MacIntyre, SheilaAlmuqrin, Abdulmajeed Mohammed A2025-04-282024-03-07https://hdl.handle.net/20.500.14154/75286Campylobacter jejuni stands as the predominant etiological agent responsible for bacterial gastroenteritis worldwide. Notably, isolates of this pathogen exhibit considerable genetic variability, thereby giving rise to discernible differences in both physiological and virulence properties. Historically, the predominant emphasis of scientific and clinical research has been directed towards C. jejuni strains procured from human clinical specimens, the agricultural milieu and the food production industry. However, it is important to acknowledge that such isolates may not unequivocally serve as true representatives of the natural populations of C. jejuni. Recent observations revealing significant diversity within Campylobacter populations colonizing wild animals have sparked increased interest among microbiologists focused on understanding mechanisms of C. jejuni host adaptation, colonization and pathogenesis. An earlier comparative genomics analysis of newly isolated C. jejuni strains from farm associated Norway rats revealed that ~20% of isolates belonged to a clearly distinct and previously unrecorded clonal complex, termed RG-1. This suggested that Norway rats harbor an unexplored C. jejuni diversity. This study established continued association of RG-1 strains with farm rats, with no evidence of recovery of related strains from other sources. By also addressing the genetic diversity and physiological properties of these strains this study has aimed to identify factors that may contribute to the apparently specialized niche association of the RG-1 clade of C. jejuni. The continued prevalence of C. jejuni RG-1 strains and the diversity of rat-associated C. jejuni were investigated by isolation from faecal samples of Norway rats inhabiting two local farms. Of the forty-four isolates identified as C. jejuni, thirty-three were found to be glc positive C. jejuni while four strains (Dg434a, Dg456, Dg471 and Dg475) were identified as RG-1 isolates. Strains were identified as RG-1 using a targeted PCR screen, developed using a comparative genomics approach that shortlisted a set of 13 potential marker genes for RG-1. Further, in-silico analyses and PCR trials further narrowed this down to 3 target genes which along with hipO could be used in an extensive rapid screen to directly assess prevalence of RG-1 strains in a wider range of geographic and animal sources. Hybrid genome sequencing followed by phylogenetic analyses confirmed assignment of the newly isolated RG-1 strains. All four clustered in a distinct clade with thirty previously isolated RG-1 strains, when compared to the diverse range of all previously sequenced rat-associated Campylobacter isolates. Notably, phylogenetic analysis within the RG-1 group highlighted divergence between farms and clustering of isolates from the same farm, regardless of the farmed animal. MLST analyses identified that three of the four RG-1 strains (Dg456, Dg471, and Dg475) are closely related to Dg147, sharing the same allelic profile for the aspA, glnA, gltA, glyA, tkt, and uncA loci but differing in the pgm locus. However, the fourth strain, Dg434a, exhibited differences in alleles for aspA, pgm, and tkt7, indicating its phylogenetic distance from most other RG-1 strains. The phylogenetic distinctions between RG-1 strains and other C. jejuni clinical and chicken isolates suggested a potentially unique ecological niche for these strains and hinted at variations in crucial metabolic and physiological traits. Functional annotations of genome sequences of RG-1 strains highlighted possession of key genes related to specific carbon source metabolism, amino acid catabolism, motility, oxidative stress response, and heat shock resistance. Comparative genomics revealed 28 unique genes in RG-1 strains absent in other C. jejuni strains and potentially relevant to niche survival and host colonization. Growth of RG-1 strains exhibited the classic microaerophilic profile of C. jejuni but generally exhibited poorer growth and lower motility than NCTC 11168 and some other rat isolates. A notable distinguishing feature of RG-1 isolates was the exceptionally poor survival at 42oC. This narrow range of growth temperature for RG-1 could be one of the reasons for successful colonisation of rats and apparently not chickens. Adaptability to different growth temperatures may be an important property in defining the habitat or host niche of more diverse C. jejuni strains. Similarly, the mucosal contents might play an important role in providing protection to RG-1 strains and suitable environment for growth and colonisation of the host gut as caecal mucus preparations from rats and chicken both provided protection to RG-1 strains. This project confirms continued association of the highly diverged clade of C. jejuni RG-1 strains with farm associated rats and identifies several factors that may contribute to niche survival and specific host colonization. This provides a solid foundation for further analysis of geographic and host prevalence of these strains together with elucidation of mechanisms involved in niche preference. Ultimately, this clade of strains may prove to be a useful example of C. jejuni with high host specificity with possibilities for development of protection against infection in the agriculture industry.310enfarm associated ratsCampylobacter jejunigastroenteritisThesis