Title Adaptive responses of campylobacter jejuni to antibiotic and biocide selection pressures in

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Adaptive responses of campylobacter jejuni to antibiotic and
Title
biocide selection pressures in chemostat culture
Author(s) Duignan, Olivia
Publication
2016-04-18
Date
Item record http://hdl.handle.net/10379/5690
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Adaptive Responses of Campylobacter jejuni to Antibiotic
and Biocide Selection Pressures in Chemostat Culture.

Thesis Presented for the Degree of Doctor of Philosophy
(Microbiology) at the National University of Ireland, Galway

by

Olivia Duignan

Microbiology Department, School of Natural Sciences,
National University of Ireland, Galway

Head of School: Professor Vincent O'Flaherty
Head of Department: Professor James P. O'Gara
Supervisors: Dr Cyril V. Carroll and Dr Gerard T. Fleming

April 2016
To my family
Michael and Gráinne

Breege and Thomas
Michelle, Seán, Lorraine, Louise, Sinéad, Leeanne, Anita, Gráinne, Aisling, Eimear,
Avril and Conor

for your constant encouragement
without whom none of my success would be possible
Table of Contents
Table of contents

List of Tables vi
List of Figures xiii
List of abbreviations xi
Thesis structure and Declaration of contribution xiv
Acknowledgments xv
Abstract xvii

CHAPTER 1 – Introduction
1.1 The genus Campylobacter 1
1.2 General characteristics 2
1.3 Campylobacterosis: post-infection sequalae and treatment 3
1.4 Sources of Campylobacter 5
1.5 Transmission of Campylobacter in the domestic environment 6
1.6 Detection and typing methods for Campylobacter spp. 8
1.7 Antimicrobial resistance in food borne pathogens 13
1.7.1 Incidences biocide resistance in food borne pathogens 14
1.8 Assessing the efficacy of antimicrobial agents 21
1.8.1 Antimicrobial resistance mechanisms and definitions 24
1.8.2 Permeability of the cell envelope 25
1.8.3 Efflux 29
1.8.4 Mutational resistance mechanisms 36
1.8.5 Enzymatic mediated resistance 37
1.8.6 Transmission of resistance genes by horizontal gene transfer 38
1.8.7 Biofilms 40
1.8.8 Stress response and multi-antimicrobial resistance 43
1.9 Ameloriating the cost of resistance 45
1.10 Ciprofloxacin and benzalkonium chloride: Mechanism of action,
resistance and role in the prevention of transmission of
Campylobacter jejuni. 46
1.10.1 Fluoroquinolones 47
1.10.2 Benzalkonium chloride 50
1.11 Development of adapted strains in vitro 53
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Table of Contents
1.11.1 Selection using serial transfer 53
1.11.2 Periodic selection 54
1.11.3 Specific selection in continuous culture 58
1.12 Elucidating antimicrobial resistance mechanisms through
proteomic investigations. 60
1.13 Aims and objectives of this project. 61
1.14 Bibliography 62

CHAPTER 2 – Adaptation of Campylobacter jejuni NCTC 11168 to
antibiotic and biocide by means of chemostat enrichment.

2.1 Abstract 101
2.2 Introduction 102
2.3 Materials and methods 104
2.3.1 Bacterial Strains 104
2.3.2 Culture Media and Antimicrobials 104
2.3.3 Antimicrobial Susceptibility Testing 104
2.3.4 Maximum specific growth rate (µmax) determinations 107
2.3.5 Adaptation of C. jejuni to ciprofloxacin/ benzalkonium
chloride by enrichment chemostat culture 107
2.3.6 Pulsed field gel electrophoresis (PFGE) 110
2.4 Results 112
2.4.1 Colony and cell morphology of C. jejuni on agar and in
chemostat culture 112
2.4.2 Minimum Inhibitory Concentration 112
2.4.3 Maximum specific growth rate (µmax) determination of
C. jejuni 113
2.4.4 Adaptation of C. jejuni to ciprofloxacin and BKC in
chemostat culture. 114
2.4.4.1 Chemostats operated using ciprofloxacin as a selection
pressure 116
2.4.4.2 Chemostats operated using BKC as a selection pressure 122
2.4.4.3 Chemostats operated without selection pressure 126

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2.4.5 Cross-resistance to antibiotics in chemostat cultures
undergoing adaptation to BKC 129
2.4.6 PFGE of C. jejuni (original strain) and adapted variants 132
2.5 Discussion 133
2.6 Bibliography 141

CHAPTER 3 – Phenotypic characterization of chemostat-derived variants of
C. jejuni NCTC 11168 adapted to ciprofloxacin or
benzalkonium chloride.

3.1 Abstract 147
3.2 Introduction 149
3.3 Materials and methods 151
3.3.1 Bacterial Strains 151
3.3.2 Culture Media and Antimicrobials 152
3.3.3 Bacterial fitness. 152
3.3.4 Stability of the resistance phenotype 153
3.3.5 Determining the effect of dilution on the efficacy of SavlonTM
against chemostat-dervived strains 153
3.3.6 Efflux inhibition assay and antimicrobial susceptibility testing 154
3.4 Results 156
3.4.1 Growth characteristics and stability of adapted phenotypes. 156
3.4.1.1 Maximum specific growth rates (µmax) of
chemostat-derived isolates compared with the original
strain 156
3.4.1.2 Stability of developed resistance in chemostat
isolates 159
3.4.2 Efficacy of SavlonTM against BKC-adapted and ciprofloxacin-
adapted C. jejuni variants 162
3.4.3 Cross-resistance and the ability of two different EPIs to reverse
cross- resistance in ciprofloxacin-adapted C. jejuni variants 168
3.5 Discussion 170
3.6 Bibliography 178

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Table of Contents

CHAPTER 4 – An investigation into the role of proteomic changes in the
adaptation of C. jejuni NCTC 11168 to ciprofloxacin and
benzalkonium chloride

4.1 Abstract 185
4.2 Introduction 186
4.3 Materials and methods 188
4.3.1 Strains and Antimicrobial Agents 188
4.3.2 Sodium Dodecyl Gel Electrophoresis (SDS-PAGE) 188
4.3.2.1 Preparation of samples 188
4.3.2.2 Electrophoretic analysis of the proteins by
SDS-PAGE 189
4.3.3 Two-Dimensional Gel Electrophoresis (2DGE) 189
4.3.3.1 Culture conditions 189
4.3.3.2 Protein extraction 190
4.3.3.3 First dimension- Isoelectric focusing (IEF) 191
4.3.3.4 Second dimension: SDS-PAGE 192
4.3.3.5 Gel analysis 192
4.3.3.6 Identification of proteins by MALDI-TOF-MS 193
4.4 Results 194
4.4.1 Analysis of C. jejuni and chemostat derived
ciprofloxacin- and BKC-adapted isolates of C. jejuni
by SDS-PAGE 194 222
4.4.2 Identification of proteins involved in BKC and
ciprofloxacin adaptation in C. jejuni 194
4.4.2.1 Identification of proteins that were altered upon
adaptation of C. jejuni to BKC or following a 25 %
MIC challenge with BKC 196
broth
4.4.2.2 Identification of proteins that were altered upon
adaptation of C. jejuni to ciprofloxacin or following
a 25 % MIC challenge with ciprofloxacin 200
broth
4.5 Discussion 205
4.6 Bibliography 215
S4.1 Representative 2D gels 223
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Table of Contents

CHAPTER 5 – An investigation into the role of the role of target site
mutations in the adaptation of C. jejuni NCTC 11168 to
antibiotic ciprofloxacin.

5.1 Abstract 225
5.2 Introduction 226
5.3 Materials and methods 228
5.3.1 Strains and Antimicrobial Agents 228
5.3.2 Antimicrobial Susceptibility Testing 228
5.3.3 Isolation of chromosomal DNA from C. jejuni 228
5.3.4 Sequencing the quinolone resistance-determining region of
gyrA 230
5.4 Results 232
5.4.1 Mutations in gyrA conferring resistance to
ciprofloxacin 232
5.5 Discussion 234
5.6 Bibliography 237

CHAPTER 6 – General Discussion

6.1 General discussion 242
6.2 Aims and objectives 244
6.3 Ciprofloxacin 244
6.4 BKC 250
6.5 Future work 253
6. 6 Bibliography 256

v
List of Tables
List of Tables

Table 1.1 Biochemical and growth characteristics of Campylobacter
species 3

Table 1.2 List of quinolone antibiotics approved for clinical use 46

Table 2.1 Summary of operating conditions of chemostats carried out
in this study and corresponding chemostat population
parameters measured 115

Table 2.2 Results from SmaI generated PFGE profiles comparisons of
antimicrobial-adapted variants generated in this study 132

Table 3.1 Summary of operating conditions of chemostats carried out
in this study 151

Table 3.2 Strain characteristics of ciprofloxacin-adapted variants
isolated from CR2 and CR3 160

Table 3.3 Strain characteristics of BKC-adapted variants
isolated from CR5 161

Table 3.4 MIC values for BKC and ciprofloxacin-adapted
broth
variants to the commercial disinfectant SavlonTM 162

Table 3.5 MIC values of the original strain and BKC-adapted variants
broth
to BKC 166

Table 3.6 Cross-resistance in ciprofloxacin (CIP) adapted C. jejuni
variants to the antibiotics - ampicillin (AMP), erythromycin
(ERY) and cefotaxime (CEF), commercial disinfectants
SavlonTM (SAV) and Dettol® (DET), the antiseptic
TCP® and the biocide benzalkonium chloride (BKC) 168

Table 4.1 Strains (and corresponding MIC values) used to elucidate
broth
the proteomic changes occuring in C. jejuni
as a result of adaptation to ciprofloxacin or BKC 190

Table 4.2 Analyses of spot intensities from proteins identified
as differentially expressed in the BKC-adapted
variant compared to the original C. jejuni strain and
following a 25 % MIC challenge with BKC 196

Table 4.3 Differentially expressed proteins identified in original and
BKC-adapted variant, using mass spectrometry 197
vi
List of Tables

Table 4.4 Analyses of spot intensities from proteins identified
as differentially expressed in the ciprofloxacin-adapted
variant compared to the original C. jejuni strain and
following a 25 % MIC challenge with ciprofloxacin 200

Table 4.5 Differentially expressed proteins identified in original and
ciprofloxacin-adapted strains ±25 % MIC challenge
concentration of ciprofloxacin using mass spectrometry 201

Table 5.1 Primers used in this study for amplification of the
quinolone resistance- determining region of gyrA 230

Table 5.2 Mutations detected in the QRDR of gyrA gene and
corresponding ciprofloxacin MIC values of
Agar
ciprofloxacin-adapted and BKC-adapted variants 232

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