Elsevier

Anaerobe

Volume 40, August 2016, Pages 50-53
Anaerobe

Pathogenesis and Toxins
Screening for enterotoxigenic Bacteroides fragilis in stool samples

https://doi.org/10.1016/j.anaerobe.2016.05.004Get rights and content

Highlights

  • Molecular identification of enterotoxigenic Bacteroides fragilis (ETBF) in stool samples.

  • Comparison of different methods of DNA preparation and ETBF amplification.

  • Selective enrichment of Bacteroides increases identification of ETBF.

  • Quantitative PCR increases sensitivity over conventional PCR.

  • Use of multiple methods offers greatest detection rates.

Abstract

Bacteroides fragilis is a commensal bacterium found in the gut of most humans, however enterotoxigenic B. fragilis strains (ETBF) have been associated with diarrhoea and colorectal cancer (CRC). The purpose of this study was to establish a method of screening for the Bacteroides fragilis toxin (bft) gene in stool samples, as a means of determining if carriage of ETBF is detected more often in CRC patients than in age-matched healthy controls. Stool samples from 71 patients recently diagnosed with CRC, and 71 age-matched controls, were screened by standard and quantitative PCR using primers specific for the detection of the bft gene. Bacterial template DNA from stool samples was prepared by two methods: a sweep, where all colonies growing on Bacteroides Bile Esculin agar following stool culture for 48 h at 37 °C in an anaerobic environment were swept into sterile water and heat treated; and a direct DNA extraction from each stool sample. The bft gene was detected more frequently from DNA isolated from bacterial sweeps than from matched direct DNA extractions. qPCR was found to be more sensitive than standard PCR in detecting bft. The cumulative total of positive qPCR assays from both sample types revealed that 19 of the CRC patients had evidence of the toxin gene in their stool sample (27%), compared to seven of the age-matched controls (10%). This difference was significant (P = 0.016). Overall, ETBF carriage was detected more often in CRC patient stool samples compared to controls, but disparate findings from the different DNA preparations and testing methods suggests that poor sensitivity may limit molecular detection of ETBF 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).

Section snippets

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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