The biology of antibiotic resistance plasmids

284 Pages·2015·7.39 MB·English

THE BIOLOGY OF ANTIBIOTIC
RESISTANCE PLASMIDS
by
HOWARD THIEN HUI SAW

A thesis submitted to the University of Birmingham for the degree of
DOCTOR OF PHILOSOPHY

School of Immunity and Infection
College of Medical and Dental Sciences
University of Birmingham
April 2015
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ABSTRACT
Plasmids confer genes encoding clinically relevant antibiotic resistance. It was hypothesised
that the AcrAB-TolC multidrug resistance efflux pump was required for clinically relevant
levels of carbapenem resistance. However, carbapenemase-producing Salmonella TolC
mutants were less susceptible to carbapenems. In the presence of the efflux inhibitor phe-arg-
β-naphthylamide (PAβN), wildtype bacteria and 36/86 non-replicate clinical isolates of
carbapenem-producing Enterobacteriaceae were ≥4-fold less susceptible to ertapenem.
Experimental data suggested that OmpF repression conferred the increased carbapenem MICs.
Two bla -encoding plasmids have been isolated in the UK; pKpQIL-UK was found in K.
KPC
pneumoniae, but its variant, pKpQIL-D2 was also found in other species. Therefore, it was
hypothesised that a region of pKpQIL-D2 either conferred a broader plasmid host range
and/or a fitness benefit to the host bacterium. Fitness studies measuring growth rates, ability
to form biofilm, conjugation frequency and plasmid persistence showed that both plasmids
affected the host bacterium but in different ways. Compared to pKpQIL-UK, pKpQIL-D2 did
not confer a significant fitness advantage to its host under the conditions tested. RNA-
sequencing showed both plasmids affected a different set of genes related to metabolism. The
understanding of the factor(s) contributing to the persistence and dissemination of successful
plasmids may help to control antibiotic resistance.
Acknowledgements

Firstly, I would like to thank both of my supervisors, Laura Piddock and Mark Webber for
their great guidance and support throughout my PhD studies.

I am also grateful for the presence of many kind colleagues in the Antimicrobial
Research Group. The help and advice provided by them during my studies are very much
appreciated.

The constant motivation from my friends in England and also those from Malaysia,
Melbourne and Singapore has kept me moving even during very harsh times.

I appreciate the help provided by many of our collaborators namely, Neil Woodford,
Michel Doumith, Shazad Mushtaq, Mark Sutton and Matthew Wand in Public Health England.

I am thankful for the help that has been provided by the staffs and colleagues in the
School of Immunity & Infection, and Institute of Microbiology & Infection.

Lastly, I would like to thank the University of Birmingham for awarding me the Elite
Doctoral Researcher Scholarship which funds my PhD studies in England.
Table of Contents
CHAPTER ONE:  INTRODUCTION ...................................................................................... 1 
1.1  Bacteria ......................................................................................................................... 1 
1.1.1  Salmonella Typhimurium as a Model Organism ......................................... 3 
1.2  β-lactam Antibiotics ..................................................................................................... 4 
1.2.1  Mechanism of Action of β-lactams .............................................................. 8 
1.2.2  Carbapenems .............................................................................................. 10 
1.3  Antibiotic Resistance .................................................................................................. 13 
1.3.1  Burden of Antibiotic Resistance ................................................................ 14 
1.3.2  Antibiotic Resistance Mechanisms ............................................................ 15 
1.3.3  Carbapenemase-mediated Antibiotic Resistance ....................................... 19 
1.3.4  Antibiotic Resistance Which Requires AcrAB-TolC ................................ 23 
1.3.5  Efflux Inhibitors ......................................................................................... 25 
1.4  Spread and Acquisition of Antibiotic Resistance ....................................................... 27 
1.4.1  Lateral Gene Transfer (LGT) in Dissemination of Antibiotic Resistance . 27 
1.5  Plasmid-mediated Antibiotic Resistance .................................................................... 29 
1.5.1  The bla -encoding pKpQIL Plasmid ..................................................... 30 
KPC
1.6  Factors Important in Plasmid Dissemination in Enterobacteriaceae .......................... 32 
1.6.1  Plasmid Stability and Segregation ............................................................. 32 
1.6.2  Inhibition of Plasmid Transfer ................................................................... 34 
1.7  Plasmid-mediated Antibiotic Resistance and Fitness ................................................. 35 
1.8  Background to This Project ........................................................................................ 37
1.8.1  Plasmid pCT .............................................................................................. 37 
1.8.2  Plasmid pKpQIL-UK & pKpQIL-D2 ........................................................ 38 
1.9  Hypotheses to be Investigated in This PhD Project ................................................... 43 
1.10  Aims & Objectives ..................................................................................................... 43 
CHAPTER TWO:  METHODS .............................................................................................. 44 
2.1  Bacterial Strains, Plasmids & Culture Conditions ..................................................... 44 
2.1.1  Bacterial Identification .............................................................................. 44 
2.2  Plasmid Extraction ..................................................................................................... 56 
2.3  Polymerase Chain Reaction ........................................................................................ 56 
2.4  DNA Gel Electrophoresis ........................................................................................... 57 
2.5  DNA Sequencing ........................................................................................................ 57 
2.6  Insertional Inactivation of Plasmidic Genes ............................................................... 62 
2.6.1  Primer design ............................................................................................. 62 
2.6.2  Electrocompetent Cell Preparation and Electroporation ........................... 62 
2.6.3  Gene Inactivation by Homologous Recombination ................................... 63 
2.7  β-lactamase Assay ...................................................................................................... 64 
2.8  Hoechst 33342 Accumulation Assay ......................................................................... 65 
2.9  Outer Membrane Protein Extraction .......................................................................... 66 
2.10  Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) ........ 67 
2.11  Selection of Rifampicin Resistant Bacteria ................................................................ 67 
2.12  P22 Transduction ........................................................................................................ 68 
2.13  Conjugation ................................................................................................................ 69
2.14  Growth Kinetics ......................................................................................................... 70 
2.15  Determination of Conjugation Frequency .................................................................. 71 
2.16  Minimum Inhibitory Concentration (MIC) Determination ........................................ 72 
2.17  Biofilm Assay ............................................................................................................. 72 
2.17.1  Biofilm Formation on Plastic in Microtitre Tray ....................................... 72 
2.17.2  Biofilm Formation on Glass Under Flow .................................................. 73 
2.18  Plasmid Persistence .................................................................................................... 75 
2.19  Pairwise Competition Assay ...................................................................................... 76 
2.20  Galleria mellonella Infection Model .......................................................................... 77 
2.21  RNA Sequencing ........................................................................................................ 77 
2.21.1  RNA Extraction ......................................................................................... 77 
2.21.2  Ribosomal RNA (rRNA) Reduction .......................................................... 79 
2.21.3  RNA Library Preparation ........................................................................... 79 
2.21.4  Sequencing ................................................................................................. 80 
2.21.5  Data Analyses ............................................................................................ 81 
CHAPTER THREE:  THE CELL ENVELOPE AND CARBAPENEM RESISTANCE ...... 83 
3.1  Background ................................................................................................................ 83 
3.2  Hypothesis and Aims .................................................................................................. 84 
3.3  Carbapenem Resistance by bla is Seen Even in the Absence of the Tripartite
KPC
AcrAB-TolC Multidrug Resistance Efflux Pump ...................................................... 84 
3.3.1  Strain Constructions ................................................................................... 84
3.3.2  The MICs of Antibiotics in Strains Lacking a Component of the AcrAB-
TolC Efflux Pump .................................................................................... 103 
3.4  The Salmonella TolC Mutant is Less Susceptible to β-lactam Antibiotics and This is
Not Specific to Carbapenemase ............................................................................... 106 
3.5  Increased Carbapenem Resistance is Not Associated with Passive Release of
Carbapenemase from the Periplasmic Space of the Salmonella Efflux Mutant ....... 108 
3.6  Efflux Inhibitor Alters Susceptibility to Some β-lactam Antibiotics ....................... 113 
3.6.1  PAβN Reduces β-lactam Antibiotic Susceptibility in K. pneumoniae, E.
coli and Salmonella .................................................................................. 113 
3.6.2  PAβN Increases Carbapenem Resistance in Clinical Isolates of
Enterobacteriaceae ................................................................................... 118 
3.7  PAβN and Lack of TolC Alter the Porin Profile and Confers Decreased
Susceptibility to β-lactam Antibiotics ...................................................................... 124 
3.7.1  Outer Membrane Protein Mutants Showed Reduced Susceptibility to
Ertapenem ................................................................................................ 124 
3.7.2  PAβN Reduces Outer Membrane Protein Expression in Salmonella Strains
................................................................................................................. 129 
3.7.3  Outer Membrane Protein Profile of Clinical Isolates of Enterobacteriaceae .
................................................................................................................. 131 
3.8  Discussion ................................................................................................................ 133 
3.9  Future Work ............................................................................................................. 142 
3.10  Key Findings ............................................................................................................ 144 
CHAPTER FOUR:  COMPARISON OF PLASMIDS pKpQIL-UK WITH pKpQIL-D2 .. 145 
4.1  Background .............................................................................................................. 145 
4.2  Hypothesis and Aims ................................................................................................ 145
4.3  Comparison of the Genetic Sequence of pKpQIL-UK with that of pKpQIL-D2 .... 145 
4.4  Transfer of Plasmid into Different Species of Enterobacteriaceae .......................... 146 
4.5  Growth Rates of Enterobacteriaceae pKpQIL-UK and -D2 .................................... 149 
4.6  Plasmid Persistence .................................................................................................. 156 
4.7  Pairwise Competition of K. pneumoniae Ecl8 carrying pKpQIL-UK and -D2 ....... 160 
4.8  Conjugation Frequencies of the Plasmids into Various Enterobacteriaceae ............ 164 
4.9  Biofilm Formation .................................................................................................... 169 
4.9.1  Biofilm Formation on Plastic ................................................................... 169 
4.9.2  Biofilm Formation under Constant Flow of Medium .............................. 169 
4.10  Infection of Galleria mellonella by Plasmid Carrying K. pneumoniae ST258 ........ 174 
4.11  Minimum Inhibitory Concentration of Antibiotics for Various Plasmid-bearing Hosts
.................................................................................................................................. 176 
4.12  Summary of Phenotypes Conferred by Carriage of pKpQIL-UK vs -D2 ................ 183 
4.13  Gene Expression Profiling of Plasmid Carrying K. pneumoniae ST258 with RNA
Sequencing ............................................................................................................... 183 
4.13.1  Determination of Growth Phase for RNA Extraction .............................. 183 
4.13.2  Optimisation of Total RNA Extraction .................................................... 187 
4.13.3  Ribosomal RNA Depletion and Library Preparation ............................... 191 
4.13.4  RNA Sequencing ..................................................................................... 191 
4.14  Discussion ................................................................................................................ 207 
4.15  Future Work ............................................................................................................. 216 
4.16  Key Findings ............................................................................................................ 218
CHAPTER FIVE:  OVERALL DISCUSSION .................................................................... 219 
REFERENCES ....................................................................................................................... 226