Comparative phylogenomics and antimicrobial resistance in livestock-associated lineages of ...

437 Pages·2017·18.77 MB·English

by  Daniel R Knight

Comparative phylogenomics and antimicrobial
resistance in livestock-associated lineages of
Clostridium difficile

Daniel Raymond Knight
BSc (Hons)

This thesis is presented for the degree of Doctor of Philosophy of
The University of Western Australia
School of Biomedical Science
2018
THESIS DECLARATION
I, Daniel R. Knight, certify that:
This thesis is the result of my own original work and has been substantially accomplished
during enrolment in the degree. The thesis does not contain any material previously published
or written by another person, or which is the outcome of collaboration, except where
specifically indicated in the text. The work described in this thesis was funded by a Research
Training Program Scholarship from the Australian Federal Government, conferred by The
University of Western Australia (UWA), and in part by a project grant from the Australian
Research Council (ARC, #DP150104670).
Daniel R. Knight
6th November 2017
Student 21413653

RESEARCH SUPERVISORS
Professor Thomas V. Riley1-4 [Principle/co-ordinating supervisor], Professor Barbara J.
Chang1 [Co-supervisor], Assoc. Professor Christopher Peacock1 [Co-supervisor] and Assoc.
Professor Timothy T. Perkins1 [Co-supervisor].
1Department of Microbiology, School of Biomedical Science, Faculty of Health and Medical
Sciences, The University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands,
Western Australia, Australia. 2Department of Microbiology, PathWest Laboratory Medicine,
Queen Elizabeth II Medical Centre, Nedlands, Western Australia, Australia. 3School of
Medical and Health Sciences, Edith Cowan University, Joondalup, Western Australia,
Australia. 4School of Veterinary and Life Sciences, Murdoch University, Murdoch, Western
Australia, Australia.

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ABSTRACT
Clostridium difficile is toxin-producing antimicrobial resistant (AMR) enteropathogen of
international One Health importance. This opportunistic bacterium can cause life-threatening
infectious diarrhoea in humans and livestock, particularly piglets, most often after
perturbation of the gut microbiota by antimicrobials. C. difficile infection (CDI) presents a
major clinical and economic challenge to public health systems worldwide, reaching epidemic
states in some regions. In recent years, there have been dramatic increases in the incidence
and severity of CDI, and its associated morbidity and mortality, in both healthcare and
community settings.
Worldwide, livestock has been identified as important amplification reservoirs for clinically
important strains of C. difficile, raising the possibility that CDI may have a zoonotic or
foodborne aetiology. PCR ribotype (RT) 014 and multilocus sequence type (ST) 11 are major
C. difficile lineages of One Health importance. In Australia, these lineages are well-
established in porcine and bovine populations, respectively, and also contribute to the burden
of CDI in humans. However, the true extent of genetic overlap and potential for zoonotic
transfer of these lineages between livestock and human populations remains unclear. This
thesis provides novel and critical insights into the genetic diversity, evolutionary history,
AMR and zoonotic potential of C. difficile isolated from livestock and humans in Australia.
First, this thesis describes the prevalence, concentration and molecular epidemiology of C.
difficile on the carcasses and in the faeces of neonatal calves in Australian abattoirs. Calves
were found to be contaminated with high levels of C. difficile spores during the slaughter
process and are reservoirs for clinically important strains of C. difficile, predominated by 4
ST11 RTs 033, 126, 127 and 288. Furthermore, the unique slaughter age of these animals
presents a significant and perhaps under-appreciated risk factor for contamination of carcasses
during the slaughter process and may compromise the quality of meat for domestic and export
markets.
Next, whole-genome sequencing (WGS) and state-of-the-art high-resolution phylogenomics
were used to investigate microevolution and zoonotic transmission in >240 strains of RT014
and ST11. Core genome analysis revealed that C. difficile common to humans and livestock in
Australia do not form distinct populations but actually share a very recent evolutionary
history. Moreover, for the first time, this approach substantiated inter-species clonal
transmission (over local and national distances) of both C. difficile RT014 and ST11 between
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livestock and humans in Australia. These findings provide compelling evidence that CDI has
a foodborne or zoonotic aetiology. Further, they challenge the existing paradigm that CDI is
primarily a healthcare-associated infection.
Finally, this thesis provides the first analysis of the pan-genomes, and the most
comprehensive investigation to date of the resistomes, prophage repertoire and key virulence
loci for these important lineages. RT014 and ST11 are characterised by ultra-conserved core
genomes and massive open pan-genomes. Such findings are indicative of a versatile sympatric
species with both the capability and high propensity to move freely between different host
populations. Much of this repertoire stems from large diverse collections of clinically
important temperate prophages of the Siphoviridae and Myoviridae present in both lineages.
Also, significant RT and ST-specific heterogeneity were found in key virulence loci such as
the S-layer and toxin gene loci, with a number of novel variants identified. Also, an extensive
array of clinically important AMR genes and mobile genetic elements were identified. These
genetic loci conferred phenotypic resistance to clindamycin, erythromycin, tetracycline, and
aminoglycosides, and in the case of ST11, fluoroquinolones. Importantly, no reduced
susceptibility to first-line human CDI therapies vancomycin, metronidazole and fidaxomicin
was observed. However, this thesis describes a cryptic vanB2-like vancomycin resistance
gene cluster in C. difficile, the first such report for this species, signalling that we may be very
close to seeing a fully vancomycin-resistant strain of C. difficile.
This thesis has made significant contributions to our understanding of the zoonotic potential
and AMR burden of this important pathogen. This knowledge will be essential for the
development of a “One Health” approach to control strategies to reduce the impact of CDI in
humans and animals.

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TABLE OF CONTENTS
CHAPTER 1 INTRODUCTION ............................................................................................. 1
1.1 SCOPE ......................................................................................................................... 2
1.2 THESIS AIM AND RESEARCH OBJECTIVES ....................................................... 3
1.3 THESIS STRUCTURE ................................................................................................ 4
CHAPTER 2 LITERATURE REVIEW ................................................................................ 6
2.1 INTRODUCTION ....................................................................................................... 7
2.2 C. DIFFICILE AND C. DIFFICILE INFECTION ..................................................... 7
2.2.1 A brief history ..................................................................................................... 7
2.2.2 Taxonomic classification .................................................................................... 8
2.2.3 Clinical and economic burden ............................................................................ 9
2.2.4 Clinical presentation and host susceptibility..................................................... 10
2.2.5 Pathophysiology ................................................................................................ 13
2.2.6 Laboratory detection ......................................................................................... 16
2.2.7 Therapeutic options ........................................................................................... 16
2.3 DIVERSITY AND EVOLUTION IN THE GENOME OF C. DIFFICILE .............. 18
2.3.1 Summary (abstract) ........................................................................................... 18
2.3.2 Methods for determining strain relatedness ...................................................... 18
2.3.3 C. difficile phylogeny and molecular epidemiology ......................................... 21
2.3.4 The C. difficile genome ..................................................................................... 23
2.3.5 Pathogenicity, binary toxin and accessory gene regulator loci ......................... 25
2.3.6 Ultra-low genome conservation ........................................................................ 28
2.3.7 Mechanisms shaping diversity and evolution in C. difficile ............................. 29
2.3.7.1 Transposable elements and AMR ..................................................................... 29
2.3.7.2 Bacteriophages .................................................................................................. 31
2.3.7.3 Homologous recombination .............................................................................. 32
2.3.7.4 Forces of selection ............................................................................................ 35
2.3.8 Deciphering the complex and dynamic epidemiology of CDI ......................... 36
2.3.8.1 Evolutionary history of epidemic lineage RT027 ............................................. 36
2.3.8.2 Microevolution and transmission in the hospital environment ......................... 39
2.3.8.3 Community-associated CDI .............................................................................. 42
2.3.8.4 Animal reservoirs and zoonotic potential ......................................................... 44
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2.3.9 Conclusions and future perspectives ................................................................. 48
2.4 C. DIFFICILE IN AUSTRALIAN LIVESTOCK ..................................................... 49
2.4.1 C. difficile in Australian cattle and calves ........................................................ 50
2.4.2 C. difficile in Australian neonatal pigs .............................................................. 51
2.4.3 Summary ........................................................................................................... 53
CHAPTER 3 MATERIALS AND METHODS ................................................................... 54
3.1 MATERIALS ............................................................................................................. 55
3.1.1 Buffers, reagents, and solutions ........................................................................ 55
3.1.2 Bacterial culture media ..................................................................................... 57
3.2 C. DIFFICILE CULTURE AND IDENTIFICATION .............................................. 58
3.2.1 Isolates .............................................................................................................. 58
3.2.2 Stool specimens ................................................................................................ 59
3.3 RECOVERY OF C. DIFFICILE FROM CALVES AT SLAUGHTER ................... 59
3.3.1 Sampling of calve carcasses .............................................................................. 60
3.3.2 Sampling of calve faeces .................................................................................. 60
3.3.3 Culture of C. difficile from carcasses and faeces and spore enumeration ........ 61
3.4 EPIDEMIOLOGICAL TYPING ............................................................................... 62
3.4.1 DNA extraction ................................................................................................. 62
3.4.2 Toxin gene profiling ......................................................................................... 62
3.4.3 PCR ribotyping ................................................................................................. 63
3.4.4 BioNumerics and C. difficile reference strain collection .................................. 64
3.4.5 Capillary electrophoresis PCR ribotyping ........................................................ 66
3.5 ANTIMICROBIAL SUSCEPTIBILITY TESTING ................................................. 66
3.5.1 Agar dilution methodology ............................................................................... 66
3.5.2 Gradient diffusion methodology ....................................................................... 68
3.6 WHOLE-GENOME SEQUENCING ........................................................................ 69
3.6.1 Detailed whole-genome sequencing protocol ................................................... 70
3.6.1.1 Genomic DNA extraction ................................................................................. 70
3.6.1.2 Quantification and normalization of genomic DNA ......................................... 71
3.6.1.3 Preparation of sequencing libraries ................................................................... 71
3.6.1.4 Illumina sequencing chemistry ......................................................................... 72
3.6.2 Summary of other whole-genome sequencing protocols .................................. 76

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3.7 BIOINFORMATICS ................................................................................................. 78
3.7.1 Computing infrastructure and data storage ....................................................... 78
3.7.2 Processing and quality control of raw sequence data ....................................... 78
3.7.3 Open access data ............................................................................................... 79
3.7.4 Comparator and reference genomes.................................................................. 79
3.7.5 Multi-locus sequence typing ............................................................................. 80
3.7.6 Single nucleotide variant analysis pipeline ....................................................... 81
3.7.6.1 Introduction ....................................................................................................... 81
3.7.6.2 Reference mapping, processing and quality control ......................................... 82
3.7.6.3 Variant calling and filtering .............................................................................. 84
3.7.6.4 Maximum-likelihood phylogenetics ................................................................. 86
3.7.6.5 Variant annotation and pairwise SNV analysis ................................................ 86
3.7.6.6 Evaluation of pipeline accuracy and sensitivity ................................................ 87
3.7.7 Genome assembly and annotation pipeline....................................................... 89
3.7.7.1 De novo assembly ............................................................................................. 89
3.7.7.2 Post assembly enhancement .............................................................................. 89
3.7.7.3 Ab initio annotation and quality assessment ..................................................... 90
3.7.8 Core ortholog phylogeny .................................................................................. 91
3.7.9 Pan-genome analysis pipeline ........................................................................... 92
3.7.10 Comparative genomic analysis ......................................................................... 93
3.7.10.1 Prophages .......................................................................................................... 93
3.7.10.2 Acquired AMR genes ....................................................................................... 93
3.7.10.3 Transposons ...................................................................................................... 94
3.7.10.4 Loci associated with pathogenicity and host-pathogen interaction .................. 94
CHAPTER 4 RECOVERY AND QUANTIFICATION OF C. DIFFICILE FROM
AUSTRALIAN CALVES AT SLAUGHTER ...................................................................... 95
4.1 INTRODUCTION ..................................................................................................... 96
4.2 STUDY DESIGN ...................................................................................................... 96
4.2.1 Objectives ......................................................................................................... 96
4.2.2 Study population ............................................................................................... 97
4.3 RESULTS .................................................................................................................. 98
4.3.1 C. difficile recovery from Australian veal calves at slaughter .......................... 98
4.3.2 Concentration of C. difficile on carcasses and in faeces ................................... 99
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4.3.3 Toxin gene profiling ......................................................................................... 99
4.3.4 PCR ribotyping ............................................................................................... 101
4.4 DISCUSSION .......................................................................................................... 105
4.4.1 High prevalence of C. difficile in Australian veal calves at slaughter ............ 105
4.4.2 The many factors influencing C. difficile prevalence in cattle ....................... 105
4.4.3 Veal calves are contaminated with high concentrations of C. difficile and
present a credible risk to the microbiological quality of meat products ......... 108
4.4.4 Australian veal calves are a reservoir for clinically important RTs................ 111
4.4.5 Chapter summary ............................................................................................ 114
CHAPTER 5 COMPARATIVE PHYLOGENOMICS AND AMR ANALYSIS OF C.
DIFFICILE RT014 ............................................................................................................... 115
5.1 INTRODUCTION ................................................................................................... 116
5.2 STUDY DESIGN .................................................................................................... 117
5.2.1 Objectives ....................................................................................................... 117
5.2.2 Study population ............................................................................................. 117
5.3 RESULTS ................................................................................................................ 119
5.3.1 C. difficile RT014 genome metrics and features ............................................. 119
5.3.2 In silico multi-locus sequence typing ............................................................. 120
5.3.3 CE-ribotyping ................................................................................................. 120
5.3.4 Phylogenetic analysis of core orthologous gene clusters ................................ 123
5.3.5 Single nucleotide variant analysis ................................................................... 124
5.3.6 In silico AMR profiling .................................................................................. 128
5.3.7 Prophage discovery ......................................................................................... 129
5.3.8 In vitro antimicrobial susceptibility ................................................................ 129
5.3.9 Genomic context for AMR ............................................................................. 130
5.3.10 Presence and sequence conservation of genes associated with virulence and
host-pathogen interaction ................................................................................ 139
5.3.11 Pan-genome and proteome analysis ................................................................ 146
5.4 DISCUSSION .......................................................................................................... 150
5.4.1 Microevolutionary analysis of C. difficile RT014 from livestock and humans
reveals signatures of long-range intra- and inter-species transmission .......... 150
5.4.2 C. difficile RT014 harbours a diverse repertoire of AMR genes and mobile
genetic elements of clinical importance .......................................................... 152
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5.4.3 RT014 from humans and pigs show similar virulence potential in silico....... 155
5.4.4 Sequence divergence in genes associated with pathogenicity and host-pathogen
interaction largely correlate with ST affiliation .............................................. 156
5.4.5 The C. difficile RT014 lineage is characterised by a large diverse pan-genome
and low levels of genome conservation .......................................................... 157
5.4.6 Chapter summary and concluding remarks..................................................... 158
CHAPTER 6 MICROEVOLUTION AND COMPARATIVE GENOMICS OF C.
DIFFICILE ST11 .................................................................................................................. 159
6.1 INTRODUCTION ................................................................................................... 160
6.2 STUDY DESIGN .................................................................................................... 161
6.2.1 Objectives ....................................................................................................... 161
6.2.2 Study population ............................................................................................. 161
6.3 RESULTS ................................................................................................................ 166
6.3.1 Genome sequence data and metrics ................................................................ 166
6.3.2 In silico multi-locus sequence typing ............................................................. 167
6.3.3 Microevolutionary analysis ............................................................................. 167
6.3.4 Case report: an RT126-ST258 clone in unrelated oncology patients in WA . 176
6.3.5 In silico AMR profiling .................................................................................. 179
6.3.6 In vitro antimicrobial susceptibility and phenotype-genotype concordance .. 183
6.3.7 Genomic context for AMR: transposon characterisation................................ 190
6.3.8 In-depth analysis of vanB2-like element in C. difficile strain AI0499 ............ 194
6.3.9 Prophage discovery ......................................................................................... 197
6.3.10 Comparative analysis of virulence- and pathogenicity-associated loci .......... 200
6.3.11 Pan-genome analysis ....................................................................................... 211
6.4 DISCUSSION .......................................................................................................... 216
6.4.1 Intra- and inter-species transmission of globally disseminated ST11 clones
provides compelling evidence that CDI may be a foodborne or zoonotic
infection .......................................................................................................... 216
6.4.2 ST11 show high levels of AMR mediated by DNA gyrase mutations and a
diverse reservoir of clinically important transposable elements ..................... 225
6.4.3 C. difficile strain AI0499 harbours a cryptic vanB2 resistance operon, the first
such report for this species .............................................................................. 229
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6.4.4 ST11 is characterised by an immense open pan-genome, a large heterogeneous
population of temperate prophages and diversity in major virulence and
pathogenicity-associated loci .......................................................................... 231
6.4.5 Chapter summary ............................................................................................ 238
CHAPTER 7 GENERAL DISCUSSION ........................................................................... 239
7.1 KEY FINDINGS ...................................................................................................... 241
7.1.1 Australian livestock are a significant reservoir of clinically important, zoonotic
lineages of C. difficile ..................................................................................... 241
7.1.2 Livestock-associated lineages of C. difficile harbour a diverse repertoire of
clinically important AMR elements ................................................................ 244
7.1.3 Livestock-associated lineages of C. difficile are characterised by huge open
pan-genomes, large heterogeneous populations of temperate prophages and
diversity in virulence and pathogenicity-associated loci ................................ 247
7.2 STUDY LIMITATIONS AND FUTURE RESEARCH PRIORITIES ................... 249
7.3 CONCLUDING REMARKS ................................................................................... 251
REFERENCES ..................................................................................................................... 252
APPENDICES ........................................................................................................................ A1
APPENDIX I THESIS PUBLICATIONS .......................................................................... A2
APPENDIX II THESIS-RELATED PUBLICATIONS ................................................... A92
APPENDIX III NON-THESIS PUBLICATIONS ........................................................... A93
APPENDIX IV PRESENTATIONS AND AWARDS .................................................... A94
APPENDIX V SUPPLEMENTARY DATA, CHAPTER 5 ............................................ A99
APPENDIX VI SUPPLEMENTARY DATA, CHAPTER 6 ......................................... A105
APPENDIX VII CUSTOM BIOINFORMATICS SCRIPTS ......................................... A129
APPENDIX VIII STUDIES USED TO DEVELOP THE SNV PIPELINE .................. A129

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