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The Silver Cation (Ag+): Antibacterial Mode of Action and Mechanisms of Resistance

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The Silver Cation (Ag+): Antibacterial Mode of Action and Mechanisms of Resistance The Silver Cation (Ag+): Antibacterial Mode of Action and Mechanisms of Resistance Christopher Paul Randall Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Molecular and Cellular Biology Faculty of Biological Sciences September 2013 ii Intellectual property and publication statements The candidate confirms that the work submitted is his/her own, except where work which has formed part of jointly authored publications has been included. The contribution of the candidate and the other authors to this work has been explicitly indicated below. The candidate confirms that appropriate credit has been given within the thesis where reference has been made to the work of others. Chapters 3 and 4 contain work based on jointly-authored publications. The publications are referenced below, together with a description of the contribution made by each author to the publication. Publication one: Randall CP, Oyama LB, Bostock JM, Chopra I & O'Neill AJ (2013) The silver cation (Ag+): antistaphylococcal activity, mode of action and resistance studies. Journal of Antimicrobial Chemotherapy 68: 131-138. CPR concieved the study, designed and carried out experiments and wrote the manuscript; LBO assisted with some experiments; JMB provided intellectual input; IC and AJO assisted in concieving the study, designing experiments and in writing the manuscript. Publication two: Randall CP, Mariner KR, Chopra I & O'Neill AJ (2013) The target of daptomycin is absent from Escherichia coli and other Gram-negative pathogens. Antimicrobial Agents and Chemotherapy 57: 637-639 CPR concieved the study, designed and carried out experiments and wrote the manuscript; KRM carried out preliminary experiments; IC and AJO assisted in concieving the study, designing experiments and writing the manuscript. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement © 2013 The University of Leeds and Christopher Paul Randall The right of Christopher Paul Randall to be identified as the Author of this work has been asserted by him in accordance with the Copyright, Designs and Patents Act 1988 The content of this thesis remains the confidential information of Smith and Nephew plc until the 31st September 2015 iii Acknowledgements I am extremely grateful to both Dr Alex O‘Neill and Professor Ian Chopra for giving me the opportunity to work in their respective laboratories for the past four years. Thank you also for the support, advice and guidance you have provided me during this time. I would also like to acknowledge both the MRC and Smith and Nephew plc. for providing the funding for this project. At Smith and Nephew I would like to thank Kiersten Vaughan, Emma Woodmansey, and all of the members of the wound care team for your assistance with this project, and for being so welcoming during my time spent working at York. Thank you to all members (past and present) of the O‘Neill and Chopra groups for making the last four years such a memorable and enjoyable experience, and also for providing me with the technical advice and guidance to make it through my PhD studies. In addition, I am immensely grateful to Kirsty Owen, Bjorn Mohl and John Heritage for their friendship during this time. Thank you also to Catherine Bladen, Laura Wetherill and Sally Harrison for their assistance with qPCR, liposome creation, and next-generation sequencing, respectively. Finally, I would like to thank and dedicate this thesis to my family and to my partner Katherine. The work presented in this thesis would not have been possible without your constant and unwavering support. iv Abstract The increasing prevalence of infections attributed to antibiotic-resistant bacteria has prompted renewed interest in exploiting the antibacterial properties of Ag+ to treat such infections. However, the antibacterial mode of action (MOA) and bactericidal activity of Ag+ are poorly understood, and there are concerns that the prolific and unrestricted use of Ag+ in consumer products will select bacterial Ag+ resistance, thus limiting the clinical utility of Ag+. Ag+ resistance already exists, although aspects of the molecular basis of Ag+ resistance, and the current prevalence of Ag+-resistant pathogens are unclear. This thesis sought to address these issues. Ag+ was found to be bacteriostatic in culture media and bactericidal in buffer, and was unable to eradicate Staphylococcus aureus biofilms in vitro. MOA studies indicated that the primary antibacterial target of Ag+ is the cell membrane. Evidence was obtained suggesting that Ag+ does not interfere with the phospholipid component of the membrane, but instead probably damages integral membrane proteins to produce an antibacterial effect. A survey of hospital staphylococcal isolates (n=1006) found universal susceptibility to Ag+, and Ag+ resistance could not be selected in S. aureus and several other pathogens in vitro. However, in Escherichia coli, high-level Ag+ resistance arose rapidly and was not associated with a fitness cost likely to prevent its emergence in the clinical setting. Ag+-resistant strains contained mutations in genes regulating expression of an Ag+ efflux mechanism and outer membrane porins. A detailed characterisation of a known Ag+-resistance determinant (the sil operon), was also conducted to provide further insights into the mechanism of Ag+ resistance conferred by this determinant. v Collectively, these studies provide further insights into the MOA of Ag+ and the mechanisms of Ag+ resistance, which could potentially be applied to optimising the future uses of Ag+ as an antibacterial agent. vi Table of contents Chapter Contents Page INTELLECTUAL PROPERTY AND PUBLICATION ii STATEMENTS ACKNOWLEDGMENTS iii ABSTRACT iv TABLE OF CONTENTS vi LIST OF TABLES xi LIST OF FIGURES xiii ABBREVIATIONS xv 1 GENERAL INTRODUCTION 1 1.1 The use of antibacterial chemotherapy in the treatment of 1 infectious disease 1.1.1 Infectious disease in the pre-antibiotic era 1 1.1.2 A brief history of antibacterial chemotherapy 2 1.1.3 Mode of action (MOA) of antibiotics 4 1.1.4 Antibiotic resistance 7 1.1.5 History and impact of antibiotic resistance 11 1.1.6 The antibiotic discovery ‗void‘ and the need for alternative 13 strategies to treat infectious disease 1.2 Silver as an antibacterial agent 16 1.2.1 History of silver 16 1.2.2 Current applications of antibacterial silver 18 1.2.3 Antibacterial activity of silver 23 1.2.3.1 Inhibitory activity of silver 23 1.2.3.2 Bactericidal activity of silver 25 1.2.4 Toxicity of silver 26 1.2.5 Antibacterial MOA of Ag+ 27 1.2.5.1 DNA 27 1.2.5.2 Protein 28 1.2.5.3 Cell membrane 29 1.2.5.4 Generation of reactive oxygen species 30 1.2.6 Bacterial resistance to Ag+ 32 vii 1.2.6.1 Exogenous Ag+ resistance 36 1.2.6.1.1 SilE 37 1.2.6.1.2 SilRS 38 1.2.6.1.3 SilCBA 38 1.2.6.1.4 SilF 39 1.2.6.1.5 SilP 39 1.2.6.2 Endogenous Ag+ resistance 40 1.3 Introduction to the research in this study; aims and 42 objectives 2 MATERIALS AND METHODS 44 2.1 Bacterial strains and plasmids 44 2.2 Antibacterial agents and chemicals 49 2.3 Bacteriological media and culture conditions 50 2.4 General microbiology techniques 51 2.4.1 Determination of susceptibilities to antimicrobial agents 51 2.4.2 Time-kill analysis 51 2.5 Resistance studies 52 2.5.1 Selection of spontaneous antibacterial-resistant mutants 52 2.5.2 Selection of endogenous Ag+ resistance 52 2.5.2.1 Repeated exposure method 52 2.5.2.2 Continuous exposure method 53 2.5.3 Determination of bacterial fitness 53 2.5.3.1 Growth rate determination 53 2.5.3.2 Determination of competitive fitness 54 2.5.6 Assessment of porin expression in E.coli outer membranes 54 2.5.7 SDS-PAGE 55 2.6 Mode of action studies 56 2.6.1 BacLight™ assay of membrane integrity 56 2.6.2 Membrane potential 57 2.6.3 Construction of liposomes 58 2.6.4 Liposome damage assay 59 2.6.5 -galactosidase leakage 59 2.6.6 Bacillus subtilis reporters 59 2.6.7 Macromolecular synthesis assays 60 2.6.8 Enzyme specificity assays 61 2.6.8.1 RNA polymerase 61 viii 2.6.8.2 Malate dehydrogenase 61 2.6.8.3 Chymotrypsin 62 2.6.8.4 -galactosidase 63 2.6.9 Transcriptiome analysis 63 2.6.10 Screening of a near saturation transposon library of S. aureus 64 2.7 Molecular biology techniques 65 2.7.1 Isolation of plasmid DNA 65 2.7.2 Isolation of chromosomal DNA 65 2.7.3 Polymerase chain reaction 65 2.7.4 Determination of DNA concentration 66 2.7.5 Colony PCR 66 2.7.6 Agarose gel electrophoresis and DNA fragment purification 67 2.7.7 DNA sequence determination (Sanger) 67 2.7.8 Next generation sequence determination 67 2.7.9 Quantitative PCR (qPCR) 68 2.7.10 Lambda red recombineering 69 2.7.10.1 Construction of disruption cassette 70 2.7.10.2 Removal of disruption cassette 71 2.7.10.3 Allelic replacement 71 3 DEVELOPMENT OF STANDARDISED 72 METHODOLOGY TO ASSESS THE ANTIBACTERIAL ACTIVITY OF AG+, AND ITS USE IN EXAMINING THE PREVALENCE OF AG+ RESISTANCE AMONGST THE STAPHYLOCOCCI Abstract 72 3.1 Introduction 73 3.1.1 Aspects of the antibacterial activity of Ag+ are unclear 73 3.1.2 The propensity for bacterial Ag+ resistance to arise is unknown 76 3.1.3 Use of the staphylococci as a model genus for this study 77 3.2.
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