Pathogenesis and ToxinsScreening for enterotoxigenic Bacteroides fragilis in stool samples
Introduction
Bacteroides fragilis are anaerobic gram negative bacteria commonly found as part of the human faecal microbiota. Infection most likely occurs in early childhood, and the inherent ability of B. fragilis to evade the host immune response suggests that colonisation may persist for life [1]. Whereas carriage of non-enterotoxigenic strains of B. fragilis may promote mucosal health [2], some strains produce a toxin that is identified as causative agent of human diarrhoea [3]. Colonic carriage of toxin-producing strains is also reportedly more prevalent in people presenting with colorectal cancer (CRC) compared to healthy controls [4], [5], [6], fuelling speculation that persistent carriage of these bacteria may “drive” colon carcinogenesis [7]. CRC rates of 37.3 per 100,000 New Zealanders are amongst the highest in the world [8]. Our aim was to establish a method for screening stool samples for evidence of the B. fragilis toxin (bft) gene, as a first step in determining if this potential marker of colon carcinogenesis is detected more frequently in stool of CRC patients than in age-matched healthy controls. This approach involved the processing of stool samples by two different methods: culture-based amplification [9], [10] and direction extraction [11], [12], and testing each of these samples by standard and quantitative PCR (qPCR).
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Study participants
Seventy-one individuals, diagnosed with CRC between 2012 and 2014, using standard endoscopic, histological or radiological criteria, provided a stool sample prior to chemo-radiation and/or surgery. Patients found to have had pre-operative chemo-radiation therapy or those with adenomas were not included in the study. Seventy-one stool samples from a cohort of 125 collected from healthy volunteers who self-reported no evidence of bowel problems at the time of sampling were age-matched to the 71
Incidence of bft gene in stool samples analysed by standard PCR
Stool samples from 71 patients recently diagnosed with CRC, and 71 age-matched controls were screened using primers specific for the detection of the B. fragilis toxin (bft) gene in both bacterial sweeps and direct DNA extractions. Standard PCR revealed that sweeps yielded more positive PCR results (16/142 (11%)) than DNA extracted from stool samples (11/142 (8%)), indicating that the sweep method is more sensitive (Table 2). This was confirmed by assaying serial dilutions of stool samples
Discussion
There is a growing awareness that certain species of gut bacteria may drive toxin- and/or inflammation-mediated colorectal carcinogenesis [20]. We have been collecting evidence of a significant association between long-term colonic carriage of ETBF and risk of low-grade colonic dysplasia in our community. This led us to evaluate two methods for use as faecal diagnostic tests for ETBF in individuals considered at risk of developing CRC.
Culture-based amplification was shown to provide greater
Conflict of interest declaration
The authors declare that they have no conflict of interest.
Acknowledgements
This study was funded by grants from Genesis Oncology Trust NZ and the Rotary Club of Christchurch Sunrise.
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