Pangenome-guided tools for investigating the role of epsilonproteobacteria in human gastroenteritis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Veterinary Science at Massey University, Manawatu, New Zealand
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Date
2018
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Massey University
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Abstract
Gastroenteritis affects billions of people every year and current testing methods fail to
identify the cause for approximately half of the samples submitted for microbiological testing.
Epsilonproteobacteria contains Campylobacter jejuni, the most commonly reported cause of
bacterial gastroenteritis in the world, and Helicobacter pylori, a gastric pathogen and class I
carcinogen. This bacterial class also contains ≥20 additional species known, or suspected, of
being human pathogens. To better understand the role some of these species play in human
gastroenteritis, novel rapid, cost effective methods are needed. The growing number of whole
genome sequences available for this class were exploited to first evaluate the classification of
the genetically heterogeneous species C. concisus and then to identify taxon-specific CDS for a
range of Epsilonproteobacterial taxa. Probes were designed to detect 28 of these CDS and
incorporated into a single multiplex ligation-dependent probe amplification (MLPA) assay which
was tested against DNA from 43 Epsilonproteobacterial species and then applied to DNA
extracts from stool samples from a childhood gastroenteritis case control study undertaken in
Belgium. The 22 C. concisus genomes consistently clustered into two genomospecies (GS)
represented by ATCC 33237T (GS1) and CCUG 19995 (GS2). Taxon-specific genes were
identified for 28 taxa, including the two C. concisus genomospecies, and concordant results
were observed for the majority of MLPA probes and DNA extracts from pure cultures. The
probes designed to detect C. lari subsp. concheus and H. pullorum failed to detect the target
DNA; all of the urease positive thermophilic Campylobacter DNA extracts were also positive for
the probe designed to detect C. subantarcticus, some probes lacked repeatability in the
presence of elevated EDTA and the size differences between some probes needs to be
optimised. C. jejuni was the most common Epsilonproteobacterial species isolated by culture
and C. concisus was the most common species detected by MLPA. Both C. jejuni and C.
concisus GS2 were detected in significantly higher numbers in cases than controls in a Belgian childhood case control study. This demonstrated the utility of the Epsilonproteobacteria MLPA
assay and provides some evidence that C. concisus GS2 may have a role in childhood
gastroenteritis.
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Keywords
Bacterial genetics, Technique, Bacterial genomes, Analysis, Campylobacter, Identification, Gastroenteritis in children, Case studies