Colonisation and virulence characteristics of Campylobacter concisus oral and clinical strains

Campylobacter concisus is a heterogenic bacterium which normally colonises the human oral cavity. It has been associated with oral cavity infections including gingivitis and periodontitis. It has also been isolated from other sites including enteric infections mainly in the elderly, immunocompromised patients, and children less than 24 months of age. To date there are little data about the mode of transmission, reservoir, and pathogenic potential of C. concisus. This bacterium is genotypically divergent with two or more genetic subgroups (genomospecies), however it is not known if a particular genomospecies is more associated with infections. This study investigated genetic diversity and colonisation of C. concisus amongst families with infants and newborn babies and other healthy individuals.<br><br>In this study, C. concisus strains were isolated from gum swabs of healthy volunteers of different age groups ranging from 1 week-50 years of age. The strains were collected from 7 different families and other unrelated healthy individuals. A total of 47 C. concisus oral strains, including 9 strains from infants before teething were isolated. The isolated strains were grouped into genomospecies (A and B) according to PCR amplification of the 23s rDNA. Interestingly, all infants were colonised with genomospecies B before teething except one infant whom was colonized with different C. concisus isolates from genomospecies A and B. Furthermore, three infants were followed up after teething and were all found to be colonised by genomospecies A isolates as well as genomospecies B. In general, 60% of C. concisus strains belonged to genomospecies A, while genomospecies B composed 40% of the strains. However, genomospecies B was found to be the dominant within children.<br><br>To investigate the phenotypic and genotypic relationship between C. concisus strains, SDS-PAGE was performed for each family on the outer membrane proteins (OMPs) of isolates from each member. Non-teething infants were also analysed separately. The dendrograms generated from SDS-PAGE profiles of the isolated C. concisus strains, showed that the OMPs were not identical even for the strains isolated from members of the same family. The only exception was the two strains isolated from twin infants within one of the families in this investigation.<br><br>This study also investigated motility as a virulence factor in C. concisus including the presence of the flagellin genes flaA, flaB and flaC and their expression and also the motility speed of selected C. concisus strains. Fifteen C. concisus clinical strains isolated from children suffering from gastroenteritis at the Royal Children’s Hospital, Melbourne, and two reference strains (ATCC 51561 and ATCC 51562) in addition to the oral strains from this study were tested. Using specific PCR primers, designed according to C. concisus 13826 published whole genome to amplify the full gene nucleotide sequence, flaA was amplified in some strains (26.5%) regardless of the genomospecies while flaB was detected in genomospecies B strains only (34.4%). In contrast, flaC was detected in all C. concisus clinical and oral isolates tested. New primer sets from shorter regions (<300 bp) of all these genes were designed from the conserved region in each gene to be used for gene expression. Conserved regions of flaA, flaB and flaC and their expression were thus studied for the first time in C. concisus grown in brain heart infusion broth (BHI). The expression of flagellin genes was investigated using RT-PCR compared to the reference strain C. concisus ATCC 51561. flaA was significantly up-regulated in only one C. concisus oral strain, while flaB was significantly up-regulated in two C. concisus strains including the strain with up-regulated flaA. However, only flaC gene expression was significantly up-regulated in all tested C. concisus strains.<br><br>Virulence characteristics related to motility, adhesion and invasion were investigated in selected C. concisus oral strains. The motility speed was measured using a modified method from Differential Dynamic Microscopy (DDM). Adhesion and invasion assays were also performed in an in vitro model using the human epithelial cell line INT-407. A C. concisus strain isolated from a healthy child (RMIT O17) was found to be significantly more invasive than the other tested strains. The motility speed of the tested strains varied, however C. concisus RMIT-O17 was found to be more motile than the other tested strains.<br><br>To conclude, Cape Town filtration protocol is suggested to be the best way to isolate C. concisus from clinical and oral samples. C. concisus genomospecies B was found to first colonise the human oral cavity. Our results suggest that there are variations in the expression of flagellin genes within C. concisus strains and these genes are heterogeneous in different genomospecies. The flaC primer set used in this study can be used as a species-specific primer set to detect new C. concisus isolates. Based on flagellin genes tested in this study, knock-out mutants of flaA, flaB and flaC genes would be necessary to investigate the role of these genes in motility.