CFU in the kidneys, 2.62–3.42 log10 CFU in the bladder, and 2.05–2.73 log10 CFU in urine. Bacterial titers for K. pneumoniae were 6.38, 3.45, and 5.19 log10 CFU in kidneys, bladders, and urine for the untreated controls on day 7. ETX0282 or CPDP alone resulted in an approx. 1–2-log CFU decrease in titers in kidneys and urine. The combination of CPDP (50 mg/kg) + ETX0282 (5–50 mg/ kg) reduced titers to 2.67–2.79 log10 CFU in the kidneys, 2.48–2.81 log10 CFU in the bladder, and 2.05–2.39 log10 CFU in urine (Table 3.3). 2.2.8  Cefepime + VNRX-5133 VNRX-5133 is a cyclic boronate BLI with inhibitory activity against serine-active and MBLs (Fig. 3.5), in combination with cefepime as a parenteral drug in Phase 1 of clinical development. The indications being pursued are cUTI, cIAI. In a study of six ceftazidime/avibactam-resistant K. pneumoniae isolates with mutations in KPC-3 that arose during therapy and four K. pneumoniae parent isolates with wild-type KPC-3, the MIC for cefepime in combination with 4 µg/mL of VNRX5133 ranged from 0.4 to 4 µg/mL for the wild-type and mutant forms of KPC-3, compared to 16 to >128  µg/mL for ceftazidime in combination with 4 µg/mL of avibactam [79]. Cefepime + 4 µg/mL of VNRX-5133 was also highly active among five P. aeruginosa clinical isolates that were ceftolozane/tazobactam sensitive (pretreatment) and ceftolozane/tazobactam resistant (posttreatment), with MICs for cefepime between 4 and 8 µg/mL for all but one isolate. In clinical isolates of P. aeruginosa that were resistant to ceftolozane/tazobactam and cross-resistant to ceftazidime/avibactam, the MIC of cefepime in the presence of 4 µg/mL of VNRX-5133 was 2–16 µg/mL. In vivo activity for the combination has been established in CD1 neutropenic mice, in the thigh infection model with MDR strains, four E. coli, three K. pneumoniae, and two P. aeruginosa with different resistance mechanisms (VIM, KPC, TEM, SHV-1, OXA1, CTX-M, AmpC, and OmpK35) and cefepime MICs of 8–256 mg/L [80]. Two hours

Antibiotics targeting Gram-negative bacteria

Table 3.3  Summary of efficacy data of CPDP in combination with ETX0282 in the murine ascending UTI model. Drug and dose

Organism

Source

Bacterial titer in log10 CFU

CPDP (50 mg/kg) +  ETX0282 (5, 10, 25, and 50 mg/kg)

Escherichia coli

Kidney Bladder Urine Kidney Bladder Urine Kidney Bladder Urine Kidney Bladder Urine

2.62–2.93 2.62–3.42 2.05–2.73 6.58 3.68 5.78 5.28 3.76 5.04 6.53 6.02 6.64

Drug and dose

Organism

Source

Bacterial titer in log10 CFU

CPDP (25 mg/ kg) + ETX0282 (5, 10, 25, and 50 mg/kg)

Klebsiella pneumoniae

Kidney Bladder Urine Kidney Bladder Urine Kidney Bladder Urine Kidney Bladder Urine

2.67–2.79 2.48–2.81 2.05–2.39 5.65 3.04 4.82 3.65 2.53 2.47 6.38 3.45 5.19

CPDP 50 mg/kg

ETX0282 10 mg/kg

Untreated control (day 7)

CPDP 25 mg/kg

ETX0282 25 mg/kg

Untreated control (day 7)

CPDP, cefpodoxime proxetill; UTI, urinary tract infections.

Figure 3.5  Structures of VNRX-5133 and vaborbactam, cyclic boronate β-lactamase inhibitors.

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after infection, cefepime (8–128 mg/kg) was given alone every 2 h and suboptimal cefepime doses were combined with VNRX-5133 (0.03–128 mg/kg) every 2, 4, and 8 h for 24 h. Each drug individually had poor activity but the static daily dose and 1-log kill were restored when combined with VNRX-5133. The q2h regimen was more effective than q4h and q8h VNRX-5133 regimens. 2.2.9  Cefepime + AAI101 Enmetazobactam, also known as AAI101, is a penicillanic acid sulfone ESBL inhibitor, similar in structure to tazobactam (Fig. 3.6). The strategic placement of a methyl group in AAI101 gives the inhibitor a net neutral charge, leading to better bacterial cell penetration [81]. It exerts potent inhibitory activity toward class A β-lactamases such as CTX-M, TEM, and SHV β-lactamases. The combined activities of cefepime and enmetazobactam extend the spectrum of activity to ESBL, AmpC, and OXA-48-producing strains of Enterobacteriaceae and Pseudomonas. The drug, currently in Phase 3 of clinical development as a parenteral drug, is positioned as a carbapenem-sparing treatment option in settings with a high incidence of ESBL-producing Enterobacteriaceae. In a study of 223 cefepime nonsusceptible isolates (22% produced carbapenemases and 67% produced ESBLs), addition of AAI101 at 8 mg/L lowered the MIC50 of cefepime from >64 to 0.13 mg/L [82]. In another study of 1993 clinical isolates of Enterobacteriaceae and P. aeruginosa collected in the United States and Europe during 2014 and 2015, in the presence of 8 µg/mL of Enmetazobactam, the MIC90 of cefepime was reduced from 16 to 0.12 µg/mL for E. coli, >64 to 0.5 µg/mL for K. pneumoniae, 16 to 1 µg/mL for E. cloacae, and 0.5 to 0.25 µg/mL for Enterobacter aerogenes [83]. For P. aeruginosa isolates, Enmetazobactam did not enhance the potency of cefepime. Of note, for ESBL-producing isolates of E. coli, enmetazobactam lowered the cefepime MIC90 from >64 to 0.12 µg/mL, and for ESBL-producing K. pneumoniae from >64 to 1 µg/mL.The activity for KPC-producing Enterobacteriaceae was not as impressive. In vivo efficacy studies, cefepime–AAI101 was tested using 20 Enterobacteriaceae strains, which included 3 clinical isolates of E. coli and 17 of K. pneumoniae from the isolate inventory of the Center for Anti-Infective Research and Development [84]. These isolates were resistant to cefepime and most were resistant to meropenem, although a molecular characterization was not provided. At 2 h after inoculation, mice were treated

Figure 3.6  Structure of AAI101 and tazobactam, penicillanic acid sulfone β-lactamase inhibitors

Antibiotics targeting Gram-negative bacteria

with humanized regimens of cefepime or cefepime–AAI101 through the subcutaneous route. The humanized cefepime–AAI101 dosing regimen resulted in bacterial reductions of ≥0.5 log10 CFU for 12 of the 20 strains, in contrast with cefepime alone, which resulted in efficacy against only 3 isolates. In a Phase 2 clinical study, different doses of AAI101 were tested in patients with cUTIs, including acute pyelonephritis (AP) [85]. In cohort 1, patients received either 500 mg of AAI101 in addition to 1 g of cefepime (n = 15) or 1 g of cefepime alone (n = 7). In cohort 2, patients were assigned 750 mg of AAI101 with 2 g of cefepime (n = 15) or 2 g of cefepime alone. Treatment was given intravenously three times a day for 7–10 days. Cefepime/AAI101 was safe and well tolerated. Overall, microbiological eradication, defined as baseline qualifying bacterial pathogen reduced to 50% of these isolates in the cefiderocol arm nonsusceptible to cefepime. Clinical studies evaluating cefiderocol’s clinical effectiveness for severe infections (bloodstream infections, hospital-acquired pneumonia, ventilator-associated pneumonia) caused by carbapenem-resistant Gram-negative pathogens are ongoing. These studies will play a key role in determining the full use of this drug in clinical practice as a truly broad-spectrum antibiotic (NCT02714595, NCT03032380, and NCT03869437). KBP-7072 belongs to the tetracycline chemical class. It is in Phase 1 of clinical development as an IV and oral agent for the treatment of community-acquired pneumonia [94]. It has largely a Gram-positive spectrum but is also active against carbapenemresistant Acinetobacter [95]. TP-271 is a novel, fully synthetic fluorocycline antibiotic in Phase 1 of clinical development for community-acquired bacterial pneumonia. TP-271 is active against

Antibiotics targeting Gram-negative bacteria

community respiratory Gram-positive and Gram-negative pathogens [96]. It has also shown antibacterial activity against A. baumannii. In a study of 25 A. baumannii isolates that included carbapenem-resistant phenotypes, the MIC50/90 was 0.13/1 µg/mL. In a lung infection model, TP-271 showed dose-dependent efficacy against four CRAB isolates. The PK/PD parameter that best predicted efficacy was the free drug ELF AUC/ MIC (Poster # 94, ASM Microbe 2017). TP-6076 is another synthetic fluorocycline antibiotic, such as eravacycline and TP271, which inhibits bacterial protein synthesis, also in Phase 1 of clinical development. It has shown potent activity against CRAB and carbapenem-resistant Enterobacteriaceae. In a study of 53 carbapenem-resistant Enterobacteriaceae from the FDA–CDC Antimicrobial Resistance Bank (Diversity panel), the MIC50/90 was 0.063/0.25 µg/mL. The isolates expressed both serine-β-lactamase and MBL. Further, the MIC50/90 of TP-6076 was conserved when tested against 19 polymyxin-resistant Enterobacteriaceae isolates [97]. In a study of Acinetobacter clinical isolates from 13 Greek hospitals (n = 121), the MIC50 and MIC90 values were 0.03 and 0.06 mg/L, respectively.There was no difference in the MIC90 value for colistin-susceptible or -resistant isolates [98]. The in vivo efficacy of TP-6076 has been demonstrated in murine thigh and lung infection models against nine A. baumannii strains with MICs for TP-6076 between 0.0078 and 0.25 µg/mL. Greater than 1-log reduction in CFU were observed at exposures equivalent to those achieved in Phase 1 studies in humans [99]. BOS-228 (LYS228) is a monobactam with activity against both serine-β-lactamase and MBL expressing carbapenem-resistant Enterobacteriaceae (CRE). It is in Phase 2 of clinical development for bacterial infections as an IV drug. In a study of 271 MDR Enterobacteriaceae strains, the MIC90 was 1 µg/mL. Against isolates expressing ESBLs (n = 37) or carbapenem resistance (n = 77), LYS228 had MIC90 values of 1 and 4 µg/mL, respectively [100]. Inducible blaDHA-1, a class C βlactamase, can decrease susceptibility to LYS228 in some K. pneumoniae clinical isolates, particularly if additional resistance mechanisms are present [101]. LYS228 has demonstrated efficacy in the neutropenic thigh model with K. pneumoniae producing KPC-2 or NDM-1 [102]. Using dose fractionation in the neutropenic murine thigh model, the percentage of the dosing interval that free drug concentrations remained above the MIC (%fT > MIC) was shown as the parameter that drives the efficacy of LYS228.This work was undertaken with Enterobacteriaceae-producing β-lactamase enzymes such as ESBLs, NDM-1, KPC, CMY-2, and OXA-48 [103].These data support future use of the drug for carbapenem-resistant infections. SPR206 is a polymyxin derivative being developed for in IV use for treating serious Gram-negative infections. The compound is in Phase 1 of clinical development. In a study of 182 P. aeruginosa, 185 Acinetobacter spp., and 22 S. maltophilia isolates, SPR206 demonstrated a MIC50/90 of 0.25/0.5 mg/L for P. aeruginosa isolates. This included 22.5% meropenem-resistant isolates. Against A. baumannii, the MIC50/90 was

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0.12/0.25 mg/L. SPR206 inhibited 93.1% of all Acinetobacter spp. isolates at ≤2 mg/L. About 64.9% of the isolates were meropenem resistant [104]. In a study of 541 Enterobacteriaceae isolates collected from 150 medical centers worldwide and an additional 32 CRE isolates, the compound had a MIC50/90 of 0.06/0.12 mg/L. Against the challenge set of 32 CRE isolates, this activity was unchanged. These values were better than those for colistin and polymyxin B [105].The efficacy of SPR206 in the ascending UTI model was tested using E. coli ATCC700928 (MIC 0.03 µg/mL) and E. coli UTI89 (MIC 0.125 µg/mL). SPR206 administered at 4 mg/kg q8h SC for 3 days reduced the burden of E. coli UTI89 by 3.0-log CFU/g and of E. coli ATCC700928 by 4.1-log CFU/g compared to the day 1 control [106].The efficacy of SPR206 has also been tested in the immunosuppressed lung and thigh infection models using a P. aeruginosa strain (Pa14, lads mutant; hypervirulent, MIC 0.13 mg/L) or A. baumannii NCTC13301 (Ab13301) (OXA-23+; carbapenem resistant, MIC 0.25 mg/L) [107]. In the lung model, SPR206 dosed at 30 mg/kg, q8h subcutaneous reduced the burden of Pa14 by 3.6-log CFU/mL compared to 2-h control. SPR206 dosed at 20 mg/kg q4h subcutaneous, in the lung model, reduced the burden of Ab13301 by 4.6-log CFU/mL compared to 2-h control. In the thigh model, SPR206 dosed at 4 mg/kg IV, q4h reduced the burden of Ab13301 by 4.3-log CFU/g compared to 2-h control. These efficacies were similar or better than efficacies mediated by polymyxin B, which was used as a comparator in these studies. A summary of the compounds in clinical development are given in Table 3.5.

3  What the future holds The pipeline for new compounds, while lean, is promising. Broadly speaking, the new drugs in development are of three types: novel chemical structures targeting novel targets, combinations of approved antibiotics with novel partner compounds, and analogs of existing classes. There are three axes against which these compounds must be evaluated. The first axis is the coverage of specific Gram-negative species and genotypes associated with resistance, the second is the indications these compounds are being developed for, and the third is their chemical diversity. A significant number of compounds target MDR Enterobacteriaceae but nonfermenters such as Acinetobacter and Pseudomonas are underrepresented. ETX2514 is being developed exclusively for Acinetobacter infections and murepavadin, for Pseudomonas infections. Others in development such as cefiderocol, SPR206, TP-6076, TP-271, and ceftazidime/avibactam/aztreonam (not covered here due to the lack of a novel component) have demonstrated coverage of one or both of these organisms in vitro but definitive clinical studies are ongoing or need to be undertaken to demonstrate their efficacy in patients. The current pipeline has multiple compounds that are able to overcome carbapenem resistance, particularly that driven by class A and C β-lactamases. This includes approved

Antibiotics targeting Gram-negative bacteria

Table 3.5  Compounds in clinical development.

Compound

Chemical class

Phase of de­ velopment

Cefiderocol (IV)

Cephalosporin

Phase 3

KBP-7072

Tetracycline Tetracycline Tetracycline

Monobactam Polymyxin

Phase 2

TP-271 TP-6076 (IV)

BOS-228 (IV) SPR-206 (IV)

Indication

Microbiological spectrum (Gramnegative)

Mechanisms of resistance covered

Enterobacteriaceae, Acinetobacter, Pseudomonas

Carbapenem resistance

Phase 1

cUTI, HABP, VABP, bloodstream infections, and sepsis CAP

Acinetobacter

Phase 1

CAP

Acinetobacter

Carbapenem resistance Carbapenem resistance Carbapenem resistance, polymyxin resistance Carbapenem resistance Carbapenem resistance

Phase 1

Phase 1

Enterobacteriaceae, Acinetobacter Bacterial infections Gramnegative infections

Enterobacteriaceae Broad Gramnegative

cUTI, complicated urinary tract infection; HABP, hospital-acquired bacterial pneumonia; IV, intravenous; VABP, ventilator-associated bacterial pneumonia.

drugs such as ceftazidime–avibactam, meropenem–vaborbactam, plazomicin and eravacycline and multiple β-lactam and BLIs in development. Several compounds, including ceftazidime–avibactam and ETX2514–sulbactam, additionally cover class D β-lactamases but very few cover class B β-lactamases. Tetracycline analogs, TP-271 and TP-6076, and monobactam analog, BOS-228, have shown in vitro activity against isolates expressing class B β-lactamases. While the tetracyclines cover Acinetobacter and Enterobacteriaceae, the monobactam is intended only for Enterobacteriaceae, pointing to the complexity of addressing diverse resistance mechanisms and a range of pathogenic species. Overall, resistance to carbapenems is being addressed, albeit with gaps. On the other hand, colistin resistance remains a challenge. In fact, most compounds have not been tested against colistin-resistant isolates, making it difficult to evaluate their potential for clinical use. Clinical trials for antibacterial compounds have mainly focused on cUTIs and intraabdominal infections. However, data for more difficult infections such as bloodstream infections are not available. Further, patients included in clinical studies with highly resistant infections remain a minority. Difficulties in recruiting such patients (leading to

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higher cost and longer duration of trials) are the main reason. The international community must come together to overcome these obstacles. Clinical trial sites in lower and middle-income countries, with higher burden of disease, must be established and regulatory agencies must join hands to speed up the development of compounds. The chemical diversity of the current pipeline is limited; compounds are clustered within a small number of chemical types. A significant number of compounds are combinations of β-lactams and BLIs. These inhibitors are either derived from DBO, cyclic boronic acids or tazobactam. Others are modifications of the aminoglycoside, cephalosporin, polymyxin, or tetracycline classes. Chemical diversity is important for preserving new compounds from cross-resistance that may emerge or already exist in the clinical isolate population. A related consideration is the appropriate hierarchy of use in the clinic. Take the case of the β-lactam and BLI combinations. Are there sufficiently differentiated therapeutic niches for these combinations? Some researchers have addressed this question through in vitro studies. For example, IMI–REL has been shown to be active against ceftazidime–avibactam-resistant K. pneumoniae isolates that carried variant KPC-3 genes, while ceftazidime-avibactam was active against IMI–REL-resistant K. pneumoniae isolates that carried OXA-48-type carbapenemases. The presence of major OmpK36 porin mutations among KPC-producing K. pneumoniae isolates was associated with higher IMI–REL MICs and a trend toward higher ceftazidime–avibactam MICs [46]. Similarly, studies conducted with K. pneumoniae isolates resistant to ceftazidime–avibactam show greater sensitivity to cefepime–VNRX-5133. Such differences in activity at a molecular level need to be validated in clinical studies. The same is true for the multiple tetracycline compounds in development. It is hoped that clinical use and differentiation are established during the clinical development phase. Finally, additional considerations need to be taken into account for future discovery efforts: (1) susceptibility studies tend to be conducted on isolates sourced from North America and Europe. Preclinical testing must extend to other geographies to allow a true assessment of compounds against a diverse range of clinical isolates and resistance mechanisms. (2) There is a need for oral drugs that can be step-down therapies as patients recover and leave the hospital. In the current pipeline, only one drug is being developed as an oral formulation. (3) There is growing recognition that the microbiome plays an important role in maintaining good health. The focus on broad-spectrum antibiotics has meant that adverse effects on the microbiota have been unavoidable. Focusing on narrow-spectrum drugs, or better still, undertaking a holistic approach to addressing infections needs to become a priority. (4) As new agents get approved and go to market, we know that the forces of evolution will eventually lead to resistance to these agents. While we cannot predict how soon this will happen, we know from history that it does not take long. For example, among the newly approved antibiotics, resistance development to ceftazidime/avibactam during or following therapy has already been reported [108,109]. It is, therefore, essential that the judicious use of the new agents is ensured.

Antibiotics targeting Gram-negative bacteria

This is possible if antibiotic prescribing is predicated on molecular diagnostics that identify the organism and its genotype. (5) Finally, the goal of keeping new antibiotics accessible must be a cornerstone of the global strategy to combat infections. It is estimated that 10 times more people die from lack of access to antibiotics, rather than resistance. Thus new antibiotics that go to market must be priced affordably. This calls for a high level of cooperation and collaboration across national borders.

References [1] WHOWHO publishes list of bacteria for which new antibiotics are urgently needed, WHO, Geneva, (2017). [2] F.S. Codjoe, E.S. Donkor, Carbapenem resistance: a review, Med Sci 6 (1) (2018) 1. [3] M.J.Trimble, P. Mlynárcˇ ik, M. Kolárˇ, R.E. Hancock, et al. Polymyxin: alternative mechanisms of action and resistance, Cold Spring Harb Perspect Med 6 (10) (2016) a025288. [4] É. Ruppé, P.L. Woerther, F. Barbier, Mechanisms of antimicrobial resistance in Gram-negative bacilli, Ann Intensive Care 5 (1) (2015) 61. [5] Y. Zhang, Q. Wang,Y.Yin, H. Chen, L. Jin, B. Gu, et al. Epidemiology of carbapenem-resistant Enterobacteriaceae infections: report from the China CRE Network, Antimicrob Agents Chemother 62 (2) (2018) e01882-17. [6] M. Akova, Epidemiology of antimicrobial resistance in bloodstream infections,Virulence 7 (3) (2016) 252–266. [7] M. Kazi L. Drego, C. Nikam, K. Ajbani, R. Soman, A. Shetty, et al. Molecular characterization of carbapenem-resistant Enterobacteriaceae at a tertiary care laboratory in Mumbai, Eur J Clin Microbiol Infect Dis 34 (3) (2015) 467–472. [8] D. van Duin,Y. Doi, The global epidemiology of carbapenemase-producing Enterobacteriaceae,Virulence 8 (4) (2017) 460–469. [9] G. Jacoby, AmpC β-lactamases, Clin Microbiol Rev 22 (1) (2009) 161–182. [10] L. Poirel, P. Nordmann, Carbapenem resistance in Acinetobacter baumannii: mechanisms and epidemiology, Clinical Microbiology and Infection 12 (9) (2006) 826–836. [11] A. Ramette, A. Kronenberg, and the Swiss Centre for Antibiotic Resistance (ANRESIS)et al. Prevalence of carbapenem-resistant Acinetobacter baumannii from 2005 to 2016 in Switzerland, BMC Infect Dis 18 (1) (2018) 159. [12] H.A. Zowawi, A.L. Sartor, H.E. Sidjabat, H.H Balkhy, T.R. Walsh, S.M. Al Johani, et al. Molecular epidemiology of carbapenem-resistant Acinetobacter baumannii isolates in the Gulf Cooperation Council States: dominance of OXA-23-type producers, J Clin Microbiol 53 (3) (2015) 896–903. [13] M.D. Zilberberg, M.H. Kollef and A.F. Shorr, et al. Secular trends in Acinetobacter baumannii resistance in respiratory and blood stream specimens in the United States, 2003 to 2012: a survey study, J Hosp Med 11 (1) (2016) 21–26. [14] S.N. Bulens, S.H. Yi, M.S. Walters, J.T. Jacob, C. Bower, J. Reno, et al. Carbapenem-nonsusceptible Acinetobacter baumannii, 8 US metropolitan areas, 2012–2015, Emerg Infect Dis 24 (4) (2018) 727–734. [15] L.Y. Hsu, A. Apisarnthanarak, E. Khan, N. Suwantarat, A. Ghafur, P.A. Tambyah, et al. Carbapenemresistant Acinetobacter baumannii and Enterobacteriaceae in South and Southeast Asia, Clin Microbiol Rev 30 (1) (2017) 1–22. [16] W. Hsieh, N.Y. Wang, J.A. Feng, L.C. Weng, H.H. Wu, et al. Types and prevalence of carbapenem-resistant Acinetobacter calcoaceticus–Acinetobacter baumannii complex in Northern Taiwan, Antimicrob Agents Chemother 58 (1) (2013) 201–204. [17] J.M. Rodríguez-Martínez, L. Poirel, P. Nordmann, et al. Molecular epidemiology and mechanisms of carbapenem resistance in Pseudomonas aeruginosa, Antimicrob Agents Chemother 53 (11) (2009) 4783–4788. [18] M.S.Walters, J.E. Grass, S.N. Bulens, E.B. Hancock, E.C. Phipps, D. Muleta, et al. Carbapenem-resistant Pseudomonas aeruginosa at US emerging infections program sites, 2015, Emerg Infect Dis 25 (7) (2019) 1281–1288.

65

66

Drug Discovery Targeting Drug-Resistant Bacteria

[19] T. Karampatakis, C. Antachopoulos, A. Tsakris, E. Roilides, et al. Molecular epidemiology of carbapenem-resistant Pseudomonas aeruginosa in an endemic area: comparison with global data, Eur J Clin Microbiol Infect Dis 37 (7) (2018) 1211–1220. [20] S. Meradji, A. Barguigua, K. Zerouali, D. Mazouz, H. Chettibi, N. Elmdaghri, et al. Epidemiology of carbapenem non-susceptible Pseudomonas aeruginosa isolates in Eastern Algeria, Antimicrob Resist Infect Control 4 (2015) 27. [21] V. Estepa, B. Rojo-Bezares, J.M. Azcona-Gutiérrez, I. Olarte, C. Torres,Y. Sáenz, et al. Characterisation of carbapenem-resistance mechanisms in clinical Pseudomonas aeruginosa isolates recovered in a Spanish hospital, Enferm Infecc Microbiol Clin 35 (2017) 137–138. [22] A.O. Olaitan, B. Thongmalayvong, K. Akkhavong, S. Somphavong, P. Paboriboune, S. Khounsy, et al. Clonal transmission of a colistin-resistant Escherichia coli from a domesticated pig to a human in Laos, J Antimicrob Chemother 70 (12) (2015) 3402–3404. [23] C.A. Aires, P.S. Pereira, M.D. Asensi, A.P. Carvalho-Assef, et al. mgrB mutations mediating polymyxin B resistance in Klebsiella pneumoniae isolates from rectal surveillance swabs in Brazil, Antimicrob Agents Chemother 60 (11) (2016) 6969–6972. [24] M. Monaco, T. Giani, M. Raffone, F. Arena, A. Garcia-Fernandez, S. Pollini, et al. Colistin resistance superimposed to endemic carbapenem-resistant Klebsiella pneumoniae: a rapidly evolving problem in Italy, Euro Surveill 19 (42) (2014) pii: 20939. [25] K. Novovic´, A. Trudic´, S. Brkic´, Z. Vasiljevic´, M. Kojic´, D. Medic´, et al. Molecular epidemiology of colistin-resistant, carbapenemase-producing Klebsiella pneumoniae in Serbia from 2013 to 2016, Antimicrob Agents Chemother 61 (5) (2017) e02550-16. [26] L. Poirel, A. Jayol, P. Nordmann, et al. Polymyxins: antibacterial activity, susceptibility testing, and resistance mechanisms encoded by plasmids or chromosomes, Clin Microbiol Rev 30 (2) (2017) 557–596. [27] G. Goel, L. Hmar, M. Sarkar De, S. Bhattacharya, M. Chandy, et al. Colistin-resistant Klebsiella pneumoniae: report of a cluster of 24 cases from a new oncology center in eastern India, Infect Control Hosp Epidemiol 35 (8) (2014) 1076–1077. [28] S Chen, F Hu, X Zhang, X Xu, Y Liu, D Zhu, et al. Independent Emergence of Colistin-Resistant Enterobacteriaceae Clinical Isolates without Colistin Treatment. Journal of Clinical Microbiology 49 (11) (2011) 4022–4023. [29] Z.A. Qureshi, L.E. Hittle, J.A. O’Hara, J.I. Rivera, A. Syed, R.K. Shields, et al. Colistin-resistant Acinetobacter baumannii: beyond carbapenem resistance, Clin Infect Dis 60 (9) (2015) 1295–1303. [30] Antibiotics Currently in Global Clinical Development. Pewtrusts.org, 2019, Available from URL: https://www.pewtrusts.org/-/media/assets/2019/03/antibiotics-currently-in-global-clinical-development.pdfla=en&hash=078238EF15FACD9753ED2C4EBAB58F16B664B59E [31] G. Poulakou, S. Lagou, D.E. Karageorgopoulos, G. Dimopoulos, et al. New treatments of multidrugresistant Gram-negative ventilator-associated, Ann Transl Med 6 (21) (2018) 423. [32] H.S. Sader, G.E. Dale, P.R. Rhomberg, R.K. Flamm, et al. Antimicrobial activity of murepavadin tested against clinical isolates of Pseudomonas aeruginosa from the United States, Europe, and China, Antimicrob Agents Chemother 62 (7) (2018) e00311-18. [33] N. Srinivas, P. Jetter, B.J. Ueberbacher, M.Werneburg, K. Zerbe, J. Steinmann, et al. Peptidomimetic antibiotics target outer-membrane biogenesis in Pseudomonas aeruginosa, Science 327 (2010) 1010–1013. [34] M.J. Melchers, J. Teague, P. Warn, J. Hansen, F. Bernardini, A. Wach, et al. Pharmacokinetics and pharmacodynamics of murepavadin in neutropenic mouse models, Antimicrob Agents Chemother 63 (3) (2019) e01699-18. [35] Wallnofer, A., Murepavadin (POL7080): A Pathogen-Specific, Novel Antibiotic for the Treatment of Infections due to P. aeruginosa in Patients with Nosocomial Pneumonia. Polyphor, Switzerland, 2017; Available from URL: https://www.fda.gov/media/103043/download. [36] A. Wach, K. Dembowsky, G.E. Dale, et al. Pharmacokinetics and safety of intravenous murepavadin infusion in healthy adult subjects administered single and multiple ascending doses, Antimicrob Agents Chemother 62 (4) (2018) e02355-17. [37] A. Apostolos, F. Frantzeskaki, C. Diakaki, S. Zakynthinos, E. Ischaki, K. Mandragos, et al. Pharmacokinetic and efficacy analysis of murepavadin (POL7080) co-administered with standard-of-care (SoC) in a phase II study in patients with ventilator-associated pneumonia (VAP) due to suspected or documented Pseudomonas aeruginosa infection, ECCMID,Vienna, (2017).

Antibiotics targeting Gram-negative bacteria

[38] D. Corbett, A.Wise,T. Langley, K. Skinner, E.Trimby, S. Birchall, et al. Potentiation of antibiotic activity by a novel cationic peptide: potency and spectrum of activity of SPR741, Antimicrob Agents Chemother 61 (8) (2017) e00200-17. [39] D.V. Zurawski, A.A. Reinhart, Y.A. Alamneh, M.J. Pucci, Y. Si, R. Abu-Taleb, et al. SPR741, an antibiotic adjuvant, potentiates the in vitro and in vivo activity of rifampin against clinically relevant extensively drug-resistant Acinetobacter baumannii, Antimicrob Agents Chemother 61 (12) (2017) e01239-17. [40] M. Vaara, Polymyxin derivatives that sensitize Gram-negative bacteria to other antibiotics, Molecules 24 (2) (2019) 249. [41] P.B. Eckburg, N. Farinola, L. Utley, S. Walpole, T. Keutzer, E. Kopp, et al. Safety of SPR741, a novel polymyxin potentiator, in healthy adults receiving single-and multiple-dose intravenous administrations, ECCMID, Madrid, (2018). [42] M. Shirley, Ceftazidime-avibactam: a review in the treatment of serious Gram-negative, Drugs 78 (6) (2018) 675–692. [43] S. Dhillon, Meropenem/vaborbactam: a review in complicated urinary tract infections, Drugs 78 (12) (2018) 1259–1270. [44] T.A. Blizzard, H. Chen, S. Kim, J. Wu, R. Bodner, C. Gude, et al. Discovery of MK-7655, a beta-lactamase inhibitor for combination with Primaxin(R), Bioorg Med Chem Lett 24 (3) (2014) 780–785. [45] D. Wong, D. van Duin, et al. Novel β-lactamase inhibitors: unlocking their potential in therapy, Drugs 77 (6) (2017) 615–628. [46] G. Haidar, C.J. Clancy, L. Chen, P. Samanta, R.K. Shields, B.N. Kreiswirth, et al. Identifying spectra of activity and therapeutic niches for ceftazidime-avibactam and imipenem-relebactam against carbapenem-resistant Enterobacteriaceae, Antimicrob Agents Chemother 61 (9) (2017) e00642-17. [47] D.M. Livermore, M. Warner, S. Mushtaq, et al. Activity of MK-7655 combined with imipenem against Enterobacteriaceae and Pseudomonas aeruginosa, J Antimicrob Chemother 68 (10) (2013) 2286–2290. [48] S.H. Lob, J.A. Karlowsky, K.Young, M.R. Motyl, S. Hawser, N.D. Kothari, et al. Activity of imipenem/ relebactam against MDR Pseudomonas aeruginosa in Europe: SMART 2015–17, J Antimicrob Chemother 74 (8) (2019) 2284–2288. [49] J.A. Karlowsky, K.M. Kazmierczak, K. Young, M.R. Motyl, D.F. Sahm, et al. In vitro activity of imipenem-relebactam against clinical isolates of Gram-negative Bacilli isolated in hospital laboratories in the United States as part of the SMART 2016 Program, Antimicrob Agents Chemother 62 (7) (2018) e00169-18. [50] I. Galani, M. Souli, K. Nafplioti, P. Adamou, I. Karaiskos, H. Giamarellou, et al. In vitro activity of imipenem-relebactam against non-MBL carbapenemase-producing Klebsiella pneumoniae isolated in Greek hospitals in 2015-2016, Eur J Clin Microbiol Infect Dis 38 (6) (2019) 1143–1150. [51] E.G. Rhee, M.L. Rizk, N. Calder, M. Nefliu, S.J. Warrington, M.S. Schwartz, et al. Pharmacokinetics, safety, and tolerability of single and multiple doses of relebactam, a β-lactamase inhibitor, in combination with imipenem and cilastatin in healthy participants, Antimicrob Agents Chemother 62 (9) (2018) e00280-18. [52] C. Lucasti, L.Vasile, D. Sandesc, D.Venskutonis, P. McLeroth, M. Lala, et al. Phase 2, dose-ranging study of relebactam with imipenem-cilastatin in subjects with complicated intra-abdominal infection, Antimicrob Agents Chemother 60 (2016) 6234–6243. [53] M. Sims,V. Mariyanovski, P. McLeroth, W. Akers,Y.C. Lee, M.L. Brown, et al. Prospective, randomized, double-blind, phase 2 dose-ranging study comparing efficacy and safety of imipenem/cilastatin plus relebactam with imipenem/cilastatin alone in patients with complicated urinary tract infections, J Antimicrob Chemother 72 (9) (2017) 2616–2626. [54] J. Motsch, C. de Oliveira,V. Stus, I. Koksal, O. Lyulko, H.W. Boucher, et al. RESTORE-IMI 1: a multicenter, randomized, double-blind, comparator controlled trial comparing the efficacy and safety of imipenem/relebactam versus colistin plus imipenem in patients with imipenem-non-susceptible bacterial infections, ECCMID, Madrid, (2018). [55] J. Motsch, C. Murta de Oliveira,V. Stus, I. Köksal, O. Lyulko, H.W. Boucher, et al. RESTORE-IMI 1: a multicenter, randomized, double-blind trial comparing efficacy and safety of imipenem/relebactam vs colistin plus imipenem in patients with imipenem-nonsusceptible bacterial infections, Clin Infect Dis (2019) pii: ciz530.

67

68

Drug Discovery Targeting Drug-Resistant Bacteria

[56] M.D. Barnes,V. Kumar, C.R. Bethel, S.H. Moussa, J. O’Donnell, J.D. Rutter, et al.Targeting multidrugresistant Acinetobacter spp.: sulbactam and the diazabicyclooctenone β-lactamase inhibitor ETX2514 as a novel therapeutic agent, mBio 10 (2) (2019) e00159-19. [57] W.F. Penwell, A.B. Shapiro, R.A. Giacobbe, R.F. Gu, N. Gao, J. Thresher, et al. Molecular mechanisms of sulbactam antibacterial activity and resistance determinants in Acinetobacter baumannii, Antimicrob Agents Chemother 59 (2015) 1680–1689. [58] L. Krizova, L. Poirel, P. Nordmann, A. Nemec, et al. TEM-1 beta-lactamase as a source of resistance to sulbactam in clinical strains of Acinetobacter baumannii, J Antimicrob Chemother 68 (12) (2013) 2786–2791. [59] S. Kuo, Y.T. Lee, T.L. Yang Lauderdale, W.C. Huang, M.F. Chuang, C.P. Chen, et al. Contribution of Acinetobacter-derived cephalosporinase-30 to sulbactam resistance in Acinetobacter baumannii, Front Microbiol 6 (2015) 231. [60] Durand-Reville, T, S. Guler, J. Comita-Prevoir, B. Chen, N. Bifulco, H. Huynh. et al., ETX2514 is a broad-spectrum beta-lactamase inhibitor for the treatment of drug-resistant Gram-negative bacteria including Acinetobacter baumannii. Nat Microbiol., 2017. 2: p. 17104. [61] M. Hackel, S. Bouchillon, B. DeJonge, K. Lawrence, J. Mueller, R. Tommasi, et al. Global surveillance of the activity of sulbactam combined with the novel β-lactamase inhibitor ETX2514 against clinical isolates of Acinetobacter baumannii from 2014, IDWeek, New Orleans, (2016). [62] A. Miller, ETX2514 restoration of sulbactam activity against multidrug resistant Acinetobacter baumannii correlates with β-lactamase inhibition in vitro and in vivo, ECCMID,Vienna, (2017). [63] J. Lickliter, K. Lawrence, J. O’Donnell, R. Isaacs, et al. Safety and pharmacokinetics (PK) in humans of intravenous ETX2514, a β-lactamase inhibitor (BLI) which broadly inhibits ambler class A, C, and D β-lactamases, IDWeek, San Diego, (2017). [64] O. Sagan, R.Yakubsevitch, K.Yanev, R. Fomkin, E. Stone, J. O’Donnell, et al. A double-blind, randomized, placebo-controlled study to evaluate the safety and efficacy of intravenous sulbactam-ETX2514 in the treatment of hospitalized adults with complicated urinary tract infections, including acute pyelonephritis, ECCMID, Amsterdam, (2019). [65] A. Morinaka,Y. Tsutsumi, M.Yamada, K. Suzuki, T. Watanabe, T. Abe, et al. OP0595, a new diazabicyclooctane: mode of action as a serine beta-lactamase, antibiotic and β-lactam ‘enhancer’, J Antimicrob Chemother 70 (10) (2015) 2779–2786. [66] R. Okujava, F. Garcia, A. Haldimann, C. Zampaloni, I. Morrissey, S. Magnet, et al. Activity of meropenem/nacubactam combination against Gram-negative clinical isolates: ROSCO Global Surveillance 2017, IDWeek, San Francisco, (2018). [67] S. Mushtaq, A. Vickers, N. Woodford, A. Haldimann, D.M. Livermore, et al. Activity of nacubactam (RG6080/OP0595) combinations against MBL-producing Enterobacteriaceae, J Antimicrob Chemother 74 (4) (2019) 953–960. [68] I. Morrissey, S. Magnet, S. Hawser, S. Louvel, R. Okujava, C. Zampaloni, et al. In vitro activity of Nacubactam, a novel dual action diazabicyclooctane, alone and with meropenem, against beta-lactamasepositive Enterobacteriaceae, ECCMID, Madrid, (2018). [69] M.L. Monogue, S. Giovagnoli, C. Bissantz, C. Zampaloni, D.P. Nicolau, et al. In vivo efficacy of meropenem with a novel non-β-lactam–β-lactamase inhibitor, nacubactam, against Gram-negative organisms exhibiting various resistance mechanisms in a murine complicated urinary tract infection model, Antimicrob Agents Chemother 62 (9) (2018) e02596-17. [70] T.E. Asempa, A. Motos, K. Abdelraouf, C. Bissantz, C. Zampaloni, D.P. Nicolau, et al. Efficacy of human-simulated epithelial lining fluid exposure of meropenem-nacubactam combination against class A serine β-lactamase-producing Enterobacteriaceae in the neutropenic murine lung infection model, Antimicrob Agents Chemother 63 (4) (2019) e02382-18. [71] D.M. Livermore, S. Mushtaq, M. Warner, A.Vickers, N. Woodford, et al. In vitro activity of cefepime/ zidebactam (WCK 5222) against Gram-negative bacteria, J Antimicrob Chemother 72 (5) (2017) 1373–1385. [72] H.S. Sader, M. Castanheira, M. Huband, R.N. Jones, R.K. Flamm, et al. WCK 5222 (cefepime–zidebactam) antimicrobial activity against clinical isolates of Gram-negative bacteria collected worldwide in 2015, Antimicrob Agents Chemother 61 (5) (2017) e00072-17. [73] K.S. Thomson, S. AbdelGhani, J.W. Snyder, G.K. Thomson, et al. Activity of cefepime–zidebactam against multidrug-resistant (MDR) Gram-negative pathogens, Antibiotics 8 (1) (2019) 32.

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[74] L.M. Avery, K. Abdelraouf, D.P. Nicolau, et al. Assessment of the efficacy of WCK 5222 (cefepime– zidebactam) against carbapenem-resistant Acinetobacter baumannii in the neutropenic murine lung infection model, Antimicrob Agents Chemother 62 (11) (2018) e00948-18. [75] R.A. Preston, G. Mamikonyan, S. DeGraff, J. Chiou, C.J. Kemper, A. Xu, et al. Single-center evaluation of the pharmacokinetics of WCK 5222 (cefepime–zidebactam combination) in subjects with renal impairment, Antimicrob Agents Chemother 63 (1) (2018) e01484-18. [76] Durand-Reville, T., ETX0282, a Novel Oral Agent Against Multidrug-Resistant Enterobacteriaceae, in ASM Microbe. 2017: New Orleans. [77] S. McLeod, N. Carter, M. Hackel, R. Badal, J. Mueller, R.Tommasi, et al.The novel β-lactamase inhibitor ETX1317 effectively restores the activity of cefpodoxime against extended spectrumβ-lactamase (ESBL)- and carbapenemase-expressing enterobacteriaceae isolated from recent urinary tract infections, Microbe, Atlanta, GA, (2018). [78] W.J.Weiss, K. Carter, M. Pulse, P. Nguyen, D.Valtierra, K. Peterson, et al. Efficacy of cefpodoxime proxetil and ETX0282 in a murine UTI model with E. coli and K. pneumoniae, ECCMID, Amsterdam, (2019). [79] D.M. Daigle, J.C. Hamrick, C. Chatwin, D. Lou, M. Schuster, N. Kurepina, et al. Cefepime/VNRX5133 broad-spectrum activity is maintained against emerging KPC- and PDC-variants in multidrug resistant K. pneumoniae and P. aeruginosa, IDWeek, San Francisco, (2018). [80] P. Georgiou, M. Siopi, M. Tsala, C. Lagarde, W. Kloezen, R. Donnelly, et al. VNRX-5133, a novel broad-spectrum β-lactamase inhibitor, enhances the activity of cefepime against Enterobacteriaceae and P. aeruginosa isolates in a neutropenic mouse-thigh infection model, ECCMID, Amsterdam, (2018). [81] K.M. Papp-Wallace, C.R. Bethel, J. Caillon, M.D. Barnes, G. Potel, S. Bajaksouzian, et al. Beyond piperacillin-tazobactam: cefepime and AAI101 as a potent β-lactam − β-lactamase inhibitor combination, Antimicrob Agents Chemother 63 (5) (2019) e00105-19. [82] J.L. Crandon, D.P. Nicolau, In vitro activity of cefepime/AAI101 and comparators against cefepime non-susceptible Enterobacteriaceae, Pathogens 4 (3) (2015) 620–625. [83] I. Morrissey, S. Magnet, S. Hawser, S. Shapiro, P. Knechtle, et al. In vitro activity of cefepime–enmetazobactam against Gram-negative isolates collected from U.S. and European hospitals during 2014–2015, Antimicrob Agents Chemother 63 (7) (2019) e00514-19. [84] J.L. Crandon, D.P. Nicolau, In vivo activities of simulated human doses of cefepime and cefepime– AAI101 against multidrug-resistant Gram-negative Enterobacteriaceae, Antimicrob Agents Chemother 59 (5) (2015) 2688–2694. [85] Allecra Therapeutics. Allecra Therapeutics Announces Positive Top-Line Results from Phase 2 CACTUS Study of its Lead Candidate, AAI101, in Combination with Cefepime to Treat Complicated Urinary Tract Infections (cUTI), 2018, Prnewswire.com, Weil am Rhein, Germany and Saint Louis, France. Available from URL: https://www.prnewswire.com/news-releases/allecra-therapeuticsannounces-positive-top-line-results-from-phase-2-cactus-study-of-its-lead-candidate-aai101-incombination-with-cefepime-to-treat-complicated-urinary-tract-infections-cuti-300619155.html [86] L.J. Scott, Eravacycline: a review in complicated intra-abdominal infections, Drugs 79 (3) (2019) 315– 324. [87] K.M. Shaeer, M.T. Zmarlicka, E.B. Chahine, N. Piccicacco, J.C. Cho, et al. Plazomicin: a next-generation aminoglycoside, Pharmacotherapy 39 (1) (2019) 77–93. [88] A. Ito, T. Nishikawa, S. Matsumoto, H. Yoshizawa, T. Sato, R. Nakamura, et al. Siderophore cephalosporin cefiderocol utilizes ferric iron transporter systems for antibacterial activity against Pseudomonas aeruginosa, Antimicrob Agents Chemother 60 (2016) 7396–7401. [89] A. Ito, T. Sato, M. Ota, M. Takemura, T. Nishikawa, S. Toba, et al. In vitro antibacterial properties of cefiderocol, a novel siderophore cephalosporin, against Gram-negative bacteria, Antimicrob Agents Chemother 62 (1) (2018) e01454-17. [90] J.A. Karlowsky, M.A. Hackel, M. Tsuji, Y. Yamano, R. Echols, D.F. Sahm, et al. In vitro activity of cefiderocol, a siderophore cephalosporin, against Gram-negative bacilli isolated by clinical laboratories in North America and Europe in 2015–2016: SIDERO-WT-2015, Int J Antimicrob Agents 53 (4) (2019) 456–466. [91] N. Kohira, N. Kohira, J. West, A. Ito, T. Ito-Horiyama, R. Nakamura, T. Sato, et al. In vitro antimicrobial activity of a siderophore cephalosporin, S-649266, against Enterobacteriaceae clinical isolates including carbapenem-resistant strains, Antimicrob Agents Chemother 60 (2) (2016) 729–734.

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[92] K.M. Kazmierczak, M. Tsuji, M.G. Wise, M. Hackel, Y. Yamano, R. Echols, et al. In vitro activity of cefiderocol, a siderophore cephalosporin, against a recent collection of clinically relevant carbapenemnon-susceptible Gram-negative bacilli, including serine carbapenemase- and metallo-β-lactamaseproducing isolates (SIDERO-WT-2014 Study), Int J Antimicrob Agents 53 (2) (2019) 177–184. [93] S. Portsmouth, D. van Veenhuyzen, R. Echols, M. Machida, J.C.A. Ferreira, M. Ariyasu, et al. Cefiderocol versus imipenem-cilastatin for the treatment of complicated urinary tract infections caused by Gram-negative uropathogens: a phase 2, randomised, double-blind, non-inferiority trial, Lancet Infect Dis 18 (12) (2018) 1319–1328. [94] B. Zhang,Y. Wang, C.Yanhua, F.Yang, et al. Single ascending dose safety, tolerability, and pharmacokinetics of KBP-7072, a novel third generation tetracycline, Open Forum Infect Dis 3 (Suppl_1) (2016). [95] Kojim, KBP-7072 obtained QIDP and fast track designations. KBP Biosciences. November 22, 2016. Available from URL: https://kbpbiosciences.com/kbp-7072-obtained-qidp-and-fast-track-designations/ [96] T.H. Grossman, C. Fyfe, W. O’Brien, M. Hackel, M.B. Minyard, K.B. Waites, et al. Fluorocycline TP271 is potent against complicated community-acquired bacterial pneumonia pathogens, mSphere 2 (1) (2017) e00004-17. [97] C. Fyfe, G. LeBlanc, B. Close, J. Newman, et al.TP-6076 is active against carbapenem- and polymyxinresistant Enterobacteriaceae and Acinetobacter baumannii isolates, ECCMID,Vienna, (2017). [98] M.E. Falagas, T. Skalidis, K.Z. Vardakas, G.L. Voulgaris, G. Papanikolaou, N. Legakis, et al. Activity of TP-6076 against carbapenem-resistant Acinetobacter baumannii isolates collected from Greek hospitals, Int J Antimicrob Agents 52 (2) (2018) 269–271. [99] J. Newman, J. Zhou, C. Fyfe,W.Weiss, M. Pulse, et al. In vivo efficacy of TP-6076 in murine thigh and lung infection models challenged with Acinetobacter baumannii, ECCMID, Amsterdam, (2019). [100] J. Blais, S. Lopez, C. Li, A. Ruzin, S. Ranjitkar, C.R. Dean, et al. In vitro activity of LYS228, a novel monobactam antibiotic, against multidrug-resistant Enterobacteriaceae, Antimicrob Agents Chemother 62 (10) (2018) e00552-18. [101] A.K. Jones, S. Ranjitkar, S. Lopez, C. Li, J. Blais, F. Reck, et al. Impact of inducible blaDHA-1 on susceptibility of Klebsiella pneumoniae clinical isolates to LYS228 and identification of chromosomal mpl and ampD mutations mediating upregulation of plasmid-borne blaDHA-1 expression, Antimicrob Agents Chemother 62 (10) (2018) e01202-18. [102] W.J. Weiss, M.E. Pulse, P. Nguyen, E.J. Growcott, et al. In vivo efficacy of novel monobactam LYS228 in murine models of carbapenemase-producing Klebsiella pneumoniae infection, Antimicrob Agents Chemother 63 (4) (2019) e02214-18. [103] E.J. Growcott, T.A. Cariaga, L. Morris, X. Zang, S. Lopez, D.A. Ansaldi, et al. Pharmacokinetics and pharmacodynamics of the novel monobactam LYS228 in a neutropenic murine thigh model of infection, J Antimicrob Chemother 74 (1) (2018) 108–116. [104] S.J.R. Arends, P.R. Rhomberg, T. Lister, N. Cotroneo, A. Rubio, R.K. Flamm, et al. In vitro activity evaluation of a next-generation polymyxin, SPR206, against non-fermentative Gram-negative Bacilli responsible for human infections, ASM-ESCMID, Lisbon, (2018). [105] S.J.R. Arends, P.R. Rhomberg, T. Lister, N. Cotroneo, A. Rubio2, R.K. Flamm, et al. Activity of an investigational polymyxin-B-like compound (SPR206) against a set of Enterobacteriaceae organisms responsible for human infections, ASM-ESCMID, Lisbon, (2018). [106] L. Grosser, K. Heang, A. Rubio, et al. In vivo efficacy of SPR206 in an immunocompetent murine ascending urinary tract infection model caused by Escherichia coli, ASM-ESCMID, Lisbon, (2018). [107] L. Grosser, K. Heang, J. Teague, P. Warn, D. Corbett, M.J. Dawson, et al. In vivo efficacy of SPR206 in murine lung and thigh infection models caused by multidrug resistant pathogens Pseudomonas aeruginosa and Acinetobacter baumanii, ASM-ESCMID, Lisbon, (2018). [108] R.K. Shields, B.A. Potoski, G. Haidar, B. Hao,Y. Doi, L. Chen, et al. Clinical outcomes, drug toxicity, and emergence of ceftazidime-avibactam resistance among patients treated for carbapenem-resistant Enterobacteriaceae infections, Clin Infect Dis 63 (12) (2016) 1615–1618. [109] R.K. Shields, L. Chen, S. Cheng, K.D. Chavda, E.G. Press, A. Snyder, et al. Emergence of ceftazidimeavibactam resistance due to plasmid-borne blaKPC-3 mutations during treatment of carbapenemresistant Klebsiella pneumoniae infections, Antimicrob Agents Chemother 61 (3) (2017) e02097-16.

CHAPTER 4

Recent development of antibacterial agents to combat drug-resistant Grampositive bacteria Mohini Mohan Konai, Swagatam Barman, Yash Acharya, Kathakali De, Jayanta Haldar Antimicrobial Research Laboratory, New Chemistry Unit and School of Advanced Materials, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, Karnataka, India

1 Introduction Rapid emergence of drug-resistant Gram-positive bacteria has become an escalating problem in recent years [1,2]. Considering the severity of this clinical ultimatum, the World Health Organization has recently published a report, which has identified multidrug-resistant Gram-positive bacteria, not susceptible to conventional antibiotic therapy. This report listed methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate-resistant Staphylococcus aureus (VISA), vancomycin-resistant S. aureus (VRSA), and vancomycin-resistant Enterococcus faecium, among others, as high-priority pathogens [1]. This problem of antibiotic resistance has been prevalent from the middle of the 20th century. Ever since the era of antibiotics began, bacteria started developing resistance toward an antibiotic within a couple of months or years of their use in clinic. Once bacteria become resistant, they start transferring the mutated resistance conferring genes to other bacteria; thus a chain reaction is initiated that leads to worldwide spread of resistance [3]. The first report of resistance to penicillin was documented in 1940 (even before its approval for clinical use in 1942) but presently, almost all bacteria have developed resistance against this antibiotic [4]. Methicillin, an example of later generation of β-lactam antibiotics, was introduced in the clinics in 1959, but MRSA arose in 1960 [5]. Consequently, between 1970 and 1980, MRSA-associated diseases have resulted in uncountable numbers of deaths around the world. This particular bacterium is increasingly becoming untreatable by multiple classes of existing antibiotics (e.g., mupirocin, tetracycline, clindamycin, ciprofloxacin, erythromycin, and gentamicin). At present, the situation is even worse, as MRSA has already developed resistance against the last resort antibiotic, vancomycin. While VISA was reported in 1996, the development of VRSA was observed in 2001, whereas vancomycin-resistant E. faecium (VRE) was reported much earlier, in 1988 [6-8]. In order to combat these Gram-positive superbugs, linezolid was introduced in 2000. Unfortunately, bacteria started developing resistance against Drug Discovery Targeting Drug-Resistant Bacteria. http://dx.doi.org/10.1016/B978-0-12-818480-6.00004-7 Copyright © 2020 Elsevier Inc. All rights reserved.

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this antibiotic from next year onward (2001) [9]. Later, daptomycin was launched in 2003 but resistance development against daptomycin too was reported in 2005 [10]. At last, when the antibiotic pipeline against Gram-positive bacteria was almost about to dry out, three semisynthetic glycopeptide antibiotics, telavancin, dalbavancin, and oritavancin, were approved by the FDA. Telavancin was approved in 2009 for the treatment of acute bacterial skin and skin-structure infections (ABSSSI) and later on in 2013, the FDA gave approval for the treatment of hospital-acquired pneumonia, including ventilator-associated pneumonia caused by S. aureus and Streptococcus pneumoniae [11]. In 2014 another lipoglycopeptide antibiotic, dalbavancin, was approved for the treatment of ABSSSI caused by various Gram-positive bacteria (such as S. aureus and MRSA, vancomycin-sensitive E. faecium, Enterococcus faecalis, and Streptococcus pyogenes) [12]. In the same year, another semisynthetic glycopeptide antibiotic, oritavancin, was also approved for the treatment of complicated skin and skin-structure-associated infections caused by different Gram-positive bacteria that included staphylococci, enterococci, streptococci, and anaerobic bacteria such as Clostridium difficile and Clostridium perfringens [13]. Alongside, in 2018, oritavancin was also approved for application against Gram-positive bacteria. But, the use of these antibiotics has been majorly limited for treatment against drug-sensitive Gram-positive bacteria.They have not received approval yet for the treatment of infections caused by vancomycin-resistant pathogens. Nevertheless, infections caused by various Gram-positive superbugs (such as MRSA, VISA, VRSA, and VRE), that displayed resistance to multiple antibiotics are becoming increasingly difficult to treat. Hence, the current scenario demands for urgent development of new and more effective antibacterial agents for countering the overwhelming threat of infections caused by Gram-positive superbugs.

2  Antibacterial drugs under clinical development A lot of effort has been directed toward the development of new antibacterials against drug-resistant Gram-positive bacteria, and a few drug candidates have already entered into different stages of clinical trials (Table 4.1). A couple of them are currently in Phase III clinical trials. A widely used drug, fusidic acid, is undergoing Phase III clinical trials for its application in treating MRSA-associated acute skin and skin-structure infections [14]. Though fusidic acid is being highly prescribed and marketed extensively in different countries for the treatment of various skin and skin-related infections as a topical applicant, it is not approved by the FDA yet. It is known to act on bacteria by inhibiting bacterial protein synthesis [15]. A ketolide class drug, solithromycin, showing activity against S. pyogenes and S. pneumoniae, is currently undergoing Phase III clinical trials [16,17]. This drug is found to be very effective for the treatment of community-acquired bacterial pneumonia (CABP). It acts by binding to 23S ribosome and inhibiting protein synthesis in bacteria [18].

Table 4.1  Antibiotics approved by the FDA and in advanced phases of clinical trials against Gram-positive pathogens. Antibiotics

Class

Disease

Bacteria

Status

Company

Vancomycin (Vancocin) Linezolid (Zyvox) Daptomycin (Cubicin) Ceftaroline fosamil (Teflaro) Telavancin (Vibativ) Dalbavancin (Dalvance) Oritavancin (Orbactiv) Fusidic Acid (Taksta) Solithromycin (Solithera) Nafithromycin Contezolid acefosamil Afabicin Gepotidacin

Lipoglycopeptide Oxazolidinone Lipopeptide

ABSSSI

MRSA, Staphylococcus aureus, Staphylococcus epidermidis MRSA,VRSA,VRE

ViroPharma

Cephalosporin

ABSSSI, CAPB

Lipoglycopeptide Lipoglycopeptide Lipoglycopeptide Fusidane

ABSSSI

FDA approved in 2009 FDA approved in 2014 FDA approved in 2014 Phase III

Cumberland Pharmaceuticals

ABSSSI

MRSA, S. aureus, Streptococcus pyogenes, Enterococcus faecium MRSA, S. aureus,VRSA, Haemophilus influenzae, Streptococcus pneumoniae MRSA, S. pyogenes, S. pneumoniae MRSA, S. pyogenes, S. pneumoniae MRSA, S. pyogenes, S. pneumoniae MRSA

FDA approved in 1958 FDA approved in 2000 FDA approved in 2003 FDA approved in 2010

Ketolide

CAPB

S. pyogenes, S. pneumoniae

Phase III

Cempra Pharmaceuticals

Ketolide Oxazolidinone Benzofurans Triazaacenaphthylene Spiropyrimidinetrione

CAPB ABSSSI

S. pyogenes, S. pneumoniae S. aureus, MRSA, S. pyogenes, S. pneumoniae MRSA MRSA, S. pyogenes, S. pneumoniae, Neisseria gonorrhoeae MRSA, S. pyogenes, S. pneumoniae, N. gonorrhoeae

Phase II Phase II

Wockhardt MicuRx

Phase II Phases II and III Phase II

Debiopharm International GlaxoSmithKline

ABSSSI ABSSSI

ABSSSI GC, ABSSSI, CAPB GC

Pfizer Cubist Pharmaceuticals Forest Laboratories

Durata Therapeutics The Medicines Company Arrevus

Global Antibiotics Research and Development Partnership

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ABSSSI, acute bacterial skin and skin-structure infections; CABP, community-acquired bacterial pneumonia; GC, gonococcal infection; MRSA, methicillin-resistant Staphylococcus aureus; VRSA, vancomycin-resistant S. aureus.

Recent development of antibacterial agents to combat drug-resistant Gram-positive bacteria

Zoliflodacin

ABSSSI, CAPB ABSSSI

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Nafithromycin, another ketolide class of drug, is under Phase II clinical trials for the treatment of ABSSSI caused by Gram-positive bacteria S. pyogenes and S. pneumoniae [19]. This antibiotic displayed activity against Gram-negative bacteria as well, such as Legionella pneumophila and Haemophilus influenzae. Contezolid acefosamil, a drug belonging to the oxazolidinone class, is under Phase II trials for treatment of ABSSSI, caused by various Gram-positive bacteria, S. aureus, S. pyogenes, S. pneumoniae, including drugresistant bacteria, such as MRSA [20]. The mechanism of action of this antibiotic too involved the inhibition of protein synthesis [21]. In another example, afabicin is undergoing Phase II clinical trials for the treatment of ABSSSI caused by MRSA [22]. It acts by inhibiting bacterial fatty acid biosynthesis [23] that is extremely crucial for their growth. Another drug, gepotidacin, is currently in different phases of clinical trials (Phase II and Phase III) for treating the infections caused by various Gram-positive bacteria (MRSA, S. pneumoniae, and S. pyogenes) as well as Gram-negative bacteria (Neisseria gonorrhoeae) [24].The mechanism of action involved inhibition of bacterial Topoisomerase II [25]. A similar drug candidate, zoliflodacin, is in Phase II of clinical trials for the treatment of gonorrhoea and is also effective for the treatment of infections caused by MRSA, S. pneumoniae, and S. pyogenes [26]. It acts on bacteria by inhibiting DNA gyrase and topoisomerase IV [27]. Even though a few drug candidates are undergoing clinical trials, the extent of threats created by antibiotic resistance in Gram-positive bacteria demands for more number to be entering the antibiotic pipeline. In this direction, extensive research has been carried out by various research groups in the recent past. In an approach a series of new antibiotics have been discovered from natural sources, which showed great promise as future drugs. In another approach, efforts have been dedicated toward the development of semisynthetic analogs of existing classes of antibiotics. Moreover, a plethora of synthetic antibacterial agents have also been discovered in the recent past, which can be further developed as future antibacterial drugs. This chapter aims to provide an overview of all three approaches with an emphasis on recent developments in the field.

3  Nature-derived antibiotics Nature has always served as the major source of antibiotics as therapeutics of bacterial infections. Penicillin was the first class of naturally occurring antibiotics, discovered by Alexander Fleming in 1928. Penicillins belong to the large family of β-lactam antibiotics that also include cephalosporins and carbapenems. Followed by β-lactams, a plethora of new classes of antibiotics were discovered from nature and introduced in the clinic [2836]. Aminoglycosides were introduced in 1944 and in the decade that followed, novel branches of antibiotics entered in the clinical pipeline. Tetracyclines were introduced in 1950 and macrolides in 1952 followed by glycopeptides (vancomycin) in 1956. It was during the period of 1940-1970 that most of the new classes of antibiotics were invented and approved for clinical application. This period is known as the “golden

Recent development of antibacterial agents to combat drug-resistant Gram-positive bacteria

era” of antibiotic discovery. However, the rapid emergence of resistance development made all classes of antibiotics ineffective against bacterial infections. In order to address this clinical threat, a group of researchers have continued their efforts in search for new antibiotics from natural sources being inspired by the successful past of nature-derived antibiotics. In the following section, we focus our discussion on novel classes of antibiotics that have been discovered recently from natural sources and showed great potential for further development as future drugs.

3.1  Antibiotics discovered from the soil microbiome 3.1.1 Teixobactin Among the recent classes of antibiotics discovered from nature, teixobactin has drawn a lot of attention of the scientific community. Teixobactin is a nonribosomal depsipeptide synthesized by the Gram-negative bacteria, Eleftheria terrae, isolated from soil (Fig. 4.1A) [37]. This displayed excellent broad-spectrum activity against Gram-positive bacteria, including drug-resistant superbugs, MRSA, and VRE. Teixobactin was active against MRSA and VRE with the MIC values of 0.25 and 0.5 µg/mL, respectively, and was also effective against Mycobacterium tuberculosis, Bacillus anthracis, and C. difficile [37]. Although teixobactin was not able to kill Gram-negative bacteria, a significant activity was noted against outer membrane-defective Escherichia coli mutants. Therefore it indicates

Figure 4.1  Antibiotics discovered from the soil microbiome.

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that the outer membrane of Gram-negative bacteria poses as the barrier for teixobactin to enter into this class of bacteria, thereby making it ineffective against them. However, this newly developed antibiotic displayed great potential for further development as a drug for treating Gram-positive infections. It revealed excellent in vivo efficacy in three different murine models of infections, which is equivalent to or even better than the antibiotics, vancomycin and amoxicillin. For an example, in a mouse model of intraperitoneal MRSA infection, teixobactin had a PD50 (which is defined as the protective dose at which half the animals survive) value of 0.2 mg/kg, wherein vancomycin displayed a higher value of 2.75 mg/kg [37]. More importantly, S. aureus and M. tuberculosis failed to develop resistance to teixobactin even after multiple passages in the laboratory, which thereby indicates its great potential as an antibacterial agent with sufficient longevity. An investigation into the mechanism of actions suggested that a strong inhibition of peptidoglycan biosynthesis is the primary cause of antibacterial activity of teixobactin, which was confirmed by the studies where teixobactin was found to bind to the pyrophosphate motifs of various bacterial cell wall substrates (e.g., lipid II, a precursor of peptidoglycan, and lipid III, a precursor of cell wall teichoic acid). Further studies suggested that synergistic inhibition of peptidoglycan as well as teichoic acid synthesis lead to cell wall damage, delocalization of autolysins. These thereby resulted in lysis of bacterial cells [38].This multimode of action explains the lack of resistance development against teixobactin. 3.1.2 Malacidins Brady et al. [39] have reported the discovery of malacidins in 2018. Malacidins belong to the family of negatively charged lipopeptide antibiotics that act on bacteria through a calcium-dependent manner (Fig. 4.1B). Daptomycin, the well-known member of this family, is one of the last resort antibiotics for treating Gram-positive infections. Previously known all the antibiotics of this family, including daptomycin, consist of a conserved Asp-X-Asp-Gly motif in their chemical structure that is responsible for binding calcium and thereby essential for activity of this class of antibiotics. However, this newly discovered antibiotic, malacidin, is the first member, wherein Asp-X-Asp-Gly is replaced by (Asp-OH)-Asp-Gly as the calcium-binding motif [39].There are two different forms of malacidins that have been discovered from nature, malacidins A and B. The only difference between them is the presence of a single methylene group at N-terminal lipid chain (Fig. 4.1B). Malacidin A displayed excellent activity against different Grampositive bacteria, including multidrug-resistant strains; however, no activity was found against Gram-negative bacteria. It was effective to kill MRSA and VRE with the MIC values of 0.2-0.8 and 0.8-2.0 µg/mL, respectively [39]. In addition, malacidin A showed almost no toxicity toward mammalian cells and the propensity of resistance development against this antibiotic was not seen even after multiple passages of bacterial exposure in the laboratory [39]. More importantly, it revealed good in vivo efficiency in a rat model

Recent development of antibacterial agents to combat drug-resistant Gram-positive bacteria

of cutaneous wound-infection caused by MRSA, thereby indicating the possibility of the further development of this antibiotic as a drug. The existing calcium-dependent antibiotic, for example, daptomycin, acts on bacteria through binding with the cytoplasmic membrane phospholipids followed by oligomerizing in the membrane.This leads to destabilization of bacterial membrane integrity, followed by cell death. Few other members of this class of antibiotics (such as laspartomycin and friulimicins), however, known to bind with the lipid II precursor undecaprenyl phosphate (C55-P), thereby function as a cell wall biosynthesis inhibitor [39]. The mechanistic studies for malacidin too suggested that cell wall biosynthesis inhibition is the primary cause of antibacterial activity, which involves binding to lipid II through calcium-dependent manner instead of binding to C55-P. Further studies suggested no cross-resistance to the lipid II-targeting antibiotic, vancomycin, that therefore confirms a different binding site of malacidin. However, further studies need to be performed to investigate if malacidin shares similar binding mode with the other lipid II-targeting antibiotics, such as teixobactin. 3.1.3 Lysocins Lysocins are depsipeptides that share a common structural backbone consisting of 12 amino acid residues. This class of antibiotics was first identified from soil bacteria Lysobacter sp. for inhibiting growth of S. aureus. The most potent analog, lysocin E (Fig. 4.1C), exhibited good antibacterial activity (MIC = 4 µg/mL) against different Gram-positive bacteria, including drug-resistant superbugs such as MRSA, but remained ineffective against Gram-negative bacteria. More importantly, this showed excellent in vivo activity against S. aureus in mice model of intraperitoneal infection (ED50 = 0.5 mg/kg body weight, subcutaneous administration) and also showed low in vivo toxicity. A dose of 400 mg/kg of intraperitoneal administration of lysocin was found to be tolerated in mice. A mechanistic investigation suggested that lysocin E causes an immediate termination of all cellular biosynthesis pathways in bacteria, which is therefore indicative of membrane-disruptive mechanism of action that is responsible for its strong bactericidal activity. Moreover, S. aureus-resistant mutants to lysocin E acquired an altered gene of menaquinone biosynthesis and an external addition of menaquinone was found to rescue wild-type bacteria back. Further studies suggested that lysocin E exerted antibacterial activity upon binding menaquinone directly within the cytoplasmic membrane, which is the cause of membrane disruption [40]. In a recent study, Santiago et al. have reported that lipid II is a molecular binding partner of lysocins that is likely responsible for the bacteriolytic nature [41].This mode of action was established through a novel genome-wide mutant profiling approach, wherein peptidoglycan synthesis assays suggested the inhibition through an antibiotic/substrate binding stoichiometry of 2:1. Furthermore, the binding between lysocin E and lipid II was directly verified through an innovative affinity capture assay, wherein menaquinone remained ineffective toward this interaction. Therefore the

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menaquinone-dependent antibiotic susceptibility could be explained due to the fact that menaquinone-deficient S. aureus mutants consist of very low levels of lipid II. However, further studies need to be performed to understand if lysocin E binds to both menaquinone and lipid II simultaneously in a noncompetitive manner or not. This might provide more insights into the exact mode of action of this class of antibiotics.

3.2  Antibiotics discovered from human microbiome In order to search for new class of antibacterial drugs, human microbiome has also attracted immense attention of researchers as an alternative source. The human microbiome plays a significant role toward controlling the pathogenesis, in addition to fulfilling multiple beneficial purposes in humans [42]. As a result of permanent interspecies competition, they generated diverse classes of antibacterial compounds that have been poorly investigated to date. Hence, there is a high possibility that many novel classes of antibacterial drugs might originate from the human microbiota [43]. In the recent past a significant effort was therefore dedicated in this direction, which resulted in identification of several new classes of antibacterial compounds that displayed great potential for developing as future drugs. As the first example of this kind, Chu et al. [43] have identified a class of N-acetylated linear peptides, named humimycins (humimycins A and B), consisting of four l- and three d-amino acids in peptide sequence (Fig. 4.2A). Humimycins and their synthetic analogs were found to target the lipid II flippase MurJ of S. aureus, thereby being able to sensitize β-lactam antibiotics against resistant pathogens as well [43]. Similarly, Zipperer et al. [44] have identified lugdunin, a macrocyclic thiazolidine peptide antibiotic, that is produced by the human nasal bacterium, Staphylococcus lugdunensis (Fig. 4.2B). Lugdunin displayed antibacterial efficacy against different Grampositive bacteria, including drug-resistant superbugs MRSA and VRE with MIC values of 1.5 and 12 µg/mL, respectively. More importantly, it displayed appreciable efficacy in murine model of skin infection, and no propensity of resistance development was seen in S. aureus even after 30 days serial passaging. However, the details of the mechanism of action are yet to be explored to understand further antibacterial efficacy of lugdunin. In another example a novel thiopeptide antibiotic, lactocillin, was discovered by Fischbach et al. [45] (Fig. 4.2C). Lactocillin, produced by the vaginal microbiota (Lactobacillus spp.), is exhibited a broad-spectrum activity against different Gram-positive bacteria such as S. aureus and E. faecalis. Even though the preliminary studies indicate lactocillin as a promising antibacterial agent, however, further efficacy as well as detailed mode of antibacterial action needs to be investigated to understand the therapeutic potential.

4  Semisynthetic antibiotics The rapid emergence of bacterial resistance motivated the scientific community to search for a robust alternative to naturally derived conventional antibiotics. In addition to this, huge pressures of human pharmacokinetics, pharmacodynamics, drug metabolism, safety

Recent development of antibacterial agents to combat drug-resistant Gram-positive bacteria

Figure 4.2  Antibiotics discovered from human microbiome.

concern, efficacy, and of course synthetic complexity led the foundation stone for the chemists and pharmacologists to modify either the naturally derived obsolete antibiotics or the naturally abundant crucial intermediates with an aim of achieving more safe and efficacious antibiotics. Primarily, this approach has been considered as semisynthetic strategy where naturally occurring antibiotics are used as the starting point of chemical synthesis. This section is mainly focused on the recent development of various semisynthetic antibiotics (such as β-lactam, tetracycline macrolides, and glycopeptides) that either target mainly Gram-positive bacteria or display broad-spectrum activity.

4.1 Semisynthetic β-lactams β-Lactam antibiotics are one of the widely prescribed antibiotics in clinic for several years from 1941 when blockbuster drug penicillin was introduced in clinical settings. This class of antibiotics primarily targets penicillin binding proteins (PBPs) to prevent the formation of cell wall in both Gram-positive and negative bacteria [46,47]. After the

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discovery of penicillin, many other generations of semisynthetic β-lactam antibiotics have been developed by using 6-aminopenicillanic acid as a nature-derived intermediate within the last few decades [48]. First-generation penicillins (such as penicillins G and V) were used as a long-term treatment option for mild-to-moderate infections caused by susceptible Staphylococcus spp. [49,50]. However, occurrence of bacterial resistance has restricted the use of first generation forever. Bacteria mainly developed a high level of resistance by producing β-lactamase enzymes that basically destroy the β-lactam antibiotic through the opening of four-membered lactam ring [51]. Therefore in order to tackle bacterial resistance, further β-lactamase stable semisynthetic analogs of the same class (such as methicillin, oxacillin, cloxacillin, and nafcillin) were developed [52]. These new generations of penicillins were effective for treating infections caused by penicillin-resistant Staphylococci. Nevertheless, the emergence of MRSA limited the usages of the aforementioned β-lactam antibiotics [53,54]. Contemporarily, the discovery of penicillinase stable naturally occurring cephalosporin-C initiated a new direction toward developing several semisynthetic analogs of cephalosporin to combat penicillinase harboring Grampositive bacteria. Naturally derived 7-aminocephalosporanic acid (7-ACA) was used as the key intermediate for semisynthetic cephalosporin production (Fig. 4.3A) [55]. Firstgeneration cephalosporins (such as cephalothin) displayed appreciable activity against Gram-positive pathogens (Fig. 4.3B). They were pretty active against Gram-negative bacteria as well [56]. Further modification through insertion of different side chains at C7 and C3′ of 7-ACA produced second-generation cephalosporins (such as cefuroxime) that were significantly active against Gram-negative bacteria (Fig. 4.3C). However, they were less active against Gram-positive pathogens in comparison to the first-generation cephalosporins [57]. Subsequent structural modification of second-generation cephalosporins resulted in third-generation antibiotics (such as ceftazidime) that were also less efficient to tackle Gram-positive bacterial infections (Fig. 4.3D) [58,59]. Over the

Figure 4.3  Precursor and semisynthetic analogs of cephalosporin.

Recent development of antibacterial agents to combat drug-resistant Gram-positive bacteria

period the emergence of cephalosporinase (which destroys third-generation cephalosporins) initiated the development of fourth-generation analogs such as cefepime that displayed equally potent activity against both the Gram-positive and negative bacteria (Fig. 4.3E) [60]. In addition to penicillin and cephalosporin class, carbapenems (such as imipenem and meropenem) were also found to be effective to combat multidrugresistant Gram-positive bacteria. However, the more recent development of β-lactam antibiotics and β-lactamase inhibitors is being carried out mainly to tackle multidrugresistant Gram-negative pathogens.

4.2  Semisynthetic tetracyclines Tetracycline class of antibiotics has been used for more than five decades as a frontline weaponry to tackle infections caused by both the Gram-positive and Gram-negative bacteria. The first antibiotic of this class, chlorotetracycline, was isolated from the metabolic component of Streptomyces aureofaciens (Fig. 4.4A) [61,62]. Soon after the isolation of chlorotetracycline, catalytic hydrogenolysis of carbon-chlorine bond of this compound generated first semisynthetic analog of this antibiotic class, tetracycline itself (Fig. 4.4A) [63]. It inhibits protein synthesis upon binding with the bacterial ribosomal subunit [64].

Figure 4.4  Semisynthetic analogs of tetracycline.

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However, bacteria have already developed a high level of resistance against tetracycline, majorly by extruding the antibiotic through efflux machinery [65]. On the other hand, ribosomal protection proteins developed by the bacteria reduced tetracycline binding affinity with the ribosomal subunit by six times [66]. This rapid emergence of bacterial resistance motivated the scientific community for the development of semisynthetic derivatives of tetracycline. The semisynthesis was initiated with the removal of the C6 hydroxyl group of tetracycline and this modification resulted in sancycline that retained the broad-spectrum antibacterial activity with better pharmacokinetics and pharmacodynamics (Fig. 4.4B) [67]. Another semisynthetic approach involves nitration at C7 position of sancycline followed by reductive amination-generated minocycline (Fig. 4.4C) that displayed broader spectrum antibacterial activity (active against tetracycline-resistant Gram-positive Staphylococci) in comparison to tetracycline [68,69]. Unfortunately, along with the subsequent time span, minocycline has also become ineffective owing to the widespread bacterial-resistance development. In order to overcome such resistance, further semisynthetic modification of minocycline at C9 position through a subsequent nitration, reduction followed by acylation with glycine analog resulted in tigecycline (Fig. 4.4D) [70]. An extended spectrum activity against multidrug resistant bacteria (both Gram-positive and negative) and the severe side effects brought tigecycline in the FDA “black box” warning drug list [71,72]. Hence, this highly active antibiotic is only recommended in the case of severe life-threatening situation due to bacterial infections. In recent days, further modification of glycine moiety of tigecycline produced other two broad-spectrum antibiotics, omadacycline (Fig. 4.4E) and eravacycline (Fig. 4.4F) [73,74]. They were approved by the FDA in 2018 for the treatment of CABP, complicated skin, and intraabdominal infections caused by both the Gram-positive and negative bacteria.

4.3  Semisynthetic macrolides Macrolides are one of the most widely used antibiotics that primarily target the Grampositive bacteria and usages of these antibiotics are very common in the case of respiratory tract, throat, and skin infections [75,76]. Erythromycin is the first ever macrolide antibiotic and it was discovered in 1949 by a group of scientists at Eli Lilly from a culture broth of fungus Saccharopolyspora erythraea (Fig. 4.5A) [77]. Essentially, it is a Streptomyces metabolic product that consists of a 14-membered macrolactone ring [78]. In addition to the sole broad-spectrum Gram-positive activity, this class of antibiotics was found to be selectively active against few limited Gram-negative pathogens such as H. influenzae and Helicobacter pylori [79]. Macrolides basically inhibit the bacterial protein synthesis through binding with the 50S ribosomal subunit and this results in the early or premature release of nascent peptide from m-RNA-ribosome complex [80]. Unfortunately, overwhelming usages of macrolides promoted Gram-positive organisms to develop extensive resistance propensity primarily through drug efflux machinery [81]. Alongside, point

Recent development of antibacterial agents to combat drug-resistant Gram-positive bacteria

Figure 4.5  Erythromycin and its degraded product spiroketal.

mutations at the ribosomal binding site and the drug degradation by bacterial erythromycin esterase are also the major contributors for the resistance development [82,83]. Nevertheless, prior to the bacterial resistance development, clinical uses of erythromycin revealed its limited oral bioavailability and short in vivo half-life. Instability in acidic conditions (spiroketal formation at the C9 ketone from C6 and C12 hydroxyl groups in acidic condition) gave rise to the unpleasant side effects such as stomach pain (Fig. 4.5) [84,85]. Hence, these mentioned obstacles either poor PK/PD, instability, or bacterial resistance development were the key motivations for the scientists to modify the existing structure through semisynthetic strategy. In order to address the problem of acid instability, a group of scientists from Taisho Pharmaceuticals,Tokyo introduced clarithromycin (Fig. 4.6A) where C6 hydroxyl group of erythromycin was selectively capped with a methyl group. Acid stable and orally active clarithromycin had slightly expanded spectrum activity in comparison to the erythromycin [86,87]. Alongside, deletion of C9 ketone through subsequent semisynthetic steps by Gorjana Djokic′ et al. at Pliva in Croatia resulted in one more acid stable and orally active macrolide, azithromycin (Fig. 4.6B) [88]. After the approval of FDA in 1991, azithromycin was found to be the seventh most widely prescribed drug in 2010 in United States. According to a current report, about 76% MRSA isolates are found to be resistant against azithromycin [89]. Hence, the on growing resistance propensity created enough spaces for the scientist to move on from the first-generation macrolides (erythromycin, clarithromycin, and azithromycin) to the newer generation. Eventually, the cocrystal structure of erythromycin with bacterial ribosome suggested that the cladinose sugar attached to the adjacent oxygen of C3 did not play any role in controlling antibacterial activity of macrolides [90]. Removal of this sugar moiety with C3 ketone and incorporation of cyclic carbamate at C11 and C12, with the included nitrogen generated first ketolide antibiotics telithromycin that displayed activity against many macrolide-resistant Gram-positive organisms (Fig. 4.6C) [75,91]. This ketolide consists of a biaryl group tethered by a short alkyl chain responsible for additional interaction with bacterial ribosome through π-stacking. Hence, this additional interaction is solely responsible for its improved efficacy against a

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Figure 4.6  Semisynthetic analogs of macrolides.

broad spectrum of resistant Gram-positive pathogens [87]. It displayed antibacterial activity (MIC) against erm(B) and mef containing mutants of S. pneumoniae in the range of 0.008-1 g/mL, whereas first-generation macrolides were ineffective against these isolates [92]. Although telithromycin was approved by the FDA in 2004, unfortunately its usages have been restricted owing to the severe side effects (neuromuscular disease, visual disturbances, and hepatotoxicity) caused by the presence of pyridyl-imidazole functionality [93,94]. Furthermore, solithromycin has been introduced as another next-generation ketolide where aniline ring was placed for pyridyl ring of telithromycin along with the incorporation of fluorine moiety at C2 site (Fig. 4.6D) [95]. Solithromycin showed MIC against drug-resistant isolates of S. aureus, S. pneumoniae, and E. faecalis in the range of 256 µg/mL) [52]. Ebselen and its analog were found to be potently active against B. subtilis (0.12 µg/mL), Bacillus cereus (0.86 µg/mL), and Mtb (10 µg/mL) [53]. Additionally, ebselen has demonstrated antibiofilm activity and reduced the alreadyestablished staphylococcal biofilms [54]. Ebselen does not interact negatively with other FDA-approved tested drugs and exhibits synergy with daptomycin, retapamulin, fusidic acid, and mupirocin. Further, ebselen demonstrates synergy with silver and this combination causes rapid depletion of glutathione and inhibits thioredoxin system, resulting in inhibition of ribonucleotide reductase (RR) and DNA synthesis [55]. The in vivo therapeutic efficacy of ebselen was evaluated in a mice model of staphylococcal skin infections. Ebselen (1% and 2% in petroleum jelly) significantly reduced the mean bacterial counts compared with the control vehicle–treated group (P ≤ .01). The severity of S. aureus skin infection is mediated by host pro-inflammatory cytokines rather than by bacterial burden. Ebselen is already known for its immune-modulatory, antiinflammatory, and antioxidant activities, thus, can be a good option for treatment of skin infections. In the skin model of S. aureus infection, ebselen exhibited reduction in levels of the pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-6, IL-1 beta, and monocyte chemo attractant protein-1 in MRSA USA300 skin lesions [56]. In mice peritonitis model, a combination of ebselen and silver was tested for the in vivo efficacy against E. coli infection since combination demonstrated potent antibacterial activity in vitro. Briefly, mice were infected intraperitoneally with ∼107 cfu of MDR E. coli to mimicking acute peritonitis. After infection, the mice were treated with ebselen, silver, and ebselen + silver intraperitoneally. The combination of silver and ebselen led to a significant reduction (100-fold reduction) in bacterial load compared to the ebselen alone or untreated. Furthermore, 80% mice survived at end of the experiment in combination-treated group as compared to 30% in the control group [55]. Ebselen acts as a glutathione peroxidase mimetic and is thereby able to prevent cellular damage induced by reactive oxygen species. It interferes with proton-translocation function and ATPase activity in yeast [51]. In E. coli, it competitively inhibits thioredoxin reductase. Ebselen exhibited potent activity in those bacteria in which thioredoxin reductase and thioredoxin are essential for DNA synthesis and are lacking glutathione reductase, and glutaredoxin [57]. However, in Gram-positive bacteria, the exact mechanism of action of ebselen is still unclear. In addition, ebselen acts by inhibition of protein synthesis and also inhibits toxin production [54]. However, further work is needed to identify the cellular target of ebselen in S. aureus. In addition, ebselen exhibits a high barrier against resistance development.

Repurposing nonantibiotic drugs as antibacterials

Taken together, ebselen demonstrates broad-spectrum antibacterial activity against Gram-positive bacteria and Gram-negative bacteria either alone or in combination. Furthermore, it exhibited excellent in vivo efficacy as topical antimicrobial agent targeting Staphylococcal lesion and in murine peritonitis model of E. coli. All together, these findings suggest ebselen can be repurposed and developed as a potential treatment for MDR bacterial infections.

8 Niclosamide Niclosamide is a chlorinated salicylanilide possessing anthelmintic and potential antineoplastic activity. It is currently used against most tapeworm infections such as intestinal nematodes, filarial nematodes, flukes, and tapeworms. Niclosamide has been approved for nearly 50 years for the treatment of such infections in humans [58]. Niclosamide has been identified as a potential anticancer agent by various high-throughput screening campaigns [59]. The antibacterial activity of niclosamide has been investigated against ESKAPE pathogens. It demonstrated potent activity against the Gram-positive bacteria such as S. aureus (0.125 µg/mL) and E. faecium (0.25 µg/mL), while it failed to exhibit the antibacterial activity against Gram-negative pathogen (MIC 64 µg/mL) [4]. Surprisingly, it has exhibited potent antibacterial activity against Helicobacter pylori (0.25 µg/mL) with immunomodulatory effect by decreasing secretion of IL-8 in a gastric cancer cell line after H. pylori infection [60]. The antibacterial activity of niclosamide was not limited to only the Gram-positive members of the ESKAPE pathogens, it also exhibited potent activity against S. epidermidis and S. pyogenes with an MIC of 0.125 µg/mL. Further, it has demonstrated synergy with colistin against colistin-resistant strains of A. baumannii and K. pneumoniae. Niclosamide alone exhibited a range of MIC from 6.25 to 400 µM for colistin-susceptible and colistin-resistant A. baumannii strains and from 400 to >800 µM for K. pneumoniae strains. Niclosamide at 1–4 µM in a combination with colistin increased the activity of colistin significantly. In these bacteria, niclosamide increased the proportion of negative charges on their cell walls and thus was able to potentiate the activity of colistin against colistin-resistant A. baumannii and K. pneumoniae [61]. Intriguingly, niclosamide appeared to have no antibacterial activity against P. aeruginosa, but it has been observed to inhibit P. aeruginosa quorum sensing (QS) and thus inhibits biofilms [62]. In addition, with strong antibiofilm activity against Gram-positive bacteria, niclosamide has been evaluated as a versatile antimicrobial surface coating against device-associated, hospital-acquired bacterial infections [63]. Broad spectrum antibacterial activity in Gram-positive bacteria, those associated with hospital-acquired and device-associated infections, was assessed, and niclosamide-coated device was found to prevent and treat bacterial infections even at low concentration. Interestingly, no

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resistance was developed even after an exposure of H. pylori bacteria to niclosamide for 30 days. The in vivo efficacy of niclosamide was investigated in Galleria mellonella model of H. pylori infection. Briefly, randomly selected G. mellonella larvae weighing 300–350 mg were infected with 10 µL H. pylori cells (OD600 = 0.3) in the last left proleg using a 10 µL Hamilton syringe. After 2-h post infection, compounds were administered at into the last right proleg and the larvae were incubated at 37°C and monitored afterward. They were considered dead if they lacked response to external stimuli.The niclosamidetreated group significantly rescued larvae with a survival rate up to ∼70% compared to the no treatment group (P  256 µg/mL). However, loperamide demonstrated potent antibacterial activity against Gram-negative bacteria in combination with minocycline, including against the MDR strains. Loperamide demonstrated synergistic interaction with many antibiotic classes. It potentiates the activity of several classes of antibiotics such as tetracyclines and polymyxin B, but it demonstrates its antagonistic activity

Repurposing nonantibiotic drugs as antibacterials

toward aminoglycosides because it dissipates proton motive force and likely interferes with aminoglycoside uptake [68]. Loperamide + minocycline combination was also found active against Salmonella enterica serovar typhimurium. The combination was also evaluated for in vivo efficacy in a mice infection model. Mice were treated with loperamide (50 mg/kg), minocycline (100 mg/kg), and combination (loperamide 100 mg/ kg + minocycline 1 mg/kg). Loperamide or minocycline alone had no impact on the infection while its combination exhibited excellent efficacy and demonstrated 105–106 fold reduction in cecum. Loperamide exhibited synergy with the outer membrane permeating antibiotic polymyxin B that increased membrane permeability in E. coli and P. aeruginosa [68]. Loperamide restricts intracellular growth of Mtb in alveolar macrophages through induction of autophagy in vitro and in vivo mice experiments. The effect of loperamide therapy was evaluated in the autophagosome formation in MDMs (Murine derived macrophages) after determining the recruitment of LC3 to vesicles and exhibited significant enhancement in autophagosome formation in Mtb H37Rv–infected human and murine macrophages [69]. Taken together, loperamide demonstrated synergy with tetracycline and polymyxin B, which can be a good option for treatment of MDR Gram-negative infections. Loperamide also demonstrates potential as an adjunctive therapy for the treatment of TB by an induction of autophagy.

10 Antidepressants In the 1950s the first antidepressant used was an anti-TB drug isoniazid due to its euphoric effects in TB patients. Later, it was reported to be a monoamine-oxidase inhibitor (MAOI). Afterward in 1959, chlorpromazine, a psychotropic drug, demonstrated antimicrobial activity for the first time [70]. Since then, newer classes of antidepressants comprising tricyclic antidepressants (TCAs) and the serotonin-specific reuptake inhibitors (SSRIs) have demonstrated weak-to-strong antimicrobial effects alone or in combination [71–73]. Some of the examples with antibacterial activity include sertraline, fluoxetine, paroxetine, escitalopram (SSRIs), and MAOIs and TCAs such as tranylcypromine, phenelzine, amitriptyline, imipramine [74]. These antidepressants, SSRIs in particular, have intrinsic antimicrobial activity against Gram-positive bacteria, most probably acting as efflux pump inhibitors [70]. Numerous studies have been done to study their antibacterial activity against multiresistant Gram-positive and Gram-negative bacteria especially in combination with standard antibiotics where they have shown remarkable results [72,75]. The differences between the serum Cmax reached by these drugs, and their MICs make it difficult for them to attain the concentrations required to inhibit even the most susceptible bacteria and be used as single therapy clinically. However, in combination,

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activity is much more potent [70]. Also, these drugs have demonstrated resistance reversal in quinolone-resistant bacterial strains acting synergistically with ciprofloxacin, suggesting immense potential for further investigation into their efflux pump inhibition properties and as antibiotic adjuvants [75]. Thus combination therapy is most feasible for psychotropic drugs, and they can also aid in development of derivatives with better pharmacokinetic profiles and lesser neurological effects.

11 Sertraline Sertraline is a third-generation selective serotonin reuptake inhibitors (SSRI) type antidepressant. In humans, SSRIs act by inhibiting the pump that directs serotonin toward the presynapse neurone, thus increasing its quantity at the synapse space. The antibacterial activity of sertraline is thought to be due to probable efflux pump inhibition in bacteria. However, further studies are warranted in this area [70]. The in vitro activity of sertraline alone and in combination has been reported against various Gram-positive and Gram-negative bacterial and fungal species in numerous studies [75–79]. Its combination with tetracycline and ciprofloxacin against Corynebacterium urealyticum resulted in a 4–64-fold decrease in the MIC value of ciprofloxacin and 2–32-fold for tetracycline at up to 1/256 MIC and 1/32 MIC of sertraline, respectively. Also, ciprofloxacin synergy with sertraline against the quinolone-resistant strains (MIC > 1 mg/L) brought them within the sensitive (MIC 0.5–1.0 >1.0–≤4.0 >4.0 FICI, fractional inhibitory concentration index.

Table 11.6  Synergy between P128 and antibiotics.

1 2 3 4 5 6 7 8

P128 + antibiotics

FICI range

Vancomycin Daptomycin Oxacillin Linezolid Ciprofloxacin Cotrimethoxazole Azithromycin Tigecycline

0.1–0.5 0.3–0.5 0.15–0.27 0.15–0.5 0.28–0.3 0.4 0.37 0.15–0.3

FICI, fractional inhibitory concentration index.

respectively, and C A and C B are the concentrations of the drugs in combination, respectively. FICs are interpreted as shown in Table 11.5. P128 exhibited significant synergistic effects with a variety of antibacterials irrespective of the MOA of the antibiotic or the chemical class (Table 11.6). Time–kill curves show P128 and daptomycin (DAP) at sublethal concentrations caused almost complete killing of the culture and did not allow regrowth through the final 8 h time point, demonstrating the superiority of the combination (Fig. 11.15). Synergy was observed across a variety of clinical isolates from S. aureus and S. epidermidis, which allows exploring the use of P128 against clinical resistant strains. As mentioned earlier using similar assays, “synergy” has also been demonstrated for several lysins tested in combination with different classes of antimicrobials, especially antibiotics [43]. Synergy—potential for reversal of drug resistance For several clinical isolates of S. aureus or S. epidermidis, the individual MICs of oxacillin, cephazolin,VAN, DAP, linezolid, and ciprofloxacin were found to be several folds higher than the clinical sensitivity cutoff values, confirming their drug resistance phenotype. These strains were exposed to a combination of sub-MIC concentrations of P128 and the antibiotic to which the strain has been shown to be resistant (Fig. 11.16). Interestingly, at a concentration within its clinical sensitivity range, the respective antibiotic in the presence of P128 could inhibit the growth of bacteria, thus reversing the

Phage therapy—bacteriophage and phage-derived products as anti-infective drugs

Figure 11.15  Time–kill curve of P128 and daptomycin combination.

Figure 11.16  Resensitization of drug-resistant bacteria to antibiotics, in the presence of P128.

resistance phenotype [44]. The ability of P128 to resensitize resistant staphylococci to standard of care (SoC) antibiotics reinforces the ability of a combination with P128 to prevent emergence of drug resistance and supports the use of P128 in combination with first-line antibiotics under conditions where antibiotic sensitivity tests are not readily available.

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The entire set of Tier 2 assay results of P128 established the lysins a potent antistaphylococcal protein with novel properties: • potentiating in the presence of serum • synergy with SoC antibiotics • ability to reverse resistance. P128 was rapidly progressed through the next tier of tests to enable building and testing the lysin in the various animal models of interest. Tier 3 assays: activity in different biological niches in vitro models: Lysin molecules that have been found to have satisfactory properties in the Tier 1 and 2 assays were subjected to the Tier 3 assays, which probe specific properties of the molecule that are relevant in vivo. It is well established that a staphylococcal infection can result in the formation of biofilms that represent different environmental adaptations of the microbe [45]. Thus it would enhance the therapeutic efficiency of P128 if the antistaphylococcal lysin molecules would have activity on staph biofilms. Several relevant growth models of S. aureus and S. epidermidis, which represent these physiological niches, have been reported [45] and were included as assays for testing in this tier. Testing of anti-biofilm activity of lysins: A biofilm consists of bacterial communities encased within a self-produced protective matrix of sugars and proteins. Compared to free-floating (planktonic) forms of bacteria, biofilm-associated bacteria are more difficult to eradicate using antibiotics due to the presence of an extracellular matrix that acts as a protective matrix and an adaptive response that leads to a change in the metabolic activity of the microbe. It is becoming increasingly evident that many infectious pathogens are capable of forming this structure in vivo and hence becoming resilient to the action of antibiotics. P128 is a potential drug for the treatment of serious S. aureus infections, such as chronic wounds, bacteremia, infective endocarditis (IE), and device-associated infections. All these indications involve biofilm formation as a part of the pathogenesis. P128 was tested for its anti-biofilm efficacy in eradication of preformed biofilms and in the prevention of biofilms. Microtiter plate-biofilm assay format: The anti-biofilm activity of P128 and other lysins can be assessed by the ability to eliminate preformed biofilms in microtiter plate wells. The presence and density of the biofilm is indicated by the intensity of the crystal violet staining of biofilms growing in the microplate wells. As seen in Fig. 11.17, a visual inspection of crystal violet-stained wells showed that P128 added to 24-h-old biofilm at 1× MIC could destroy the biofilms of S. aureus and S. epidermidis within 2 h. In contrast, none of the SoC antibiotics, VAN, DAP, or linezolid, showed any significant change in the biomass removal even at >100×, and even after an exposure of up to 24 h [32,35]. In vitro catheter surface-biofilm model: P128 was also tested for its ability to eliminate preformed biofilms from the luminal surface of catheters. In vivo conditions of biofilm formation in device-associated infections of S. aureus biofilms can be simulated using

Phage therapy—bacteriophage and phage-derived products as anti-infective drugs

Figure 11.17  Effect of P128 on 24-h preformed Staph biofilms.

catheters. These biofilms can be detected using safranin stain and by scanning electron microscopy (SEM). In this experimental setup, biofilms of MRSA strain MW2 were formed on the surface of catheters. Treatment of the biofilms with P128 at 1× MIC (8 µg/mL) led to their eradication, as no biofilm mass was visible upon safranin staining (Fig. 11.18A). Similar observations were made by visualization of P128-treated biofilms. The stained catheters were washed in PBS and allowed to dry. After drying, samples were fixed on aluminum stubs with a double-sided carbon-adhesive tape, coated with 5- to 7-nm-thick gold by use of a sputter-coating system (Q150T; Quorum Technologies)

Figure 11.18  Comparison of activity of P128 and vancomycin on biofilm Staphylococcus aureus MW2 on the surfaces of catheters (A) and SEM images of biofilm on catheter surface treated with P128 or vancomycin (B). SEM, scanning electron microscopy.

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and were examined by SEM (Neon 40; Carl Zeiss) for the presence of biofilm structures (Fig. 11.18B). It was observed that P128 at 8 µg/mL (1× MIC) eradicated biofilms, whereas VAN had a minimal effect on the biofilms even at 90× MIC (90 µg/mL) [35]. Testing of lysin-antibiotic synergy on biofilms: Planktonic cells of S. aureus and S. epidermidis showed significant synergy when treated with combinations of P128 with several individual antibiotics. Similar experiments were designed and carried out with combinations of antibiotics with P128 against S. aureus biofilms formed on the insides surface of the catheter. The results showed significant synergy in the ability of these combinations to eradicate preformed biofilms as compared to the individual components. The significant lowering of the minimum biofilm inhibitory concentration (MBICs) (>100fold) of antibiotics of various classes, such as linezolid and DAP, resulted in low FICI values for combinations of P128 with antibiotics. The experimental design in Fig. 11.19 is as follows: biofilms of S. aureus on the catheter were formed with a VAN-resistant S. aureus and the presence of the biofilm on the catheter was confirmed by safranin staining (Fig. 11.19A). As seen in Fig. 11.19B, an exposure of the biofilm on the catheter to VAN at 10 µg/mL or P128 at 1 µg/mL (sub-MIC concentration) had no effect on the biofilm; however, a combination of these at the same sub-MIC concentrations resulted in complete eradication of the biofilm. The catheters were processed for SEM and examined for the presence of biofilm structures. The ability of P128 to potentiate the effects of antibiotics on biofilms could play a role in eliminating infections more rapidly than either P128 alone or the antibiotic

Figure 11.19  Activity of P128 on vancomycin-resistant Staphylococcus aureus biofilm formed on catheter surface (A) and SEM image showing eradication of biofilm by P128 and vancomycin combination at sub-MIC concentrations (B). MIC, minimum inhibitory concentration; SEM, scanning electron microscopy.

Phage therapy—bacteriophage and phage-derived products as anti-infective drugs

alone. Interestingly, the lysine CF301 has also been shown to have potent anti-biofilm activity [33]. Test of lysins for the prevention of multispecies biofilm: Biofilm communities in most environments, including those present in human infections, tend to be polymicrobial. S. aureus and Pseudomonas aeruginosa are known to cause biofilm-related infections and are frequently found together in chronically infected wounds, cystic fibrosis, otitis media, and on in-dwelling medical devices such as prostheses, stents, implants, catheters, and endotracheal tubes. Studies using a wound infection model suggested that polymicrobial infections of Staphylococcus and Pseudomonas were more virulent than single-species infections [46]. Findings also support the precolonization hypothesis whereby persons precolonized with S. aureus are predisposed to secondary colonization with P. aeruginosa. A recent study also demonstrated S. aureus to be the pioneer colonizer in biofilm growth and that it augments the attachment of P. aeruginosa [47].This raises the question whether a lysin such as P128, which is specific to and highly effective against staph, would be able to break this virulent association by selectively killing staph and thus prevent formation of multispecies biofilm. In an in vitro model, S. aureus, P. aeruginosa, and Enterococcus faecalis were cultured either singly or in mixed cultures, allowing biofilm formation on a solid support. This model mimics the wound environment for growth of biofilms under in vitro conditions [48]. The ability of P128 to prevent biofilm formation was tested in this model. Briefly, P. aeruginosa, E. faecalis, and S. aureus cultures were individually diluted to 1 × 106 CFU/ mL, mixed in equal volumes and added to LCWPB (Lubbock chronic wound pathogenic biofilm, supplemented with 50% bovine plasma and 5% hemolyzed horse blood) medium containing a sterile pipette tip. In this model, the pipette tip acts as a surface for biofilm formation. P128 was added to the mixed culture at 10, 50, or 250 µg/mL and incubated at 37°C, 150 rpm for 24 h. Upon completion of incubation, the tips were observed for biofilm formation. In the absence of P128, a confluent and thick mass of biofilm could be seen. For enumeration of bacteria, the biofilms formed on the tips were washed, transferred to clean test tubes, and macerated with sterile scissors. Pipette tips, on which biofilm formation was not visible, were also processed the same way. The contents were vortexed thoroughly, diluted, and plated on trypticase soy agar plates for CFU enumeration [32]. The combination of S. aureus, P. aeruginosa, and E. faecalis cultures led to the formation of a thick and leathery biofilm carrying approximately equal numbers (107–108 CFU/ mL) of all the organisms. P128, added at a concentration of as low as 10 µg/mL, was found to prevent the formation of biofilm in this model. The lack of biofilm formation was reflected in very low bacterial counts of P. aeruginosa, E. faecalis, and S. aureus, obtained after processing the pipette tips used for the growth of biofilms (Table 11.7). The results demonstrated that inhibition of S. aureus growth was sufficient to prevent biofilm formation by P. aeruginosa and E. faecalis, suggesting possible use of P128

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Table 11.7  Effect of P128 on formation of multispecies biofilms. P128 concentration (µg/mL)

0 (cell control)

10

50

250

Isolate

No. of CFU/mL

Pseudomonas aeruginosa Staphylococcus aureus Enterococcus faecalis P. aeruginosa S. aureus E. faecalis P. aeruginosa S. aureus E. faecalis P. aeruginosa S. aureus E. faecalis

7 × 108 1 × 108 3 × 107 2.2 × 105 2 × 104 9 × 105 7 × 104 2 × 103 1.8 × 104 1.7 × 105 70% Protection of animals from fatality at ≤1/4× ED

Remarks

Mouse peritoneal infection These data aid the design of clinical studies and positioning of the molecule Mouse intravenous infection

≥1 Log reduced CFU in blood compared to input bacterial dose, up to 8 h after dosing No detectable ADA 2 In rats, which are a weeks after a single standard toxicology dose. No or 10× Safety margins and extrapolation from efficacy and PK/PD studies In vivo, intravenous Indicative maximum administration in tolerated dose arrived at rat and minipig In vivo, rat and Systemic toxicity Daily dosing for 14 minipig evaluated, and target days organs identified, if any. NOAEL established In vivo, minipigs PK profile over 24 h, PK parameters and immune response and ADA titers—data available In vivo, intravenous No anaphylaxis or signs of administration in hypersensitivity minipig No hypersensitivity reaction to readministration after a washout period of 15 days Higher animals for NOAEL to be established These are moleculetoxicity, rat for specific CNS toxicity; dog requirements, often for telemetry, etc. defined during preIND discussions, as needed by the regulatory agency

ADAs, antidrug antibodies; ED, effective dose; MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration; MHB, Muller Hinton broth; PK, pharmacokinetics; PD, pharmacodynamics; GLP, good laboratory practices, CNS, central nervous system; IND, investigational new drug, NOAEL, no observed adverse effects level; PAE, pharmacologically active exposure; RBC, red blood cell; SoC, standard of care.

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the bacterial surface, followed by destruction of the cellular barriers. Lysins are selective to bacteria, and concomitantly, can be expected to pose a lower risk of interaction with eukaryotic cells. However, this must be rigorously tested and established through in vitro and in vivo evaluation. 1. Potential for drug-induced hemolysis Lysis of red blood cells (RBCs) is considered to be a serious toxicity liability. The test of potential to cause hemolysis becomes the first step in determining the safety of any drugs or materials that are administered into blood. Hemolysis occurs if the RBC membrane ruptures and hemoglobin is released, which may lead to adverse health effects ranging from anemia, hypertension to renal toxicity [49]. This test involves contacting RBCs harvested from whole blood, with the lysin, at various concentrations. It assesses the propensity of lysins to bind to RBC and cause damage or lysis of the delicate cellular membrane. Hemoglobin release in the plasma is the indicator of RBC lysis. While lysis, or the absence of it, can easily be distinguished even visibly, the percentage of lysis can be measured more accurately by reading the absorbance of 100% lysed RBCs and comparing with the test supernatants, at 540 nm. Fig. 11.21 shows that lysin P128 had no adverse effect on RBCs. 2. Potential for cytotoxic effects The potential for toxicity of a lysin can be probed effectively using mammalian cells in culture, as the first step. This can predict potential cellular toxicities if associated with the lysin, prior to testing it in animals. The cell-viability assay in a standard microplate format is ideal to evaluate the protein that is added to the culture media. Cytotoxicity would be evident through cell detachment and cell death. Cells grown in monolayers in culture plates can be visualized under a microscope for signs of cytotoxicity and changes in the physiology and structure of the cells, such as appearance of

Figure 11.21  Absence of cytotoxicity in RBCs exposed to P128. RBC, red blood cell.

Phage therapy—bacteriophage and phage-derived products as anti-infective drugs

vacuoles, blebbing of membranes, etc. In order to evaluate cytotoxicity of P128, one normal mammalian cell line (Vero, monkey kidney) and one cancer cell line (Hep G2, human liver cancer) were used [50]. Cells were exposed to varying concentrations of P128 ranging from 0.04 to 2.5 mg/mL in the culture media for 24 h. Exposure to P128 at the highest tested dose of 2.5 mg/mL did not result in any reduction in viability in either of the cell lines, and there were no signs of cytotoxicity [50]. 3. Safety to animals For assessing in vivo safety, groups of 5–7 mice were administered single-bolus intravenous (IV) doses of the lysin, in an ascending-dosing design. P128 administered in the range of 0.5–90 mg/kg in mice was well tolerated, no adverse effects were observed at the highest concentration tested, and animals survived until the end of the study (14 days). Level 2: qualifying efficacy in vivo Efficacy in experimental animal models of MRSA infection: It is appropriate to test lead lysins in multiple models of systemic infection, beginning with the simplest model of peritoneal infection, with rescue from mortality as the end point. Subsequently, the lysin is tested in more complex models that clinically simulate the human infection, for example, models to evaluate the efficacy under conditions of bacteremia, concomitant with infection of tissues and organs. Mouse peritoneal infection model: The mouse peritonitis model is considered to be highly reproducible and well suited for evaluation of antibacterial agents for efficacy in bacteremia [51,52]. Dose–response studies of P128 and synergy with SoC antibiotics were evaluated in this model.Variants of the standard model were used to assess the efficacy of P128 under conditions simulating more severe infection: enhancement of virulence of S. aureus with the use of mucin in the infection inoculum, which coats the bacterial surface, rendering the bacteria less susceptible to phagocytosis [53], and S. aureus infection in neutropenic mice, in which the immune response to the infection is impaired [54]. In normal mice, infection with MRSA COL strain resulted in 80%–100% mortality within 72 h when administered intraperitoneally as an inoculum of 109 CFU/animal. MRSA USA300 in normal mice with the use of 5% unresolved in >80% mortality at 109 CFU. Neutropenic mice were more susceptible and >80% of the animals succumbed to an infective inoculum of 108 CFU/mouse [55]. Normal mice infected with MRSA COL were treated with a single intraperitoneal dose of 0.2, 1.0, or 5.0 mg/kg P128, 3 h after intraperitoneal bacterial challenge; and survival of animals was monitored for 72 h. Survival data from 2 independent experiments, using a total of 20 animals per group, were analyzed to assess efficacy and significance. While 100% of untreated animals succumbed to the infection within 24 h, P128-treated animals showed an improved survival of 20% at 0.2 mg/kg, 40% at 1 mg/kg, and 70% at 5 mg/kg after 3 days and animals continued to survive until end of an observation of 7 days (Fig. 11.22).This study served for preliminary evaluation of the therapeutic range of

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Figure 11.22  Dose–response of P128 in MRSA (strain COL) infection in normal mice. MRSA, methicillinresistant Staphylococcus aureus.

P128 and indicated the minimum effective dose (MED) and also enabled an estimation of its ED50 (“median effective dose” or “effective dose for 50% of the population”). The ED50 of P128 was estimated to be 3 mg/kg [56]. Synergy with SoC drugs: Based on the synergy observed in vitro [32], the effect of combining P128 with SoC antibiotics,VAN, and DAP was evaluated in the mouse peritonitis model in normal mice.VAN and DAP were tested independently at the mouseequivalent of the human therapeutic dose (110 mg/kg for VAN and 50 mg/kg for DAP in mice) and at 50% (55 mg/kg for VAN and 25 mg/kg for DAP) and 25% (27.5 mg/kg for VAN and 12.5 mg/kg for DAP) of the therapeutic dose. Importantly, for combination therapy, subtherapeutic doses of the drugs were used, rather than the therapeutic equivalent dosage. In combination with 0.2 mg/kg of P128, 55 mg/kg (1/2× therapeutic dose) of VAN and 12.5 mg/kg (1/4× therapeutic dose) of DAP were used. Survival of animals was monitored over 72 h to assess efficacy and synergy. Combined results from three experiments showed that untreated control animals infected with MRSA COL succumbed to the infection with a mortality of 88%. In all treatment groups, mortality was delayed. At the subtherapeutic dose, P128 alone reduced mortality to 50%–70%, which in itself could be considered significant, while the subtherapeutic doses of VAN or DAP given alone, reduced mortality to 50%–55%. P128 in combination with VAN or DAP further reduced mortality to 15%–20% (80%–85% survival; Fig. 11.23), indicative of a significant shift in the ED50 of the combination, compared to both the drugs when administered singly. Superior combination-efficacy of P128 with antibiotics has clinical implications for the treatment of S. aureus bacteremia. The rapid bactericidal effect of P128 is likely to cause a rapid reduction of bacterial load, this would facilitate the action of the SoC because of the reduced bacterial load, and this would result in improved clinical profile and faster resolution of the infection. An early clinical response could

Phage therapy—bacteriophage and phage-derived products as anti-infective drugs

Figure 11.23  Effect of combination of subtherapeutic dose levels of P128 and SoC antibiotics in MRSA COL infection and bacteremia in mice. Vancomycin (A) and daptomycin (B). MRSA, methicillin-resistant Staphylococcus aureus; SoC, standard of care.

in turn reduce the duration of antibiotic therapy and hospitalization. Such synergy in animal infection models has also reported for other lysins, including CF301 and SAL200 [33,42,57]. P128 is efficacious both as a monotherapy and in combinations with several antibiotics in different animal models of bacteremia. Level 3: PK/PD profiling To plan human studies and to minimize risk, it is necessary to make predictions about the fate of the drug candidate in the human body after administration. Relevant pharmacological information on the “ADME” (administration, distribution, metabolism, and excretion) characteristics must be obtained in animal studies before administration to humans. Pharmacokinetic parameters—including bioavailability after oral or other systemic administration, plasma half-life, and volume of distribution, clearance, and exposure—are determined in two or three animal species. Using established interspecies conversation factors, these data are than used to predict the human parameters. The science of PK and PD of “biotherapeutics/proteins” has been advancing, especially for human-derived recombinant therapeutic proteins and MAbs, since the launch of rhInsulin in 1982.The interpretation of PD of lysins would however vary in principle from human proteins, as lysins are not expected to have any pharmacological action on the human organism itself; instead, they act on bacteria that have taken residence within the human host. The translation of PK data from animals to humans and extrapolation from in vitro assays to in vivo readouts is critical for the successful development of safer and more effective drugs however, while the PK parameters of a lysin molecule can be compared to known biotherapeutics of a similar size, the PD knowledge base of this class of molecules is nonexistent. Thus it is imperative that detailed PD studies are carried out to understand the PD drivers. The route of administration impacts PK and generally, therapeutic proteins are administered by parenteral routes, such as IV, subcutaneous (SC), or intramuscular injection. While gastrointestinal (GI) absorption is precluded owing to a molecular size, and

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mainly gastric degradation, pulmonary delivery in the form of aerosol formulations or inhalable dry powders may be needed in the case of pulmonary infection. Administration by this route has been effected for select proteins [58,59]. Although SC administration is a preferred route for proteins, the clinical indications that are targeted by P128 involve an acute medical illness; hence, the route of administration of the molecule is envisaged as an IV infusion route of delivery. The PK of P128 was characterized in mouse and rat, and the PK/PD correlation was derived in the mouse thigh infection (MTI) model [60]. Following single IV bolus dose administrations of P128 at 10, 30, or 60 mg/kg, the concentration of P128 available in plasma was determined over 24 h. Blood samples were collected from groups of five mice at multiple time intervals starting with 1-min post-administration. A sensitive sandwich ELISA method was used to determine P128 concentrations in blood for evaluating bioavailability. The lower limit of quantification of the assay was 1 ng/mL. PK parameters were estimated using noncompartmental analysis in Phoenix WinNonlin software. Determination of the PK profile allowed determination of its residence time at >MIC levels, in plasma. Owing to the size and charge properties of P128, its route of elimination could be hypothesized to renal clearance. Binding to plasma components such as albumin was evaluated, as these can modulate the PK characteristics; however, P128 showed poor or no binding to mouse albumin and other components. Although P128 was largely confined to the plasma compartment, the ability of P128 to reduce the bacterial load at the infection site, the thigh, indicated that a theta fraction of P128 was distributed and reached tissue spaces. This correlated well in subsequent detailed efficacy studies in the mouse thigh tissue infection. The PK profile showed a multiexponential decline with a low systemic clearance and a low volume of distribution at steady state (Fig. 11.24A). The t > MIC was about 45 min for 10 mg/kg, about 2 h for 30 mg/kg and in the case of 60 mg/kg, the concentration remained above MIC for over 8 h (Fig. 11.24B) [60]. The characteristics of the PK profile indicated in the following: • P128 (Cmax) occurred within 5 min (tmax) of the start of the 5 min IV injection (mean 0.15 h). Concentration fell to less than 10% of maximum within 30 min. • An early rapid decline phase of the concentration–time curve could be delineated, conforming to a two-compartment model. • The volume of distribution of P128 was relatively low, consistent with its being confined largely to systemic circulation. • Clearance was rapid, and in conjunction with a small volume of distribution, resulted in a longer elimination phase. The pharmacokinetic parameters are summarized in Table 11.9. The pharmacodynamics correlation was performed using the standard neutropenic MTI model. P128 showed dose-dependent antibacterial activity following single IV

Phage therapy—bacteriophage and phage-derived products as anti-infective drugs

Figure 11.24 Pharmacokinetics (PK)  profile of intravenous P128 in mice over 24 h (A); and expanded profile over first 8 h (B).

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Table 11.9  PK parameters estimated for P128 following single IV doses of 10, 30, and 60 mg/kg in naïve BALB/c mice. PK parameter

10 mg/kg

30 mg/kg

60 mg/kg

t1/2 (h) Cmax (µg/mL) AUC0–∞ (h µg/mL) AUC (%Extrap) CL (mL/h/kg) Vss (mL/kg) AUC/Dose Cmax/Dose

0.83 167 11.6 0.082 859 157 1.16 16.7

5.5 459 47.6 0.074 629 146 1.59 15.3

5.2 2212 225 0.071 266 59.7 3.75 36.9

IV, intravenous, PK, pharmacokinetics, AUC, area under the curve, CL, clearance.

doses. Maximum bactericidal effect was seen within 30 min of administration. Fig. 11.25 shows time versus plasma concentrations of P128 (mean ± s.d.) following a single IV injection of 60 mg/kg, interposed with CFU load in thigh tissue, in mice treated with P128, or left untreated. Briefly, BALB/c mice were rendered neutropenic with cyclophosphamide and MRSA MW2 cells (105 CFU/mouse) were injected into the thigh muscle. Bacterial load in thigh tissue was determined by sacrificing subsets of mice at designated time points over 24 h and harvesting the thigh tissue, and plating the pro-

Figure 11.25  Bacterial load in the thigh tissue of infected neutropenic mice administered 60 mg/kg dose of P128.

Phage therapy—bacteriophage and phage-derived products as anti-infective drugs

cessed samples for enumeration of CFU. The time course of bacterial CFU in thigh tissue, posttreatment with P128, can be seen. A significant (P  100 mg. At this stage the process for the expression and purification needs to be scaled up. The process used is finely optimized so that a similar/identical protocol can be used when the protein is required in much larger quantities as well as during GMP production. Lastly, it is the requirement of the protein for GLP Toxicology and Clinical studies. As the protein is required in quantities exceeding 1 g, the expression and purification is scaled up to a fermenter-based production. At this stage the manufacturing process requires documentation as per GMP standards and the process is scaled and optimized to facilitate manufacturing of the product even for marketing purposes too . Table 11.10  Manufacturing process—progression chart. Progression chart: Protein manufacturing—quantity and quality requirements Stage 1—in vitro evaluation studies in discovery

Stage 2—in vitro and in vivo characterization studies

Stage 3— preclinical toxicology studies

Stage 4— clinical studies

Typical protein quantity requirements Seed culture requirement

50 g

>500 g

Glycerol Research cell stock of bank transformants

Research cell bank

Fermentation

Shake flask expression

Batch mode fermentation

High cell density fermentation

Manufacturing facility

Lab scale

Lab scale

Lab scale

Cell bank manufactured in a cGMP certified facility High cell density fermentation GMP certified facility

Phage therapy—bacteriophage and phage-derived products as anti-infective drugs

Table 11.10  Manufacturing process—progression chart.(Cont.) Progression chart: Protein manufacturing—quantity and quality requirements Stage 1—in vitro evaluation studies in discovery

Typical yields after purification Formulation In-process controlsa Analytical methods and batch release parametersb Storage buffers/ Container closure systemc Stability indicating testsd

Stage 2—in vitro and in vivo characterization studies

Stage 3— preclinical toxicology studies

Stage 4— clinical studies

∼50–100 mg/L ∼100– 250 mg/L Compatible with assays Initiate development Initiate development

>1 g/L

>1 g/L

Initiate development

Development to be complete

Initiate development

Development to be complete

Compatible with end use Development to be complete Development to be complete

cGMP, Current Good Manufacturing Practice a In-process controls: Development of in-process control checks is initiated during the manufacturing progression stages 1 and 2, by identifying critical parameters in the manufacturing process, which when not met, can lead to failures or inconsistent product.The in-process control tests need to be identified, monitored and controlled during the manufacturing process. The in-process control tests need to be in place with established acceptance limits before the protein is manufactured for preclinical toxicology studies. These tests are identified in both the fermentation and the purification process to monitor and ensure the consistency of process intermediates, so as to provide a consistent final product. b Analytical methods and batch release parameters: Analytical methods and batch release parameters are developed to assess the quality and quantity of the protein being manufactured. These analytical methods are usually orthogonal methods and their development is initiated in parallel to the manufacturing process optimization and scale-up. The choice of the analytical methods and stringency depends on the intended end use/route of administration of the therapeutic protein. Typically, the analytical methods developed include tests to provide assurance on quality, potency, strength, purity, identity, and safety of the protein drug substance and drug product manufactured. These analytical tests are comprehensive and cover the quality attributes of the protein-like appearance, pH, protein concentration, purity profile, identity, biological activity, sterility as well as process- and product-related impurities. As a regulatory requirement, the analytical methods are usually qualified or validated for specificity, linearity, accuracy, precision, quantification, and detection limit as applicable to ensure that they are adequate to detect significant deviations from the specifications. c Storage buffers/container closure system: The choice of the final storage buffer for the protein and the container closure system for final packing/storage of the protein drug substance or the formulated protein drug product are important aspects of the final manufacturing process as they need to be compatible with the final dosing forms, whether it is for toxicological studies, clinical studies, or finally in the market. A container closure system refers to all the packaging components that together contain and protect the dosage form. Typical components are containers (e.g., ampoules, vials, bottles) closures (e.g., screw caps, stoppers).The dosage forms need to be evaluated for compatibility with the container closure system. Preliminary studies are initiated during the early stages of in vitro/in vivo evaluation of the protein to screen buffers and test protein compatibility with a different container closure system, to zero in on the optimal storage buffer and compatible container closure system. The container closure system and storage buffers finalized can thus be implemented during the manufacture of protein for the preclinical toxicology studies. d Stability indicating tests: During the development of analytical methods to assess the quality attributes of the protein for batch release, a suitable subset of these tests that can indicate protein degradation by assessing the protein profile, physical attributes, protein quality, and protein quantity are selected using real time and accelerated temperature stress studies with the protein. These selected analytical tests are then included as a part of formal stability studies. The stabilityindicating analytical tests with defined acceptance limits are finalized during the early stages of protein evaluation, so that they can be implemented to ensure the stability of the protein when used in the preclinical toxicology studies.The information gathered from these stability studies helps in defining the shelf life of the protein drug substance and drug product manufactured for clinical studies.

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Manufacture of P128—The manufacturing process, the in-process controls, and the product testing methods were developed and defined to ensure that the drug characteristics are maintained consistently across batches, thereby making the drug safe and effective in humans.These manufacturing activities are collectively defined in regulatory terminology as “chemistry, manufacturing, and controls” (CMC). Every drug being developed needs to have a robust CMC to achieve regulatory approvals. The CMC for P128 have been developed to ensure that the drug presents no unusual potential risk when administered to humans. 3.3.1  The chemistry, manufacturing, and controls for lysin P128 3.3.1.1 Overview P128, acting functionally as an endoprotease, has a theoretical molecular weight of 26,488.9 Da (27 kDa). It is produced as a recombinant protein in E. coli expression system, under the control of the T7 promoter.The production process involves seed inoculum preparation where the expression host from a characterized cell bank vial manufactured in a current good manufacturing practice ( cGMP) certified facility is inoculated into culture media and grown to be used as an input to the upstream process (fermentation). The fermentation process is followed by a downstream process for purification of the expressed protein from the other contaminating host cell proteins, host cell DNA, endotoxins, and media components. 3.3.1.2  Manufacturing process development The optimization of the manufacturing process for P128 occurred over more than 70 in-house developmental batches. These batches were used to produce the unformulated protein referred to as the protein drug substance (DS). A number of process changes were adopted at different levels in the development of the entire manufacturing process. Briefly, these included changes made in the following: 1. Cell substrate: Change of the antibiotic selection marker; introduction of two-tiered research cell bank consisting of a first tier master cell bank (MCB) and a second tier working cell bank system (WCB) progressing on to a two-tiered GMP cell bank. 2. Fermentation process: Changes in culture media, fermenter mode of operation, and scale of operation. 3. Purification process: Changes in the chromatographic resin, the purification process, and scale of operation. 4. Analytical methods and specifications: Changes as needed for improvement in analytical procedures and DS specifications in tune with the process changes and regulatory requirement. The impact of changes made in the cell substrate, the fermentation process, and the purification process on (1) intermediates of the fermentation process, (2) intermediates of the purification process, and on the (3) final DS were individually evaluated with reference to the prechange status through a set of “DS comparability records” to ensure

Phage therapy—bacteriophage and phage-derived products as anti-infective drugs

that the process change resulted in an improved process or product and did not have any adverse impact on the quality of the DS. 1. Cell substrate: P128 DS manufacture does not use any cell substrate of animal origin.The MCB was prepared in a cGMP certified facility from the characterized research cell bank. Each of the raw materials used for master cell bank manufacture is individually tested and released before use. The media used for culturing the cell bank is prepared from genetically modifed organisms (GMO) free vegetable proteins replacing animal-based peptones and is free of bovine spongiform encephalopathy/transmissible spongiform encephalopathy risks. The cell substrate used to prepare the inoculum for the DS manufacture is tested and characterized, to confirm the absence of microbial contamination and to ensure identity, purity, viability of the cell substrate, and quality of the protein to be produced from the cell substrate, before use in the batch manufacture as per ICH guidelines. In addition, the cell banks are subjected to stability testing at periodic intervals and at a designated frequency to ensure its stability. 2. Fermentation process: The changes in the fermentation process were aimed at increasing protein yield. These include (1) changes that were made to optimize culture media and duration of growth, in order to increase culture optical density (OD600) or biomass, (2) changes in the concentration of the inducer and the duration of induction to optimize P128 expression, and (3) changes to the scale and mode of operation of fermentation. The upstream protein expression protocol was initiated in batch mode, shake flask operation, at 50 mL culture volume, and then successfully scaled up to a high cell density fed batch mode operation in a fermenter, at 2, 10 L at lab scale, and further progressed to a pilot scale of 30 L in a cGMP certified facility. The yields also increased from 0.16 g/L in shake flasks to >8 g/L in a fermenter. These increases in yields were needed to comply with the requirements of P128 in the early preclinical studies as well as to meet the requirement of the P128 protein for late stage preclinical and clinical studies. Various “in-process” tests were established with acceptance limits and performed to monitor and ensure the process control of the fermentation process.The “in-process” tests and their limits were established based on data gathered and trends observed in the developmental batches. Some of the in-process controls of fermentation include microscopy of the culture, optical density, pH, temperature, and agitator RPM, total gas flow through the sparger, protein expression profile, and in vitro biological activity of the expressed protein. 3.3.1.3  Purification process The changes in the purification process were primarily implemented to meet the challenges of handling higher biomass and thereby increased load of contaminants and impurities generated due to the higher cell density, generated as a result of the optimized fermentation process.

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These included (1) scale of operation, (2) use of a micro-fluidizer in place of a sonicator to handle higher biomass, (3) introducing additional purification steps to remove the increased load of nucleic acid and endotoxins due to higher biomass, (4) changes in chromatographic resins and purification system to handle increased levels of contaminating proteins due to higher biomass, (5) optimizing the final storage buffer for the purified P128 DS, (6) improvements in methods of dialysis/concentration to handle higher operating volumes, and (7) the use of depyrogenated glassware and water for injection throughout the process to prevent introduction of additional endotoxin. The purification process was successfully scaled up from laboratory scale to pilot scale of 30 L in a cGMP certified facility, without compromising on the quality of the protein manufactured. As the protein was intended for parenteral administration in humans, the purification process was developed with stringency compatible with allowed limits of contaminating host protein, DNA, and endotoxin. Various “in-process” tests were established with acceptance limits and performed to monitor and ensure the process control of the purification process.The “in-process” tests and their limits were established based on data gathered and trends observed in the developmental batches. Some of the in-process controls of the purification process include resin volume, bed height, linear flow rate, protein quantity limits at different stages of the process, and in vitro biological activity of the purified protein. 3.3.1.4  Analytical methods and specifications Identification of analytical methods appropriate for testing of the DS and development of these methods was carried out alongside the development of the manufacturing process. The majority of the analytical tests and their attributes (appearance, pH, protein content, identity, purity, protein profile, biological activity, and determination of sterility as per United States Pharmacopeia) were standardized during the early development batches. Additional analytical tests (product-related impurities: tests for integrity/purity and process-related impurities: endotoxin content, host protein content, and host DNA content) were developed during the later batches. As the DS was intended for parenteral administration in humans, the analytical methods and specifications for the impurities were defined for the P128 final product. These analytical tests and specification limits were used as go/no criteria for batch release to ensure consistency of batches. A subset of these analytical tests and limits was also used to confirm the “stability” of the drug substance. Purified P128 was now ready for development studies, which progresses from a detailed animal safety testing followed by a stepwise advance through the different phases of trials starting with healthy volunteers and finally into trials treating infected patients.

3.4  Stage 4: clinical development 3.4.1  Preclinical studies: pharmacology/toxicology Preclinical toxicology studies in animals constitute a key milestone to be crossed in the development of a new drug candidate [69–71]. In contrast with the high attrition

Phage therapy—bacteriophage and phage-derived products as anti-infective drugs

rate observed with small molecule drugs during safety testing in animals, biological drugs are at an advantage. Biologics, including lysins, are less likely to show unpredictable off-target activities compared to small molecules. This is especially true of lysins that are specific to bacteria and are not expected to act on mammalian cells. Also, proteins tend to have more predictable ADME pathways and therefore the failure rate of biologics in animal toxicity studies tends to be concomitantly lower. An important factor that has to be considered and investigated thoroughly as part of toxicology studies for proteins is the potential for immunogenicity and consequent hypersensitivity or immunotoxicity [72,73]. This assumes even more significance for proteins of heterologous origin, such as lysins, which are derived from phages or bacteria [74]. The scope of the toxicology program to support initial human trials are typically based on the Phase 1 clinical plan, which in turn is a reflection of the subsequent development plans and the positioning of the molecule in terms of the end use in therapy. The target indication, route of administration, and the duration of treatment influence the design of the studies, choice of test animal species, age, etc. The preclinical safety program for P128 included support for the proposed clinical regimen that was planned; a combination study with either of the current SoC drugs, VAN, or DAP. The toxicology plan had also to include repeat dosing of P128 as this was also a reflection of the clinical plans. This translated to the need of dosing animals for up to 14 days at a dose frequency simulating or exceeding that proposed for human trials. Studies have to be conducted following both the ICH M3 (R2) for general principles, and ICH S6 + addendum R1 for protein-based therapeutics [75–77]. P128 was evaluated for systemic exposure in rat and minipig, in advance of human trials. Maximum feasible dose considerations, based on formulation characteristics, solubility, optimum repeat daily dose volume, and dose frequency limitations in the toxicology plan, equate to roughly 1000 µg/kg/day intravenously in rats, and 30,000 µg/kg/day by IV infusion over 1 h, in minipigs. The minipig in place of dog is considered a suitable model for safety testing owing to numerous anatomical, physiological, and genetic and biochemical similarities to humans. In addition, the minipig model is acceptable to regulatory authorities and presents with a practical and flexible modality [78]. Mini pigs meeting the quality required for regulatory studies are available; all procedures necessary in toxicity studies are feasible to implement; and information on normal basis values of pharmacology, hematology, and clinical chemistry are available. The minipig has several advantages over nonhuman primates and dogs. Dogs present with gastro-intestinal lesions or cardiovascular toxicity in reaction to some drugs especially for anti-infectives at high doses, leading to exaggerated pharmacodynamic effects. Such reactions are not as pronounced in minipigs, making them a better predictor of the human situation [79].

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Since P128 is a protein of heterologous origin, it could elicit an immune response, which could include production of neutralizing antibodies, allergenic hypersensitivity reactions, and immunotoxicity. The risk of drug-neutralization was evaluated in vitro using hyperimmune serum raised against purified P128. The potential for systemic hypersensitivity was characterized in non-GLP studies in rats. In the formal (GLP) preclinical studies of P128 in rats and minipigs, ADA assessments were included. In addition, assessment of the pharmaco/toxicokinetics of the protein was included to determine comparative exposure by IV infusion and to extrapolate safety margins, for going on to human studies. In GLP studies in rats, P128 was proven safe at the highest tested dose level of 1.0 mg/kg, given once daily for 14 days. In pilot non-GLP studies in minipigs, the highest tested dose of 30 mg/kg, administered as an IV infusion over 1 h, daily for 7 days, was found to be well tolerated. Formal GLP studies in minipigs are in progress. In the case of both lysins CF301 and SAL200, preclinical safety studies were conducted in rats and dogs. The NOAEL (no observed adverse effects level) of CF301 was 2.5 mg/kg in dogs [80]. Consequently, following the FDA guidance [81] for the calculation of human equivalent dose (HED), the maximum single dose tested in humans was 0.4 mg/kg in the case of CF301 [82]. SAL200 was tested in rats and dogs [83], and subsequently in primate (rhesus monkey), where the study design focused also on evaluating daily dosing of SAL200 for a short duration, of less than a week, along with PK characterization. Up to 80 mg/kg administered as a single dose, and up to 20 mg/kg, administered twice a day at 12-h intervals for 5 days were found to be well tolerated in monkeys [84]. Residence time, as defined by the time of last quantifiable concentration, was found to be 1 h. Just as in the case of P128, Cmax was achieved within minutes. Also, Cmax and systemic exposure to SAL200 increased in a dose-dependent manner, demonstrating linear kinetics [84]. 3.4.2  Clinical development The transition of a lysin into clinical development as a “candidate drug” depends on the successful completion of preclinical studies that establish its efficacy, and the pharmacological properties that establish its safety at the drug exposure levels required to effect a cure for the indication in humans. While the target product profile (TPP) is defined earlier during the preclinical development stages, the target clinical indication is generally a well defined “unmet medical need,” which is typically the starting point for clinical development. The lysin family of proteins P128, CF301, and SAL200 exemplifies a paradigm shift in terms of the kind of molecules that can be used for treating infection. Unique properties such as rapidity of kill, specificity for Staphylococcus spp. as well as robust in vitro and in vivo synergy with conventional antibiotics, warranted a careful reconsideration of the clinical positioning of lysins to maximize their potential. This section describes the specific strategy adopted for the clinical development P128.

Phage therapy—bacteriophage and phage-derived products as anti-infective drugs

3.4.2.1  Defining target medical condition Any proposed medical intervention can be viewed from different angles—from a patient’s perspective in terms of both the clinical need and cost of the treatment; from a clinician’s perspective, the ease of use and effectiveness; from a regulatory angle, the need, comparison with available treatments, quality and control of use; and from the innovator’s view, the clinical impact and economic value. Lysins being a new class, the regulatory aspects are the paramount factor that would be influencing the progress of such a therapeutic into the clinic. S. aureus and CoNS such as S. epidermidis are major human pathogens that cause a wide range of clinical infections. S. aureus, which is both a commensal bacterium and an opportunistic human pathogen, causes up to 25% of hospital-acquired infections, resulting in longer stays in the hospital, higher costs, and increased mortality. It is the leading cause of life-threatening clinical conditions such as bacteremia and IE, osteoarticular infections, skin and soft tissue infections, pleuropulmonary, and device-related infections. In terms of the epidemiology of S. aureus, there has been a clear shift since the past decades: the organisms are seen to associate with a growing number of health-care-associated infections, such as IE and prosthetic device infections; and community-associated skin and soft tissue infections [85]. MRSA poses a significant threat to health care as it is resistant to most of the antibiotics. Also, CoNS such as S. epidermidis lead to significant debilitating clinical infections, such as prosthetic device and implants infections in the elderly as well as in the immune-compromised patient populations [86,87]. S. aureus bloodstream infections are prevalent both in the hospital and in the community settings, resulting in an estimated 150,000–200,000 hospitalizations each year in the United States alone, where the mortality rates are over 20% [88]. Among invasive infections, the annual incidence rate of S. aureus bacteremia varies from 3.6 to 6.0 per 100,000 person–years [89]. Further complicating the treatment, drug-resistant strains of S. aureus such as MRSA have now been reported to be resistant to second-line SoC antibiotics such as VAN and DAP [90]. The potential unmet medical indications involving drug-resistant Staphylococci, which lysin P128 can target, are listed in Table 11.11, along with a glimpse of the type of clinical trials they would entail. 3.4.2.2  Target product profile TPP takes into account all the characteristics of the product under development and enables strategic placement of the drug for specific clinical utility or market. The TPP for P128 is described in Table 11.12. 3.4.2.3  Target product profile for the systemic use of P128 Positioning of P128 lysin: The clinical positioning of lysine P128 is dictated both by the efficacy of the molecule in appropriate infection models and the regulatory acceptance of a first in class molecule that has no precedence for clinical use for treating bacterial

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Table 11.11  Targeting infections involving drug-resistant Staphylococcus aureus and other staphylococci with lysin P128. Sl. no

Clinical indication

Complexity of clinical trial

End points in the clinical trial

1

SAB including infective endocarditis

Complex trial design. Recruitment may be difficult

2

Ventilator associated, community acquired and nosocomial pneumonia due to S. aureus

Recruitment is more feasible compared to endocarditis

3

Musculoskeletal infections such as septic arthritis, osteomyelitis

Complexity of the trial high. Recruitment will be a challenge

4

Prosthetic joints and implants infections

Complexity of the trial high. Recruitment will be a challenge

5

Skin and soft tissue infections such as cellulitis, impetigo, carbuncle, and folliculitis

Relatively less complex

6

Infections due to immunocompromised conditions: such as peritonitis, febrile neutropenia during chemotherapy

Complexity is high. Clinical condition mainly depends on the underlying immunocompromised condition

Clinical response and clinical cure rate compared to standard antibiotics Microbiological cure also needed but is not sufficient as the sole parameter for registration Clinical cure rate compared to standard antibiotics. Microbiological cure alone would not be valid as an end point Clinical response and cure compared to standard antibiotics. Microbiological cure not valid as end point Clinical response and clinical cure compared to standard antibiotics. Microbiological cure is also accepted as end point Clinical cure and clinical response compared to standard antibiotics. Wound healing in applicable condition. Microbiological cure rate is not accepted as end point Clinical cure and clinical response rate compared to standard antibiotics

SAB, Staphylococcus aureus bacteremia.

Phage therapy—bacteriophage and phage-derived products as anti-infective drugs

Table 11.12  TPP of P128.

Title

Disease indication

Patient segment

Pathogen spectrum requirements Value-adding parameters Differentiation from general antibiotics Product description Administration

Mechanism of action

Indication of use

P128 (StaphTAME) for the treatment of Staphylococcus aureus and Staphylococcus epidermidis bacteremia and infective endocarditis in combination with SoC P128 for treatment of S. aureus and S. epidermidis bacteremia and infective endocarditis in combination with SoC irrespective of resistance status of strains (i.e., including resistant strains like MRSA) Hospital in-patients with bacteremia and/or infective endocarditis having one or more blood cultures that were positive for S. aureus or S. epidermidis regardless of its antibiotic resistance status (i.e., including resistant strains) S. aureus, including MRSA Effective on S. epidermidis and other coagulase negative Staphylococcus Very specific effect, no effect on any normal flora P128 is a purified chimeric protein consisting of 239 amino acids with a molecular mass of 26,488.9 Da, designated “StaphTAME” In a PoC, intravenous infusion over 1 h, of P128 in solution formulation, either using central line or peripheral line, daily, for a period of 3–7 days would be tested Rapid bactericidal effect by cleaving staphylococcal cell wall, leading to killing of S. aureus or S. epidermidis and significant reduction of Staph cells in the blood; and in the case of the presence of biofilms, sufficient disruption of integrity of biofilm and rapid killing of biofilm-embedded cells For the treatment of bacteremia and endocarditis in patients having one or more blood cultures positive for S. aureus or S. epidermidis, regardless of antibiotic resistance status, and administered in combination with antibiotics (SoC), in patients aged 18 and above

MRSA, methicillin-resistant Staphylococcus aureus; PoC, proof of concept study; SoC, standard of care; TPP, target product profile.

infections. Positioning will also depend upon clinical developability, market need, and the profile of the patients on whom the molecule has the best chance of activity.The preclinical experimental data on the PK and the PD parameters derived from the various animal models indicated that P128 administered by IV route was able to eradicate the microbe from different tissues in the mouse and rat infection models. As the PK was characterized in mouse, the data from the mouse model of bacteremia were used to estimate the efficacious dose and the pharmacologically active exposure (PAE). By allometric scaling and extrapolation, the PAE in humans was calculated to be in the range of 1.0–1.5 mg/kg, when

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administered as monotherapy. The preliminary predicted HED, based on the minimum efficacious dose (MED) in mouse, was in the region of 0.4 mg/kg. From a regulatory aspect, the positioning P128 as a stand-alone drug for the treatment of “skin and soft tissue infection” as a topical application was a viable option, especially considering the promising results from using P128 formulated as a hydrogel in canine pyoderma. The infective organisms belonged to different drug-resistant Staphylococcal species, including MRSA, S. epidermidis, Staphylococcus pseudintermedius, Staphylococcus equorum, S. haemolyticus, and others. In these studies, clinical efficacy of P128 was examined by application of the P128 hydrogel to affected areas of the skin twice daily for 8 days. In all the P128 gel-treated dogs, improvement was evident by reduced redness and discharge and the lesions dried up subsequently, as a sign of complete healing. Also, no allergic reactions were observed in any of the dogs after 8 days of application of the gel [91].These results indicated that P128 would be efficacious in the case of similar skin infections in humans. Use of P128 topically for more serious wounds however may still present the challenge of being able to penetrate in sufficient quantities into the deeper parts of the infected tissue owing to limitations imposed by the size of the molecule. This consideration led to positioning P128 for systemic application with bacteremia as the indication of choice. The clinical positioning of P128 as adjunct therapy along with SoC antibiotics rather than as a monotherapy was decided based on the following aspect: • From allometric scaling, the extrapolated predicted dose of P128 as monotherapy in man for the treatment of bacteremia was 0.4 mg/kg, a pretty high dose for a protein therapeutic. • On the other hand, the ability of P128 to synergize with the SoC was a unique property whereby the dose of P128 required to achieve efficacy in different animal models of staph infection was significantly reduced, affording an estimated HED of 0.02 mg/kg, and faster cure rates could also be achieved. Taking advantage of this effective synergy and building it into the bacteremia indication, P128 was positioned as an adjunct therapy along with an SoC such as VAN for the treatment of staph bacteremia with endocarditis—this indication having a significant unmet medical need. Design of clinical trials of lysins: As P128 is in early development, details of clinical trials and the drivers of the design are discussed for the lysins CF301 and SAL200, in this section. One of the options for developing an antibacterial is in demonstrating the efficacy of the new drug in a noninferiority trial design where it has to be proved that the new investigational therapy is not less efficacious to the current standard therapy. Noninferiority trials are ideal when the new therapy may have certain advantages over current treatment such as better safety profile in one set of patients, or convenience of dosing or even cost benefit. With few exceptions, most antibacterial drugs that are currently used are approved based on the noninferiority trials [92].

Phage therapy—bacteriophage and phage-derived products as anti-infective drugs

The second alternative is in demonstrating superiority of an investigational drug over current active antibacterial therapies, which is often challenging, time-consuming, and clearly of higher risk. Superiority trials seem to be the most obvious choice for progressing a new antibacterial by demonstrating efficacy of the new drug against infections that are resistant to the SoC. However, in such a case, the control group would have to be patients treated with SoC despite knowing that the infective strain is resistant to the drug—this is an unacceptable ethical issue, thereby requiring a more complex design. In the case of adjunct therapy, where the concept is to enhance the cure rate of SoC by using it in a combination with a second therapeutic molecule, it becomes mandatory to have superiority-based clinical trial design in order to demonstrate that the performance of combination of the SoC and lysin is superior to SoC alone. CF301 and SAL200 are being positioned as adjunct therapies and a superiority-based clinical design has been used to establish efficacy [93,94]. CF301 (Exebacase) developed by ContraFect Corporation is a phage-derived lysin that also acts against S. aureus.The Phase 1 safety trial on healthy volunteers was completed by the end of 2015, where it was shown that CF301 was generally well tolerated at 0.04–0.4 mg/ kg and no clinical adverse safety signals were observed [95]. The Phase 2 trial evaluating CF301 in patients with S. aureus bacteremia was a randomized, double blind, multicentric, comparative study for the treatment of adult patients with single dose of CF301 (0.25 mg/ kg, given as a single 2 h IV-infusion) in conjunction with SoC antibacterial therapy for the treatment of S. aureus bloodstream infections (bacteremia), including IE. The results [96] of this trial showed that the clinical responder rates on day 14 in the subset of patients with bacteremia, including right-sided endocarditis treated with Exebacase was 80.0% compared to 59.5% in the patients treated with antibiotics alone, a 20.5% improvement (P = .028). The lysin SAL200 (Tonabacase) is also an endolysin and is being progressed in South Korea. The Phase 1 safety trial on healthy volunteers conducted for SAL200 showed that the IV infusion of single ascending doses of SAL200 (0.1, 0.3, 1, 3, and 10 mg/kg of body weight) were well tolerated, and no serious adverse events were observed in this clinical study [97]. The Phase 2 study is also being designed as a trial to demonstrate superiority, where a single IV dose of Tonabacase (SAL200) (3 mg/kg) in addition to the conventional standard treatment is administered, for persistent S. aureus bacteremia. Patients having staph bacteremia even after 48 h of treatment with an antibiotic to which the infecting S. aureus is susceptible have been enrolled in this study.The trial is currently in progress [94]. Approach of regulatory bodies to lysins: The damage being inflicted by AMR has been addressed by regulatory agencies, within their own limits and capabilities. The US FDA has developed four distinct modes for the process of accelerating approval of new drugs, which can be applied to anti-infectives in general and to lysins also: 1. Priority review 2. Breakthrough therapy 3. Accelerated approval 4. Fast track

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FDA granted Fast Track Designation to ContraFect’s CF301, for the treatment of S. aureus infections, including MRSA. Established under the FDA Modernization Act of 1997, the Fast Track Drug Development Program is intended to facilitate the development as well as expedite the review of drugs to treat serious conditions and potentially fill an unmet medical need. The Fast Track Designation provides earlier access to and more frequent communication with the FDA regarding all aspects of a designated drug’s clinical development program. Additionally, the designated drug may be eligible for the submission of the New Drug Application (NDA) on a rolling basis as well as Accelerated Approval and Priority Review if supported by clinical data at the time of the NDA submission.

4 Conclusion Table 11.13 lists the key properties of the three lysins that are at various stages of drug development. These molecules represent a paradigm shift in the approach to treat “infection”—there are many open questions on how well these molecules will behave as “drugs”; however, the overwhelming threat of AMR warrants novel solutions to be explored. Table 11.13  Profile of three clinical-stage anti-Staph lysins. Sl. no

1 2 3

4

Company Therapeutic indication Protein

5

Target coverage Synergy

6

Positioning

7

Clinical status

P128 (StaphTAME)

CF301 (Exebacase)

SAL200 (Tonabacase)

GangaGen 1. Nasal carriage 2. Bacteremia Chimeric protein-binding domain—lysostaphin Catalytic domain—phage K ectolysin S.aureus and CONS

ContraFect Bacteremia

Intron Bacteremia

Endolysin of Streptococcus suis prophage

Endolysin of Staph phage SAP-1

Staph and CONS

Staphylococcus aureus

Significant, in vitro and in Significant, in vitro Significant, in vitro vivo and in vivo and in vivo Adjunct therapy with SoC— Adjunct therapy Adjunct therapy with multiple doses of P128 with daptomySoC—single dose cin—single dose of SAL200 of CF301 Phase 2a completed for nasal Phase 2 completed Phase 1 completed carriage; for systemic infection, GLP tox Pilot tox in minipigs—no vasculitis seen

SoC, standard of care; CONS, Coagulase-negative Staphylococcus; GLP, Good Laboratory Practice.

Phage therapy—bacteriophage and phage-derived products as anti-infective drugs

The majority of anti-infective agents currently in the market as well as in the pipeline are small molecules. The present AMR crisis needs to be tackled by encompassing novel therapeutic modes that have not been already primed to develop resistance. One such approach is the field of therapeutics drawn from other biological resources, including large molecule biological agents best represented today by the bacteriophages or phage-derived protein entities like the lysins. This chapter has discussed a possible pathway to bring lysins through the drug discovery path, highlighting the properties and challenges that need to be addressed along the path.

5 Summary Bacteriophage-encoded proteins, like the lysins for example, combine several novel properties of a phage-based therapeutic as well as overcome some of the disadvantageous that a bacteriophage therapy encounters. Given the dire need for novel therapies for the treatment of serious infection, lysins offer a novel mode of action with no preexisting or primed resistance. P128, a phage-derived lysin that has been detailed in this chapter, is a chimeric protein that is active on both MRSA and methicillin-sensitive staph aureus strains. P128 and the lysins, in general, are rapidly bactericidal to the pathogen of interest and have a “narrow spectrum” of activity. Both these properties are well suited for an anti-infective, the rapidity of the action helps in reducing the bacterial burden swiftly and the narrow spectrum leaves the normal microbiome undisturbed. Another key feature of the three lysins that have characterized has been the synergistic effect the lysins show when combined with antibiotics, irrespective of the chemical class or the MOA of the antibiotic. This synergy could also be demonstrated in a unique strategy where combinations with P128 resulted in reversing the antibiotic resistance of several resistant strains of S. aureus, this property was also independent of the chemical class or the mode of action of the antibiotics. These properties provided a niche clinical positioning possibility for P128. P128 was positioned as an adjunct therapy to be used in combination with SoC; animal model studies showed that significantly reduced doses of P128 were required when in combination with antibiotics to demonstrate efficacy superior to that seen with either of entities singly. In fact, one of the lysins CF301 has been studied as a combination in a human Phase 2b clinical trials where it has been shown to be effective. One of the challenges in developing P128 or other lysins is their potential “immunogenicity.” While P128 is currently undergoing toxicity studies, no hypersensitivity issues were encountered with CF301 or SAL200 in human studies. Thus even though immunogenicity is always a consideration with foreign proteins, the benefits of lysin as therapeutic agents seem to outweigh the potential issues of immunogenicity. The field of “nontraditional antibiotics” is a very much understudied and underutilized area—several novel approaches, including vaccines and peptides along with lysins,

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offer unique strategies to challenge the “super bug.” Success with any one of these will surely go a long way in helping this area to become a mainstream treatment possibility.

Acknowledgments For us authors, writing this chapter and describing the development of GangaGen’s P128 has been a rewarding experience and an opportunity to recall and recount the journey of progressing the molecule. This research would not have been possible without the hard work and dedication of many, many people who have worked at various levels in the project over the years.We acknowledge each and every member of team GangaGen who has contributed intellectually and practically in this effort.We gratefully thank Dr. Ramachandran, the founder of GangaGen, for his suggestions, constant encouragement, and support.

References [1] F.L. Gordillo Altamirano, J.J. Barra, Phage therapy in the post antibiotic era, Clin Micro Rev 32 (2) (2019) e00066-18. [2] U. Sharma, et al. Phage-derived lysins as potential agents for eradicating biofilms and persisters, Drug Discov Today 23 (4) (2018) 848–856. [3] M. Delbruck, The growth of bacteria and lysis of the host, J Gen Physiol 20 (1940) 643–660. [4] J. Caldentey, D.H. Bamford,The lytic enzyme of the Pseudomonas phage φ6. Purification and biochemical characterization, Biochim Biophys Acta 1159 (1992) 44–50. [5] M. Moak, I.J. Molineux, Role of the Gp16 lytic transglycosylase motif in bacteriophage T7 virions at the initiation of infection, Mol Microbiol 37 (2000) 345–355. [6] P.S. Rydman, D.H. Bamford, Bacteriophage PRD1 DNA entry uses a viral membrane-associated transglycosylase activity, Mol Microbiol 37 (2000) 356–363. [7] S.H. Kao, W.H. McClain, Baseplate protein of bacteriophage T4 with both structural and lytic functions, J Virol 34 (1980) 95–103. [8] V.D. Paul, et al. A novel bacteriophage tail-associated muralytic enzyme (TAME) from phage K and its development into a potent antistaphylococcal protein, BMC Microbiol 11 (2011) 226. [9] M. Rashel, et al. Tail-associated structural protein gp61 of Staphylococcus aureus phage φMR11 has bifunctional lytic activity, FEMS Microbiol Lett 284 (2008) 9–16. [10] R.Young, Bacteriophage lysis: mechanism and regulation, Microbiol Rev 56 (1992) 430–481. [11] R.Young, Bacteriophage holins: deadly diversity, J Mol Microbiol Biotechnol 4 (2002) 21–36. [12] M. Schmelcher, et al. Bacteriophage endolysins as novel antimicrobials, Future Microbiol 7 (2012) 1147–1171. [13] A. Marchler-Bauer, S.H. Bryant, CD-Search: protein domain annotations on the fly, Nucleic Acids Res 32 (2004) W327–W331. [14] S.C. Becker, et al. Differentially conserved staphylococcal SH3b_5 cell wall binding domains confer increased staphylolytic and streptolytic activity to a streptococcal prophage endolysin domain, Gene 443 (2009) 32–41. [15] I. Fernández-Ruiz, et al.Thousands of novel endolysins discovered in uncultured phage genomes, Front Microbiol 9 (2018) 1033. [16] J.E. Schmitz, et al. Identifying active phage lysins through functional viral metagenomics, Appl Environ Microbiol 76 (2010) 7181–7187. [17] R. Schuch, et al. A genetic screen to identify bacteriophage lysins, Methods Mol Biol 502 (2009) 307–319. [18] Y. Huang, et al. CD-HIT Suite: a web server for clustering and comparing biological sequences, Bioinformatics 26 (2010) 680–682. [19] R.D. Finn, et al. The Pfam protein families database: towards a more sustainable future, Nucleic Acids Res 44 (2016) D279–D285. [20] R.D. Finn, et al. InterPro in 2017—beyond protein family and domain annotations, Nucleic Acids Res 45 (2017) D190–D199.

Phage therapy—bacteriophage and phage-derived products as anti-infective drugs

[21] P.M. Rountree,The serological differentiation of staphylococcal bacteriophages, Microbiology 3 (1949) 164–173. [22] S.O. Flaherty, et al. Genome of staphylococcal phage K: a new lineage of myoviridae infecting Grampositive bacteria with a low G+C content, J Bacteriol 186 (2004) 2862–2871. [23] A. Bateman, N.D. Rawlings,The CHAP domain: a large family of amidases including GSP amidase and peptidoglycan hydrolases, Trends Biochem Sci 28 (2003) 234–237. [24] G. Xia, et al. Wall teichoic acid-dependent adsorption of staphylococcal siphovirus and myovirus, J Bacteriol 193 (2011) 4006–4009. [25] H. Nakagawa, et al. Isolation and characterization of the bacteriophage T4 tail-associated lysozyme, J Virol 54 (2) (1985) 460–466. [26] A. Fomenkov, et al. EcoBLMcrX, a classical modification-dependent restriction enzyme in Escherichia coli B: characterization in vivo and in vitro with a new approach to cleavage site determination, PLoS One 12 (2017) 1–21. [27] S. Sundarrajan, et al. Bacteriophage-derived CHAP domain protein, P128, kills Staphylococcus cells by cleaving interpeptide cross-bridge of peptidoglycan, Microbiology 160 (2014) 2157–2169. [28] S. Rohrer, B. Berger-Bächi, FemABX peptidyl transferases: a link between branched-chain cell wall peptide formation and beta-lactam resistance in Gram-positive cocci, Antimicrob Agents Chemother 47 (2003) 837–846. [29] H.Wisplinghoff, et al. Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study, Clin Infect Dis 39 (2004) 309–317. [30] R. Zakhour, et al. Catheter-related infections in patients with haematological malignancies: novel preventive and therapeutic strategies, Lancet Infect Dis 16 (11) (2016) e241–e250. [31] H. Oliveira, et al. Phage-derived peptidoglycan degrading enzymes: challenges and future prospects for in vivo therapy,Viruses 10 (6) (2018) 292. [32] S. Nair, et al. Antibiofilm activity and synergistic inhibition of Staphylococcus aureus biofilms by bactericidal protein P128 in combination with antibiotics, Antimicrob Agents Chemother 60 (2016) 7280– 7289. [33] R. Schuch, et al. Combination therapy with lysin CF-301 and antibiotic is superior to antibiotic alone for treating methicillin-resistant Staphylococcus aureus-induced murine bacteremia, J Infect Dis 209 (2014) 1469–1478. [34] S.Y. Jun, et al. Antibacterial properties of a pre-formulated recombinant phage endolysin, SAL-1, Int J Antimicrob Agents 41 (2) (2013) 156–161. [35] N. Poonacha, et al. Efficient killing of planktonic and biofilm-embedded coagulase-negative staphylococci by bactericidal protein P128, Antimicrob Agents Chemother 61 (8) (2017) e00457-17. [36] National Committee for Clinical Laboratory StandardsMethods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard—ninth edition, NCCLS, Wayne, PA, (2012). [37] A.A. Vipra, et al. Antistaphylococcal activity of bacteriophage derived chimeric protein P128, BMC Microbiol 12 (2012) 41. [38] M. Balouiri, et al. Methods for in vitro evaluating antimicrobial activity: a review, J Pharm Anal 6 (2016) 71–79. [39] National Committee for Clinical Laboratory Standards: Methods for determining Bactericidal Activity of Antimicrobial Agents; Approved Guideline M26-A. NCCLS, Wayne, PA; 1999. [40] S. Schmidt, et al. Effect of protein binding on the pharmacological activity of highly bound antibiotics, Antimicrob Agents Chemother 52 (11) (2008) 3994–4000. [41] C. Indiani, et al. The antistaphylococcal lysin, CF-301, activates key host factors in human blood to potentiate methicillin-resistant Staphylococcus aureus bacteriolysis, Antimicrob Agents Chemother 63 (4) (2019) e02291-18. [42] N.H. Kim, et al. Effects of phage endolysin SAL200 combined with antibiotics on Staphylococcus aureus infection, Antimicrob Agents Chemother 62 (10) (2018) e00731-18. [43] M. Pastagia, et al. Lysins: the arrival of pathogen-directed anti-infectives, J Med Microbiol 62 (2013) 1506–1516. [44] S. Nair, et al. Restoration of sensitivity of a diverse set of drug-resistant Staphylococcus clinical strains by bactericidal protein P128, J Med Microbiol 67 (2018) 296–307.

357

358

Drug Discovery Targeting Drug-Resistant Bacteria

[45] D. Lebeaux, et al. From in vitro to in vivo models of bacterial biofilm-related infections, Pathogens 2 (2) (2013) 288–356. [46] M.K.Yadav, et al. In vitro multi-species biofilms of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa and their host interaction during in vivo colonization of an otitis media rat model, Front Cell Infect Microbiol 7 (2017) 125. [47] P.M. Alves, et al. Interaction between Staphylococcus aureus and Pseudomonas aeruginosa is beneficial for colonisation and pathogenicity in a mixed biofilm, Pathog Dis 76 (2018) 1. [48] Y. Sun, et al. In vitro multispecies Lubbock chronic wound biofilm model, Wound Repair Regen 16 (2008) 805–813. [49] R.P. Rother, et al. The clinical sequelae of intravascular haemolysis and extracellular plasma haemoglobin: a novel mechanism of human disease, JAMA 293 (2005) 1653–1662. [50] S.E. George, et al. Biochemical characterization and evaluation of cytotoxicity of antistaphylococcal chimeric protein P128, BMC Res Notes 5 (2012) 280. [51] N. Frimodt-Møller, et al. The mouse peritonitis/sepsis model, Handbook of animal models of infection, Elsevier BV, Amsterdam, The Netherlands, 1999, pp. 127–136. [52] A.J. Lewis, et al. Current murine models of sepsis, Surg Infect 17 (4) (2016) 385–393. [53] L. Olitzki, Mucin as a resistance-lowering substance, Bacteriol Rev 12 (2) (1948) 149–172. [54] A.F. Zuluaga, et al. Neutropenia induced in outbred mice by a simplified low-dose cyclophosphamide regimen: characterization and applicability to diverse experimental models of infectious diseases, BMC Infect Dis 6 (2006) 55. [55] Channabasappa S et al. 2017. In vivo synergy and efficacy of P128 in combination with standard of care antibiotics in Methicillin resistant Staphylococcus aureus (MRSA) bacteremia, ECCMID conference, 2017. [56] S. Channabasappa, et al. Efficacy of chimeric ectolysin P128 in drug-resistant Staphylococcus aureus bacteraemia in mice, J Antimicrob Chemother 73 (12) (2018) 3398–3404. [57] A. Daniel, et al. Synergism between a novel chimeric lysin and oxacillin protects against infection by methicillin-resistant Staphylococcus aureus, Antimicrob Agents Chemother 54 (4) (2010) 1603–1612. [58] J.S. Patton, Mechanisms of macromolecular absorption by the lungs, Adv Drug Deliv Rev 19 (1996) 3–36. [59] A. Adjei, P. Gupta, Pulmonary delivery of therapeutic peptides and proteins, J Control Release 29 (3) (1994) 361–373. [60] Sriram B et al. 2017. Pharmacokinetics and efficacy of ectolysin P128 in a mouse model of systemic Methicillin resistant Staphylococcus aureus (MRSA) infection. ASM Microbe conference, 2017. [61] Rotolo JA et al. 2016. PK PD driver of efficacy for CF-301, a novel anti-staphylococcal lysin: implications for human target dose. ASM Microbe, 2016. [62] Xiong YQ, et al. 2014. Efficacy of single-dose lysin CF-301 in combination with vancomycin or daptomycin (DAP) in experimental endocarditis due to Methicillin resistant staphylococcus aureus (MRSA). ICAAC, 2014. [63] A.G. Cheng, et al. A play in four acts: Staphylococcus aureus abscess formation, Trends Microbiol 19 (2011) 225–232. [64] S.D. Kobayashi, et al. Pathogenesis of Staphylococcus aureus abscesses, Am J Pathol 185 (2015) 1518–1527. [65] A.G. Cheng, et al. Genetic requirements for Staphylococcus aureus abscess formation and persistence in host tissues, FASEB J 23 (10) (2009) 3393–3404. [66] S. Channabasappa, et al. Efficacy of novel antistaphylococcal ectolysin P128 in a rat model of methicillin-resistant Staphylococcus aureus bacteremia, Antimicrob Agents Chemother 62 (2) (2017) e01358-17. [67] H. Yang, et al. Novel chimeric lysin with high-level antimicrobial activity against methicillin-resistant Staphylococcus aureus in vitro and in vivo, Antimicrob Agents Chemother 58 (2014) 536–542. [68] L. Zhang, et al. LysGH15 kills Staphylococcus aureus without being affected by the humoral immune response or inducing inflammation, Sci Rep 6 (2016) 29344. [69] M.A. Dorato, L.A. Buckley, Toxicology in the drug discovery and development process, Curr Protoc Pharmacol (2006) Chapter 10: Unit 10.3.1 - 10.3.35. [70] Y.Vugmeyster, et al. Pharmacokinetics and toxicology of therapeutic proteins: advances and challenges, World J Biol Chem 3 (4) (2012) 73–92.

Phage therapy—bacteriophage and phage-derived products as anti-infective drugs

[71] F. Giordanetto, Early drug development: bringing a preclinical candidate to the clinic, volume 1,WileyVCH Verlag GmbH & Co. KGaA, (2018) eISBN:9783527801756. [72] R.P. Evens, Pharma success in product development—does biotechnology change the paradigm in product development and attrition, AAPS J 18 (1) (2016) 281–285. [73] M.P. Baker, et al. Immunogenicity of protein therapeutics: the key causes, consequences and challenges, Self Nonself 1 (4) (2010) 314–322. [74] M. Fenton, et al. Recombinant bacteriophage lysins as antibacterials, Bioeng Bugs 1 (1) (2010) 9–16. [75] ICH S7A—harmonized tripartite guideline safety pharmacology studies for human pharmaceuticals, 2000. [76] ICH M3(R2)—nonclinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals, 2009. [77] Guidance for industry—S6 addendum to preclinical safety evaluation of biotechnology-derived pharmaceuticals, 2009. [78] R. Forster, et al. The RETHINK project on minipigs in the toxicity testing of new medicines and chemicals: conclusions and recommendations, J Pharmacol Toxicol Methods 62 (3) (2010) 236–242. [79] H. Lehmann, The minipig in general toxicology, Scand J Lab Anim Sci 25 (1998) 59–62. [80] ContraFect FORM S-1—SEC.gov. https://www.sec.gov/Archives/edgar/data/1478069/000119312 514149004/d609128ds1.htm. [81] FDA guidance for industry: estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers, 2005. [82] Cassino C et al. 2016, Results of the first in human study of lysin CF-301 evaluating the safety, tolerability and pharmacokinetic profile in healthy volunteers. ECCMID conference, 2016. [83] S.Y. Jun, et al. Preclinical safety evaluation of intravenously administered SAL200 containing the recombinant phage endolysin SAL-1 as a pharmaceutical ingredient, Antimicrob Agents Chemother 58 (2014) 2084–2088. [84] S.Y. Jun, et al. Pharmacokinetics of the phage endolysin-based candidate drug SAL200 in monkeys and its appropriate intravenous dosing period, Clin Exp Pharmacol Physiol 43 (2016) 1013–1016. [85] S.Y.C.Tong, et al. Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management, Clin Microbiol Rev 28 (3) (2015) 603–661. [86] M.E. Rupp and P.D. Fey, P. D., Staphylococcus epidermidis and Other Coagulase-Negative Staphylococci. In Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases, Elsevier Inc. 2014,Vol. 2, pp. 2272-2282.e5. [87] Mandell, Douglas, and Bennett’s principles and practice of infectious diseases, 2015 (8th ed.),Saunders, an imprint of Elsevier Inc. ISBN: 978-1-4557-4801-3. [88] R.M. Klevens, M.A. Morrison, J. Nadle, et al. Invasive methicillin-resistant Staphylococcus aureus infections in the United States, JAMA 298 (2007) 1763–1771. [89] M.L. Landrum, C. Neumann, C. Cook, et al. Epidemiology of Staphylococcus aureus blood and skin and soft tissue infections in the US military health system, 2005–2010, JAMA 308 (2012) 50–59. [90] A.J. Brink, Does resistance in severe infections caused by methicillin-resistant Staphylococcus aureus give you the ‘creeps’?, Cur Opin Crit Care 18 (2012) 451–459. [91] R.P. Junjappa, et al. Efficacy of anti-staphylococcal protein P128 for the treatment of canine pyodermapotential applications,Vet Res Commun 37 (3) (2013) 217–228. [92] http://www.fda.gov/AdvisoryCommittees/Calendar/ucm385727.htm. US Food and Drug Administration. Anti-Infective Drugs Advisory Committee meeting, March 31, 2014. [93] https://clinicaltrials.gov/ct2/show/NCT03163446?cond=CF301&rank=2. [Accessed July 2, 2019]. [94] https://clinicaltrials.gov/ct2/show/NCT03089697. Phase IIa clinical study of N-Rephasin® SAL200. [Accessed July 2, 2019]. [95] https://www.escmid.org/escmid_publications/escmid_elibrary/material/?mid=49244. [Accessed June 12, 2019]. [96] https://ir.contrafect.com/press-releases/detail/248/contrafects-exebacase-cf-301-improved-clinical. [Accessed June 12, 2019]. [97] S.Y. Jun, et al. Pharmacokinetics and tolerance of the phage endolysin-based candidate drug SAL200 after a single intravenous administration among healthy volunteers, Antimicrob Agents Chemother 61 (2017) e02629-16.

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Drug discovery targeting drug-resistant nontuberculous mycobacteria Sven Hoffnera, Michael M. Chanb,c,d, Edward D. Chanb,c,d, Diane Ordwaye

Department of Public Health Sciences, Karolinska Institutet, Stockholm, Sweden Department of Medicine, Rocky Mountain Regional Veterans Affairs Medical Center, Denver, CO, United States Departments of Medicine and Academic Affairs, National Jewish Health, Denver, CO, United States d Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Anschutz Medical Campus, Denver, CO, United States e Mycobacteria Research Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, United States a

b c

1  The need for drug discovery targeting drug-resistant nontuberculous mycobacteria Pulmonary disease caused by nontuberculous mycobacteria (NTM) is increasing globally, creating a serious public health problem. Patients with NTM lung disease currently require lengthy multidrug treatment regimens that are frequently associated with drug intolerance, toxicity, and often poor clinical outcome. Thus there is an urgent need to develop more effective drugs to counter NTM drug resistance as well as new approaches to reduce NTM-associated morbidity and mortality. It is also paramount to bridge research gaps in understanding NTM pathogenesis and host risk factors, develop evidence-based regimens to improve clinical outcomes, minimize drug toxicity, mitigate drug resistance, and prevent reinfections. This section covers the current advances of NTM drug discovery with the ultimate goal of improving the clinical outcomes of patients with NTM lung disease.

1.1  Innate resistance of nontuberculous mycobacteria to antimicrobials In general, antimicrobial resistance may occur by several mechanisms, including reduced cell wall permeability, enzymatic degradation, efflux of the antibiotics, as well as mutation of the protein target of the antimicrobial agent [1]. Millar and Moore [1] noted that whereas environmental NTM have evolved several antimicrobial resistance mechanisms, their ability to produce antimicrobial compounds to kill neighboring but competing microorganisms is limited; in contrast, the opposite is seen with environmental Streptomyces species. Thus through natural selection, NTM have evolved mechanisms to survive in the oftentimes harsh environment, which likely accounts for their innate resistance to antimicrobials and disinfectants.

Drug Discovery Targeting Drug-Resistant Bacteria. http://dx.doi.org/10.1016/B978-0-12-818480-6.00012-6 Copyright © 2020 Elsevier Inc. All rights reserved.

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1.2  Challenges of nontuberculous mycobacteria drug development Falkinham [2] outlined the challenges that must be addressed in the development of new antibiotics that target NTM. He broadly divided these challenges into overcoming (1) innate and physiologic traits of NTM such as their hydrophobic nature, slow growth, and adaptation to physical properties of the environment, all of which create greater innate resistance to antimicrobials and (2) factors that complicate microbiologic analysis in the clinical laboratory, resulting in less reliability of drug susceptibility testing—such as colony variation, biofilm formation, persistence, and residence inside phagocytes. The lipid-rich, nonpolar NTM surface hampers both the binding and intracellular transport of antibiotics that carry positive or negative charges [2]. Based on the basic chemistry principle that “like dissolves in like,” one approach in developing antibiotics against NTM is to add hydrophobic side groups to a new core molecule or existing antibiotic; one successful example is that hydrophobic derivatives of erythromycin— clarithromycin and azithromycin—are significantly more effective against NTM than erythromycin. Combining different antibiotics with dissimilar mechanisms of action may also improve efficacy; an example of this is that ethambutol—by reducing synthesis of the arabinogalactan macromolecule—may allow other antibiotics to penetrate the NTM cell membrane. NTM are slow growing in large part due to their time spent in synthesizing their complex lipid-rich outer membrane [2]. Since the rate of replication of NTM is likely to increase susceptibility to antibiotics, it stands to reason that NTM grown in nutrientrich medium are more sensitive to antibiotics than the same strain grown in nutrientpoor medium or in vivo. In addition, Mycobacterium abscessus subspecies abscessus, exposed to clarithromycin or azithromycin, may induce production of a methylase—encoded by a functional erm41 gene—that causes methylation of the 23S rRNA target of clarithromycin or azithromycin, inducing resistance to the macrolide [3]. Laboratory challenges include not recognizing differences in antibiotic susceptibility between strains of the same species as well as other characteristics that may negatively affect the antibiotic susceptibility testing. For example, Mycobacterium avium complex (MAC) strains may produce two colony morphotypes that may revert back and forth: (1) transparent (T) colonies that are relatively more slow growing, hydrophobic, virulent, and antibiotic resistant and (2) opaque (O) colonies that emerge during cultivation on laboratory medium and are faster growing, less hydrophobic, and display greater antibiotic sensitivity. A problematic issue may occur if an (O) strain is sensitive to antibiotic X but the antibiotic X-resistant (T) MAC organism is the morphotype that infects the patient; testing antibiotic susceptibility by traditional methods with the (O) strain will likely demonstrate a disconnect of drug susceptibility in vitro and yet a demonstrable lack of efficacy of antibiotic X in the NTM patients. This potential scenario may be a reason for the lack of correlation between MIC (minimum inhibitory concentration) and prediction of response to therapy. Indeed, it has been recommended, prior to testing the strains against

Drug discovery targeting drug-resistant nontuberculous mycobacteria

various antibiotics, that the frequency of (O) to (T) colonies be ≤1:1000. Because of their hydrophobic nature, it is estimated that >99% of NTM in liquid medium adhere to the surface of the container that holds them. Thus testing antibiotic susceptibility in tubes or tissue culture plates may not be an accurate reflection; rather, it was recommended that MIC of NTM be measured in biofilms. A related example of this recommendation for antibiotic testing is that NTM in biofilms are also able to become resistant to disinfectants used to clean indoor plumbing and medical equipment. Most bacterial infections are treated with only one antibacterial agent and in vitro drug susceptibility tests are thus carried out to examine the activity of this drug against the infecting bacteria. In NTM infections the situation is different since the patients are always offered combined drug therapy. If, and only if, we are sure that the components of this drug combination act separately and express no interaction, testing the drugs separately in vitro could be expected to give clinically relevant information. If on the other hand, we cannot rule out an interaction such as synergisms, antagonism, or an additive effect, we must consider the need to test also the combined drugs to better reflect their combined antibacterial capacity in a patient. Several in vitro studies have investigated such combined drug effects and shown that these are frequently occurring events. In a number of early studies from Sweden, mainly based on the radiometric Bactec 460 culture system (B&D), showed a role of ethambutol to potentiate/act synergistically against M. avium and several other clinically relevant species of NTM [4,5].This is most likely explained by a reduced permeability barrier for the second drug limiting its reach of the drug target within the bacteria [6]. It should of course be questioned if in vitro DST (drug susceptibility testing) results of combined drugs rather than single drugs better could predict the clinical efficacy of a specific drug combination, a minority of patients infected with M. avium strains initially shown to be in vitro resistant both ethambutol and rifampicin, but susceptible to their combination, failed when treated with the combination of these drugs. Interestingly, in these nonresponders resistance to the combined drugs had developed during therapy and could be identified when the two drugs were used in combination for repeated in vitro testing [7]. The area of combined effects of drugs, and how to test them are discussed in a review by van Ingen [8]. The recommended antibiotic treatments for pulmonary MAC and M. abscessus subspecies are demonstrated in Table 12.1. Multiple clinical studies have shown that only 70% of patients with MAC and 50% of patients infected with M. abscessus subspecies who complete the recommended therapy resulted in cure [9]. Moreover, recurrent rates are as high as 25%–45%, albeit some recurrent cases are due to new infections rather than to recrudescence [10]. The need for not only better combination therapies but also approaches to confront the high potential for reinfection of pulmonary NTM disease has led to multiple preclinical and clinical studies expanding the potential for building a successful treatment regimen.

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Table 12.1  Recommended treatment regimens. Species

Recommended treatment regimen

Mycobacterium avium complex

Standard regimen is a macrolide (azithromycin or clarithromycin), rifampin, and ethambutol. For relatively mild disease with cavitation, the drugs may be given thrice weekly. For more severe disease, especially with cavitation, daily therapy is recommended ± thrice weekly aminoglycoside (amikacin) for the first 2–3 months For M. abscessus complex species with a functional erm41 gene, a recommended regimen includes daily clofazimine and cefoxitin (or imipenem) plus thrice weekly amikacin for the first 2–3 months. For species without the functional erm41 gene, daily macrolide (azithromycin or clarithromycin) and cefoxitin (or imipenem) plus amikacin thrice weekly for the first 2–3 months

Mycobacterium abscessus complex

1.3  New compounds tackling drug resistance New efforts using compounds developed for Gram-positive pathogens, such as linezolid, have been studied for efficacy against NTM (Fig. 12.1). Linezolid is an oxazolidinone class antibiotic typically reserved for drug-resistant pathogens such as methicillin-resistant Staphylococcus aureus. Linezolid has shown some efficacy for refractory cases of disseminated NTM, although with long-term use, it is associated with serious adverse effects such as myelosuppression, serotonin syndrome, and lactic acidosis [11]. Tedizolid, another

Figure 12.1  Drug regimens therapeutic vaccine strategies to stop NTM. NTM, nontuberculous mycobacteria.

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oxazolidinone, has lower toxicity than linezolid and is also being studied against NTM, but it is still in early development [12]. Oxazolidinones are synthetic antibacterial agents with a distinctive mechanism of action, have a wide spectrum of activity, oral bioavailability, and well-established “structure–activity relationship” [13]. Oxazolidinones act at the transcriptional level by inhibiting the initiation phase of protein synthesis [13]. Combination treatment with tedizolid and clofazimine has the potential to improve treatment efficacy and diminish development of drug resistance. Compared to linezolid, tedizolid has superior bioavailability, longer half-life, and has better accumulation in lung macrophages [12,14]. Several oxazolidinones are currently in development (e.g., sutezolid) for their possible use in tuberculosis (TB) treatment; an important limiting factor for their development and long-term use is their toxicity, especially myelosuppression [15]. Clofazimine, typically used for leprosy, has been shown to be effective in NTM patients (primarily MAC patients) [16]. Presently, clofazimine is used for NTM infections, although application through Novartis and the provider’s Institutional Review Board is required. Clofazimine is a riminophenazine dye that preferentially binds to mycobacterial DNA and also has antiinflammatory properties [17]. In recent years, many insights into the process of achieving drug synergy aimed at NTMs have been gained, leading to the understanding that this approach can be successful and lead to patient’s cure [9]. Recently, preclinical animal models comparing the antimycobacterial activity of clarithromycin, clofazimine, bedaquiline, and clofazimine–bedaquiline combinations against M. abscessus treatment of interferon-gamma (IFN-gamma) knockout and severe combined immunodeficiency mice demonstrated that clofazimine and bedaquiline were the most effective in decreasing the organ burden of M. abscessus [18–20]. This study highlights the importance of synergy among compounds in optimizing therapeutic efficacy against M. abscessus. Additional studies using the zebrafish model demonstrated that bedaquiline was highly efficacious against M. abscessus infection, and treatment was sufficient to protect the infected larvae from M. abscessus–induced killing [21]. Clofazimine alone was bacteriostatic for both M. abscessus and M. avium, whereas the clofazimine– amikacin combination was synergistic against both NTM [21]. The finding that addition of clofazimine to a regimen of amikacin and clarithromycin led to suppression of emergence of resistance was made by evaluation of time kill kinetic assays [22]. Bedaquiline is an antitubercular medication that shows bacteriostatic activity against M. avium and M. abscessus in vitro. However, bedaquiline did not reduce mortality of M. abscessus-infected mice in one study [23]. The mechanism of synergy of the clofazimine and bedaquiline combination has been investigated in an animal model of drug-resistant strains of Mycobacterium tuberculosis (MTB) [24,25]. These studies demonstrated that clofazimine acts as a prodrug that is then reduced by NADH (nicotinamide adenine dinucleotide hydrogen) nicotinamide dinucleotide hydrogen (NDH-2), leading to the release of reactive oxygen species upon reoxidation by molecular oxygen [24]. Importantly, clofazimine likely competes with menaquinone (MK-4), a main cofactor in the mycobacterial

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electron transfer chain, for its reduction by NDH-2 [24]. Additional studies examined the effect of MK-4 administration on the action of clofazimine against MTB and discovered a direct competition between clofazimine and MK-4 such that MK-4 reduced the killing of clofazimine in nonreplicating and replicating mycobacteria [24]. Mycobacteria form biofilms composed of an extracellular DNA matrix that creates a barrier to effective drug penetration. One recent study found that the addition of DNase to amikacin and gatifloxacin increased the efficacy of these antibiotics [26], and increasing evidence suggests that targeting biofilm formation is crucial to combating NTM disease [18,27]. Glycopeptidolipids are a class of glycolipids produced by several NTM that are, as a group, important biofilm pioneers and formers [28]. NTM biofilm formation results in greater resistance to most antibiotics, and likely more recalcitrance to host immunity [28–30]. Preclinical studies, evaluating the use of new drugs for treatment for MAC and M. abscessus species, have also discovered inhibitors of the trehalose monomycolate transporter MmpL3, indicating that this transmembrane protein may be an attractive target for NTM [31]. The MmpL family of proteins translocates complex glycol-lipids and siderophores across the cell envelope of mycobacteria and plays an important role in the biogenesis of the outer membrane of NTM [32]. Piperidinol-based compound 1 (PIPD1) and indole-2-carboximides are two in development compounds that target a transporter molecule in mycobacteria, inhibiting the transport of mycolic acid, preventing the mycolylation of arabinogalactan [31]. In vitro studies have shown that indolecarboxamides lead compounds, 6 and 12, exhibited strong activity against a wide panel of M. abscessus isolates alone as well as in infected macrophages [31]. The indolecarboxamides strongly inhibited the transport of trehalose monomycolate, resulting in the loss of trehalose dimycolate production and abrogating mycolylation of arabinogalactan constituents of the biofilm. These studies explored new compounds against M. abscessus infections along with promising translational development for the treatment of NTM infections [33]. With the exception of cefoxitin and imipenem for M. abscessus complex organisms, NTM possess high levels of intrinsic drug resistance to most β-lactams due to the presence of mycobacterial β-lactamases. However, combining the β-lactamase inhibitors avibactam or clavulanic acid with a β-lactam or monobactam has been shown to significantly lower the MICs of several β-lactams (cefoxitin, amoxicillin, ceftaroline, and ceftazidime) or carbapenems (meropenem and imipenem) [34–38]. Additionally, rifampin has also been shown to have synergy with various carbapenems (faropenem, biapenem, doripenem, and meropenem) against M. abscessus [39].

1.4  New targets for compound development Millar and Moore [1] have published an encyclopedic work on the compounds that have been investigated to kill or antagonize growth of NTM, and readers are encouraged to study their magnum opus.The general groups of compounds they have well-documented

Drug discovery targeting drug-resistant nontuberculous mycobacteria

include (1) plant-derived extracts and compounds from land plants, berries, fungi, and marine fauna; for example, essential oils; extracts obtained from various ethnic medical plants using hexane, various alcohols, and other solvents for the extraction; curcumin; persimmon-derived tannin; (2) venom-derived antimicrobial peptides from scorpion, cobra, and wasps; (3) novel synthetic or modified existing drugs; for example, nitrogen heterocycles derivatives; 3-phenylquinolone efflux pump inhibitors; Nicotinamide adenine, dinucleotide phosphate (NADPH)/NADH oxidase inhibitors; indole-2-carboxamides to inhibit the mycolic acid transporter MmpL3; and various forms of oxazolidinone; (4) repurposing of existing drugs; for example, bedaquiline; clofazimine; and zafirlukast, a leukotriene receptor antagonist; and (5) host-directed approaches, including the cyclic peptides ohmyungsamycins to induce autophagy and the antimycobacterial phages. Research studies have evaluated the effects of soluble persimmon-derived tannins in vitro and in the murine model of MAC infection as tannins are known to have potent antioxidant and bacteriostatic properties [40]. Soluble tannin hydrolysate displayed high bacteriostatic activity against MAC in vitro. Mice fed with a soluble tannin-containing diet demonstrated reduced MAC burden in the mouse lungs, less pulmonary granuloma formation, and reduction in proinflammatory cytokines. Additional compounds of interest are SPR719, a benzimidazole that has been studied as an antimicrobial against NTM [41]. The orally available prodrug SPR720 was studied against 161 isolates of NTM and found to be potent against multiple clinical strains of NTM with an MIC50 of 0.25–4 µg/mL for several NTM species [41]. A new antioxidant mimetic, MnTE-2PyP, increased phagosome–lysosome fusion in macrophages and reduced the number of viable M. abscessus in human THP-1-differentiated macrophages [42]. Also, thiacetazone is an antitubercular drug that is no longer used in most cases; however, derivatives of thiacetazone have shown efficacy against M. avium and M. abscessus in murine models [43]. While not a pharmacologic agent per se, intravenous injection of mesenchymal stem cells into mice enhanced clearance of M. abscessus in their lungs and spleens, increased recruitment of T cells, macrophages, and monocytes, and increased several proinflammatory cytokines and molecules such as IFN-gamma, tumor necrosis factor-alpha (TNFalpha), and nitric oxide [44].

1.5  Existing antimicrobials being repurposed for nontuberculous mycobacteria treatment A major hurdle of treating NTM infections is that long-term use of antibiotics is required and associated with significant adverse drug effects or intolerance such as neurotoxicity and nephrotoxicity as seen with amikacin. Amikacin is an aminoglycoside that is recommended to treat cavitary and/or more severe NTM lung disease. The typical dose for amikacin in those with normal renal function is 10–25 mg/kg IV thrice weekly, typically for the first 2–3 months of initiation of therapy. The major side effects of all aminoglycosides—particularly with prolonged use as employed to treat chronic NTM lung

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disease—are ototoxicity, vestibulotoxicity, renal toxicity, and hypomagnesemia. Other approaches to reduce the toxicity associated with long-term use of antibiotics are to reformulate preexisting drugs for inhalation treatment. NTM have relatively few drugs in development compared to TB, many of which are repurposed or reformulations of older drugs, such as linezolid and clofazimine [45]. Since NTM can reside intracellularly in host phagocytes, the liposomal form of amikacin was studied, as liposome-encapsulated drugs often have greater penetration intracellularly than free drugs as well as, perhaps, less toxicity. Bermudez et al. [46] compared the efficacy of free and liposomal-encapsulated intravenous amikacin or gentamicin against MAC infection of beige mice and found marked decrease in bacterial burden in various organs in mice treated with liposomal aminoglycosides compared to those treated with either free drug. In another liposomal amikacin preparation, it was also shown to have much greater efficacy against a systemic MAC infection in the beige mouse [47]. Similarly, in murine peritoneal macrophages, liposomal amikacin showed significantly greater inhibitory activity against intracellular MAC than free amikacin [48]. Inhalation treatment targets the drug of interest directly to the airways, potentially lowering the dose of the compound required and thus limiting toxicity while maintaining optimal efficacy [49]. Inhaled administration will also obviate the need for long-term intravenous catheter placement for the parenteral antibiotic administration. A randomized trial adding liposomal amikacin by inhalation to patients with MAC pulmonary disease who were failing prior treatment regimens resulted in a considerable higher rate of culture conversion compared to the placebo group [50]. Additionally, a second clinical trial was conducted to study inhaled amikacin therapy in patients with difficult-to-treat NTM pulmonary diseases [51]. A total of 23 patients with NTM lung disease—mostly due to MAC and few due to M. abscessus—were treated with combination therapy, including inhaled amikacin, that showed a sputum conversion rate of 38% in the 21 MAC lung disease subjects and 100% in the two subjects infected with M. abscessus [51]. Because there was no control arm without inhaled amikacin, it is difficult to say with any degree of certainty that inhaled amikacin helped albeit it was suggestive [51]. However, the results of liposomal inhaled amikacin in patients with refractory MAC lung disease were recently published [52]. Adults with MAC lung disease who were still sputum positive after at least 6 months of standard therapy were randomized to either continued standard therapy or standard therapy plus inhaled liposomal amikacin. Those who also received the inhaled liposomal amikacin daily were significantly more likely to achieve culture conversion albeit dysphonia, cough, and dyspnea were also more likely [52]. While not clinically available or used at the present time, other potential antiNTM drugs that have been incorporated into liposomes or potential new formulations include the fluoroquinolones, clarithromycin, rifamycin, ethambutol, and clofazimine [53]. These clinical studies support the development of new combination inhaled therapies that may lead to improved clinical patient outcome.

Drug discovery targeting drug-resistant nontuberculous mycobacteria

An initial phase I preclinical study, evaluating the safety and efficacy of ciprofloxacin dry powder for inhalation (DPI) for patients with noncystic fibrosis bronchiectasis with two or more exacerbations in the previous year with a predefined bacteria in sputum, has been completed [54]. Although ciprofloxacin was found to be safe, the differences in the treated and placebo groups were not significant [54]. However, a subsequent phase II trial aimed to evaluate the efficacy of ciprofloxacin DPI in prolonging the time to first exacerbation and reducing the frequency of exacerbations with two treatment regimens (28 days on/off and 14 days on/off) in patients with noncystic fibrosis bronchiectasis using a stringent definition of exacerbation [55]. Ciprofloxacin DPI reduced NTM bacterial burden and demonstrated a trend toward fewer exacerbations. Although the phase I and II trials targeted reduced bacterial burden in seven different pathogens, the RESPIRE studies could provide insight into the potential use of other anti-NTM compounds formulated in a dry power inhalation treatment. In addition, preclinical evaluation of the safety and efficacy of inhaled tobramycin in cystic fibrosis patients was recently conducted [56]. Although reformulation of currently utilized NTM drugs is being developed, there still exists the need for new bactericidal therapeutic agents. The role of nitric oxide in mycobacterial infections is controversial, particularly in humans [57]. Mice with disruption for inducible nitric oxide synthase (iNOS) are more susceptible to MTB [58]. Nitric oxide is thought to kill MTB via direct toxicity as well as induction of apoptosis of MTB-infected macrophages [59]. With pulmonary TB, reduced availability of lung nitric oxide has been shown to be associated with more severe disease and delayed sputum conversion [59]. In an in vitro murine macrophage model of MAC infection, reactive nitrogen intermediates (RNI) such as nitric oxide and free fatty acids, but not reactive oxygen intermediates, are important anti-MAC effectors employed by the macrophages [60]. Both the smooth opaque and transparent variants of Mycobacterium fortuitum are derived from the same strain and yet the transparent variant is considered to be more virulent than the opaque strain. In mouse macrophages that were stimulated with IFN-gamma, the opaque variant induced 5–9× higher levels of nitric oxide than the transparent strain [61], indicating that virulence is inversely proportional to the amount of nitric oxide induced; that is, nitric oxide, by killing the NTM strain that induces it at greater level, makes that particular NTM strain less virulent. Others have also shown that a pathogenic strain of M. avium produced lower levels of IFN-gamma, IL-1-beta, and iNOS than macrophages infected with a nonpathogenic species [62]. In human endothelial cells, nitric oxide production was greatest with M. abscessus infection, followed by MTB and Mycobacterium smegmatis [63]. The mycobacterial cell wall comprises a myriad of lipoglycans and lipoproteins that could potentially induce nitric oxide production. Interestingly, the lipomannan of Mycobacterium chelonae, Mycobacterium kansasii, and Mycobacterium bovis Bacillus Calmette–Guérin (BCG) not only induced production of TNF-alpha and nitric

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oxide in primary murine macrophages but also paradoxically inhibited nitric oxide production by lipopolysaccharide (LPS)-activated macrophages [64]. On the other hand, others showed that RNI were not involved in controlling growth of M. fortuitum in murine peritoneal macrophages [65]. Estrogen may also mediate a host-protective effect against NTM via nitric oxide. Tsuyuguchi et al. [66] examined the effects of estrogen on controlling MAC infection, since a disproportionate number of postmenopausal women are affected by NTM lung disease.They showed that ovariectomized mice were more susceptible to NTM infection and replacement with estrogen conferred greater protection [66]. They demonstrated in vitro that 17-beta-estradiol increased production of RNI and iNOS mRNA expression by macrophages prestimulated with IFN-gamma and infected with MAC [66]. Interestingly, mycobacteria have developed evasive mechanisms against nitric oxide, indirectly suggesting that nitric oxide is important in host immunity against them. For example, M. smegmatis possesses flavohemoglobin I that is known to detoxify nitric oxide by acting as a nitric oxide dioxygenase [67]. Expression of the MTB hypoxic response protein-1 (Hrp1, aka Rv2626c) gene into M. smegmatis enhanced NTM survival under hypoxic and nitric oxide stress conditions in macrophages and in mice [68]. In addition, the Hrp1-containing M. smegmatis produced more proinflammatory cytokines as well as iNOS. In the zebrafish model of mycobacterial infection, stabilization of HIF1-alpha and reduction in HIF2-alpha are known to induce iNOS and RNI in neutrophils prior to infection and a decrease in Mycobacterium marinum burden in the infected fish larvae [69]. Mutation of the meIF gene in the mel2 locus of M. marinum resulted in decreased growth in murine macrophages; this was abrogated by NOS inhibition, indicating that meIF/mel2 locus of this NTM conferred resistance to RNI [70]. Mycothiol in M. smegmatis was shown to protect the bacteria from being killed by nitric oxide [71]. A phase I study in healthy adults showed that nitric oxide at 160 parts per million (ppm) for 30 min, five times daily, and for 5 days was shown to be safe and well-tolerated [72]. In a subsequent study of eight cystic fibrosis patients who were given 160 ppm of inhaled nitric oxide for 30 min thrice daily for 2- to 5-day periods, it was noted that nitric oxide treatment significantly reduced the bacterial and fungal number in their sputa [72]. In the context of NTM infection, two cystic fibrosis patients with recalcitrant M. abscessus lung infection inhaled nitric oxide at 160 ppm and were given as a compassionate use [73]. One patient was treated for a total of 26 days (72 total inhalations with a minimal time interval of 3.5 h between treatments); the second patient was treated for 21 days (90 total inhalations). In these two patients, addition of inhaled nitric oxide resulted in a significant reduction in M. abscessus load in their sputa, as measured by qPCR (quantitative polymerase chain reaction) [73]. Three additional studies published as abstracts also revealed promising results with inhaled nitric oxide with NTM lung disease in regard to improvement in lung function and/or reduction or elimination of M. abscessus/NTM [73,74].

Drug discovery targeting drug-resistant nontuberculous mycobacteria

1.6  Therapeutic vaccine approaches The development of highly efficacious new treatment regimens against NTMs is achievable. However, NTM patients have a high chance of being reinfected with NTMs. Thus to control opportunistic NTM and to cease subsequent reinfection, a therapeutic vaccine is urgently needed against NTMs. Many approaches may be exploited to develop and advance vaccines against NTM. For example, similar virulence factors are expressed during infection and disease progression with other pathogens.These cross-reactive antigens produced by other pathogens may allow development of NTM preventative vaccines or therapeutic vaccine given in combination with effective antimicrobial treatment and postexposure vaccine to prevent reinfections [75]. In Sweden a significant increase in NTM infection in children was observed after the finalization of general BCG vaccination in 1975. The average yearly incidence of NTM disease in children under 5 years of age increased from 0.06 during a 5-year period, 1969–1974, with BCG vaccination to a maximum of 5.7 during 5 years, 1981–1985, after its discontinuation [76,77]. Studies have shown that NTM-based preexisting immunity could lead to lack of BCG vaccine efficacy against MTB [78]. However, if BCG is given prior to NTM exposure in a murine model, there is some protection against M. kansasii and M. avium [79]. Additional studies have shown that BCG was not protective against Mycobacterium intracellulare and Mycobacterium simiae, possibly because BCG lacks cross-reactive antigens against these NTM species [79]. BCG-vaccinated mice infected with Mycobacterium ulcerans developed transient protection as evinced by reduced clinical disease accompanied by increased CD4+ TH1 responses [80]. Recently, unexpected cross-reactivity of NTM/MTB-specific CXCR3+CCR6+ memory T cells in non-TB-exposed healthy control donors has been observed [81], including three of the four antigens (Rv3619c, Rv3620c, and Rv2608) that make up the ID93 fusion protein. Rv3619c and Rv3620c were shown to be TB-reactive (notably, these antigens are not included in BCG), whereas Rv2608 was cross-reactive in individuals with both latent TB infection and nonexposed household contacts [81]. An adjuvant is likely to play a role in a robust vaccine response against NTMs [82]. Early IFN-gamma induction has been shown to be critical for immunity against M. ulcerans infection as shown by the delayed progression of infection and reduced bacterial burden in wild type compared to IFN-gamma knockout mice [83]. Other data demonstrate promising protective responses in mice, including increased survival, decreased pathology, and reduced bacterial load compared to BCG, using a recombinant BCG expressing M. ulcerans, Ag85A, in the Buruli ulcer mouse model [84]. The Infectious Disease Research Institute (IDRI) has engineered a vaccine against MTB, ID93 (a clinical TB antigen) + glucopyranosyl lipid adjuvant (GLA)–stable nanoemulsion (SE) that is currently being tested in a phase IIa trial in South Africa for safety, immunogenicity, and dose selection in 60 TB patients administered posttreatment [85].

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The IDRI developed the synthetic Toll-like receptor 4, GLA, formulated in an oil-inwater SE to enhance adaptive immune responses [82]. Additional research is required to develop vaccines against NTM infections [86,87].

References [1] B.C. Millar, J.E. Moore, Antimycobacterial strategies to evade antimicrobial resistance in the nontuberculous mycobacteria, Int J Mycobacteriol 8 (1) (2019) 7–21. [2] J.O. Falkinham 3rd., Challenges of NTM drug development, Front Microbiol 9 (2018) 1613. [3] K.A. Nash, B.A. Brown-Elliott, R.J. Wallace Jr., A novel gene, erm(41), confers inducible macrolide resistance to clinical isolates of Mycobacterium abscessus but is absent from Mycobacterium chelonae, Antimicrob Agents Chemother 53 (4) (2009) 1367–1376. [4] S.E. Hoffner, S.B. Svenson, A.E. Beezer, Microcalorimetric studies of the initial interaction between antimycobacterial drugs and Mycobacterium avium, J Antimicrob Chemother 25 (3) (1990) 353–359. [5] S.E. Hoffner, S.B. Svenson, G. Kallenius, Synergistic effects of antimycobacterial drug combinations on Mycobacterium avium complex determined radiometrically in liquid medium, Eur J Clin Microbiol 6 (5) (1987) 530–535. [6] U. Hjelm, J. Kaustová, M. Kubín, S.E. Hoffner, Susceptibility of Mycobacterium kansasii to ethambutol and its combination with rifamycins, ciprofloxacin and isoniazid, Eur J Clin Microbiol Infect Dis 11 (1) (1992) 51–54. [7] S.E. Hoffner, N. Heurlin, B. Petrini, S.B. Svenson, G. Källenius, Mycobacterium avium complex develop resistance to synergistically active drug combinations during infection, Eur Respir J 7 (2) (1994) 247–250. [8] J. van Ingen, et al. Resistance mechanisms and drug susceptibility testing of nontuberculous mycobacteria, Drug Resist Updat 15 (3) (2012) 149–161. [9] J. van Ingen, B.E. Ferro, W. Hoefsloot, M.J. Boeree, D. van Soolingen, Drug treatment of pulmonary nontuberculous mycobacterial disease in HIV-negative patients: the evidence, Expert Rev Anti Infect Ther 11 (10) (2013) 1065–1077. [10] L. Lande, J. George, T. Plush, Mycobacterium avium complex pulmonary disease: new epidemiology and management concepts, Curr Opin Infect Dis 31 (2) (2018) 199–207. [11] P. Chetchotisakd, S. Anunnatsiri, Linezolid in the treatment of disseminated nontuberculous mycobacterial infection in anti-interferon-gamma autoantibody-positive patients, Southeast Asian J Trop Med Public Health 45 (5) (2014) 1125–1131. [12] B.A. Brown-Elliott, R.J. Wallace Jr., In vitro susceptibility testing of tedizolid against nontuberculous mycobacteria, J Clin Microbiol 55 (6) (2017) 1747–1754. [13] P.S. Jadhavar, D.M. Vaja, T.M. Dhameliya, A.K. Chakraborti, Oxazolidinones as anti-tubercular agents: discovery, development and future perspectives, Curr Med Chem 22 (38) (2015) 4379–4397. [14] R.M. Raju, et al. Leveraging advances in tuberculosis diagnosis and treatment to address nontuberculous mycobacterial disease, Emerg Infect Dis 22 (3) (2016) 365–369. [15] P.K. Singh, O. Silakari, Current status of O-heterocycles: a synthetic and medicinal overview, ChemMedChem 13 (11) (2018) 1071–1087. [16] S.L. Martiniano, B.D. Wagner, A. Levin, J.A. Nick, S.D. Sagel, C.L. Daley, Safety and effectiveness of clofazimine for primary and refractory nontuberculous mycobacterial infection, Chest 152 (4) (2017) 800–809. [17] W. Levis, T. Rendini, Clofazimine mechanisms of action in mycobacteria, HIV, and cancer, J Infect Dis 215 (9) (2017) 1488. [18] I.M. Orme, D.J. Ordway, Host response to nontuberculous mycobacterial infections of current clinical importance, Infect Immun 82 (9) (2014) 3516–3522. [19] A. Bernut, J.L. Herrmann, D. Ordway, L. Kremer,The diverse cellular and animal models to decipher the physiopathological traits of Mycobacterium abscessus infection, Front Cell Infect Microbiol 7 (2017) 100. [20] A. Obregon-Henao, K.A. Arnett, M. Henao-Tamayo, L. Massoudi, E. Creissen, K. Andries, A.J. Lenaerts, D.J. Ordway, Susceptibility of Mycobacterium abscessus to antimycobacterial drugs in preclinical models, Antimicrob Agents Chemother 59 (11) (2015) 6904–6912.

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[21] C. Dupont, A.Viljoen, S. Thomas, F. Roquet-Banères, J.L. Herrmann, K. Pethe, L. Kremer, Bedaquiline inhibits the ATP synthase in Mycobacterium abscessus and is effective in infected zebrafish, Antimicrob Agents Chemother 61 (11) (2017) e01225–17. [22] B.E. Ferro, J. Meletiadis, M. Wattenberg, A. de Jong, D. van Soolingen, J.W. Mouton, J. van Ingen, Clofazimine prevents the regrowth of Mycobacterium abscessus and Mycobacterium avium type strains exposed to amikacin and clarithromycin, Antimicrob Agents Chemother 60 (2) (2016) 1097–1105. [23] I. Lerat, E. Cambau, R. Roth Dit Bettoni, J.L. Gaillard, V. Jarlier, et al. In vivo evaluation of antibiotic activity against Mycobacterium abscessus, J Infect Dis 209 (6) (2014) 905–912. [24] B. Lechartier, S.T. Cole, Mode of action of clofazimine and combination therapy with benzothiazinones against Mycobacterium tuberculosis, Antimicrob Agents Chemother 59 (8) (2015) 4457–4463. [25] S. Shang, C.A. Shanley, M.L. Caraway, E.A. Orme, M. Henao-Tamayo, L. Hascall-Dove, et al. Activities of TMC207, rifampin, and pyrazinamide against Mycobacterium tuberculosis infection in guinea pigs, Antimicrob Agents Chemother 55 (1) (2011) 124–131. [26] T.T. Aung, WHJ Chor, JKH Yam, M Givskov, L Yang, RW Beuerman, et al. Discovery of novel antimycobacterial drug therapy in biofilm of pathogenic nontuberculous mycobacterial keratitis, Ocul Surf 15 (4) (2017) 770–783. [27] E.D. Chan, X Bai, M Kartalija, IM Orme, DJ Ordway, et al. Host immune response to rapidly growing mycobacteria, an emerging cause of chronic lung disease, Am J Respir Cell Mol Biol 43 (4) (2010) 387–393. [28] J.S. Schorey, L. Sweet, The mycobacterial glycopeptidolipids: structure, function, and their role in pathogenesis, Glycobiology 18 (11) (2008) 832–841. [29] X. Xiang, W Deng, M Liu, J Xie, et al. Mycobacterium biofilms: factors involved in development, dispersal, and therapeutic strategies against biofilm-relevant pathogens, Crit Rev Eukaryot Gene Expr 24 (3) (2014) 269–279. [30] L. Hall-Stoodley, et al. Mycobacterium marinum biofilm formation reveals cording morphology, FEMS Microbiol Lett 257 (1) (2006) 43–49. [31] J.L. Low, et al. Screening of TB actives for activity against nontuberculous mycobacteria delivers high hit rates, Front Microbiol 8 (2017) 1539. [32] J.M. Belardinelli, et al. Structure-function profile of MmpL3, the essential mycolic acid transporter from Mycobacterium tuberculosis, ACS Infect Dis 2 (10) (2016) 702–713. [33] A.P. Kozikowski, et al. Targeting mycolic acid transport by indole-2-carboxamides for the treatment of Mycobacterium abscessus infections, J Med Chem 60 (13) (2017) 5876–5888. [34] V. Dubee, et al. Beta-lactamase inhibition by avibactam in Mycobacterium abscessus, J Antimicrob Chemother 70 (4) (2015) 1051–1058. [35] V. Dubée, et al. Impact of β-lactamase inhibition on the activity of ceftaroline against Mycobacterium tuberculosis and Mycobacterium abscessus, Antimicrob Agents Chemother 59 (5) (2015) 2938–2941. [36] A. Kaushik, et al. Combinations of avibactam and carbapenems exhibit enhanced potencies against drug-resistant Mycobacterium abscessus, Future Microbiol 12 (2017) 473–480. [37] D. Deshpande, et al. The discovery of ceftazidime/avibactam as an anti-Mycobacterium avium agent, J Antimicrob Chemother 72 (Suppl_2) (2017) i36–i42. [38] A.L. Lefebvre, et al. Inhibition of the beta-lactamase BlaMab by avibactam improves the in vitro and in vivo efficacy of imipenem against Mycobacterium abscessus, Antimicrob Agents Chemother 61 (4) (2017). [39] A. Kaushik, et al. Carbapenems and rifampin exhibit synergy against Mycobacterium tuberculosis and Mycobacterium abscessus, Antimicrob Agents Chemother 59 (10) (2015) 6561–6567. [40] Y. Matsumura, M. Kitabatake, N. Ouji-Sageshima, S.Yasui, N. Mochida, R. Nakano, et al. Persimmonderived tannin has bacteriostatic and anti-inflammatory activity in a murine model of Mycobacterium avium complex (MAC) disease, PLoS One 12 (8) (2017) e0183489. [41] B.A. Brown-Elliott, A. Rubio, R.J. Wallace Jr., In vitro susceptibility testing of a novel benzimidazole, SPR719, against nontuberculous mycobacteria, Antimicrob Agents Chemother 62 (11) (2018). [42] R.E. Oberley-Deegan,Y.M. Lee, G.E. Morey, D.M. Cook, E.D. Chan, J.D. Crapo, et al. The antioxidant mimetic, MnTE-2-PyP, reduces intracellular growth of Mycobacterium abscessus, Am J Respir Cell Mol Biol 41 (2) (2009) 170–178. [43] L.E. Bermudez, R. Reynolds, P. Kolonoski, P. Aralar, C.B. Inderlied, L.S.Young, et al.Thiosemicarbazole (thiacetazone-like) compound with activity against Mycobacterium avium in mice, Antimicrob Agents Chemother 47 (8) (2003) 2685–2687.

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[44] J.S. Kim, C. Sang-Ho, W.S. Kim, S.J. Han, S.B. Cha, H.M. Kim, et al. A novel therapeutic approach using mesenchymal stem cells to protect against Mycobacterium abscessus, Stem Cells 34 (7) (2016) 1957–1970. [45] M.L.Wu,V. Dartois,T. Dick, et al. NTM drug discovery: status, gaps and the way forward, Drug Discov Today 23 (8) (2018) 1502–1519. [46] L.E. Bermudez, et al. Treatment of disseminated Mycobacterium avium complex infection of beige mice with liposome-encapsulated aminoglycosides, J Infect Dis 161 (6) (1990) 1262–1268. [47] E.A. Petersen, et al. Liposomal amikacin: improved treatment of Mycobacterium avium complex infection in the beige mouse model, J Antimicrob Chemother 38 (5) (1996) 819–828. [48] L. Kesavalu, J.A. Goldstein, R.J. Debs, N. Düzgünes, P.R. Gangadharam, et al. Differential effects of free and liposome encapsulated amikacin on the survival of Mycobacterium avium complex in mouse peritoneal macrophages, Tubercle 71 (3) (1990) 215–217. [49] S.J. Rose, et al. Delivery of aerosolized liposomal amikacin as a novel approach for the treatment of nontuberculous mycobacteria in an experimental model of pulmonary infection, PLoS One 9 (9) (2014) e108703. [50] K.N. Olivier, J.B. Grayson, E.M. Hersh, R.T. Dorr, S.M. Chiang, M. Oka, R.T. Proffitt, et al. Randomized trial of liposomal amikacin for inhalation in nontuberculous mycobacterial lung disease, Am J Respir Crit Care Med 195 (6) (2017) 814–823. [51] K. Yagi, et al. The efficacy, safety, and feasibility of inhaled amikacin for the treatment of difficult-totreat non-tuberculous mycobacterial lung diseases, BMC Infect Dis 17 (1) (2017) 558. [52] D.E. Griffith, et al. Amikacin liposome inhalation suspension for treatment-refractory lung disease caused by Mycobacterium avium complex (CONVERT): a prospective, open-label, randomized study, Am J Respir Crit Care Med 198 (12) (2018) 1559–1569. [53] I.I. Salem, N. Duzgunes, Efficacies of cyclodextrin-complexed and liposome-encapsulated clarithromycin against Mycobacterium avium complex infection in human macrophages, Int J Pharm 250 (2) (2003) 403–414. [54] A. De Soyza,T. Aksamit,T.J. Bandel, M. Criollo, J.S. Elborn, E. Operschall, et al. RESPIRE 1: a phase III placebo-controlled randomised trial of ciprofloxacin dry powder for inhalation in non-cystic fibrosis bronchiectasis, Eur Respir J 51 (1) (2018). [55] T. Aksamit, T.J. Bandel, M. Criollo, J.S. Elborn, E. Operschall, E. Polverino, et al. The RESPIRE trials: two phase III, randomized, multicentre, placebo-controlled trials of ciprofloxacin dry powder for inhalation (ciprofloxacin DPI) in non-cystic fibrosis bronchiectasis, Contemp Clin Trials 58 (2017) 78–85. [56] S. van Koningsbruggen-Rietschel, H.E. Heuer, N. Merkel, H.G. Posselt, D. Staab, C. Sieder, et al. Pharmacokinetics and safety of an 8 week continuous treatment with once-daily versus twice-daily inhalation of tobramycin in cystic fibrosis patients, J Antimicrob Chemother 71 (3) (2016) 711–717. [57] C. Nathan, M.U. Shiloh, Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens, Proc Natl Acad Sci USA 97 (16) (2000) 8841–8848. [58] J.D. MacMicking, R.J. North, R. LaCourse, J.S. Mudgett, S.K. Shah, C.F. Nathan, et al. Identification of nitric oxide synthase as a protective locus against tuberculosis, Proc Natl Acad Sci USA 94 (10) (1997) 5243–5248. [59] J. Chan, K. Tanaka, D. Carroll, J. Flynn, B.R. Bloo, et al. Effects of nitric oxide synthase inhibitors on murine infection with Mycobacterium tuberculosis, Infect Immun 63 (2) (1995) 736–740. [60] T. Akaki, K. Sato, T. Shimizu, C. Sano, H. Kajitani, S. Dekio, et al. Effector molecules in expression of the antimicrobial activity of macrophages against Mycobacterium avium complex: roles of reactive nitrogen intermediates, reactive oxygen intermediates, and free fatty acids, J Leukoc Biol 62 (6) (1997) 795–804. [61] T.R. Da Silva, J.R. De Freitas, Q.C. Silva, C.P. Figueira, E. Roxo, S.C. Leão, et al.Virulent Mycobacterium fortuitum restricts NO production by a gamma interferon-activated J774 cell line and phagosomelysosome fusion, Infect Immun 70 (10) (2002) 5628–5634. [62] S.K. Roach, J.S. Schorey, Differential regulation of the mitogen-activated protein kinases by pathogenic and nonpathogenic mycobacteria, Infect Immun 70 (6) (2002) 3040–3052. [63] B.E. Garcia-Perez, D.A.Villagómez-Palatto, J.I. Castañeda-Sánchez, R.M. Coral-Vázquez, I. RamírezSánchez, R.M. Ordoñez-Razo, et al. Innate response of human endothelial cells infected with mycobacteria, Immunobiology 216 (8) (2011) 925–935.

Drug discovery targeting drug-resistant nontuberculous mycobacteria

[64] V.J. Quesniaux, D.M. Nicolle, D. Torres, L. Kremer, Y. Guérardel, J. Nigou, et al. Toll-like receptor 2 (TLR2)-dependent-positive and TLR2-independent-negative regulation of proinflammatory cytokines by mycobacterial lipomannans, J Immunol 172 (7) (2004) 4425–4434. [65] P.H. Nibbering, S.I.Yoshida, M.T. van den Barselaar, R. van Furth, et al. Bacteriostatic activity of BCG/ PPD-activated macrophages against Mycobacterium fortuitum does not involve reactive nitrogen or oxygen intermediates, Scand J Immunol 40 (2) (1994) 187–194. [66] K. Tsuyuguchi, K. Suzuki, H. Matsumoto, E. Tanaka, R. Amitani, F. Kuze, et al. Effect of oestrogen on Mycobacterium avium complex pulmonary infection in mice, Clin Exp Immunol 123 (3) (2001) 428–434. [67] N. Thakur, A. Kumar, K.L. Dikshit, Type II flavohemoglobin of Mycobacterium smegmatis oxidizes dlactate and mediate electron transfer, Int J Biol Macromol 112 (2018) 868–875. [68] C. Sun, G. Yang, J. Yuan, X. Peng, C. Zhang, X. Zhai, et al. Mycobacterium tuberculosis hypoxic response protein 1 (Hrp1) augments the pro-inflammatory response and enhances the survival of Mycobacterium smegmatis in murine macrophages, J Med Microbiol 66 (7) (2017) 1033–1044. [69] P.M. Elks, S. Brizee, M. van der Vaart, S.R. Walmsley, F.J. van Eeden, et al. Hypoxia inducible factor signaling modulates susceptibility to mycobacterial infection via a nitric oxide dependent mechanism, PLoS Pathog 9 (12) (2013) e1003789. [70] S. Subbian, P.K. Mehta, S.L. Cirillo, L.E. Bermudez, J.D. Cirillo, et al. A Mycobacterium marinum mel2 mutant is defective for growth in macrophages that produce reactive oxygen and reactive nitrogen species, Infect Immun 75 (1) (2007) 127–134. [71] C.C. Miller, M. Rawat, T. Johnson, Y. Av-Gay, et al. Innate protection of Mycobacterium smegmatis against the antimicrobial activity of nitric oxide is provided by mycothiol, Antimicrob Agents Chemother 51 (9) (2007) 3364–3366. [72] C. Miller, B. McMullin, G. Regev, L. Serghides, K. Kain, et al. A phase I clinical study of inhaled nitric oxide in healthy adults, J Cyst Fibros 11 (4) (2012) 324–331. [73] K. Yaacoby-Bianu, M. Gur, Y. Toukan, V. Nir, F. Hakim, Y. Geffen, et al. Compassionate nitric oxide adjuvant treatment of persistent mycobacterium infection in cystic fibrosis patients, Pediatr Infect Dis J 37 (4) (2018) 336–338. [74] R.P. Howlin, K. Cathie, L. Hall-Stoodley,V. Cornelius, C. Duignan, R.N. Allan, et al. Low-dose nitric oxide as targeted anti-biofilm adjunctive therapy to treat chronic pseudomonas aeruginosa infection in cystic fibrosis, Mol Ther 25 (9) (2017) 2104–2116. [75] V. Le Moigne, J.L. Gaillard, J.L. Herrmann,Vaccine strategies against bacterial pathogens in cystic fibrosis patients, Med Mal Infect 46 (1) (2016) 4–9. [76] V. Romanus, et al. Atypical mycobacteria in extrapulmonary disease among children. Incidence in Sweden from 1969 to 1990, related to changing BCG-vaccination coverage, Tuber Lung Dis 76 (4) (1995) 300–310. [77] Idiopathic pulmonary fibrosis: diagnosis and treatment. International consensus statement. Am J Respir Crit Care Med 2000;161(2):646–664. [78] H.C. Poyntz, E. Stylianou, K.L. Griffiths, L. Marsay, A.M. Checkley, H. McShane, et al. Non-tuberculous mycobacteria have diverse effects on BCG efficacy against Mycobacterium tuberculosis, Tuberculosis (Edinb) 94 (3) (2014) 226–237. [79] I.M. Orme, F.M. Collins, Prophylactic effect in mice of BCG vaccination against nontuberculous mycobacterial infections, Tubercle 66 (2) (1985) 117–120. [80] A.G. Fraga,T.G. Martins, E.Torrado, K. Huygen, F. Portaels, M.T. Silva, et al. Cellular immunity confers transient protection in experimental Buruli ulcer following BCG or mycolactone-negative Mycobacterium ulcerans vaccination, PLoS One 7 (3) (2012) e33406. [81] C.S. Lindestam Arlehamn, et al. Immunological consequences of intragenus conservation of Mycobacterium tuberculosis T-cell epitopes, Proc Natl Acad Sci USA 112 (2) (2015) E147–E155. [82] R.N. Coler, S. Bertholet, M. Moutaftsi, J.A. Guderian, H.P. Windish, S.L. Baldwin, et al. Development and characterization of synthetic glucopyranosyl lipid adjuvant system as a vaccine adjuvant, PLoS One 6 (1) (2011) e16333. [83] R. Bieri, M Bolz, MT Ruf, G Pluschke, et al. Interferon-gamma is a crucial activator of early host immune defense against Mycobacterium ulcerans infection in mice, PLoS Negl Trop Dis 10 (2) (2016) e0004450.

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[84] B.E. Hart, L.P. Hale, S. Lee, Recombinant BCG expressing mycobacterium ulcerans Ag85A imparts enhanced protection against experimental Buruli ulcer, PLoS Negl Trop Dis 9 (9) (2015) e0004046. [85] IDRI-TBVPX-203. Available from: https://clinicaltrials.gov/ct2/show/NCT02465216?term=ID93 &rank=2. [86] A.M. Checkley, D.H. Wyllie, T.J. Scriba, T. Golubchik, A.V. Hill Hanekom, et al. Identification of antigens specific to non-tuberculous mycobacteria: the Mce family of proteins as a target of T cell immune responses, PLoS One 6 (10) (2011) e26434. [87] S.J. Lee, J.H. Jang, G.Y. Yoon, D.R. Kang, H.J. Park, S.J. Shin, et al. Mycobacterium abscessus d-alanyl-dalanine dipeptidase induces the maturation of dendritic cells and promotes Th1-biased immunity, BMB Rep 49 (10) (2016) 554–559.

CHAPTER 13

New strategies to combat drug resistance in bacteria Taru Singha, Sajad Ahmad Dara,b, Shukla Dasa, Shafiul Haqueb

Department of Microbiology, University College of Medical Sciences (University of Delhi) and GTB Hospital, Delhi, India b Research and Scientific Studies Unit, College of Nursing and Allied Health Sciences, Jazan University, Jazan, Saudi Arabia a

Appendices and Nomenclatures AGNPs  silver nanoparticles AHLs  acyl-homoserine lactones AMP  adenosine monophosphate AMR  antimicrobial resistance AuNPs  gold nanoparticles BGCs  biosynthetic gene clusters BFP  bundle-forming pilus DNA  deoxyribonucleic acid EPs  efflux pumps EPIs  efflux pump inhibitors et al.  at alia (all others) GMP  guanosine monophosphate GNR  Gram-negative rods HCI  high-content imaging HGT  horizontal gene transfer Ial  invasion-associated locus LEE  locus of enterocyte effacement mRNA  messenger ribonucleic acid MS  mass spectrometry NPs  nanoparticles NGS  next-generation sequencing OMP  outer membrane protein PCR  polymerase chain reaction QS  quorum sensing RPMA  reverse-phase protein microarray rRNA  ribosomal RNA sRNAs  small RNAs ST  heat-stable toxin SLT  shiga-like toxin STEC  shiga toxin-producing E. coli UNGA  United Nations General Assembly WGS  whole genome sequencing

Drug Discovery Targeting Drug-Resistant Bacteria. http://dx.doi.org/10.1016/B978-0-12-818480-6.00013-8 Copyright © 2020 Elsevier Inc. All rights reserved.

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1 Introduction Antimicrobial resistance (AMR) of enteric pathogen is of great public health concern in the developing world [1]. AMR has emerged as an important public health problem in clinical settings as well as in the community. It was one of the three main agendas in the United Nations General Assembly, 2016. With more and more organisms that are becoming resistant to the currently present antibiotics, the possibility of finding an alternative to these antibiotics is becoming a challenge [2]. Prolonged exposure to high levels of antibiotics may lead to the development of novel resistance mechanisms by providing the necessary selective pressure to increase latent intrinsic resistance. A positive correlation between antibiotic use and an increase in the number of antibiotic-resistant strains has been reported [3–7]. Antibiotic misuse can increase the number of deaths as predicted in Fig. 13.1.

1.1  Development of antimicrobial drugs After the discovery of antibiotics in the late 1920s, the widespread use of antimicrobial substances has been approved for the treatment of many infectious diseases. Many antimicrobials were described within a few years after the introduction of penicillin serendipitously in 1928 by Alexander Fleming. Time line of the discovery of different antibiotic classes is depicted in Fig. 13.2. Most antimicrobials were discovered within a few years after the introduction of penicillin in 1941—sulphonamide (1938), aminoglycoside (1944), cephalosporin (1945), chloramphenicol (1949), tetracycline (1950), macrolides (1952), glycopeptides (1956), rifamycin (1957), nitroimidazole (1959), quinolones

Figure 13.1  Deaths attributable to antimicrobial resistance every year by 2050. (Source: Data taken from: Antimicrobial resistance: tackling a crisis for the health and wealth of nations. The review on antimicrobial resistance chaired by Jim O’Neill; December 2014).

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Figure 13.2  Time line of the discovery of different antibiotic classes in clinical use. The period from 1987 until today was void as the last antibiotic class that has been successfully introduced as treatment was discovered in 1987. (Source: Modified from: Silver LL. Challenges of antibacterial discovery. Clin Microbiol Rev 2011;24:71–109).

(1962), and trimethoprim (1968) [8]. The discovery of penicillin has encountered many drawbacks, such as low yield, instability, and purification. The history of the introduction of the differently known classes of antimicrobials had started in the 1920s, and since 1980 scientific researchers have invested a lot in generating new kinds of antimicrobials that are active against harmful organisms. However, development of new antimicrobial drugs is still wanting [9]. The wide use of antibiotics resulted in the appearance of antibiotic-resistant strains in the 1940s and it led to the development of new evolutionary

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selective pressure on bacterial populations [5]. Such selective pressures have forced bacteria to respond by adaptation or, otherwise, death. Since the targets of antibiotics were conserved, in the process of their action susceptible strains were also killed, allowing an increase in the number of resistant strains. Some bacteria produce antibiotics naturally; therefore they possess intrinsic, low-level, nonspecific resistance to antibiotic substances. It has been suggested that these low-level mechanisms may signify the source of the antibiotic resistance determinants [10,11]. It can be proved by the fact that during the preantibiotic era, resistant bacterial strains were completely absent, and even that naturally occurring resistance was extremely low [12].

1.2  Spread of antibiotic resistance Antibiotic resistance development occurs by spontaneous mutation of chromosomal genes and through deoxyribonucleic acid (DNA) transfer via transposition and horizontal transfer of extrachromosomal mobile genetic elements [13,14]. Acquisition and transfer of antibiotic resistance determinants via horizontal gene transfer (HGT) through conjugation, transformation, and recombination events are the main cause of antibiotic resistance. It was found that resistance in Escherichia coli is mostly due to horizontal transfer of antibiotic resistance genes present on integrons [15,16]. Most of the resistant organism’s harbor antibiotic-resistance genes inserted into mobile genetic elements (plasmids, transposons, and integrons) in the form of gene cassettes [17]. Nonpathogenic bacteria could serve as a reservoir of resistance genes where the habitats act as platforms for genetic exchanges. The acquisition and spread of antibiotic resistance in bacteria may occur by various means [18]; for example, modification of existing genetic material or the acquisition of new genetic material from another source by the following means: (1) vertical gene transfers: the process of transfer of developed resistance (mutation frequency in the order of 108–109) directly to all the bacteria’s progeny during DNA replication is known as vertical gene transfer or vertical evolution; (2) HGTs: lateral or HGT is a process where genetic material contained in small packets of DNA can be transferred between bacteria of the same species or different species. Transduction, transformation, and conjugation are the three methods of HGT in bacteria. Characterization of AMR genes is a powerful tool for understanding various aspects of bacterial pathogens. For example, it can be used to study various mechanisms of their resistance, their mode of transmission in clinical as well as in healthy isolates, to examine population dynamics of isolates, and spatial and temporal distribution of resistance genes [19]. Nowadays, various molecular methods have been employed to characterize AMR genes in bacteria [19,20].

1.3  Types of antibiotic resistance Antibiotic resistance acquired by an organism can be divided into two types: natural or acquired [21].The natural resistance refers to the inherent ability of an organism to resist

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the effect of an antibiotic, for example, resistance of E. coli to penicillin G because there is no reaction site of penicillin G in its structure [22]. Acquired resistance is defined as the alteration in the genetic material of an organism to eliminate the effectiveness of drugs through mutations, for example, mutation in the gyrA gene of E. coli unable ciprofloxacin to bind to an essential bacterial enzyme required for DNA replication. This allows E. coli to continue to undergo DNA replication in the presence of ciprofloxacin [23]. Chromosomal mutations lead to structural changes in the bacterial cell, and transferable resistance codes for the enzymes that metabolize antimicrobials. Chromosomal resistance is a gradual process, while transferable resistance is a fast process. Antibiotics action can be bactericidal or bacteriostatic. There are many mechanisms by which antibiotic acts like by disrupting the integrity of the cellular membrane or by inhibiting the synthesis of bacterial wall components, bacterial proteins (tetracyclines, chloramphenicol, and macrolides), and nucleic acids (sulphonamides, rifamycin’s, and quinolones). There are many complex biochemical processes in the development of AMR which are controlled by multiple antibiotic-resistant genes. Resistance either affects antibiotic uptake or its action [24]. Modified cell permeability, enzymatic modification or destruction, target alteration or bypass, and efficient efflux pumps act as a barrier for the action of antibiotics [13].

1.4  Mechanisms of multidrug resistance Antibiotic resistance has rapidly increased in the last three decades with the appearance of superbugs in recent years [25]. Factors that facilitate the process of resistance are the physical linkage of plasmid-borne resistance genes in integrons and crossresistance [10,26]. Resistance to one antibiotic may be mediated by a large number of resistance genes in different locations of a genome and product of this particular gene may be responsible for developing resistance to a class of related or unrelated antibiotics. As a result of which, antibiotic resistance patterns of distribution cannot be traced easily in the absence of its genetic background [27]. Identical resistance patterns can be obtained by routine phenotypic methods but they cannot reflect the actual image of underlying resistance mechanisms [28]. In Enterobacteriaceae, particularly E. coli, integrons have been recognized as a prime agent for the development and spread of multidrug resistance [29–31]. Some studies expected that E. coli isolates from human and nonhuman hosts would present similar resistance profiles [12]. Characterization of resistance mechanisms can be easily understood through the use of molecular research tools. There are four general mechanisms of resistance known so far as shown in Fig. 13.3: 1. decreased concentration of intracellular drug 2. inactivation of drug 3. modification of drug target 4. target bypass

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Figure 13.3  Mechanisms of antibiotic resistance. (Source: Data taken from: https://www.reactgroup. org/toolbox/understand/antibiotic-resistance/resistance-mechanisms-in-bacteria/).

Increased efflux of an antibiotic from a bacterial cell can decrease the intracellular drug concentration, for example, E. coli’s tetracycline efflux system. Knowledge of the basic genetics of the microbial resistance enables us to understand the evolution and spread of resistance and hence it is suitable to start with the recognition of the different DNA elements that play a role in the evolution and spread of resistance. Plasmids that are extrachromosomal, self-replicating double-stranded DNA molecules, and depend on the selection of their hosts, encode an AMR gene under the presence of the relevant antimicrobial selective pressure [16]. There are also mobilizable (nonselftransmissible) plasmids that can transfer resistance in association with the conjugative (self-transmissible) plasmid [32]. Bacteriophages are defined as viruses infecting only a small number of strains of related bacteria (specific host range). They have a very narrow host that greatly limits their role as a vector transferring the resistance genes [32]. DNA sequences in transposons serve as recognition sites for transposase enzymes catalyzing their transfer, which is an extremely important mechanism for the natural transfer of antibiotic resistance genes from one bacterial replicon and recombination into another.

1.5  Antibiotics in the management of infections due to Gram-negative bacteria Bacteria are considered to be the most adaptable organisms that are capable of acquiring a vast competence to survive under unfavorable conditions. β-Lactam antibiotics are considered as the most successful antimicrobial agents in the treatments of Gram-negative bacterial infections. β-Lactamase enzymes hydrolyze (i.e., break open) the β-lactam

New strategies to combat drug resistance in bacteria

ring, destroying the antibacterial properties of the molecule [33,34]. Antibiotics are regularly used for empiric therapy of serious community-onset infections, such as the thirdgeneration cephalosporins or fluoroquinolones. This multiple drug resistance has major implications for selection of adequate empiric therapy regimens. Better knowledge of microbial resistance mechanisms is essential for the decision-making processes required for better management of the antibiotic resistance problem. Selecting an empiric treatment with an adequate activity against the infecting organism is the main barrier to the synthesis of new drug agents. Empirical antibiotic choices should be designed based on a person’s needs or antibiotic resistance pattern of a particular area or country. The lack of innovation in antibiotic discovery and increased reliance on existing antibiotics have contributed to increased prevalence of resistance and the reduced efficacy of existing treatment options.

2  Combating drug resistance Most of the strategies currently employed to develop new antibiotics point towards novel approaches for drug design based on prodrugs or rational design of new molecules. However, targeting crucial bacterial processes by these means will keep creating evolutionary pressure toward drug resistance. With the decreasing efficacy of antibiotics, hospitals around the world are seeing ever-increasing numbers of infections due to drug-resistant bacteria.

2.1  Antibiotic research in the age of omics Recent advanced developments in the fields of omics, namely, transcriptomics, genomics, proteomics, metabolomics, and image-omics (high-content imaging) have provided an unrivalled opportunity in identifying host–pathogen interactions and hence characterizing gene functions in bacteria. Importantly, technological advances related to assay miniaturization, high-throughput and automated image acquisition, and quantitative analysis have made it possible to extract hundreds of functional and morphological features that are associated with bacterial infections [35]. Analysis of the data derived from the omics studies will require bioinformatics tools that range from simple statistical analysis to sophisticated algorithms for large-scale analysis such as machine-learning approaches that will help one to assign functional and biological information to the data set. Combining of information and data generated from different omics platforms will help in providing robust mechanistic models of the bacteria and their interactions [36]. The introduction of omics technologies with bioinformatics will provide the details of key signaling pathways in the host–pathogen interaction and will help in the identification of host proteins for therapeutic targeting and the discovery of host-directed small molecules that will regulate bacterial infection. The details of these omics technologies are mentioned in Fig. 13.4.

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Figure 13.4  Steps of omics approach. (Source: Data taken from: Mack SG, Turner RL, Dwyer DJ. Achieving a predictive understanding of antimicrobial stress physiology through systems biology. Trends Microbiol 2018;26:296–312).

2.1.1 Transcriptomics The transcriptome is defined as the complete set of transcripts in a specific type of cell or tissue. In general, there are two strategies for determining a transcriptome. The term transcriptome can be applied to the total of all the transcripts in a given organism, or more often to the specific subset of transcripts present in a particular cell type. Unlike the genome, which is roughly fixed for a given cell line, the transcriptome can vary with external environmental conditions. Since it includes all messenger ribonucleic acid (mRNA) transcripts of the cell, it can clearly identify the genes that are being expressed either upregulation or downregulation in real time. Transcriptomics enables to detect the quantitative expression of the mRNA molecules in a particular organism and their variations at the genome scale [37,38]. Unfortunately, indigenous data about mRNA profiling and drug resistance in bacteria strains is very limited. However, such data is of immense importance in formulating hospital-based antimicrobial policy, surveillance, and treatment as well as in outbreak control. This lack of information may lead to inappropriate empiric. This approach of mRNA expression can demonstrate the exact prediction of a resistance gene not only its resistance phenotype that directly interferes with the treatment options in a clinical setting.Very few studies have been done on this aspect of antibiotic resistance [39–42]. 2.1.2 Proteomics Proteomics is defined as the study of the proteome (protein) and it involves technologies dealing with the identification and quantification of various proteins and their

New strategies to combat drug resistance in bacteria

interactions in an organism, and various other factors that affect protein activity [43]. Recent proteomic technologies enable scientists to screen large numbers of proteins within a time that helps to synthesize biomarkers for multiple diseases, identify, validate, design more drug targets, and to check drug efficacy. Proteomics is a multidisciplinary science that is required for safer, more effective, and more cost-effective future drugs target [44]. Proteomics studies (e.g., mass spectrometry and reverse-phase protein microarray) facilitate the characterization and quantitation of proteome changes from complex samples [45,46]. Proteomics can be used to identify biomarkers and identification of targets and hence, it facilitates computational drug design. 2.1.3 Genomics The increasing number of infections caused by antibiotic-resistant strains of pathogens challenges modern technologies of drug discovery. Synthesis or discovery of new ideal antibiotics is very much in demand due to increased resistance even to new-generation drugs. This necessity demands alternative high-throughput approaches to narrow down the most potential drug agents. In this regard, microfluidic technologies are of great interest due to their ability to screen at single cell level using ultrahigh-throughput screening, next-generation sequencing (NGS), and genome mining, thus opening up unique opportunities for antibiotic discovery. It is well established that foreign genes propagated in E. coli can have undesirable toxic effects, and this has been a long-standing problem for clone-based whole genome sequencing (WGS) approaches, where toxic genes cause gaps in sequence assemblies [47,48]. At the genome-scale the scope of expression of heterologous DNA in a bacterial host cell remains largely uncharacterized. The biological relevance of ubiquitous transcription is unknown. To quantify the heterogeneity and the biological significance of gene expression, quantitative methods are needed. The gene expression heterogeneity of a microbial community suggests that by simply harvesting mRNA from populations, the unique patterns of gene expression related to specific regions of the consortia or distinct functional subpopulations in the community might be lost. The establishment of new methods that enable gene expression study would be useful and appreciated. Metabolomics studies reveal metabolites (including lipids and small molecules) that are generated in response to infection [49]. Image-omics have been applied in host-directed therapeutic discovery to study the effects of perturbations in the bacterial infection cycle [50,51].

2.2  Bioinformatics and high-throughput screening for antibiotics discovery Novel microbial secondary metabolite, including previously unknown antibiotics with high potential for drug design, is one of the genome mining strategies that can be adapted for the identification of the potential drug. For example, de novo prediction of antibiotics is obtained by bioinformatics and gene clustering methods. Quorum sensing (QS) can be

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used for activation of silent genes in antibiotic-producing bacteria to find unknown antimicrobials. Although it is not an optimal mechanism due to its difficult prediction task, however, recombinant expression strategy can be used for the same with ease. 2.2.1  Genomics-based applications To combat multidrug-resistant bacterial infections, novel classes of antibiotics with new mechanisms of action are urgently required. There are numerous genomics-based applications for the identification and discovery of new antimicrobials, some of which are mentioned next: 1. Genomics-driven natural antiinfective discovery: Naturally occurring microorganisms exhibit a wide range of metabolites or synthetic analogs. These specialized metabolites possess distinguished biological activities having antibiotics, anticancer agents, and agrochemicals applications. Several of these metabolites have been approved as potential drugs to treat emerging health threats, such as daptomycin (to treat methicillin-resistant Staphylococcus aureus), fidaxomicin (for Clostridium difficile), and carfilzomib (for multiple myeloma). Bioinformatics-based predictions suggested that several were likely to encode products with novel structures. NGS and WGS technologies have provided a platform to explore the microbial genome of an organism that along with bioinformatics tools enabled biosynthetic gene clusters encoding specialized metabolites to be identified. 2. Molecular phylogenetics to guide new natural antiinfective discovery: Genomics approaches have helped us to display a clear picture of multiple cellular and molecular networks in systems biology such as metabolism, genetic interactions, protein–protein interactions (PPIs), and transcriptional regulation. Remarkably, it can be noted that these networks are conserved during evolution for different organisms of different species, which enables us to better define and understand mammalian molecular networks based on homology with their counterparts in the hierarchy. 3. Mining genomes for antiinfectives: Genome mining for novel natural products is quickly replacing traditional approaches to antibiotic discovery. Natural products and their derivatives contribute to a great number of clinical drugs. Future drug-resistant pathogens can be too identified and expressed by combing our vast genomics resources and innovative bioinformatics search with our validated genomes-to-drugs platform for natural products discovery.

2.3  Synthesis of nanomaterials World Health Organization and the Centre for Disease Control and Prevention have raised an alarming situation against multidrug-resistant bacteria. Over a while, bacteria have acquired multiple ways to become resistant such as decreased cell permeability, change in the target site, and increased efflux, although other phenomena such as biofilm formation and QS are induced by antibiotic exposure. Nanomaterials (metallic, organic,

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carbon nanotubes) can be used in the delivery of drugs [52]. Nanoparticles (NPs) are useful in effective delivery of the drug to the site of infection by binding them to their large surface area, their drug solubility and stability [53], their ease of synthesis [54,55], their biocompatibility with target with their multi-target action as compared to traditional antibiotics [56], and their modulated release [57]. NPs may be used to inhibit drug resistance mechanisms by inhibiting important processes in bacteria. Apart from that, combined use of antimicrobials and NPs is also being investigated. Strategies involved in nanotechnology are based on the type of NPs (gold, silver copper, etc.) with different sizes and shapes and their varied antimicrobial properties. Gold NPs are weakly bactericidal and nontoxic to the host [58,59]. Instead, silver NPs are more promising for the treatment of bacterial infections [60] with their specific shape of spheres and rods for Gram-positive and Gram-negative bacteria, respectively [61]. Although some studies also reported the development of bacterial resistance against silver NPs that may increase its antibiotic tolerance also [62,63], NPs perform their activity by multiple mechanisms such as disrupting the bacterial cell wall and inhibiting biofilm formation [64].

2.4  Phage therapy Antibiotic resistance is a slow process that is acquired through a change in bacterial genomes during evolution and cross-resistance to other antimicrobials as well. Phage therapy is an old strategy that is now gaining popularity. It utilizes lytic viruses as “drugs” that target and kill susceptible cells more efficiently by their capability of self-amplification, while lysogenic phage is used in certain biotechnology applications, for example, T4 phage bind to susceptible E. coli using tail fibers [65]. Some phages require a virulence factor to attach to and infect a bacterium that further may lead to the development of antibiotic resistance [66]. The introduction of phage therapy in combination with drugs can be proved to be a suitable option to tackle resistance and needs to be validated by both in vitro and in vivo studies [67]. Apart from the bactericidal lytic cycle of phage, they also play a vital role in essential cellular processes of central dogma [68]. Phage can be employed for a single bacterial species or even to a strain making them an ideal therapeutic to selectively target and killing pathogens [68].Treatment of biofilms by phages may prove to be more promising [69]. Phage therapy can be used in a wide number of bacterial infections such as gastrointestinal infections where they reduce or prevent the colonization of virulent bacteria without disrupting the natural gut flora [70]. There are some limitations of phage that should be addressed before its wide use. Phage can be present everywhere and neutralizing antibodies against certain phage involving the human microbiome appears as a hurdle for the phage therapy. Phage cocktails are one of the approaches involving combining multiple phages that can be adapted to expand the phage host range.

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2.5  Molecular targets and strategies to combat drug resistance in bacteria Bacterial resistance has emerged over the period to many commonly used antibiotics, including ampicillin, vancomycin, and even to last-resort antibiotics, such as linezolid [71]. Over- and misuse of antibiotics for various infectious diseases are the main drivers of resistance apart from their use in the food industry or in animals either for infection prevention or growth is an additional reason for increased resistance [72]. Developed drug resistance is exerting an extra burden on the scientific community to develop novel antibiotics because broad-spectrum antibiotics eradicate the normal gut microbiota that decreases the thickness of the protective intestinal mucus layer. This allows for specific outgrowth of antibiotic-resistant bacteria [73]. After the emergence of bacterial resistance, several approaches have been developed to fight against bacterial infections. Each of these strategies targets different sites of bacteria, for example, some of them focus on the bacterial cell wall, the cell membrane, or essential bacterial enzymes (same sites as that of first-generation antibiotics), although second-, third-, or fourth-generation antibiotics also possess the same mechanism of action but with improved pharmacological properties. This is a matter of great concern as the most common site of action of antibiotics is directly related to essential bacterial processes; however, combinations of single-target antimicrobials are a common mode of treatment for Mycobacterium tuberculosis [74]. The finding of novel mechanisms or novel target sites in bacteria is a cumbersome process as the new target has to be present in a specific spectrum of bacteria and it must be absent in humans in any homolog form, although finding a new target or drug is less successful as compared to modification of the classic antibiotics using various tools [75]. However, old target sites mainly related to the essential bacterial processes and bacteria will adapt to a new environment due to adaptation pressure.Treatment against bacterial resistance can be achieved by two mechanisms—either by targeting or altering their essential cellular processes or by targeting nonessential processes. 2.5.1  Targeting essential processes New possibilities for the development of antibacterial drugs have increased by targeting pathways and processes that are vital for the survival of bacteria. Keeping this in mind, various new strategies and techniques have been deployed that are summarized next: 1. First-generation antibiotics and their analogs: Since first-generation antibiotics were synthesized by targeting important pathways or processes of bacteria, hence, chemical and structural modification of conventional drugs will provide the most efficient way to develop novel drugs against resistant bacteria [76]. For example, rifamycin of the family of ansamycin proved to be effective [77,78]. New antimicrobial agents vary in their antibacterial activities and also their targets in a bacterium.There seems to be a long gap between the activity of a drug in laboratory and

New strategies to combat drug resistance in bacteria

in the clinic. Some of the new identified antibacterial agents include vancomycin analogs, teixobactin, lipopeptides, oxazolidinones, and toxins.Tedizolid and linezolid are good examples of oxazolidinone which function by interfering with the bacterial ribosome [79]. Some variants of vancomycin such as telavancin act by disrupting the membrane integrity [80]. Oritavancin is also structurally similar to vancomycin and it interferes with cell wall biosynthesis of bacteria by binding to the 23S ribosomal RNA (rRNA) of the bacterial ribosome [81,82]. All these above mentioned drugs have a strong synergistic effect with methicillin and not with vancomycin [83].This provides new possibilities for future drug research. Till now, daptomycin is the only lipopeptide antibiotic used clinically which works by disrupting bacterial cell wall and hence lysis [84]. Surotomycin is structurally similar to daptomycin, with the presence of an aromatic ring [85]. 2. Development of inhibitors: Development of inhibitors requires the synthesis of either substrate or product analogs that can not only bind but also turn off the key enzymes in the cell. At higher concentration, vitamin C proved to be a good inhibitor of M. tuberculosis [86]. This property of vitamin C opens up windows for other bacterial infections as well. Bacteria require nucleotide derivatives such as cyclic adenosine monophosphate (AMP), cyclic-di-guanosine monophosphate (GMP), cyclic-di-AMP, and cyclic GMP-AMP as signaling molecules that are activated under particular stress conditions [87]. These signaling molecules regulate several important pathways such as cell division, QS, virulence, biofilm formation, motility, and antibiotic resistance required for normal growth and maintenance. Interrupting these signaling pathways would lead to poor bacterial survival and in the future, more research should be undertaken to develop inhibitors of these signaling nucleotides. Therefore structural analogs of these compounds or effectors, which can be either proteins or RNA, could work as effective inhibitors. For example, sortase transpeptidase inhibitors (targeting the sortase enzyme), alanine racemase inhibitors, and lipoteichoic acid synthesis inhibitors (targeting LtaS). 3. Role of efflux pumps: Efflux pumps (EPs) have identified as principal determinants of bacterial resistance. In Gram-negative bacteria, outer membranes serve as permeability barrier for druginflux but not in Gram-positive bacteria and hence they show more resistance [88]. Gram-negative bacteria show resistance to multiple antibiotics by lowering their outer membrane permeability that can be obtained by reducing the number of porins and inducing EPs [89]. Apart from EPs key role in combating drug resistance, they are also involved in other physiological processes such as virulence, pathogenicity, transportation of nutrients, and adaptation to stress [90]. In order to restore the mechanism of the current antibiotics, synthesis of potent EP inhibitors is in demand [91]. 4. Prodrugs and antibiotic derivatives: Prodrugs are defined as the inactive derivatives of a known active molecule or drug that have been chemically modified by altering their pharmacological activity. Multiple

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changes can be done in the properties of actual molecule (without compromising its antimicrobial activity) like interfering with their permeability, bioavailability, absorption, and addition or removal of parts of its structure [92]. Prodrug further requires incorporation in the body in order to convert it into active form, for example, pivampicillin, a derivative of ampicillin, was one of the first prepared prodrug [93]. Another useful antibiotic derivative is to combine two active molecules by a linker, for example, β-lactam antibiotics are linked with their inhibitors [94], sultamicillin provided a link between ampicillin and sulbactam by a methylene group that enhances ampicillins microbial activity by multiple folds [95]. 5. Riboswitches: Targeting riboswitches would help one to combat drug-resistant infections in the near future. A riboswitch is defined as a regulatory segment of an mRNA molecule that binds a small molecule, resulting in a change in production of the proteins encoded by the mRNA. Most of the antibiotics act by targeting protein translation that involves ribosomal proteins and rRNA. Riboswitches can act as potential drug targets because of their presence in most of the bacteria such as M. tuberculosis and Vibrio cholerae but not in mammals [96]. Riboswitches play an important role in regulation of gene expression at the transcriptional or translation level by regulating important cellular pathways, especially metabolic pathways [96]. 6. Small RNAs (sRNAs): sRNAs are defined as the regulatory noncoding RNAs that are 40–500 nucleotides in length [97]. They are involved in regulation (either positive or negative) of gene expression at translation level. Although not essential, they regulate pathways associated mainly with biofilm formation, virulence, and antibiotic resistance [98]. Because of limited structural complexity of sRNAs, it is difficult to design inhibitors for them. 7. Antibiofilm peptides: Biofilms are defined as the communities of microorganisms that attach to each other and to the surfaces by bacterial adherence. They are one of the adaptation mechanisms of bacteria toward stress and eradicating biofilm would help one in combating bacterial resistance [99]. Antibiofilm peptides are one of the class of inhibitors against biofilms and most of them are broad-spectrum [100]. The main advantage of these peptides is that they are capable of removing mature biofilms and they act in synergism with conventional antibiotics. Moreover, peptide sequences can be designed to target key proteins crucial for virulence and persistence in bacteria. 8. Protein epitopes: Drug designing has become easy with the introduction of protein epitopes. PPIs were one of the early pathways that were targeted by protein epitope mimetic design [101]. ß-Hairpin-shaped peptides are also significantly used in the field of antibiotic research highlighting its important role in outer membrane biogenesis of bacteria. Recently, naturally occurring ß-hairpin peptidomimetics and ß-hairpin-shaped peptides

New strategies to combat drug resistance in bacteria

have also been discovered with novel mechanisms of action against bacteria targeting outer membrane protein. 9. Combinatorial chemistry and high-throughput screening: A vast number of molecules that are structurally related can be produced by a quick and random combinatorial way [102]. There are multiple molecules that share a common basic structure and by substituting different components of the structure, we can check how a particular component affects the activity of the molecule [103,104]. It then uses a computer-based program that analyzes and chooses the best suited molecule from the library according to the specific requirements. After that the selected molecules are artificially synthesized to determine their physicochemical properties. This synthesized molecule is then studied in detail to obtain a suitable drug candidate that later will be investigated in in vitro and in vivo studies. 10. Use of bioinformatics and rational design: Rational design is defined as a tool used to create new molecules with specific characteristics based on how the molecule’s structure will affect its behavior. For example, linezolid an alternative treatment for vancomycin-resistant bacteria [105]. 11. Immunomodulation: Synthesis of peptides targeting immunomodulation also offers a suitable platform for drug designing by targeting the host, rather than the pathogen. Therefore it decreases the selective pressure for the evolution of microbial resistance. Designed peptides have a nonspecific nature and could be used as broad-spectrum protection against a range of microbial pathogens. 2.5.2  Targeting nonessential processes Apart from the essential processes within a bacterium, understanding of various nonessential processes such as bacterial adhesion, host–pathogen interactions, and bacterial communication during an infection provides new and advanced opportunities for the development of ideal drug candidate. Development of drug resistance during nonessential bacterial processes is reduced due to the decreased evolutionary pressure for adaptation [106].Various nonessential processes occurring within bacteria are discussed next: 1. Bacterial communication: QS—in bacteria small hormone-like molecules called autoinducers are necessary for chemical communication between large groups of cells [107]. This chemical communication can be intraspecies or interspecies that helps in evolution of bacteria with its host cell [106]. QS is one of the main mechanisms by which bacteria communicate. Moreover, QS also facilitates bacteria to move to a more suitable environment or can also enhance biofilm production when required. Therefore it can be concluded that QS gives bacteria power to behave as multicellular organism. QS in Gram-negative bacteria depends on derivatives of acyl-homoserine lactones [106,108,109] or other molecules that can diffuse freely through the bacterial membrane, while Gram-positive bacteria

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typically uses oligopeptides. Unlike in Gram-positive bacteria, oligopeptide autoinducers are a variation of a single core molecule in Gram-negative bacteria and thus each species of bacteria is capable of producing a peptide signal with a unique sequence [110]. Minimum selective pressure and least resistance can be developed by these unique sequenced peptides as QS does not involve essential processes of a bacterium [106]. This ability of QS serves as an ideal platform to develop narrow-spectrum drugs. Cell communication can be interrupted by hijacking of autoinducers, thereby, slowing down the onset of the disease [110]. To reduce infections caused by biofilms around the devices in hospital settings, systemic antibiotic prophylaxis as well as local administration of antimicrobial agents are given [111]. 2. Host–pathogen interactions: Host–pathogen interactions are defined as the phenomenon by which microbes or viruses sustain themselves within host organisms on a molecular, cellular, organismal, or population level [112]. The pathogenicity of an organism depends on its genetic material, either chromosomal and plasmid DNA, while some bacteria possess genes encoding for toxins or virulence factors, and also on proteins involved in cell adhesion or colonization. 3. Cell adhesion and colonization: Phenomenon like host cells adhesion of bacterial pathogens and colonization are the important drivers of the establishment of bacterial infection in the host cell. In Gramnegative bacteria, attachment of cell receptors to the host cell membrane and their effacement is one of the first steps toward disease onset. Based on the activity, structure, and composition, several classes of bacterial secretion systems have been identified with Gram-negative bacteria, four for Gram-positive bacteria, and also two common systems [113]. Pili are one of the crucial structural parts of bacteria which facilitate cell adhesion and colonization [114,115]. 4. Virulence factors and toxins: Virulence factors and toxins are encoded by bacterial DNA. Apart from QS, development of small monoclonal antibodies and peptides are also being studied for the synthesis of new drugs [116,117]. Till now, only three monoclonal antibodies have been licensed such as palivizumab, obiltoxaximab, and raxibacumab for the treatment of respiratory syncytial virus in high-risk infants, for prophylaxis and treatment of anthrax, respectively [118–120]. As these antibodies have no direct antibacterial activity, the selective pressure for resistance to appear should be low.Therapeutic antibacterial antibodies can either bind to the pathogen or can bind to toxins or other virulence factors to neutralize them and giving the host a chance to clear the infection immunologically. The antibodies against toxins are currently undergoing clinical trials, for example, shiga toxin [121,122]. 5. Signaling: Bacterial signaling plays an important role in the occurrence of any infection by deactivating the host immune system. Naturally occurring cell death pathways are

New strategies to combat drug resistance in bacteria

disrupted by the pathogen [123], and other host cell pathways are arrested or modulated by the bacteria during the progression of infection [124]. Alternative for the successful treatment of bacterial infection is to target the bacterial proteins or the interaction of the bacteria and the host proteins. 6. Small ribosomal subunit: Most of the clinically relevant or conventional antibiotics target the ribosome because of their high chemical variability. Although most of these antibiotics bind to the small ribosomal subunit, only a few have been reported to inhibit the process of protein synthesis till now, limiting their availability commercially. The initiation step of protein synthesis has a great potential for drug designing because of its higher variability of protein factors in prokaryotes and eukaryotes, and because of no common resistance pattern to antibiotics targeting this step.

3  Conclusion and future perspectives Traditional and conventional antibiotics work by interfering with essential cellular processes of the bacteria, causing increased selective pressure toward the development of drug resistance. During the last decade, most of the strategies and technologies to combat antibiotic resistance were mainly focussed on the inactivation of bacteria by multiple ways such as neutralizing their toxins and inhibiting PPI, instead of killing them. New strategies using various omics techniques, essential and nonessential bacterial pathways, and host-bacterial interactions, which are based on the detailed understanding of the molecular and cellular bacterial pathways, have the potential to contribute in the development of new drug candidates. The nonessential bacterial processes experience a very low evolution and selection pressure, hence, can provide a new generation of drugs with a long life that may take decades to develop resistance and can fulfill the current antibiotic crisis. Bacterial cell wall biosynthesis pathway is also an attractive target in the current scenario, as it is essential for bacteria but do not exist in human host.

References [1] M.F. Coker, S. Berky, C. Pandou, New development in acute diarrhoea current problem, Paediatrics 24 (1998) 15–17. [2] M.L. Cohen, Antimicrobial resistance: prognosis for public health,Trends Microbiol 2 (1994) 422–425. [3] D.J. Austin, K.G. Kristinsson, R.M. Anderson, The relationship between the volume of antimicrobial consumption in human communities and the frequency of resistance, Proc Natl Acad Sci USA 96 (1999) 1152–1156. [4] M.H. Kollef, V.J. Fraser, Antibiotic resistance in the intensive care unit, Ann Intern Med 134 (2001) 298–314. [5] I. Gustafsson, M. Sjölund, E.Torell, M. Johannesson, L. Engstrand, O. Cars, et al. Bacteria with increased mutation frequency and antibiotic resistance are enriched in the commensal flora of patients with high antibiotic usage, J Antimicrob Chemother 52 (2003) 645–650. [6] H. Goossens, M. Ferech, R. Vander Stichele, M. Elseviers, ESAC Project GroupOutpatient antibiotic use in Europe and association with resistance: a cross-national database study, Lancet 365 (2005) 579–587.

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Drug Discovery Targeting Drug-Resistant Bacteria

[7] J. Davies, D. Davies, Origins and evolution of antibiotic resistance, Microbiol Mol Biol Rev 74 (2010) 417–433. [8] J. Conly, B. Johnston, Where are all the new antibiotics? The new antibiotic paradox, Can J Infect Dis Med Microbiol 16 (2005) 159–160. [9] R.P. Rapp, Pharmacy infectious diseases practices, Ann Pharma 40 (2006) 304–306. [10] D.A. Rowe-Magnus, D. Mazel, Resistance gene capture, Curr Opin Microbiol 2 (1999) 483–488. [11] F. Van-Bambeke, Y. Glupczynski, P. Plesiat, J.C. Pechere, P.M. Tulkens, Antibiotic efflux pumps in prokaryotic cells: occurrence, impact on resistance and strategies for the future of antimicrobial therapy, J Antimicrob Chemother 51 (2003) 1055–1065. [12] T. Houndt, H. Ochman, Long-term shifts in patterns of antibiotic resistance in enteric bacteria, Appl Environ Microbiol 66 (2000) 5406–5409. [13] K. Poole, Mechanisms of bacterial biocide and antibiotic resistance, J Appl Microbiol 31 (2002) 55S–64S. [14] D.M. Livermore, Bacterial resistance: origins, epidemiology, and impact, Clin Infect Dis 36 (2003) S11–S23. [15] M. Sherley, D.M. Gordon, P.J. Collignon, Evolution of multi-resistance plasmids in Australian clinical isolates of Escherichia coli, Microbiology 150 (2004) 1539–1546. [16] A. Carattoli, Resistance plasmid families in Enterobacteriaceae, Antimicrob Agents Chemother 53 (2009) 2227–2238. [17] A. Alonso, P. Sanchez, J.L. Martinez, Environmental selection of antibiotic resistance genes, Environ Microbiol 3 (2001) 1–9. [18] D.S. Toder, M.J. Gambello, B. Iglewski, Bacterial resistance to antibiotics in toddlers, Textbook of bacteriology, University of Wisconsin, Medison, (2008). [19] S. Chen, S. Zhao, D.G. White, C.M. Schroeder, R. Lu, H. Yang, et al. Characterization of multipleantimicrobial-resistant Salmonella serovars isolated from retail meats, Appl Environ Microbiol 70 (2004) 1–7. [20] D. Boyd, A. Cloeckaert, E. Chaslus-Dancla, M.R. Mulvey, Characterization of variant Salmonella genomic island 1 multidrug resistance regions from serovars Typhimurium DT104 and Agona, Antimicrob Agents Chemother 46 (2002) 1714–1722. [21] K. Todar, Antimicrobial agents used in the treatment of infectious disease, Textbook of bacteriology, University of Wisconsin, Madison, 2002, pp. 1–11. [22] J.Y. Je, S.K. Kim, Antimicrobial action of novel chitin derivative, Biochim Biophys Acta 1760 (2006) 104–109. [23] C.N. Rodriguez, A.J. Rodriguez-Morales, A. Garcia, B. Pastran, P. Meijomil, R.A. Barbella, et al. Quinolone antimicrobial resistance in some enterobacteria: a 10-year study in a Venezuelan general hospital, Int J Antimicrob Agents 25 (2005) 546–550. [24] E.S. Azucena, S. Mobashery, Aminoglycoside-modifying enzymes: mechanisms of catalytic processes and inhibition, Drug Resist Updat 4 (2001) 106–117. [25] P. Nordmann, T. Naas, N. Fortineau, L. Poirel, Superbugs in the coming new decade; multidrug resistance and prospects for treatment of Staphylococcus aureus, Enterococcus spp. and Pseudomonas aeruginosa in 2010, Curr Opin Microbiol 10 (2007) 436–440. [26] G.D. Recchia, R.M. Hall, Gene cassettes: a new class of mobile element, Microbiology 141 (1995) 3015–3027. [27] I. Phillips, M. Casewell,T. Cox, B. De Groot, C. Friis, R. Jones, et al. Does the use of antibiotics in food animals pose a risk to human health? A critical review of published data, J Antimicrob Chemother 53 (2004) 28–52. [28] A.C. Fluit, M.R. Visser, F.J. Schmitz, Molecular detection of antimicrobial resistance, Clin Microbiol Rev 14 (2001) 836–871. [29] R.M. Hall, C.M. Collis, Antibiotic resistance in gram-negative bacteria: the role of gene cassettes and integrons, Drug Resist Updat 1 (1998) 109–119. [30] P.A. White, C.J. McIver, W.D. Rawlinson, Integrons and gene cassettes in the Enterobacteriaceae, Antimicrob Agents Chemother 45 (2001) 2658–2661. [31] M. Rijavec, M.S. Erjavec, J.A. Avguštin, R. Reissbrodt, A. Fruth, V. Križan-Hergouth, et al. High prevalence of multidrug resistance and random distribution of mobile genetic elements among uropathogenic Escherichia coli (UPEC) of the four major phylogenetic groups, Curr Microbiol 53 (2006) 158–162.

References

[32] A. Carattoli, Importance of integrons in the diffusion of resistance,Vet Res 32 (2001) 243–259. [33] J.K. Rasheed, F. Cockerill, F.C Tenover, Detection and characterization of antimicrobial resistance genes in pathogenic bacteria, In Murray P.R. Murray, E.J. Baron, J.H. Jorgensen, M.L. Landry, M.A. Pfaller (Eds.), Manual of clinical microbiology, 9th ed. vol. 1, American Society for Microbiology, Washington, DC 67, 2007 1250-120. [34] G.V. Sanchez, R.N. Master, R.B. Clark, M. Fyyaz, P. Duvvuri, G. Ekta, et al. Klebsiella pneumoniae antimicrobial drug resistance, United States, 1998–2010, Emerg Infect Dis 19 (2013) 133–136, doi: 10.3201/eid1901.120310. [35] M.A. Bray, S. Singh, H. Han, C.T. Davis, B. Borgeson, C. Hartland, et al. Cell painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes, Nat Protoc 11 (2016) 1757–1774, doi: 10.1038/nprot.2016.105. [36] C.S. Poultney, A. Greenfield, R. Bonneau, Integrated inference and analysis of regulatory networks from multi-level measurements, Methods Cell Biol 110 (2012) 19–56 https://doi.org/10.1016/B9780-12-388403-9.00002-3. [37] Q. Leroy, D. Raoult, Review of microarray studies for host intracellular pathogen interactions, J Microbiol Methods 81 (2010) 81–95 https://doi.org/10.1016/j.mimet.2010.02.008. [38] A.J. Westermann, S.A. Gorski, J. Vogel, Dual RNA-seq of pathogen and host, Nat Rev Microbiol 10 (2012) 618–630 https://doi.org/10.1038/nrmicro 2852. [39] N. Nagano, Y. Nagano, C. Cordevant, N. Shibata, Y. Arakawa, Nosocomial transmission of CTX-M-2 b-lactamase-producing Acinetobacter baumannii in a neurosurgery ward, J Clin Microbiol 42 (2004) 3978–3984. [40] J.A. Di Conza, G.O. Gutkind, M.E. Mollerach, J.A. Ayala, Transcriptional analysis of the blaCTX-M-2 gene in Salmonella enterica serovar infantis, Antimicrob Agents Chemother 49 (2005) 3014–3017. [41] M.J. Casadaban, Regulation of the regulatory gene for the arabinose pathway, araC, J Mol Biol 104 (1976) 557–566. [42] S.E. Chuang, D.L. Daniels, F.R. Blattner, Global regulation of gene expression in Escherichia coli, J Bacteriol 175 (1993) 2026–2036. [43] R.M. Hewick, Z. Lu, J.H.Wang, Proteomics in drug discovery, Adv Protein Chem 65 (2003) 309–342. [44] J. Burbaum, G.M. Tobal, Proteomics in drug discovery, Curr Opin Chem Biol 6 (2002) 427–433. [45] P.M. Jean Beltran, J.D. Federspiel, X. Sheng, I.M. Cristea, Proteomics and integrative omic approaches for understanding host-pathogen interactions and infectious diseases, Mol Syst Biol 13 (2017) 922, doi: 10.15252/msb.20167062. [46] C.Y. Chiang, I. Uzoma, D.J. Lane,V. Memiševic, F. Alem, K.Yao, et al. A reverse-phase protein microarray-based screen identifies host signaling dynamics upon Burkholderia spp. infection, Front Microbiol 6 (2015) 683, doi: 10.3389/fmicb.2015.00683. [47] R.A. Holt, R.L. Warren, S. Flibotte, P.I. Missirlis, D.E. Smailus, Rebuilding microbial genomes, BioEssays 29 (2007) 580–590. [48] R. Sorek, Y. Zhu, C.J. Creevey, M.P. Francino, P. Bork, E.M. Rubin, Genome-wide experimental determination of barriers to horizontal gene transfer, Science 318 (2007) 1449–1452. [49] W. Eisenreich, J. Heesemann, T. Rudel, W. Goebel, Metabolic host responses to infection by intracellular bacterial pathogens, Front Cell Infect Microbiol 3 (2013) 24, doi: 10.3389/fcimb.2013.00024. [50] C.Y. Chiang, R.L. Ulrich, M.P. Ulrich, B. Eaton, J. Ojeda, F.D.J. Lane, et al. Characterization of the murine macrophage response to infection with virulent and avirulent Burkholderia species, BMC Microbiol 15 (2015) 259, doi: 10.1186/s12866-015-0593-3. [51] A. Casanova, S.H. Low, M. Emmenlauer, R. Conde-Alvarez, S.P. Salcedo, J.P. Gorvel, et al. Microscopybased assays for high-throughput screening of host factors involved in Brucella infection of Hela cells, J Vis Exp 5 (2016)doi: 10.3791/54263. [52] L. Wang, C. Hu, L. Shao, The antimicrobial activity of nanoparticles: present situation and prospects for the future, Int J Nanomed 12 (2017) 1227–1249, doi: 10.2147/IJN.S121956. [53] A.J. Huh, Y.J. Kwon, “Nanoantibiotics”: a new paradigm for treating infectious diseases using nanomaterials in the antibiotic’s resistant era, J Control Release 156 (2011) 128–145, doi: 10.1016/j.jconrel.2011.07.002. [54] D. Pissuwan, T. Niidome, M. Cortie, The forthcoming applications of gold nanoparticles in drug and gene delivery systems, J Control Release 149 (2011) 65–71, doi: 10.1016/j.jconrel.2009.12.006.

395

396

Drug Discovery Targeting Drug-Resistant Bacteria

[55] M. Gholipourmalekabadi, M. Mobaraki, M. Ghaffari, A. Zarebkohan, V. Omrani, A. Urbanska, et al. Targeted drug delivery based on gold nanoparticle derivatives, Curr Pharm Des 23 (2017) 2918–2929, doi: 10.2174/1381612823666170419105413. [56] P. Jagtap,V. Sritharan, S. Gupta, Nanotheranostic approaches for management of bloodstream bacterial infections, Nanomedicine 13 (2017) 329–341, doi: 10.1016/j.nano.2016.09.005. [57] Z. Wang, K. Dong, Z. Liu,Y. Zhang, Z. Chen, H. Sun, et al. Activation of biologically relevant levels of reactive oxygen species by Au/g-C3N4 hybrid nanozyme for bacteria killing and wound disinfection, Biomaterials 113 (2017) 145–157, doi: 10.1016/j.biomaterials.2016.10.041. [58] T.P. Shareena Dasari, Y. Zhang, H. Yu, Antibacterial activity and cytotoxicity of gold (I) and (III) ions and gold nanoparticles, Biochem Pharmacol 4 (2015) 199, doi: 10.4172/2167-0501.1000199. [59] U. Rajchakit, V. Sarojini, Recent developments in antimicrobial-peptide-conjugated gold nanoparticles, Bioconjug Chem 28 (2017) 2673–2686, doi: 10.1021/acs.bioconjchem.7b00368. [60] M. Natan, E. Banin, From nano to micro: using nanotechnology to combat microorganisms and their multidrug resistance, FEMS Microbiol Rev 41 (2017) 302–322, doi: 10.1093/femsre/fux00. [61] D. Acharya, K.M. Singha, P. Pandey, B. Mohanta, J. Rajkumari, L.P. Singha, Shape dependent physical mutilation and lethal effects of silver nanoparticles on bacteria, Sci Rep 8 (2018) 201, doi: 10.1038/ s41598-017-18590-6. [62] C. Kaweeteerawat, P. Na Ubol, S. Sangmuang, S. Aueviriyavit, R. Maniratanachote, Mechanisms of antibiotic resistance in bacteria mediated by silver nanoparticles, J Toxicol Environ Health A 80 (2017) 1276–1289, doi: 10.1080/15287394.2017.1376727. [63] S. Kulshrestha, S. Qayyum, A.U. Khan, Antibiofilm efficacy of green synthesized graphene oxidesilver nanocomposite using Lagerstroemia speciosa floral extract: a comparative study on inhibition of Gram-positive and Gram-negative biofilms, Microb Pathog 103 (2017) 167–177, doi: 10.1016/j.micpath.2016.12.022. [64] A. Panácˇek, M. Smékalová, R.Vecˇerˇová, K. Bogdanová, M. Röderová, M. Kolár, et al. Silver nanoparticles strongly enhance and restore bactericidal activity of inactive antibiotics against multiresistant Enterobacteriaceae, Colloids Surf B Biointerfaces 142 (2016) 392–399, doi: 10.1016/j.colsurfb.2016.03.007. [65] H. Furukawa, S. Mizushima, Roles of cell surface components of Escherichia coli K-12 in bacteriophage T4 infection: interaction of tail core with phospholipids, J Bacteriol 150 (1982) 916–924. [66] E. Geisinger, R.R. Isberg, Antibiotic modulation of capsular exopolysaccharide and virulence in Acinetobacter baumannii, PLoS Pathog 11 (2015) e1004691, doi: 10.1371/journal.ppat.1004691. [67] W.E. Huff, G.R. Huff, N.C. Rath, J.M. Balog, A.M. Donoghue, Therapeutic efficacy of bacteriophage and Baytril (enrofloxacin) individually and in combination to treat colibacillosis in broilers, Poult Sci 83 (2004) 1944–1947. [68] C. Loc-Carrillo, S.T. Abedon, Pros and cons of phage therapy, Bacteriophage 1 (2011) 111–114. [69] B.K. Chan, P.E. Turner, S. Kim, H.R. Mojibian, J.A. Elefteriades, D. Narayan, Phage treatment of an aortic graft infected with Pseudomonas aeruginosa, Evol Med Public Health 2018 (2018) 60–66, doi: 10.1093/emph/eoy005. [70] M. Galtier, L. De Sordi, A. Sivignon, A. de Vallée, D. Maura, C. Neut, et al. Bacteriophages targeting adherent invasive Escherichia coli strains as a promising new treatment for Crohn’s disease, J Crohns Colitis 11 (2017) 840–847, doi: 10.1093/ecco-jcc/jjw224. [71] J.K. Long, T.K. Choueiri, G.S. Hall, R.K. Avery, M.A. Sekeres, Daptomycin-resistant Enterococcus faecium in a patient with acute myeloid leukemia, Mayo Clin Proc 80 (2016) 1215–1216. [72] D.I. Andersson, D. Hughes, Microbiological effects of sublethal levels of antibiotics, Nat Rev Microbiol 12 (2014) 465–478. [73] C. Ubeda, Vancomycin-resistant Enterococcus domination of intestinal microbiota is enabled by antibiotic treatment in mice and precedes blood stream invasion in humans, J Clin Invest 120 (2010) 4332–4341. [74] L.L. Silver, Multi-targeting by monotherapeutic antibacterials, Nat Rev Drug Discov 6 (2007) 41–56. [75] N. Kardos, A.L. Demain, Penicillin: the medicine with the greatest impact on therapeutic outcomes, Appl Microbiol Biotechnol 92 (2011) 677. [76] J. Fernebro, Fighting bacterial infections—future treatment options, Drug Resist Updat 14 (2011) 125–139. [77] K.L.J. Rinehart, L.S. Shield, Chemistry of the ansamycin antibiotics, Fortschr Chem Org Naturst 33 (1976) 231–307.

References

[78] W.Wehrli, Ansamycins. Chemistry, biosynthesis and biological activity,Top Curr Chem 72 (1977) 21–49. [79] V. Ong, S. Flanagan, E. Fang, H.J. Dreskin, J.B. Locke, K. Bartizal, et al. Absorption, distribution, metabolism, and excretion of the novel antibacterial prodrug tedizolid phosphate, Drug Metab Dispos 42 (2014) 1275–1284. [80] D.L. Higgins, R. Chang, D.V. Debabov, J. Leung, T. Wu, K.M. Krause, et al. Telavancin, a multifunctional lipoglycopeptide, disrupts both cell wall synthesis and cell membrane integrity in methicillinresistant Staphylococcus aureus, Antimicrob Agents Chemother 49 (2005) 1127–1134. [81] K.L. Leach, S.M. Swaney, J.R. Colca, W.G. McDonald, J.R. Blinn, L.M. Thomasco, et al. The site of action of oxazolidinone antibiotics in living bacteria and in human mitochondria, Mol Cell 26 (2007) 393–402. [82] K.D. Brade, J.M. Rybak, M.J. Rybak, Oritavancin: a new lipoglycopeptide antibiotic in the treatment of gram-positive infections, Infect Dis Ther 5 (2016) 1–15. [83] W. Sinko, Y. Wang, W. Zhu, Y. Zhang, F. Feixas, C.L. Cox, et al. Undecaprenyl diphosphate synthase inhibitors: antibacterial drug leads, J Med Chem 57 (2014) 5693–5701. [84] F.P. Tally, M.F. De Bruin, Development of daptomycin for Gram-positive infections, J Antimicrob Chemother 46 (2000) 523–526. [85] A. Deshpande, K. Hurless, J.L. Cadnum, L. Chesnel, L. Gao, L. Chan, et al. Effect of surotomycin, a novel cyclic lipopeptide antibiotic, on intestinal colonization with vancomycin-resistant enterococci and Klebsiella pneumoniae in mice, Antimicrob Agents Chemother 60 (2016) 3333–3339. [86] C.Vilchèze, T. Hartman, B. Weinrick, W.R. Jacobs Jr., Mycobacterium tuberculosis is extraordinarily sensitive to killing by a vitamin C-induced Fenton reaction, Nat Commun 4 (2013) 1881. [87] R. Hengge, A. Grundling, U. Jenal, R. Ryan, F.Yildiz, Bacterial signal transduction by cyclic Di-GMP and other nucleotide second messengers, J Bacteriol 198 (2016) 15–26. [88] M. Exner, S. Bhattacharya, B. Christiansen, J. Gebel, P. Goroncy-Bermes, P. Hartemann, et al. Antibiotic resistance: what is so special about multidrug-resistant Gram-negative bacteria?, GMS Hyg Infect Control 12 (2017)doi: 10.3205/dgkh000290. [89] M. Masi, M. Refregiers, K.M. Pos, J.M. Pages, Mechanisms of envelope permeability and antibiotic influx and efflux in Gram-negative bacteria, Nat Microbiol 2 (2017)doi: 10.1038/nmicrobiol.2017.1. [90] J. Sun, Z. Deng, A. Yan, Bacterial multidrug efflux pumps: mechanisms, physiology and pharmacological exploitations, Biochem Biophys Res Commun 453 (2014) 254–267, doi: 10.1016/j. bbrc.2014.05.090. [91] G. Spengler, A. Kincses, M. Gajdacs, L. Amaral, New roads leading to old destinations: efflux pumps as targets to reverse multidrug resistance in bacteria, Molecules 22 (2017) 468, doi: 10.3390/molecules22030468. [92] J. Top, R. Willems, M. Bonten, Emergence of CC17 Enterococcus faecium: from commensal to hospitaladapted pathogen, FEMS Immunol Med Microbiol 52 (2008) 297–308. [93] B. Perichon, P. Reynolds, P. Courvalin, Van D-type glycopeptide-resistant Enterococcus faecium BM4339, Antimicrob Agents Chemother 41 (1997) 2016–2018. [94] D.A. Boyd, B.M. Willey, D. Fawcett, N. Gillani, M.R. Mulvey, Molecular characterization of Enterococcus faecalis N06-0364 with low-level vancomycin resistance harboring a novel d-Ala-d-Sergene cluster, vanL, Antimicrob Agents Chemother 52 (2008) 2667–2672. [95] D. Nichols, N. Cahoon, E.M. Trakhtenberg, L. Pham, A. Mehta, A. Belanger, et al. Use of ichip for high-throughput in situ cultivation of “uncultivable” microbial species, Appl Environ Microbiol 76 (2010) 2445–2450. [96] A. Serganov, E. Nudler, A decade of riboswitches, Cell 152 (2013) 17–24. [97] S. Colameco, M.A. Elliot, Non-coding RNAs as antibiotic targets, Biochem Pharmacol 133 (2017) 29–42. [98] C. Michaux, A. Hartke, C. Martini, S. Reiss, D. Albrecht, A. Budin-Verneuil, et al. Involvement of Enterococcus faecalis small RNAs in stress response and virulence, Infect Immun 82 (2014) 3599–3611. [99] G. O’Toole, H.B. Kaplan, R. Kolter, Biofilm formation as microbial development, Annu Rev Microbiol 54 (2000) 49–79. [100] D. Pletzer, R.E. Hancock, Antibiofilm peptides: potential as broad-spectrum agents, J Bacteriol 198 (2016) 2572–2578. [101] M.R. Arkin,Y. Tang, J.A. Wells, Small-molecule inhibitors of protein protein interactions: progressing toward the reality, Chem Biol 21 (2014) 1102–1114, doi: 10.1016/j.chembiol.2014.09.001.

397

398

Drug Discovery Targeting Drug-Resistant Bacteria

[102] F. Castiglione, A. Lazzarini, L. Carrano, E. Corti, I. Ciciliato, L. Gastaldo, et al. Determining the structure and mode of action of microbisporicin, a potent lantibiotic active against multiresistant pathogens, Chem Biol 15 (2008) 22–31. [103] D. Jabés, C. Brunati, G. Candiani, S. Riva, G. Romanó, S. Donadio, Efficacy of the new antibiotic NAI107 in experimental infections induced by multidrug-resistant Gram-positive pathogens, Antimicrob Agents Chemother 55 (2011) 1671–1676. [104] D. Münch, A. Müller, T. Schneider, B. Kohl, M. Wenzel, J.E. Bandow, et al. The lantibiotic NAI-107 binds to bactoprenol-bound cell wall precursors and impairs membrane functions, J Biol Chem 289 (2014) 12063–12076. [105] S.T. Hsu, E. Breukink, E. Tischenko, M.A. Lutters, B. de Kruijff, R. Kaptein, et al. The nisin-lipid II complex reveals a pyrophosphate cage that provides a blueprint for novel antibiotics, Nat Struct Mol Biol 11 (2004) 963–967. [106] G. Garufi, A.P. Hendrickx, K. Beeri, J.W. Kern, A. Sharma, S.G. Richter, et al. Synthesis of lipoteichoic acids in Bacillus anthracis, J Bacteriol 194 (2012) 4312–4321. [107] J. Shen, Y. Wang, S. Schwarz, Presence and dissemination of the multiresistance gene cfr in Grampositive and Gram-negative bacteria, J Antimicrob Chemother 68 (2013) 1697–1706. [108] T. He,Y. Shen, S. Schwarz, J. Cai,Y. Lv, J. Li, et al. Genetic environment of the transferable oxazolidinone/phenicol resistance gene optrA in Enterococcus faecalis isolates of human and animal origin, J Antimicrob Chemother 71 (2016) 1466–1473. [109] G.G. Zhanel, R. Love, H. Adam, A. Golden, S. Zelenitsky, F. Schweizer, et al. Tedizolid: a novel oxazolidinone with potent activity against multidrug-resistant Gram-positive pathogens, Drugs 75 (2015) 253–270. [110] D. Zhulenkovs, Z. Rudevica, K. Jaudzems, M. Turks, A. Leonchiks, Discovery and structure-activity relationship studies of irreversible benzisothiazolinone-based inhibitors against Staphylococcus aureus sortase A transpeptidase, Bioorg Med Chem 22 (2014) 5988–6003. [111] L.B. Rice, Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE, J Infect Dis 197 (2008) 1079–1081. [112] A. Casadevall, L. Pirofski, Host-pathogen interactions: redefining the basic concepts of virulence and pathogenicity, Infect Immun 67 (1999) 3703–3713. [113] E.R. Green, J. Mecsas, Bacterial secretion systems—an overview, Microbiol Spectr 4 (2016)doi: 10.1128/microbiolspec.VMBF-0012-2015. [114] S.K. Mazmanian, H.Ton-that, Sortase-catalysed anchoring of surface proteins to the cell wall of Staphylococcus aureus, Mol Microbiol 40 (2001) 1049–1057. [115] H. Ton-That, O. Schneewind, Assembly of pili in Gram-positive bacteria, Trends Microbiol 12 (2004) 228–234. [116] S.T. Rutherford, B.L. Bassler, Bacterial quorum sensing: its role in virulence and possibilities for its control, Cold Spring Harb Perspect Med 2 (2012) 1–25. [117] P. MDowell, Z. Affas, C. Reynolds, M.T.G. Holden, S.J. Wood, S. Saint, et al. Structure, activity and evolution of the group I thiolactone peptide quorum sensing system of Staphylococcus aureus, Mol Microbiol 41 (2001) 503–512. [118] E. Sparrow, M. Friede, S. Torvaldsen, Therapeutic antibodies for infectious diseases, Bull World Health Organ 95 (2017) 235–237. [119] S.L. Greig, Obiltoxaximab: first global approval, Drugs 76 (2016) 823–830. [120] S. Mazumdar, Raxibacumab, MAbs 1 (2009) 531–538. [121] E.L. López, M.M. Contrini, E. Glatstein, S. González Ayala, R. Santoro, D. Allende, et al. Safety and pharmacokinetics of urtoxazumab, a humanized monoclonal antibody, against Shiga-like toxin 2 in healthy adults and in pediatric patients infected with Shiga-like toxin-producing Escherichia coli, Antimicrob Agents Chemother 54 (2010) 239–243. [122] M. Bitzan, R. Poole, M. Mehran, E. Sicard, C. Brockus, C. Thuning-Roberson, et al. Safety and pharmacokinetics of chimeric anti-Shiga toxin 1 and anti-Shiga toxin 2 monoclonal antibodies in healthy volunteers, Antimicrob Agents Chemother 53 (2009) 3081–3087. [123] H. Ashida, H. Mimuro, M. Ogawa, T. Kobayashi, T. Sanada, M. Kim, et al. Cell death and infection: a double-edged sword for host and pathogen survival, J Cell Biol 195 (2011) 931–942. [124] B.K. Davis, H. Wen, J.P.Y. Ting, The inflammasome NLRs in immunity, inflammation, and associated diseases, Annu Rev Immunol 29 (2011) 707–735.

Index Note: Page numbers followed by “f ” indicate figures, “t” indicate tables.

A ABSSSI. See acute bacterial skin and skin-structure infections (ABSSSI) Acinetobacter, 43 Acquired resistance, definition, 277 Acquired resistance, subtypes of, 23t Acute bacterial skin and skin-structure infections (ABSSSI), 71 Adalimumab, 230 ADAs. See antidrug antibodies (ADAs) ADME (absorption, distribution, metabolism, and excretion), 223 Aerosolization, 176 Afabicin, 74 AIs. See autoinducers (AIs) Alphaviruses, 177 AMEs. See aminoglycoside-modifying enzymes (AMEs) Aminoacyl-transfer RNA synthases, 265 Aminoglycoside, 59 Aminoglycoside-modifying enzymes (AMEs), 16 Aminoglycosides, 74 Amitriptyline, 117 AmpC β-lactamase, 280 AMPs. See Antimicrobial peptides (AMPs) AMR. See antimicrobial resistance (AMR) Animal Rule, 208 Anthrax disease, 149 Antibacterial discovery, strategies and targets for antimicrobial targets, 233 approaches to mining natural compounds silent or cryptic secondary metabolic pathways, 226 traditional antibiotics antimicrobial peptides, 230 bacteriophages, 231 nanoparticles, 231 Antibacterial drug discovery automation in molecule design, 235 biosensors role in drug discovery, 237 natural products and drug discovery, 224  

quorum sensing and drug discovery, 231 stem cells role in drug discovery, 239 target-based drug discovery, 229 Antibacterial drug resistance/approaches drug action cell-wall synthesis, interference with, 10 metabolic pathways of essential metabolites, inhibition of, 14 nucleic acid synthesis, interference with, 13 permeability of cytoplasmic membrane structure, 15 protein synthesis, inhibition of, 12 harmful multidrug-resistant strains, 25 mechanisms of developing changes in target sites, 20 decreased antibiotic penetration and efflux, 17 destruction of antibiotic molecule, 17 by global cell adaptations, 22 modification of antibiotic molecule, 15 mechanisms of spread of emergence of, 23 genetic basis and, 22 new antibacterial drug targets and novel approaches to drug development, 28 Anti-biofilm activity of lysins, 324 Antibiofilm peptides, definition, 390 Antibiotic molecule destruction of, 17 modification of, 15 Antibiotic mupirocin, 265 Antibiotic-producing organism, 1 Antibiotic resistance mechanisms, 202t Antibiotics. See also Nature-derived antibiotics; Semisynthetic antibiotics antimicrobials, 1 surveillance system to monitor, 5 dwindling drug discovery pipeline, 3 enzymatic inactivation of, 148 introducing new chemical entities and managing, 7 one health approach, 6 399

400

Index

Antibiotics targeting Gram-negative bacteria classes of antibiotics cefiderocol, 59 combating multidrug resistance with new drugs combinations of approved antibiotics, 47 compounds with targets, 46 Gram-negative infections mechanisms of resistance and their epidemiology, 40 Antibody-based drug discovery, 230 Antibody therapy, 205 Antidepressants, 115 Antidrug antibodies (ADAs), 340 Antimicrobial drugs, development of, 378 Antimicrobial peptides (AMPs), 229, 256 traditional antibiotics, 230 Antimicrobial resistance (AMR), 2, 105, 378 Antimicrobial susceptibility, 155 Antimicrobial targets, 233, 259 Antipathogenic applications of QSI, 234t Antipathogenic effect of Quorum sensing inhibition, 233 Antistaphylococcal chimeric ectolysin, 310 Anti two-component signal transduction systems, 263 Antiviral medical countermeasures, 206 Anti-virulence agents, 118 Approved antibiotics, combinations of β-lactam and β-lactamase inhibitors, 48 cefepime + AAI101, 58 cefepime + VNRX-5133, 56 cefepime + zidebactam (WCK-5222), 54 ETX0282 cefpodoxime proxetil, 55 imipenem/cilastin + relebactam, 48 meropenem + nacubactam, 53 SPR741 combination, 47 sulbactam + ETX2514, 52 Aquaculture, 233 Arthropod-transmitted alphaviruses, 177 Auranofin, 120 Autoinducers (AIs), 231 Automated de novo design, 236 Automation in molecule design, 235 Azidothymidine (AZT), 126 AZT. See azidothymidine (AZT)

B Bacterial biothreat agents, 174 Bacterial communication, 391 Bacterial efflux system, 276

Bacterial plasmids, definition, 280 Bacterial signaling, 392 Bacterial two-component signal transduction systems, 263 Bacteriophages, 256 code for peptidoglycan, 305 traditional antibiotics, 231 Bedaquiline, 365 BGCs. See biosynthetic gene clusters (BGCs) Bicyclic nitroimidazole delamanid, 155 Biofilms, definition, 390 Biology-oriented synthesis (BIOS), 235 BIOS. See biology-oriented synthesis (BIOS) Biosensor operative action, 237 Biosynthetic gene clusters (BGCs), 227 activation of, 227 Bioterrorism, concept of, 172 BioThrax, 149 Biothreat agents bacterial biothreat agents, 174 development of countermeasures to antibody therapy, 205 antiviral medical countermeasures, 206 combination therapies, 207 host-directed therapy, 187 viral biothreat agents, 177 Biothreat organism, 172 β-lactam antibiotics, 12, 79 and β-lactamase inhibitors, 48 BoNT. See Botulinum neurotoxin (BoNT) Botulinum neurotoxin (BoNT), 149 Broad-spectrum antibiotics, 14 Bryophytes, 226 Bunyaviruses, 177

C CABP. See community-acquired bacterial pneumonia (CABP) Calcium-dependent antibiotics, 77. See also Daptomycin CAMHB. See cation-adjusted Mueller-Hinton broth (CAMHB) CAMPs. See Cationic antimicrobial peptides (CAMPs) Carbapenemase-resistant Enterobacteriaceae (CRE), 40 Carbapenem-resistant Enterobacteria is K. pneumonia (CRKP), 27

Index

Carbapenems, 280 Carbapenems, Gram-negative infections resistance to, 40 Cathepsin L (Cat L), 183 Cation-adjusted Mueller-Hinton broth (CAMHB), 319 Cationic antimicrobial peptides (CAMPs), 22 Cat L. See cathepsin L (Cat L) CATs. See chloramphenicol acetyltransferases (CATs) CBDs. See cell-wall binding domains (CBDs) Cefepime + AAI101, 58 Cefepime + VNRX-5133, 56 Cefepime + zidebactam (WCK-5222), 54 Cefiderocol, 59 Cefpodoxime proxetil (CPDP), 55 Ceftazidime, 79 Cefuroxime, 79 Celecoxib, 125 Cell adhesion, 392 Cell penetrating peptides (CPPs), 28 Cellular biosynthesis pathways, in bacteria, 77 Cell-wall binding domains (CBDs), 305 Cell-wall-degrading enzymes, 305 Cell-wall synthesis, interference with, 10 Cephalothin, 79 CF301 (Exebacase), 353 Chimeric lysin P128, 307 Chimeric replication-deficient viruses, 181 Chloramphenicol, 13 Chloramphenicol, 17 Chloramphenicol acetyltransferases (CATs), 17 Ciclopirox, 127 Clofazimine, 365 Cocultivation techniques, 255 Colistin resistance, 45 Colonization, 392 Colorimetric-based assay, 178 Combat drug resistance, in bacteria antibiotic research in age of omics, 383 antibiotic resistance spread of, 380 types of, 380 antibiotics in management of infections due to Gram-negative bacteria, 382 antimicrobial drugs, development of, 378 bioinformatics and high-throughput screening for antibiotics discovery, 385 molecular targets and strategies to, 388 multidrug resistance, mechanisms of, 381

phage therapy, 387 synthesis of nanomaterials, 386 Combat drug-resistant Gram-positive bacteria, antibacterial agents to antibacterial drugs under clinical development, 72 nature-derived antibiotics antibiotics discovered from human microbiome, 78 antibiotics discovered from soil microbiome, 75 semisynthetic antibiotics semisynthetic β-lactam, 79 semisynthetic glycopeptides, 84 semisynthetic macrolides, 82 semisynthetic tetracyclines, 81 synthetic antibacterial agents derived from screening process, 88 inspired from antibacterial peptides, 90 Combating biothreat pathogens biothreat agents bacterial biothreat agents, 174 viral biothreat agents, 177 clinical trials and animal rule, 208 development of countermeasures to biothreat agents antibody therapy, 205 antiviral medical countermeasures, 206 combination therapies, 207 host-directed therapy, 187 screening strategies to identify therapeutics against high-throughput screening approaches to, 178 knowledge-based or multiomics screening, 183 screening platforms for biothreat viral agents, 181 Combating drug resistance, 383 Combating multidrug resistance, with new drugs, 46 Combination therapies, 207 Combinatorial chemistry, 391 Combined-culture methods, 255 Community-acquired bacterial pneumonia (CABP), 72 Conventional de novo drug discovery, 105 CPDP. See cefpodoxime proxetil (CPDP) CPPs. See cell penetrating peptides (CPPs) CRE. See Carbapenemase-resistant Enterobacteriaceae (CRE) CRKP. See carbapenem-resistant Enterobacteria is K. pneumonia (CRKP)

401

402

Index

Cryptic biosynthetic gene clusters, 227 C-terminal of vancomycin, 87 Cultivation-independent approach, 252 Cystic fibrosis, 110 Cytoplasmic membrane structure, disruption and increased permeability of, 15 Cytotoxic effects, 332

D Daptomycin, 15, 76 D-Cycloserine, 153 Decreased permeability, 17 Dexamethasone, 125 DHPS. See dihydropteroic acid synthase (DHPS) Diethylenetriaminepentaacetic acid (DTPA), 118 Diflunisal, 119 Dihydropteroic acid synthase (DHPS), 21 Dimer Terizidone, 153 Diphenyleneiodonium chloride (DPIC), 109 Disulfiram, 108 Disulfiram, in vivo efficacy of, 109 Diverse QS systems, 262 Diversity-oriented synthesis (DOS), 235 DNA gyrase, 13 DNA topoisomerase enzymes, 13 DOS. See diversity-oriented synthesis (DOS) DPIC. See diphenyleneiodonium chloride (DPIC) Drug action, mechanisms of antibacterial cell-wall synthesis, interference with, 10 metabolic pathways of essential metabolites, inhibition of, 14 nucleic acid synthesis, interference with, 13 permeability of cytoplasmic membrane structure, 15 protein synthesis, inhibition of, 12 Drug discovery targeting drug-resistant nontuberculous mycobacteria existing antimicrobials being repurposed for, 367 innate resistance of nontuberculous mycobacteria to antimicrobials, 361 new compounds tackling drug resistance, 364 new targets for compound development, 366 therapeutic vaccine approaches, 371 Drug-induced hemolysis, 332 Drug-loaded hydroxyapatite, 117 Drug repurposing, 105 Drugs against Mycobacterium tuberculosis drug discovery and development of treatment regimens, 143

drugs currently under development for, 156 drug susceptibility testing and importance, 155 host-directed therapies for treating tuberculosis, 158 intrinsic and acquired drug resistance in resistance against first-line drugs, mechanisms of, 149 resistance against second-line drugs, mechanisms of, 152 Drug susceptibility testing (DST), 155 DST. See drug susceptibility testing (DST) DTPA. See Diethylenetriaminepentaacetic acid (DTPA)

E Ebola virus (EBOV), 176 EBOV. See Ebola virus (EBOV) EBOV VLP-based assay, 181 EBP. See ethyl bromopyruvate (EBP) Ebselen, 111 Ectolysin ORF56, identification of, 307 Ectolysins, 305 Efflux pumps (EPs), 263 of important bacteria, 288 regulation of multidrug bacteria, 289 role of, 389 in subjugating antibiotic resistance antibiotic resistance genes, 282 bacterial efflux system, 276 inhibitors based on their mechanism of action, 286 inhibitors based on their on their origin, 287 integrons, 281 mechanism of, 275 mediated multidrug resistance, 278 resistance mechanisms in escherichia coli, 283 Ehrlich, Paul, 1 Energy dissipation, 286 Engulfed bacteria, 140 EPs. See Efflux pumps (EPs) ERM. See erythromycin ribosomal methylation (ERM) Erythromycin, 83f Erythromycin ribosomal methylation (ERM), 21 ESBL. See Extended spectrum β-lactamase (ESBL) ESC. See extended-spectrum cephalosporins (ESC) Escherichia coli, 289 ESKAPE pathogens, 107 Estrogen, 370

Index

Ethyl bromopyruvate (EBP), 107 ETX0282 cefpodoxime proxetil, 55 Extended spectrum β-lactamase (ESBL), 279 Extended-spectrum cephalosporins (ESC), 279

F FASII pathway. See FA synthesis type II (FASII) pathway FA synthesis type II (FASII) pathway, 264 Fatty acid biosynthesis pathways as antimicrobial targets, 264 First-generation antibiotics, 388 First-generation cephalosporins, 79 First-line of drugs (FLD), 14 resistance against, 149 Floxuridine, 122 Fluorescence resonance energy transfer (FRET), 238 Fluorescent-dye-based assay, 178 Fluorescent-protein biosensors, 238 5-fluorouracil, 128 Folic acid metabolism, 14 FOS. See function-oriented synthesis (FOS) Fragment-based drug discovery, 230 FRET. See fluorescence resonance energy transfer (FRET) FTRAP. See target of RNAIII-activating protein (TRAP) Functional genetic screening, to identify lysins, 307 Functional genomics screening technologies, 184t Function-driven approach, 252 Function-oriented synthesis (FOS), 235

G Gallium compounds, 127 Gemcitabine, 122 Gene cassettes, 281 Genetic target identification, 259 Gene trapping approach, 183 Genome mining, 251 Genomics, 385 based applications, 386 Global cell adaptations, 22 Glycopeptides antibiotics, 84 Glycopeptidolipids, 366 Gram-negative infections resistance and their epidemiology, mechanisms of resistance to carbapenems, 40 resistance to polymyxins, 44 Guanidinium-functionalized polycarbonates, 93

H HABP. See hospital-acquired bacterial pneumonia (HABP) Halichondrin B, 226 Harmful multidrug-resistant strains, 25 HDTs. See host-directed therapies (HDTs) Heterologous expression, in engineered hosts, 254 HGT. See horizontal gene transfer (HGT) High-throughput screening (HTS) approaches, 178, 391 Homomeric divalent vancomycin conjugation, 85 Horizontal gene transfer (HGT), 22, 380 Hospital-acquired bacterial pneumonia (HABP), 46 Hospital-acquired pneumonia, 71 Host-directed therapies (HDTs), 187 for treating tuberculosis, 158 Host lysosomal protease cathepsin L, 183 Host-pathogen interactions, definition, 392 HTS approaches. See High-throughput screening (HTS) approaches Human microbiome, 78 Humimycins, 78 Hydrogenation, 237 Hydroxypyridone class, topical antifungal drug of, 127

I Ibuprofen, 106 IChip technology. See isolation chip (iChip) technology IDRI. See Infectious Disease Research Institute (IDRI) IGRA. See interferon-gamma release assay (IGRA) Imipenem/cilastin + relebactam, 48 Immunomodulation, 391 Immunomodulators, 187 Immunosuppressant rapamycin, 158 Indole-2-carboximides, 366 Induced pluripotent stem cells (iPSCs), 239 Infectious Disease Research Institute (IDRI), 371 Inhaling aerosolized droplets, 140 Innate resistance in M. tuberculosis, 149 of nontuberculous mycobacteria to antimicrobials, 361 Integrons, 281 Interferon-gamma release assay (IGRA), 142 Intracellular bacteria, 174

403

404

Index

Intrinsic resistance, 277 Intrinsic resistance, subtypes of, 23t In vitro catheter surface-biofilm model, 324 IPSCs. See induced pluripotent stem cells (iPSCs) Isolation chip (iChip) technology, 226, 250 Ivacaftor, 110

K Ketolide class drug, 72

L Lactocillin, 78 Latent TB infection (LTBI), 142 LiaFSR, 22 Light-driven structure diversification, 228 Linezolid, 21, 154, 364 Loperamide, 114 Low-molecular-hydrophilic molecules, 10 LTBI. See latent TB infection (LTBI) Lugdunin, 78 Lysin-antibiotic synergy on biofilms, 326 Lysin drugs, 306 Lysocins, 77

M MACB. See mycolic acid-containing bacteria (MACB) Macrolides, 82 Macromolecular antibacterial peptide mimics, 92 MAC strains. See Mycobacterium avium complex (MAC) strains Malacidins, 76 Manufacturing process development, 344 MAOI. See monoamine-oxidase inhibitor (MAOI) Mapping-resistant mutants, 314 Marburg virus, 176 Marine organisms, 226 MBC. See minimum bactericidal concentration (MBC) MDR. See multidrug resistance (MDR) Membrane-disruptive mechanism of action, 77 Membrane permeabilization, 88 Meropenem + nacubactam, 53 Metabolic pathways of essential metabolites, inhibition of, 14 Metagenomics-guided antimicrobial discovery, 252 Metallo β-lactamase, 280 Metformin, 119

Methicillin-resistant Staphylococcus aureus (MRSA), 2, 71 MGEs. See Mobile genetic elements (MGEs) MIC. See minimum inhibitory concentration (MIC) Microfluids-based synthesis, 237 Microtiter plate-biofilm assay format, 324 Minigenome assays, 181 Minimum bactericidal concentration (MBC), 318 Minimum inhibitory concentration (MIC), 178, 318 definition, 155 in serum, 319 Mining genomes, for antiinfectives, 386 MNGCs. See multinucleated giant cells (MNGCs) Mobile genetic elements (MGEs), 20 Modifying regulatory elements, 254 Molecular antibacterial drug targets, 11f Molecular beacons, 156 Molecular docking, 236 Molecule design, automation in, 235 Monoamine-oxidase inhibitor (MAOI), 115 Mouse peritoneal infection model, 333 MRSA. See methicillin-resistant Staphylococcus aureus (MRSA) Multidrug resistance (MDR), 2 Multinucleated giant cells (MNGCs), 174 Multiple antibiotic-resistant genes, 381 Multiple-virulence factors, 118 Murepavadin, 46 Murepavadin (POL7080), 46 Murine systemic infection model, 93 Mycobacterium avium complex (MAC) strains, 362 Mycolic acid-containing bacteria (MACB), 255

N Nafithromycin, ketolide class drug, 72 Nanoparticles, 258 traditional antibiotics, 231 Natural antiinfective discovery, 386 Natural products, 224 Nature-derived antibiotics antibiotics discovered from human microbiome, 78 soil microbiome, 75 Neutropenia, 111 Neutropenic murine cUTI model, 54 Niclosamide, 113 Nicotinamide, 124 Nitric oxide, in mycobacterial infections, 369

Index

Nonantibiotic-based strategy, 233 Non-β-lactam class of antibiotics, 89 Nonmotile bacteria, 176 Nontuberculous mycobacteria (NTM), 361 drug development, 362 innate resistance of, 361 Nucleic acid synthesis, interference with inhibit DNA synthesis, 13

O One health approach, 6 One strain-many compounds (OSMAC) approach, 253 OSMAC approach. See One strain-many compounds (OSMAC) approach Outer membrane of bacteria, 276 Oxadiazoles, 89 Oxazolidinones, 364 Ozonation, 237

Phagoburn project, 257 Piperidinol-based compound 1 (PIPD1), 366 Plant-derived efflux pump inhibitors, 287 Plant-derived extracts, 366 Plasmids, 280 Polymyxin, 15 Polymyxin, 59 Gram-negative infections resistance to, 44 Porin-mediated mechanism, 18 Preventing microbial communication, 262 Probing antibacterial activity, 316 Prodrugs, definition, 389 Promoter regions, 254 Protein epitopes, 390 Protein synthesis, inhibition of, 12 Proteomics, 384 PRRs. See pattern recognition receptors (PRRs) Pseudomonas aeruginosa, 289 Purification process, 345

P

Q

P128 antimicrobial activity of, 315 on formation of multispecies biofilms, 328t mechanism of action of, 311 preclinical efficacy and safety testing of, 328 spectrum of, 316 PAMPs. See pathogen-associated molecular patterns (PAMPs) Pathogen-associated molecular patterns (PAMPs), 140 Pathogens deploy, 260 Pattern recognition receptors (PRRs), 140 PBPs. See penicillin binding proteins (PBPs) See penicillin-binding proteins (PBPs) PCR-based sequence-guided screening technique, 252 Penicillin binding proteins (PBPs), 79 Penicillin-binding proteins (PBPs), 12 Pentamidine, 125 Pentetic acid, 118 Pentetic acid, 118 Peptide-based drug discovery, 229 Peptide-based drugs, 229 Permeability of cytoplasmic membrane structure, 15 Phage display libraries, 229 Phage K genome, 307 Phage therapy, 301 lysins, 305 Phage therapy, 387

Q-fever, 176 QNR. See Quinolone resistance (QNR) QSI. See Quorum sensing inhibition (QSI) Quinolone resistance (QNR), 20 Quinolones, 14 Quorum sensing inhibition (QSI) mechanism, 231

R Raxibacumab, 230 Recommended treatment regimens, 364t Remote-controlled robotic synthesis, 236 Replication competent minigenome systems, 181 Repurposing nonantibiotic drugs, as antibacterials 5-fluorouracil, 128 amitriptyline, 117 antidepressants, 115 anti-virulence agents, 118 auranofin, 120 celecoxib, 125 ciclopirox, 127 dexamethasone, 125 diflunisal, 119 diphenyleneiodonium chloride, 109 disulfiram, 108 ebselen, 111 ethyl bromopyruvate, 107 floxuridine, 122 gallium compounds, 127

405

406

Index

Repurposing nonantibiotic drugs, as antibacterials (cont.) gemcitabine, 122 ibuprofen, 106 ivacaftor, 110 loperamide, 114 metformin, 119 niclosamide, 113 nicotinamide, 124 pentamidine, 125 pentetic acid, 118 sertraline, 116 statins, 123 streptozotocin, 123 zidovudine, 126 Resistance to carbapenems, 40 mechanisms in escherichia coli aminoglycosides, 283 β-lactams, 283 fluoroquinolones, 284 sulfonamides, 284 trimethoprim, 283 to polymyxins, 44 Retinoid antibiotics, 88 Reverse genetic systems, 181 Reverse pharmacology approach, 229 Ribosome engineering, 253 Riboswitches, 390 Rifampicin resistance determining region, 151 Rifamycins, 14 RNA polymerase, 14

S SAL200 (Tonabacase), 353 SAR. See structure-activity relationship (SAR) Second-generation cephalosporins, 79 Second-line drugs, resistance against, 152 Semisynthetic antibiotics semisynthetic β-lactam, 79 semisynthetic glycopeptides, 84 semisynthetic macrolides, 82 semisynthetic tetracyclines, 81 Sequence-driven approach, 252 Serotonin-specific reuptake inhibitors (SSRIs), 115 Sertraline, 116 Simvastatin, 124 Small molecular antibacterial peptide mimics, 90 Small molecule antiviral therapeutics, 206f

Small molecule therapeutics, 178 Small ribosomal subunit, 393 Small RNAs (sRNAs), 390 SoC drugs, 334 Soil microbiome, antibiotics discovered from, 75 lysocins, 77 malacidins, 76 teixobactin, 75 Solithromycin, ketolide class drug, 72 Spot-on-lawn method, 316 SPR741 combination, 47 SSRIs. See serotonin-specific reuptake inhibitors (SSRIs) Staphylococcus aureus, 288 Statins, 123 Stem cells, role in drug discovery, 239 Streptozotocin, 123 Structure-activity relationship (SAR), 88 Sulbactam + ETX2514, 52 Sulfa drugs, 1 Synthetic antibacterial agents derived from screening process oxadiazoles, 89 retinoids, 88 inspired from antibacterial peptides, 90 Synthetic chemistry approaches, 229

T T3SS. See Type 3 Secretion System (T3SS) Target-based drug discovery, 229 Target-based whole-cell combination screening approaches, 260 Targeting riboswitches, 390 Targeting two-component signal transduction systems, 263 Targeting virulence, 260 Target of RNAIII-activating protein (TRAP), 231 Target product profile (TPP), 186 Target protection, 20 TB triple therapy, 143 TCAs. See tricyclic antidepressants (TCAs) TCSs. See two-component signal transduction systems (TCSs) Teixobactin, 75, 226, 251 Telithromycin, 82 Tetracycline, 59, 74 antibiotics, 81 efflux pump, 19 Therapeutic vaccine approaches, 371

Index

Third-generation cephalosporins, 79 Time kill kinetics (TKK) assays, 318 TKK assays. See time kill kinetics (TKK) assays TMP-SMX. See trimethoprim-sulfamethoxazole (TMP-SMX) Topical antifungal drug of hydroxypyridone class, 127 Toxins, 392 TPP. See target product profile (TPP) Traditional antibiotics antimicrobial peptides, 230 bacteriophages, 231 nanoparticles, 231 Transcriptomics, 384 Translocate cell wall precursor, 87 TRAP. See target of RNAIII-activating protein (TRAP) Tricyclic antidepressants (TCAs), 115 Trimethoprim-sulfamethoxazole (TMP-SMX), 21 Turbidimetry assay, 316 Two-component signal transduction systems (TCSs), 263 Twofold dilutions of lysin, 319 Type 3 Secretion System (T3SS), 178

U Unexplored natural sources, 226

V Vancomycin-intermediate-resistant Staphylococcus aureus (VISA), 71 Vancomycin-resistant enterococci (VRE), 84

Vancomycin-resistant S. aureus (VRSA), 71 Van gene clusters, 21 Variola virus (VARV), 177 VARV. See Variola virus (VARV) Vascular endothelial growth factor (VEGF), 161 VEGF. See vascular endothelial growth factor (VEGF) Venom-derived antimicrobial peptides, 366 Ventilator-associated pneumonia, 71 Vertical gene transfer, 380 VGCC. See Voltage-gated calcium channel (VGCC) Viral biothreat agents, 177 Virulence factors, 392 Virus-like particles (VLPs), 181 VISA. See vancomycin-intermediate-resistant Staphylococcus aureus (VISA) VLPs. See virus-like particles (VLPs) Voltage-gated calcium channel (VGCC), 183 VRE. See vancomycin-resistant enterococci (VRE)

W Waksman, Selman, 1 WGS. See whole-genome sequencing (WGS) WHO. See World Health Organization (WHO) Whole-genome sequencing (WGS), 149 World Health Organization (WHO), 3, 9

Z Zidovudine, 126 Zoliflodacin, 74

407