Date of Award

2002

Document Type

Doctoral Thesis

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Dr. Seamus Fanning

Abstract

The purpose of this study was to investigate the epidemiology of thermophilic Campylobacter infections in Ireland, and to assess antimicrobial resistance among animal isolates and its relevance to human enteric infection with Campylobacter. The first part of the study focused on 84 isolates of human, poultry and porcine origins, isolated between 1996 and 1998 in the Cork region. These isolates were identified to species level using a combination of biochemical and molecular methods. Campylobacter jejuni (C. jejuni) was the predominant organism among human and poultry isolates, whilst all of the porcine isolates examined were identified as C. coli. DNA amplification fingerprinting (DAT) of the collection demonstrated a high degree of genomic heterogeneity among isolates. No DAT banding pattern observed for any of the human isolates matched those profiles observed among the animal-derived isolates. Furthermore, the genomic diversity of 36 human isolates appeared to demonstrate 32 possible different sources of infection. Antimicrobial resistance profiling of the collection showed that higher levels of drug resistance were encountered among isolates of porcine origin compared with those from either human or poultry sources.

Antimicrobial resistance occurs through the acquisition of new genes rather than by mutation events. The former (potential) mechanism was investigated in this study. A novel class of mobile genetic element, termed integrons, had previously been studied among pseudomonads and the Enterobacteriaceae. Their presence among Campylobacter spp. had not been noted to date. A preliminary investigation of the collection revealed the presence of integron-like structures of varying sizes in all isolates for the first time. Sequence analysis of three commonly-occurring amplicons revealed that each contained two partial open reading frames (ORF), one of unknown function and one of which was homologous with Helicobacter pylori {H pylori) tRNA synthetase. It was suggested that these structures might, in part, contribute to the plasticity of the Campylobacter genome, permitting site-specific recombination of unrelated DNA fragments from the environment by natural means. In this way, Campylobacter acquires DNA that may ultimately form part of its genetic structure.

The second part of the study focused on a larger collection of 378 strains of human and poultry origin that had been isolated during the year 2000. This section of the project was part of the first attempt to describe the molecular epidemiology of Campylobacter spp. in Ireland. Antimicrobial resistance profiling of a subset of 145 of these 378 isolates revealed a significant increase in resistance rates to ciprofloxacin and tetracycline when compared to the period 1996-1998. Ciprofloxacin resistance rates had risen from approximately 2% during the earlier period, to approximately 30% during the year 2000, among human and poultry isolates. A combination of agar diffusion and molecular methods for the detection of resistance provided a good correlation and aided our understanding of the underlying mechanism in this case. Since Campylobacter is primarily a zoonotic organism, it was likely that ciprofloxacin resistance had a foodborne origin, whether domestic or imported. Our data suggested the possibility, that Ireland was importing a ciprofloxacin resistance problem via nondomestic poultry product. By application of methods developed during this study it is clear that molecular investigation can facilitate the statutory functioning of such national bodies as the Food Safety Authority of Ireland (FSAI), which has responsibility for the protection of public health.

Furthermore, tetracycline resistance rates showed an increase from approximately 16% during the period 1996-1998 to approximately 28% during the year 2000, when tested by agar diffusion methods. Resistance to tetracycline among Campylobacter spp. is, in the great majority of cases, mediated by the tetO gene, and has been associated in some studies with a 23-kbp plasmid. Experimental work carried out in this study investigated the relationship between the presence of plasmids and resistance to tetracycline, and concluded that the presence or absence of plasmids did not correlate strongly with resistance. Polymerase chain reaction (PCR) analysis of the isolates using a primer pair derived from the published sequence of the tetO gene showed good correlation with phenotypic tetracycline resistance, however, and could provide a useful additional screening method for tetracycline resistance among Campylobacter spp. A non-isotopically-labelled tetO probe located the tetO gene to loci both on the host chromosome and on the 23-kbp plasmid when applied to a subset of strains and their corresponding plasmids.

A very large collection of isolates like this one also provided an opportunity to explore the question of transmission of specific genotypes through the food chain. In particular, this provided the opportunity to evaluate commonly-used phenotypic and molecular methods for the speciation of Campylobacter. Interestingly, the interpretation of the hippurate hydrolysis test, a major standard laboratory diagnostic method applied to differentiate C. jejuni and C coli, failed in some cases to identify these species correctly. This may be partly explained by the fact that some of the C. jejuni isolates identified either lacked this marker altogether or contained a marker that was substantially altered (by mutation). This feature necessitated a re-examination of conventional approaches to identification, at least to species level.

Genomic fingerprinting by DAF identified 145 designated profiles within this collection at 80% similarity, and it was possible to type all isolates by this method. There were 39 indistinguishable clusters, a number of which were isolated from both human and poultry sources. The majority of human infections studied, however, did not represent outbreaks. DAF analysis demonstrated a high degree of heterogeneity among this collection, in agreement with earlier studies of the 1996-1998 collection of isolates. However, at 80% similarity, the largest cluster accounted for 20% of the total collection. Within this cluster, 80% of the isolates were of human origin (the greater part of the human collection). This may suggest that certain isolates are more pathogenic to humans than others.

Finally, integron analysis was performed on the collection. Amplicons were detected in 84.1% of the isolates, which included human and poultry isolates, and both C. jejuni and C. coli. A total of 67.5% of isolates contained amplicons of not more than 700 bp in size. A fragment of 1,000 bp (along with additional smaller fragments) was detected among 15.8% of C. jejuni and C coli isolates, both from human and poultry sources. Amplicons greater than 1,000 bp in size were detected in two strains only, both of which were poultry-derived C. coli isolates. In all, several combinations of amplicons were observed. These ranged in size from approximately 150 to 1,100 bp. A total of 62 isolates was shown to have a gene cassette structure of approximately 1,000 bp, with the majority being identified as C. jejuni. Sequencing of one 700 bp fragment and four of the 1,000 bp fragments was performed. The 700 bp fragment showed a high degree of homology with glycyl tRNA synthetase of H. pylori (similar to the partial 466 bp ORF previously identified in the 1996-1998 collection).

Sequence data from all of the 1,000 bp fragments were identical and the ORF contained within this gene cassette was identified as an aadA2 gene, previously unknown in Campylobacter spp. This gene confers resistance to streptomycin and spectinomycin. Alignment studies of the corresponding deduced amino acid sequences identified three amino acid substitutions among aadA2 genes studied from Salmonella serovars and other enteric pathogens such as Aeromonas salmonicida and Vibrio cholerae. Antimicrobial genes such as this offer the possibility of using these unique markers to aid epidemiological studies in the future.

Comments

A thesis presented to the Higher Education Authority for the degree of Doctor of Philosophy

Access Level

info:eu-repo/semantics/openAccess

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