Molecular characterisation of Campylobacter concisus : a potential etiological agent of gastroenteritis in children

Istivan, T 2005, Molecular characterisation of Campylobacter concisus : a potential etiological agent of gastroenteritis in children, Doctor of Philosophy (PhD), Applied Sciences, RMIT University.

Document type: Thesis
Collection: Theses

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Title Molecular characterisation of Campylobacter concisus : a potential etiological agent of gastroenteritis in children
Author(s) Istivan, T
Year 2005
Abstract Campylobacter concisus is a human oral cavity bacterium, which has been linked to oral cavity infections such as gingivitis and periodontitis for the last twenty years. C. concisus has also been isolated from non-oral sources since 1989 and was considered as a potential aetiological agent of enteritis in children since 1994. Some patient subgroups, mainly children under 24 months of age, the elderly, and immunocompromised patients are known to be most susceptible to C. concisus enteric infections. However, C. concisus was isolated from saliva and stool of healthy individuals and was recently detected in the synovial fluid of patients with Campylobacter-associated reactive arthritis. Yet, little is known about this bacterium regarding its mode of transmission, reservoir, and its true potential as a pathogen, due to the lack of any established typing procedure and other virulence related studies.

C. concisus clinical strains isolated from children suffering from gastroenteritis at the Royal Children's Hospital (Melbourne), were grouped and characterized in this study using molecular methods to clarify the identity of these isolates and to detect any possible virulence factors that could be related to the pathogenicity of this bacterium. The clinical isolates were grouped into two molecular groups (genomospecies) using PCR amplification of the 23S rDNA of C. concisus with the majority of the isolates (>70%) belonging to genomospecies A. However, the protein profiles for these isolates were divergent even within the same molecular group, but they were different from the protein profiles of other closely related Campylobacter spp. isolates, which indicate the complex nature of this species.

Both secreted and cell-bound hemolytic activities were detected in C. concisus clinical isolates by different hemolysis assays using mammalian and non-mammalian erythrocytes. Variable levels of hemolysis were detected for all tested strains on different types of blood. The species specificity of red blood cell hemolysis and the difference in the hemolytic activity levels have been previously reported for Campylobacter spp.' and other pathogenic bacteria.

The fraction containing the membrane-bound hemolytic activity found in C. concisus clinical isolates was extracted and characterised using different physical and chemical tests. This hemolysin(s) is a stable calcium-dependent and iron regulated protein with phospholipase A2 enzymatic activity that prefers phosphatidylcholine as a substrate when compared to other lipid components of mammalian cells. Similar cell-associated hemolytic activities produced by C. coli and C. jejuni strains were previously related to pathogenicity in these bacteria. The phospholipase A hemolytic extract of C. concisus caused stable damaging effects on eRO tissue culture cell lines. Vacuolation and cytolytic effects were noticed on eRO cells after 18 hours of incubation with C. concisus diluted and concentrated hemolytic extracts respectively.
Remolysins and hemolytic phospholipases of several pathogens such as Serratia marcescens, Haemophilus ducreyi and in Legionella spp. have been reported to cause a damaging effect on human epithelial cells. Moreover, the phospholipases AI, A2 and e of H. pylori were linked to the degradation of the phospholipid components of the mucosal barrier and phospholipase A of H. pylori is thought to have a role in colonisation. Therefore, further studies are required to determine whether this hemolysin isolated from C. concisus produces a vacuolation effect on human epithelial cells similar to that produced by VacA toxin of Helicobacter pylori or VcVac from Vibrio cholerae.

The sequencing and analysis of the C. concisus pldA gene in this study indicated that the nucleotide sequence of this gene in clinical isolates of C. concisus resembles the pldA gene sequence encoding outer-membrane phospholipase A in other pathogenic campylobacters such as C. coli and C. jejuni. The pldA gene of C. concisus encodes for a 35 kDa protein with 97% similarity to the 35 kDa phospholipase A protein found in C. coli. When the nucleotide sequences of the flanking regions of the pldA gene were analysed in this study, they revealed that the pldA gene in C. concisus is located upstream of the' ceuB and ceuC genes, similar to the position of the pldA gene in the C. coli and the C. jejuni genome. Therefore, the presence of this gene in C. concisus clinical isolates indicates the possible pathogenic role for this gene and its encoded PLA protein in gastroenteritis cases caused by C. concisus. However further sequencing studies are needed to analyse both the ceuB and ceuC genes in different C. concisus strains belonging to both genomospecies and also from different geographic origins to detect any similarity between these genes and other pathogenic campylobacters. C. concisus clinical strains from different clinical sources should be screened for the presence of other virulence-related genes such as those encoding for cytotoxins, lipopolysaccharides (LPS) and flagellin to clarify the pathogenic role of this opportunistic pathogen.

It is recommended that C. concisus gastroenteritis cases in children under the age of primary dentition should be further investigated and highlighted leading to more awareness in the community. The detection of possible virulence factors in C. concisus gastroenteritis-related strains in this study, in addition to the recent findings for its possible relation to other infections such as in Campylobacter-associated reactive arthritis indicates the importance of this opportunistic pathogen in patients with low or impaired immunity such as young children, the elderly and immunocompromised patients.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Applied Sciences
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