Investigating the Regulatory Mechanisms of Antibiotic Resistance in Gram-negative Pathogens

213 Pages·2013·1.94 MB·English

by  Micheál Mac Aogáin

Investigating the Regulatory Mechanisms of
Antibiotic Resistance in Gram-negative
Pathogens

A Thesis Presented to the National University of Ireland for the Degree
of

Doctor of Philosophy

By

Micheál Mac Aogáin B.Sc. (Genetics)

Department of Microbiology
National University of Ireland
Cork

Supervisor: Professor Fergal O’Gara
Head of Department: Professor Gerald Fitzgerald

May 2011
Table of contents
Title page I
Table of contents II
Declaration III
Dedication IV
Abstract V
Peer-review publications VIII

General Introduction 1
1. Introduction 3
2. Antibiotic resistance 5
3. Molecular mechanisms of antibiotic resistance 2
4. The Resistance-Nodulation-Division family of efflux pumps 27
5. Conclusion 41
6. Aim of thesis 43
7. References 44

Chapter 1 Emergence of Extended-Spectrum β-lactamase and Fluoroquinolone
Resistance Genes among Irish Enterobacteriaceae Isolates 63
1.1 Abstract 64
1.2 Introduction 65
1.3 Materials and Methods 67
1.4 Results 71
1.5 Discussion 86
1.6 Conclusion 91
1.7 References 92

Chapter 2 Mechanisms of Carbapenem Resistance in Pseudomonas aeruginosa
Isolates from Turkey 98
2.1 Abstract 99
2.2 Introduction 100
2.3 Materials and Methods 102
2.4 Results 108
2.5 Discussion 117
2.6 References 121

Chapter 3 Investigating MexT-mediated Regulation of Antibiotic Resistance,
Virulence and the Novel ArsR-family Repressor PA4354 in
Pseudomonas aeruginosa 127
3.1 Abstract 128
3.2 Introduction 129
3.3 Materials and Methods 132
3.4 Results 144
3.5 Discussion 165
3.6 Conclusion 172
3.7 References 173

General Discussion 181

Acknowledgments 204

II
Declaration

I, the undersigned, Micheál Mac Aogáin, declare that I have not obtained a degree from
University College Cork, National University of Ireland, Cork or elsewhere on the basis of this
Ph. D. thesis and that the results presented in this thesis were derived from experiment
undertaken by myself at University College Cork with the following exceptions:

 Typing and PCR screening for resistance genes in P. aeruginosa isolates was performed by Dr.
Marlies Mooij (Chapter 2, Table 2)
 β-galactosidase assays to determine repression of exoS expression by MexT in PAO1 and
PAO1mexEF included in Chapter 3 were performed by Hazel O’Connor (Chapter 3, Figure 2C,
Figure 3C)
 Transcriptome analysis from which data is included in Chapter 3 was performed by Dr. Zhexien
Tian (Chapter 3, Table 3)

_________________________________
Micheál Mac Aogáin
May 2011
III
Do mo thuistí, Eoghan agus Síle dá gcuid ghrá agus tacaíocht, agus i gcuimhne ar
m'uncail Paddy.

To my parents, Eoghan and Síle for their love and support and in memory of my uncle
Paddy.
IV
Abstract
Resistance to diverse antibiotics has emerged globally in prevalent Gram-negative pathogens
including pathogenic members of the Enterobacteriaceae family and Pseudomonas aeruginosa.
As the treatment of infections caused by these pathogens is dependent on their susceptibility to
antibiotics, the emergence of multidrug resistance threatens our ability to treat these infections.
The major selective force behind the emergence of antibiotic resistance is antibiotic use. Ireland
and Turkey both have relatively high rates of antibiotic administration and as such, there exists a
correspondingly high level of selection for resistant clinical isolates. To investigate how
bacterial pathogens are responding to the selective pressure of antibiotic therapy in these regions,
multidrug-resistant Enterobacteriaceae from the Mercy University Hospital (MUH, Cork,
Ireland) and carbapenem-resistant Pseudomonas aeruginosa isolates from Zonguldak
Karaelmas University Hospital (ZKUH, Zonguldak, Turkey) were characterized at the
molecular level.
In Ireland, similar to other European countries, there is an emerging problem with resistant
Enterobacteriaceae which produce Extended-Spectrum β-lactamases (ESBLs). These enzymes
hydrolyze β-lactam antibiotics and confer broad-spectrum resistance to bacterial pathogens
which cannot be treated with conventional antibiotics or even broad-spectrum agents such as
third generation cephalosporins. As levels of cephalosporin resistance have consistently
increased at MUH since 2000, this suggested that Enterobacteriaceae harbouring diverse ESBL
genes could be emerging in this region, accounting for the observed increase in cephalosporin
resistance among Gram-negative isolates. Diverse ESBLs were detected among Irish
Enterobacteriaceae including bla and bla confirming the emergence of diverse
SHV-12 CTX-M-15
ESBL-producing strains in Ireland. The transferrable fluoroquinolone resistance genes aac(6′)-
IB-cr, qnrA, and qnrB were also detected in ESBL-positive isolates for the first time in Irish
clinical isolates. ESBLs and transferrable fluoroquinolone resistance genes are commonly found
on transferrable multidrug resistance plasmids where they are associated with multiple
resistance genes which further narrows treatment options. Escherichia coli isolates from MUH
V
were confirmed to harbour transferrable plasmids conferring resistance to multiple antibiotic
classes including narrow and broad-spectrum β-lactam as well as fluoroquinolone and
aminoglycoside antibiotics. Among Irish MDR Enterobacteriaceae, carbapenem antibiotics
remain a currently effective treatment option as isolates were universally susceptible to this
antibiotic class although non-fermentative Gram-negative isolates exhibiting carbapenem
resistance were observed.
Carbapenem resistance is a significant threat as carbapenem antibiotics are considered to be
agents of last resort and are generally reserved for severe infections. Hence a bacterial infection
exhibiting carbapenem resistance may have severely limited treatment options. In Turkey and
other countries in the Mediterranean Region, resistance to carbapenem antibiotics is high which
poses a serious threat to the treatment of Gram-negative infection in this region. An increased
incidence of imipenem-resistant P. aeruginosa infections was observed at ZKUH. Molecular
investigation revealed the independent emergence of imipenem resistance in diverse P.
aeruginosa via disruption of the OprD outermembrane porin suggesting that carbapenem
therapy is selecting against this porin in P. aeruginosa clinical isolates from this region. Strains
exhibited resistance profiles which suggested a role for the central transcriptional regulator of
antibiotic resistance MexT, which not only induces the MexEF-OprN multidrug efflux system
but also downregulates oprD hence conferring multidrug resistance. However, it was
demonstrated that mexE was not induced in imipenem-resistant isolates, which ruled out this
hypothesis. This finding is in agreement with other studies which report that induction of MexT
in clinical isolates is apparently rare. This suggests that MexT has a physiological role
independent of antibiotic detoxification and that its induction in clinical isolates confers a
fitness burden which outweighs the benefit of increased antibiotic resistance.
In addition to its ability to mediate antibiotic resistance, MexT also negatively influences
virulence. Activation of MexT causes reduced levels of homoserine lactone-dependent virulence
traits, including pyocyanin, elastase, rhamnolipids and PQS, reduced expression of the type
three secretion system and impaired attachment. Hence MexT regulates global physiological
VI
changes in P. aeruginosa. A subset of MexT-associated phenotypes have been attributed to the
induction of the MexEF-OprN pump itself which has a pleiotropic effect on the cell. However, a
subset of MexT-associated phenotypes which exhibit independence from the MexEF-OprN
efflux system was identified suggesting that MexT might directly regulate additional
transcriptional targets. Through transcriptome analysis it was demonstrated that MexT is in fact
a global transcriptional regulator which directly regulates expression of twelve novel targets in
addition to mexEF-oprN.
One of the novel MexT-regulated targets identified was the PA4354-PA4355-xenB operon.
PA4354 encodes a putative transcriptional repressor, thus adding an extra layer of complexity to
the MexT regulatory cascade. This provides a possible regulatory mechanism to explain the
negative effects of MexT on diverse targets. The role of PA4354 as an integrated part of the
MexT regulon was investigated at the molecular level. PA4354 was demonstrated to be an
autorepressor and its autoregulatory binding site was determined. PA4354 did not play a role in
MexT-mediated imipenem resistance and was not linked to any of the MexEF-OprN
independent virulence phenotypes controlled by MexT. Thus a direct regulatory role for
PA4354 in the repression of MexT-associated phenotypes was ruled out. In addition to its
regulation by MexT and PA4354, expression of the PA4354-PA4355-xenB operon appears to be
controlled by other regulatory elements which suggests it may be influenced by other regulatory
pathways and may function in divergent physiological processes.
VII
Peer-reviewed puplications associated with this thesis

Chapter 1
Mac Aogáin M, Mooij MJ, Adams C, Clair J, O'Gara F. Emergence of extended-spectrum β-
lactamase and fluoroquinolone resistance genes among Irish multidrug-resistant isolates. Diagn
Microbiol Infect Dis. 2010 May;67(1):106-9.

Chapter 2
The work carried out in Chapter 2 is to be submitted to for publication to Clinical Microbiology
and Infection.

Chapter 3
Tian ZX, Mac Aogáin M, O'Connor HF, Fargier E, Mooij MJ, Adams C, Wang YP, O'Gara F.
MexT modulates virulence determinants in Pseudomonas aeruginosa independent of the
MexEF-OprN efflux pump. Microb Pathog. 2009 Oct;47(4):237-41.

Tian ZX, Fargier E, Mac Aogáin M, Adams C, Wang YP, O'Gara F. Transcriptome profiling
defines a novel regulon modulated by the LysR-type transcriptional regulator MexT in
Pseudomonas aeruginosa. Nucleic Acids Res. 2009 Dec;37(22):7546-59.

Work from Chapter 3 is to be submitted for publication to the Journal of Bacteriology.

VIII
General Introduction

1
General Introduction
Table of contents
1. Introduction 3
1.1 Nosocomial infection 3
1.2 Infection control 4

2. Antibiotic resistance 5
2.1 History of antibiotic development 5
2.2 Antibiotic mode of action and structural diversity 6
2.3 Evolution of antibiotic resistance in clinical pathogens 8
2.3.1 Horizontal gene transfer 9
2.3.2 Mobile genetic elements 10
2.5 Prevalence of antibiotic resistance in nosocomial infections 12
2.4 Clinical impact of antibiotic resistance 14
2.6 Resistant Gram-positive nosocomial pathogens 15
2.7 Resistant Gram-negative nosocomial pathogens 15
2.7.1 Enterobacteriaceae 16
2.7.2 Non-fermentative species 17
2.7.3 Pseudomonas aeruginosa 18

3. Molecular mechanisms of antibiotic resistance 22
3.1 Target modification 23
3.2 Enzymatic inactivation 24
3.3 Reduced permeability 25
3.4 Antibiotic efflux 26

4. The Resistance-Nodulation-Division family of efflux pumps 27
4.1 The RND efflux systems of P. aeruginosa 27
4.1.1 MexAB-OprM 29
4.1.2 MexCD-OprJ 30
4.1.3 MexEF-OprN 30
4.1.4 MexXY 31
4.1.5 Other RND efflux systems of P. aeruginosa 31
4.2 Distribution of RND systems among diverse bacterial pathogens 32
4.3 Virulence phenotypes associated with RND efflux systems 36
4.4 Transcriptional regulation of resistance 37
4.5 Regulation of RND efflux systems in P. aeruginosa 38

5. Conclusion 41
6 Aim of thesis 43
7. References 44
2