Date of Award

2013

Document Type

Doctoral Thesis

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Dr. Roy D. Sleator

Second Advisor

Dr. Brigid Lucey

Abstract

Campylobacter jejuni is universally recognized as the leading bacterial cause of human gastroenteritis. However, within recent years a growing number of non- jejuni/coli Campylobacter species have been acknowledged as emerging human and animal pathogens. Advances in molecular diagnostics and development of pioneering culture methodologies have led to the detection of a range of under-appreciated and nutritionally fastidious Campylobacter species, including C. ureolyticus. Herein, we provide the first report of C ureolyticus in the faeces of patients presenting with gastroenteritis and suggest a role for this organism as an emerging enteric pathogen.

Employing a two-tiered molecular study, we report C. ureolyticus as the second most common Campylobacter species detected in the faeces of patients presenting with GI illness. C.ureolyticus. was detected in 83 samples, 23.8% of all positives. Of these, 30 were found as mixed isolates with Campylobacter species and 53 were detected in the absence of the other common bacterial enteric pathogens. We report a prominent seasonal distribution and bimodal age distribution whereby more than 50% of C.ureolyticus infections occurred at extremes of age;70 years, and perhaps suggests an opportunistic role for this pathogen.

Interestingly, of the 349 samples determined Campylobacter-genus positive by molecular means, 46% failed to grow under routine Campylobacter culture; highlighting the considerable contribution of non-culturable Campylobacter species to human enteritis. Additionally, the establish pathogens C. jejuni and C coli contributed to >56% of the non-culturable samples, even though >90% of theses samples were cultured within 3days from receipt. Our findings underscore the limitations of routine Campylobacter culture, for both the thermophilic Campylobacters such as C. jejuni and the emerging and atypical species such as C. ureolyticus.

The availability of the complete genome sequences for two C. ureolyticus isolates, including the type strain, facilitates the first whole genome analysis of C. ureolyticus.

We note a high degree of heterogeneity between C. ureolyticus isolates, in addition to the identification of 106 putative virulence associated factors. Such factors encompass each of the known virulence tactics of pathogenic Campylobacter spp. We provide the first virulence catalogue for C. ureolyticus, the components of which theoretically provide this emerging species with sufficient arsenal to establish pathology.

Using both molecular and biochemical analysis we provide the first characterisation of the C. ureolyticus urease operon. Phylogentic analysis of this 7 gene urease operon, indicates it shared a common ancestor with C. sputorum biovar paraureolyticus, however formed a distinct clade from Helicobacter species and C. lari (UPTC). The C. ureolyticus urease display simple Michaelis-Menten-type kinetics. Additionally, the Km (urea) value of 3.71 ± 0.21 mM, suggests an efficient enzyme. Furthermore, quantification of urease activity from C. ureolyticus clinical strains indicate a urease activity comparable to that of H. pylori, suggesting an important role in gastric transit.

To gain insight into the survival capability of C ureolyticus in both the host milieu and environmental reservoirs, we investigate C. ureolyticus biofilm formation. We report that C. ureolyticus is capable of producing two distinct forms of biofilm growth in liquid culture. Also, we demonstrate that the formation of floes and an adhering biofilm is increased in the presence of 0.5% bile at 37 °C for the C. ureolyticus gastrointestinal isolates. Additionally, we reveal that strains remain viable for >10months post inoculation. It is likely, that biofilms may play a crucial role in the survival of the bacterium under unfavourable environmental conditions and facilitate human spread.

Finally, we investigate the pathogenic potential of C. ureolyticus in an animal model of infection. We provide the first evidence that C. ureolyticus induces acute infection in ILIO knockout mice. We demonstrate that C. ureolyticus is capable of surviving gastrointestinal transit and colonising the gastrointestinal tract of the murine host. Furthermore, we identified clinical symptoms of campylobacterosis; including diarrhoea and gross pathological changes such as splenomegaly and colitis. These results strongly suggest a causal relationship between C. ureolyticus and acute gastrointestinal disease.

Access Level

info:eu-repo/semantics/openAccess

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