title: Screening for enterotoxigenic Bacteroides fragilis in stool samples.
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.
authors.affiliation: Department of Surgery, University of Otago Christchurch, Christchurch, New Zealand. Electronic address: jacqui.keenan@otago.ac.nz.
authors.forename: Jacqueline I
authors.initials: JI
authors.lastname: Keenan
authors.affiliation: Department of Surgery, University of Otago Christchurch, Christchurch, New Zealand.
authors.forename: Alan
authors.initials: A
authors.lastname: Aitchison
authors.affiliation: Department of Surgery, University of Otago Christchurch, Christchurch, New Zealand.
authors.forename: Rachel V
authors.initials: RV
authors.lastname: Purcell
authors.affiliation: Microbiology Department, Canterbury Health Laboratories, Christchurch, New Zealand.
authors.forename: Rosie
authors.initials: R
authors.lastname: Greenlees
authors.affiliation: Biostatistics and Computational Biology Unit, University of Otago Christchurch, Christchurch, New Zealand.
authors.forename: John F
authors.initials: JF
authors.lastname: Pearson
authors.affiliation: Department of Surgery, University of Otago Christchurch, Christchurch, New Zealand.
authors.forename: Frank A
authors.initials: FA
authors.lastname: Frizelle
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D001427:Bacterial Toxins / Q000032:analysis* / Q000096:biosynthesis; D001442:Bacteroides Infections / Q000175:diagnosis* / Q000378:metabolism / Q000382:microbiology / Q000473:pathology; D001441:Bacteroides fragilis / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity*; D016022:Case-Control Studies; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000378:metabolism / Q000382:microbiology / Q000473:pathology; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D055088:Early Detection of Cancer; D005243:Feces / Q000737:chemistry* / Q000382:microbiology; D005260:Female; D015870:Gene Expression; D005798:Genes, Bacterial; D006801:Humans; D008297:Male; D008666:Metalloendopeptidases / Q000032:analysis* / Q000096:biosynthesis; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D012680:Sensitivity and Specificity; D014774:Virulence
chemical_list: D001427:Bacterial Toxins; D017931:DNA Primers; D004269:DNA, Bacterial; C405031:Bacteroides fragilis toxin; D008666:Metalloendopeptidases
keywords: Colorectal cancer; Enterotoxigenic Bacteroides fragilis; PCR; Stool samples; bft gene