GENETIC EPIDEMIOLOGY AND HETEROGENEITY OF CAMPYLOBACTER SPP. STEVEN J. DUNN A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS OF NOTTINGHAM TRENT UNIVERSITY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY JUNE 2017 This work is the intellectual property of the author Steven J. Dunn. You may copy up to 5% of this work for private study, or personal, non-commercial research. Any re-use of the information contained within this document should be fully referenced, quoting the author, title, university, degree level and pagination. Queries or requests for any other use, or if a more substantial copy is required, should be directed in the owner of the Intellectual Property Rights. i Dedicated to my family. For everything, Thank you. ii Abstract Initially, this work examines clinical Campylobacter isolates obtained from a single health trust site in Nottingham. These results reveal novel sequence types, and identified a previously undescribed peak in incidence that is observable across national data. By utilising a read mapping approach in combination with existing comparative methods, the first instance of case linkage between sporadic clinical isolates was demonstrated. This dataset also revealed an instance of repeat patient sampling, with the resulting isolates showing a marked level of diversity. This generated questions as to whether the diversity that Campylobacter exhibits can be resolved to an intra-population level. To study this further, isolates from the dominant clinical lineage – C. jejuni ST-21 – were analysed using a deep sequencing methodology. These results reveal a number of minor allele variations in chemotaxis, membrane and flagellar associated loci, which are hypothesised to undergo variation in response to selective pressures in the human gut. In an expansion of this work, additional datasets were generated to include other clinically relevant CC’s, including the C. coli lineage ST-828. These results revealed a similar pattern of diversity, with common loci identified as undergoing variation in multiple samples, and in some instances the same amino acid residue. In general, ST-21 isolates exhibited more non-synonymous mutations, distributed across fewer loci, suggesting that this lineage may have a repertoire of alleles that is more adapted to the human host. To investigate whether the observed diversity is generated during infection, or is maintained from a diverse infectious source, isolates from fresh retail chicken were analysed. These samples revealed a marked decrease in overall diversity, as well as differences in the functions of variable loci. These results show that infections occurring from the consumption of retail chicken (the largest single source of campylobacteriosis) arise from genetically uniform populations, and that the diversity observed in clinical cases is generated within - and furthermore may be specific to - the human host. iii 1. Table of Contents Abstract……………………………………………………………………………...…...…iii List of Figures.…………………………………………………………………………..…vii List of Tables..…….……………………………………………………………..…………ix List of Abbreviations.………………………………………………………………….……x 1. Chapter One: Introduction ..................................................................... 1 1.1. History ....................................................................................................... 2 1.2. Campylobacteraceae ................................................................................ 6 1.2.1. Thermotolerant Species ...................................................................... 9 1.3. Clinical manifestations of Campylobacter ........................................... 12 1.3.1. Campylobacteriosis ........................................................................... 14 1.3.2. Extraintestinal Complications ............................................................ 14 1.3.3. Guillain-Barré Syndrome ................................................................... 15 1.3.4. Transmission ..................................................................................... 15 1.3.5. Incidence and Prevalence ................................................................. 20 1.3.6. Pathogenesis .................................................................................... 27 1.3.7. Genomics .......................................................................................... 33 1.3.8. Study Rationale ................................................................................. 38 2. Chapter Two: Methods ......................................................................... 40 2.1. Bacterial Strains ..................................................................................... 41 2.2. Sampling .................................................................................................. 41 2.3. Culture Media .......................................................................................... 43 2.3.1. Culture Conditions ............................................................................ 43 2.4. DNA Isolation .......................................................................................... 46 2.4.1. DNA Qualification .............................................................................. 48 2.4.2. DNA Quantification ........................................................................... 49 2.5. Whole Genome Sequencing .................................................................. 49 2.5.1. Estimating Sequencing Coverage ..................................................... 51 2.5.2. Genomic Read Curation ................................................................... 52 2.5.3. Genome Assembly and Annotation .................................................. 52 2.5.4. MLST Profiling .................................................................................. 53 2.5.5. Core Genome Phylogeny .................................................................. 53 2.5.6. Deep Sequencing ............................................................................. 54 iv 3. Chapter Three: Genetic epidemiology of clinical Campylobacter spp. ............................................................................................................... 56 3.1. Abstract ................................................................................................... 57 3.2. Introduction ............................................................................................. 59 3.2.1. CompArAtive Genomics of Campylobacter spp. ................................ 61 3.3. Methods ................................................................................................... 65 3.4. Results ..................................................................................................... 67 3.4.1. Incidence ........................................................................................... 67 3.4.2. SequencinG And Assembly QuAlity ................................................... 74 3.4.3. MLST ................................................................................................ 75 3.4.4. PhyloGeny ......................................................................................... 77 3.4.5. SinGle Patient IsolAtes ...................................................................... 82 3.5. Discussion ............................................................................................... 83 4. Chapter Four: Intrapopulation diversity amongst clinical C. jejuni isolates ......................................................................................................... 94 4.1. Abstract ................................................................................................... 95 4.2. Introduction ............................................................................................. 97 4.2.1. BacteriAl evolution ............................................................................. 97 4.2.2. Within-host variation ......................................................................... 98 4.2.3. Limitations of assembly based genomics ....................................... 100 4.2.4. MeAsurinG intrA-population diversity ............................................... 101 4.2.5. The Diversity of Campylobacter spp. .............................................. 103 4.3. Methods ................................................................................................. 105 4.3.1. SequencinG ..................................................................................... 105 4.3.2. ReAd MAppinG ................................................................................. 106 4.3.3. VariAnt CAlls .................................................................................... 107 4.3.4. StAtisticAl AnAlysis of Assembly Metrics ......................................... 108 4.4. Results ................................................................................................... 109 4.4.1. IsolAtes, sequencinG and assembly. ............................................... 109 4.4.2. Minor Allele vAriAtion ....................................................................... 111 4.4.3. ChemotAxis ..................................................................................... 112 4.4.4. FlAGellAr And MembrAne ................................................................. 116 4.4.5. Homopolymeric TrAct ...................................................................... 118 4.4.6. InterGenic .......................................................................................