The observed high level of genetic diversity amongst the isolates of C. concisus is in agreement with previous studies [1, 2, 10], and highlights the complex nature of this species. Cluster analysis of AFLP profiles indicated that the isolates examined in the current study comprised at least two distinct clusters. Similarly, Aabenhus et al.  denoted four AFLP clusters among 62 C. concisus isolates of which the majority (n = 56) were assigned to one of two main clusters. Results of PCR assays targeting the 23S rRNA and cpn60 genes largely corresponded with the AFLP grouping, and lend support to the suggested genetic relationship between the isolates.
As C. concisus is a common inhabitant of the oral cavity, it is to be expected that it may be isolated from both healthy and diarrheic individuals. Examining isolates from healthy individuals, it was observed that the majority of isolates belonged to genomospecies A and their AFLP profiles clustered together (AFLP cluster 1) along with the type stain of oral origin. This AFLP cluster also included one genomospecies A isolate (CHRB 1609) from a diarrheic individual. Further studies are needed to determine whether this group of isolates represents inhabitants of the oral cavity that have survived gastro-intestinal transit or whether they are intestinal-associated.
The majority (94%) of isolates from diarrheic individuals were assigned to ALFP cluster 2. Among these isolates, 71% were assigned to genomospecies B, while only 11% of diarrheic isolates belonged to genomospecies A. Engberg et al.  reported a similar predominance of genomospecies B isolates among diarrheic fecal isolates, of which 33% and 67% were assigned to genomospecies A and B, respectively. Likewise, Aabenhus et al.  reported that 34% and 53% of fecal isolates from diarrheic patients were assigned to genomospecies A and B, respectively. Our observation that isolates from genomospecies B were exclusively obtained from diarrheic individuals suggests a potential role for these isolates in intestinal disease. A comparative molecular examination of strains belonging to genomospecies A and B may shed light on their respective pathogenic potential.
Examination of the pathogenic properties amongst C. concisus isolates determined that epithelial invasion and translocation were higher for isolates assigned to AFLP cluster 2 (of which 94% were from diarrheic individuals). Additionally, epithelial translocation was higher for isolates belonging to genomospecies B (of which all isolates were from diarrheic individuals). This is of potential clinical relevance as invasiveness and translocation ability are the only factors definitively correlated with enteritis in C. jejuni-infected patients  and are likely associated with inflammatory responses and occasional bacteraemia observed with C. concisus infections . To our knowledge, this is the first study to report differences in pathogenicity between the two main C. concisus genomospecies, further supporting the likelihood that isolates belonging to AFLP cluster 2/genomospecies B incite enteritis in humans.
All of the clinical C. concisus isolates examined in the current study caused hemolysis of sheep erythrocytes, consistent with previous observations of hemolytic phospholipase activity in all C. concisus genomospecies A and B isolates from diarrheic children . As such, hemolytic activity appears to be a general characteristic of this species. Hemolysins are involved in pathogenesis and host colonization in other taxa , thus it was an unexpected observation that C. concisus genomospecies A isolates exhibited greater mean hemolysis than isolates belonging to genomospecies B. We also observed that hemolytic activity by C. concisus was inversely correlated with epithelial adherence and invasion. Staphylococcus aureus exhibits a similar inverse correlation that is attributed to interference of its α-hemolysin with epithelial β1-integrins that mediate host-cell interactions . Moreover, lower amounts of α-hemolysin are produced by invasive S. aureus isolates from endocarditis patients compared to less-invasive isolates from open wounds . Whether C. concisus hemolysin also interferes with epithelial receptors that promote adherence and invasion is unknown, and additional studies are warranted.
Another unexpected finding was that isolates from healthy individuals induced greater mean epithelial DNA fragmentation and metabolic activity compared to those from diarrheic individuals, and these variables were positively correlated. DNA fragmentation is used as an indicator of cell death. The two primary modes of cell death, namely apoptosis and necrosis, can be distinguished on the basis of physiologic features. DNA fragmentation can be present in both processes; however, during apoptosis, cell membranes typically remain intact, whereas during necrosis, cellular integrity is rapidly disrupted leading to the release of cytoplasmic contents (including lactate dehydrogenase) into the surrounding environment ("cytotoxicity"). Based on this definition, four isolates from healthy individuals (CHRB2004, CHRB3235, CHRB3287, and CHRB3290) and two isolates from diarrheic humans (CHRB2370 and CHRB3152) induced cell death consistent with apoptosis. While apoptosis can be mediated via CDT , it is unlikely that CDT was involved in the apoptosis given that we did not detect the CDTB gene in any of the strains included in our study. Furthermore, we did not find any genes with similar sequence to the CDTB gene using a BLAST search of the published C. concisus genome (NCBI accession number NC_009802), indicating that other factors (i.e. opposed to the CDT) may be responsible. The role that Campylobacter-induced epithelial cell death plays in pathogenesis is currently poorly understood; hence, the clinical significance of these findings for C. concisus remains to be determined.
Metabolic activity can be measured using the MTT assay in which metabolically active epithelial cells reduce a yellow tetrazolium salt (MTT) to purple formazan crystals that can be spectrophotometrically quantified. All of the isolates that we examined, except one isolate that caused epithelial sloughing (CHRB6), induced higher MTT values (> 130%) than the control, indicating that epithelial metabolic activity is increased by C. concisus. Some clinical strains of C. jejuni have also been reported to cause similar increases in epithelial MTT values . Given the short incubation period for the MTT assay, we conclude that the increased values most likely reflect an increase in metabolic activity due to cellular stress rather than an increase in epithelial cell numbers due to proliferation. The observed correlation between metabolic activity and DNA fragmentation may be a consequence of the increased energy demands required to sustain the apoptotic process (i.e., apoptotic DNA fragmentation is an ATP-dependent process ).
The chemokine, IL-8 is a major mediator of inflammation. In the current study, all C. concisus isolates induced transcription of IL-8 in epithelial monolayers (> 2-fold) as has been previously reported for C. jejuni  and C. concisus . Campylobacter jejuni induces epithelial IL-8 secretion by at least two independent mechanisms, one of which requires invasion and the other that is CDT-dependent [19, 34]. We observed that induction of IL-8 transcription by C. concisus was not correlated with invasion. Man et al. also recently showed that three C. concisus strains stimulated production of IL-8 in intestinal epithelial irrespective of their invasive ability . Thus in contrast to C. jejuni, it appears that factors other than invasion or CDT (which appears to be lacking in this species) are responsible for the up-regulation of IL-8 incited by C. concisus. The observation that expression of IL-8 mRNA was greater in epithelial cells treated with isolates from AFLP cluster 1 compared to isolates from cluster 2 was unexpected and suggests that these isolates may have pathogenic potential.
We identified genes encoding S-layer RTX and the zonnula occludins toxin in some of the isolates, confirming initial reports of these toxin genes in C. concisus . Surprisingly, the zot gene was more prevalent in isolates from healthy (80%) compared to diarrheic (22%) humans. The clinical significance of this observation remains to be determined. Although Zot has been shown to disrupt epithelial tight junctions, we did not observe any changes in permeability or TER of epithelial monolayers throughout the 3 h incubation period for any of the isolates. This is contrary to the observation of Man et al., that C. concisus caused increased epithelial permeability, decreased TER, and loss of membrane-associated zonnula occludens and occludin in epithelial monolayers . Possible reasons for this discrepancy include variation in methodology between the two studies (i.e., Man et al. inoculated Caco-2 cells with an MOI of 200, and assessed barrier function 6 h-post inoculation.).