Analysis of persistent and antibiotic resistant commensal Escherichia coli from healthy adults

279 Pages·2014·22.31 MB·English

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Analysis of Persistent and Antibiotic
Resistant Commensal Escherichia
coli from healthy adults

Sashindran Anantham

A thesis submitted in fulfilment of the requirements for the
degree of Doctor of Philosophy

School of Molecular Bioscience
The University of Sydney
NSW, Australia
2014
Declaration
The thesis presented here contains no material that has been submitted or accepted for any
other degree or qualification at any university or institution. To the best of my knowledge,
this thesis is original and contains no material previously published or written by another
person, unless an appropriate reference is stated in the text.

Sashindran Anantham
5-9-2013

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ACKNOWLEDGEMENTS
I would like to thank Professor Ruth Hall for all the intellectual and moral support that she
has provided throughout the last 4 years. Without her, this thesis would not have been
possible. I would like to thank my parents for all their moral and financial support. My life as
a student was far more comfortable than it should have been. To my partner Rachel and her
unending patience with me as I chose to work on weekends instead of spending time with her.
Thanks to Professor James Johnson and Connie Clabots who hosted me in Minneapolis as I
learnt the subtleties associated with animal based experimentation. I would like to thank the
staff and my fellow students at the University of Sydney and at the School of Molecular
Bioscience, all of whom made my tenure here highly rewarding and enjoyable. The
volunteers who willingly contributed faecal samples to this project deserve a special mention.
One man’s trash is another man’s treasure. Last but not least, I would like to thank all the past
and present members of the Hall Laboratory for all their assistance and support. Amy Cain,
Jannine Bailey, Grace Blackwell, Nick Evershed, Mohammad Hamidian, Chris Harmer,
Johanna Kenyon, Steven Nigro, Robert Moran, Ila Perera, Jeremy Pinyon, Virginia Post, Neil
Wilson, Matthew Wynn and Sheree Yau. Thank you.

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PUBLICATIONS
These publications contain work presented in this thesis:
Papers:
Anantham, S and R.M. Hall. 2012. pCERC1, a small, globally disseminated plasmid
carrying the dfrA14 cassette in the strA gene of the sul2-strAB gene cluster. Microb Drug
Resist 18: 364-371.

Bailey, J.K., J.L. Pinyon, S. Anantham and R.M. Hall. 2011. Distribution of the bla
TEM
gene and bla -containing transposons in commensal Escherichia coli. J Antimicrob
TEM
Chemother 66: 745-751.

Bailey, J.K., J.L. Pinyon, S. Anantham and R.M. Hall. 2010. Commensal Escherichia coli
of healthy humans: a reservoir for antibiotic resistance determinants. J Med Microbiol 59:
1331-1339.

Bailey, J.K., J.L. Pinyon, S. Anantham and R.M. Hall. 2010. Distribution of human
commensal Escherichia coli phylogenetic groups. J Clin Mirobiol 48: 3455-3456.

GenBank nucleotide accession numbers:
Anantham, S. and R.M. Hall. Escherichia coli strain S1.2.T2R plasmid pCERC1, complete
sequence. GenBank accession number JN012467. 6790 bp (Submitted May 2011).

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ABSTRACT
E. coli primarily resides as a commensal in the colon of humans and warm-blooded animals.
There is ample evidence to indicate that human commensal strains can infect anatomical sites
outside the colon, particularly the urinary tract. This is further complicated by the fact that the
commensal E. coli population represents a large reservoir of antibiotic resistance genes. Many
studies have examined the properties of strains that cause urinary tract infections but studies
on which commensal E. coli strains are likely to cause non-intestinal infections are somewhat
limited. The work presented in this thesis tested the hypothesis that the strains which can
persist in the human colon for a long time, are more likely than other strains, to have similar
properties to the E. coli that cause extraintestinal infections. To address this, a collection of
persistent and other commensal E. coli strains from 11 healthy Australian adults without any
recent antibiotic treatment was assembled and analysed.
Seventy-three strains were collected from 11 subjects, and 8 of these adults each
carried 1 or 2 strains that were detected in multiple faecal samples over a period of months to
years. These were persistent strains and a total of 13 were found of which 7 were from
phylogenetic group B2 and 3 each were from groups A and D. Of the 19 virulence and
1
siderophore receptor genes screened for, the group II capsule [kpsMT II] and the
yersiniabactin receptor [fyuA] correlated with strain persistence. The 13 persistent strains each
contained kpsMT II and 12 carried fyuA. Nine of the 13 strains belonged to ST95, ST69,
ST405 or ST131, the clones that are often found in UTI and other non-intestinal infections. In
contrast, a group of 36 strains found here that were not detected in multiple samples included
only 12 with kpsMT II and 14 with fyuA, and a single ST95 and one ST131 strain.
The 73 strains and few additional commensal strains were characterised for their
resistance to 11 antibiotics and 46 resistant strains were found. Seven of the persistent strains
were resistant to at least one antibiotic, showing that resistant strains can persist in the

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absence of antibiotic selection. One of the resistant strains was found to carry a 6.8 kb
plasmid that had a dfrA14 gene cassette, which confers resistance to trimethoprim, in the strA
streptomycin resistance gene. This plasmid, named pCERC1, was completely sequenced and
its replication region was identified. Four other very closely related plasmids were found
among the 46 resistant strains. A large plasmid carrying a large resistance island that
contained a class 1 integron, appeared to be present in 3 ST69 strains that were found here.
Variants of this island were present in 2 further ST69. Hence, plasmids appeared to be the
main agent in the dissemination of antibiotic resistance genes amongst the commensal strains.
The resemblance shared between persistent strains and those that cause extraintesintal
infections indicated that persistent strains, which are successful commensals, are the subset of
commensal strains that are most likely to cause non-intestinal infections. Moreover, the
acquisition and accumulation of resistance genes by persistent strains could explain the
emergence of multiply antibiotic resistant clones like ST131 and ST69.

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TABLE OF CONTENTS
Page no.
Declaration II
Acknowledgements III
Publications IV
Abstract V
Table of contents VII-XII
List of figures XIII-XIV
List of tables XV-XVII
Abbreviations XVIII-XIX

CHAPTER 1: INTRODUCTION 1
1.1 General overview 2
1.1.1 Escherichia coli 2
1.1.1.1 Commensal E. coli 3
1.1.1.2 Antibiotic resistant commensal E. coli 3
1.1.1.3 Commensal E. coli can cause infections 4

1.2 The population structure of E. coli 6
1.2.1 Identification of E. coli phylogenetic groups 8

1.3 Commensal E. coli population in adult humans 9
1.3.1 Early studies on commensal E. coli colonisation of humans 10
1.3.2 MLEE analysis of strains 12
1.3.3 Recent studies on strain persistence 12

1.4 Virulence factors 14
1.4.1 P fimbriae 16
1.4.2 Type 1 fimbriae 16
1.4.3 Other fimbriae and adhesins 17
1.4.4 Group II capsular polysaccharide 17
1.4.5 The ibeA invasin 18
1.4.6 Iron-uptake systems 18
1.4.7 Hemolysin and colibactin toxins 20

1.5 The colon as a reservoir for harmful E. coli 20
1.5.1 Special pathogenicity versus prevalence theory 22
1.5.2 VF carriage by commensals and isolates from infections 24
1.5.3 VF carriage of long and short-term intestinal colonisers 28
1.5.4 Phylogenetic group distribution among commensal isolates 30

1.6 E. coli that cause non-intestinal infections 31
1.6.1 The clonality of isolates that cause infections 31
1.6.1.1 CC131 33
1.6.1.2 CC95 33
1.6.1.3 CC69 34
1.6.1.4 CC405 35
1.6.2 VF carriage is variable 35

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1.6.3 Using VF carriage to identify potential ExPEC isolates 36

1.7 Antibiotic resistance 37
1.7.1 Commensal E. coli and antibiotic resistance 37
1.7.1.1 Persistence of antibiotic resistant strains 38
1.7.2 Antibiotic resistance in isolates from infections 39
1.7.3 Horizontal dissemination of resistance genes 40
1.7.3.1 Insertion sequences 40
1.7.3.2 Transposons 41
1.7.3.3 Gene cassettes and integrons 43
1.7.3.4 Complex transposons 44
1.7.3.5 Multiple antibiotic resistance islands 45
1.7.3.6 Plasmids 47

1.8 Aims 48

CHAPTER 2: MATERIALS AND METHODS 49
2.1 Materials 50
2.1.1 Chemicals and reagents 50
2.1.2 Buffers and solutions 50
2.1.3 Bacterial growth media 50
2.1.4 Sterilisation 51
2.1.5 Enzymes 51
2.1.6 Antibiotics 52

2.2 Isolation and characterisation of E. coli strains 52
2.2.1 Isolation of E. coli 52
2.2.2 Grouping of E. coli isolates by antibiotic susceptibilities 54
2.2.3 Identification of E. coli 55
2.2.3.1 API20E biotyping 55
2.2.4 Strain identification 56
2.2.4.1 Identifying persistent strains 57

2.3 Genetic analysis 57
2.3.1 Resistance gene analysis 57
2.3.2 VF analysis 58
2.3.3 MLST analysis 59
2.3.3.1 Clone specific PCRs 60

2.4 Bacterial manipulation 61
2.4.1 Bacterial growth conditions 61
2.4.2 Picking and patching 61
2.4.3 Disc diffusion 62
2.4.4 Bacterial storage 62
2.4.5 Iron uptake assay 63

2.5 DNA preparation and manipulation 64
2.5.1 DNA extraction 64
2.5.1.1 Whole cell DNA extraction 64
2.5.1.2 Boiled preparation 65

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2.5.1.3 Plasmid DNA 65
2.5.2 PCR 66
2.5.2.1 Primers 66
2.5.2.2 General PCR 66
2.5.2.3 Long-range PCR 67
2.5.2.4 RAPD 67
2.5.3 Agarose gel electrophoresis 68
2.5.4 DNA purification techniques 69
2.5.5 Estimation of DNA concentration 69

2.6 Construction and analysis of plasmid deletions 69
2.6.1 Preparation of CaCl competent cells 69
2
2.6.2 Restriction endonuclease digestion 70
2.6.3 Ligation 70
2.6.4 Transformation 71
2.6.5 Sequencing plasmids 71
2.6.6 Extended growth experiments 71

2.7 DNA sequencing analysis 72
2.7.1 Sequencing reaction 72
2.7.2 Sequencing analysis 73

CHAPTER 3: ISOLATION AND 74
CHARACTERISATION OF COMMENSAL E. COLI
3.1 Introduction 75

3.2 Strain isolation 76
3.2.1 Extended sampling 77
3.2.1.1 Subject 1 77
3.2.1.1.1 E. coli strain 1-R1 77
3.2.1.1.2 Other resistant E. coli strains 80
3.2.1.1.3 Susceptible strains 81
3.2.1.1.4 1-R1 is present 2 years later 82
3.2.1.1.5 The abundance of E. coli 82
3.2.1.1.6 K. pneumoniae in subject 1 84
3.2.1.2 Subject 2 85
3.2.1.2.1 Strain 2-S1 is still present in subject 2 86
3.2.1.2.2 A second persistent susceptible strain in subject 2 87
3.2.1.2.3 Resistant isolates 89
3.2.1.2.4 Other Enterobacteriaceae detected 90
3.2.1.3 Subject 3 90
3.2.1.3.1 Can 3-R1 and 3-S1 persist? 91
3.2.1.3.2 3-S1R persists 92
3.2.1.3.3 Enterobacteriaceae carriage 93
3.2.1.4 Subject 11 93
3.2.2 New subjects 94
3.2.2.1 Strains isolated from subjects 14, 21, 22 and 23 94
3.2.2.2 Strain sharing 96
3.2.2.3 Enterobacteriaceae in subjects 14, 21, 22 and 23 97

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