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Evolution of Fluoroquinolone Resistance in Burkholderia Cepacia

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Evolution of Fluoroquinolone Resistance in Burkholderia Cepacia Evolution of fluoroquinolone resistance in Burkholderia cepacia Cassie Francesca Pope A thesis submitted to University College London in fulfilment of the requirement for the degree of Doctor of Philosophy Centre for Medical Microbiology, Royal Free and University College Medical School, Hampstead Campus, Rowland Hill Street, London NW3 2PF UMI Number: U592601 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U592601 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Declaration ‘I, Cassie Pope, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis’. 2 Abstract Abstract This study investigates the evolution of fluoroquinolone resistance in Burkholderia cepacia and assesses fitness of clinical isolates of the B. cepacia complex. B. cepacia was chosen as a clinically relevant model of antibiotic resistance because these bacteria cause chronic infections in cystic fibrosis patients, are highly resistant to killing by many antimicrobials and consequently require long term antibiotic treatment. Fluoroquinolones are a widely used class of antimicrobials, increasingly used in medical and veterinary practice. A method was optimised and used to determine the rate of mutation occurring in topoisomerase genes that confer resistance to fluoroquinolones. The fitness cost associated with fluoroquinolone resistance mutations was assessed as a measure of the stability of resistance in the bacterial population. Clinical isolates were assessed for hypermutability using mutation to fluoroquinolone resistance as a selective tool. In Gram-negative bacteria resistance to fluoroquinolones occurs via three major mechanisms; drug efflux, reduced permeability and target alteration. The spectrum of fluoroquinolone resistance mutations occurring in vitro, the rate at which they arise, and the fitness costs of characterised topoisomerase mutations was investigated, using models relevant to transmission of the Burkholderia cepacia complex. Previous studies have shown that single point mutations in DNA gyrase, conferring resistance, have no or low cost. Only double mutations in gyrA and parC conferred a fitness cost. Second step mutations occur at a faster rate than first step mutations. Mutation in gyrA, therefore, may predispose the genome to mutation in topoisomerase genes. 3 Acknowledgments I am indebted to many people who have either helped me personally or have contributed to the work in this thesis. I gratefully acknowledge the support of my supervisors Dr. Timothy McHugh and Prof. Stephen Gillespie and thank them for having faith in me. I am indebted to Dr. Tim McHugh for his kindness, generosity and positivity and to Prof. Stephen Gillespie for his encouragement. Dr Bambos Charalambous assisted me with statistical analysis and I am grateful for his advice. Additionally I am grateful to Dr. Jonathan Pratten for help with biofilm work and his patience and good humour. Also to Dr. John Moore for providing clinical isolates. I thank Fitzroy Hall and Betty Thaine for providing an autoclave service. Also thanks to Anne Dickens and Marina Bogovac for ordering of reagents. Among other things Clare Ling and Claire Jenkins have provided sequencing training and I am grateful to them both. My colleagues have provided daily support and have provided a fun working environment. In this regard I am grateful to the clinical scientists and research staff in the department. In particular I have relied on Dr Denise O’Sullivan for advice and friendship. I thank Marcus for his patience and support. I am grateful to all for making this experience a happy one. 4 LIST OF CONTENTS Title Page 1 Declaration 2 Abstract 3 Acknowledgements 4 List of Contents 5 List of Figures 12 List of Tables 13 Abbreviations 15 CHAPTER ONE: INTRODUCTION 16 1.0 General Introduction 16 1.1 Antibiotic Resistance 16 1.1.1 Significance of antibiotic resistance 16 1.1.2 Mechanisms of antibiotic resistance 17 1.1.2.1 Alteration of target 17 1.1.2.2 Modification of antibiotic 18 1.1.2.3 Reduction in permeability 20 1.1.2.4 Efflux 21 1.1.2.5 Metabolic bypass 22 1.1.3 Genetic basis of antibiotic resistance 22 1.1.3.1 Transformation 23 1.1.3.2 Conjugation 23 1.1.3.3 Transduction 24 1.1.3.4 Stability of acquired elements 25 1.1.3.5 Persister Cells 26 1.1.4 Use of antibiotics and antibiotic resistance 26 1.2 Mutation Rates 28 1.2.1 Mutations and mutagens 28 1.2.2 Mutation rate versus mutation frequency 30 1.2.3 The fluctuation test of Luria and Delbriick 30 1.2.4 Determination of mutation rate 32 1.2.5 Fluctuation Analysis 3 3 1.2.5.1 The po method 35 1.2.5.2 Lea and Coulsons method of the median 36 1.2.6 Parameters 36 1.2.7 Assumptions of fluctuation analysis 38 1.2.8 Directed mutation controversy 39 1.2.9 Deviations from the assumptions 40 1.2.10 Detection of resistant mutants 43 1.2.11 Hypermutability 44 1.2.12 High mutation rate leads to adaptation 44 5 1.2.13 Mutator phenotypes select for antibiotic resistance 45 1.2.14 Fitness of mutators 46 1.2.15 Stability of mutators 47 1.3 Fitness 47 1.3.1 Importance of fitness 47 1.3.2 Fitness and antibiotic resistance 48 1.3.3 Measuring fitness 48 1.3.4 Cost of fitness 50 1.3.4.1 Chromosomal mutations 50 1.3.4.2 Plasmids 51 1.3.4.3 Other genetic elements 52 1.3.4.4 Compensatory mutations 53 1.3.5 Fitness Landscapes 54 1.3.6 Muller’s Ratchet 54 1.4 Bacterial Biofilms 55 1.4.1 Definition 55 1.4.2 Biofilms in human disease 56 1.4.3 Biofilm development 56 1.4.4 Adhesion 57 1.4.5 Quorum sensing 58 1.4.6 P. aeruginosa grows as a biofilm in the CF fibrosis lung 59 1.4.7 Biofilm resistance to antimicrobial killing 60 1.4.8 Genetic diversity in biofilms 62 1.6 Fluoroquinolone antibiotics 63 1.5.1 Fluoroquinolones 63 1.5.2 History of the fluoroquinolones 64 1.5.3 Mechanisms of action 67 1.5.4 Induction of the SOS response 68 1.5.5 Resistance mechanisms 69 1.5.5.1 Target alteration 69 1.5.5.1.1 GyrA 70 1.5.5.1.2 GyrB 71 1.5.5.1.3 ParC 72 1.5.5.1.4 ParE 72 1.5.5.2 Efflux 73 1.5.5.3 Multiple Antimicrobial Resistance Phenotype (MAR) 73 1.5.5.4 Plasmid mediated resistance 74 1.5.5.5 Inactivation 75 1.6 The Burkholderia cepacia complex 75 1.6.1 Taxonomy of Burkholderia 75 1.6.2 The Burkholderia cepacia complex 77 1.6.3 Burkholderia pseudomallei and Burkholderia mallei 78 6 1.6.4 Clinical significance 79 1.6.4.1 Cystic fibrosis 79 1.6.4.2 Bcc species distribution 81 1.6.4.3 Chronic granulomatous disease 82 1.6.5 Transmission 83 1.6.5.1 Environmental transmission 83 1.6.5.2 Person to person transmission 83 1.6.5.3 Transmissible strains of the Bcc 84 1.6.6 Identification 8 5 1.6.7 Treatment 88 1.6.8 Immunity 89 1.6.9 Resistance to human antimicrobial peptides 90 1.6.10 Genome 91 1.6.11 Virulence of the Bcc 92 1.6.11.1 Invasion 92 1.6.11.2 Quorum sensing 93 1.6.11.3 Exopolysaccharide 94 1.6.11.4 Proteases 94 1.6.11.5 Type III Secretion 95 1.6.11.6 Siderophores 96 1.6.11.7 Virulence models 97 1.6.12 Use in agriculture 97 1.7 Aims of thesis 99 CHAPTER 2: GENERAL MATERIALS AND METHODS 100 2.1 Culture Conditions 100 2.2 Preparation of media 101 2.2.1 Muller Hinton broth 101 2.2.2 Luria Bertani broth 101 2.2.3 Muller Hinton Agar 101 2.2.4 Commercially available agar plates 101 2.3 Preparation of buffers and solutions 102 2.3.1 lM T ris 102 2.3.2 0.5 M EDTA 102 2.3.3 Tris-Borate EDTA buffer (TBE) 102 2.3.4 5MNaCl 102 2.3.5 Phosphate buffered saline 102 2.3.6 Ciprofloxacin 103 2.3.7 Clinafloxacin 103 2.4 Growth Curve 103 7 2.5 Miles and Misra viable cell count 103 2.6 Determination of Minimum Inhibitory Concentration (MIC) 104 2.6.1 E-test 104 2.6.2 Agar dilution 104 2.6.3 Method for determination of mutation rate by the method of the median 105 2.6.5 Detection of efflux 106 2.7 DNA Extraction 107 2.7.1 Crude extraction 107 2.7.2 DNA extraction 107 2.8 Polymerase Chain Reaction (PCR) and sequencing 108 2.8.1 Polymerase chain reaction 10 8 2.8.2 Agarose gel electrophoresis 108 2.8.3 Gel photography 109 2.8.4 PCR product purification 109 2.8.5 Cycle sequencing 109 2.8.6 Ethanol precipitation 110 2.9 Fitness assays 111 2.9.1 Biofilm growth 111 2.9.1.1 Constant depth film fermenter (CDFF) 111 2.9.1.1.1 Conditions 111 2.9.1.1.2 Inoculum 111 2.9.1.1.3 Sampling 111 2.9.1.1.4 Confocal laser scanning microscopy (CLSM) 112 2.9.1.2 Crystal Violet Assay 113 2.9.2 Planktonic growth 113 2.9.3 Competition assay 114 2.9.4 Survival in water 115 2.9.5 Survival on dry surfaces 116 CHAPTER 3: ESTIMATION OF MUTATION RATE IN 117 TOPOISOMERASE GENES OF B.
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