Keenan Jacqueline I, Aitchison Alan, Purcell Rachel V, Greenlees Rosie, Pearson John F, Frizelle Frank A
Department of Surgery, University of Otago Christchurch, Christchurch, New Zealand.
Department of Surgery, University of Otago Christchurch, Christchurch, New Zealand.
Anaerobe. 2016 Aug;40:50-3. doi: 10.1016/j.anaerobe.2016.05.004. Epub 2016 May 7.
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.
脆弱拟杆菌是一种存在于大多数人肠道中的共生菌，然而产肠毒素脆弱拟杆菌菌株（ETBF）与腹泻和结直肠癌（CRC）有关。本研究的目的是建立一种在粪便样本中筛查脆弱拟杆菌毒素（bft）基因的方法，以此来确定CRC患者携带ETBF的情况是否比年龄匹配的健康对照者更常见。使用特异性检测bft基因的引物，通过标准PCR和定量PCR对71例近期诊断为CRC的患者及71例年龄匹配的对照者的粪便样本进行筛查。粪便样本中的细菌模板DNA通过两种方法制备：一种是刮取法，即在厌氧环境下于37℃对粪便进行48小时培养后，将在胆汁七叶苷琼脂平板上生长的所有菌落刮入无菌水中并进行热处理；另一种是直接从每个粪便样本中提取DNA。从细菌刮取物中分离的DNA比匹配的直接DNA提取物更频繁地检测到bft基因。发现定量PCR在检测bft方面比标准PCR更敏感。两种样本类型的定量PCR阳性检测累计总数显示，19例CRC患者的粪便样本中有毒素基因证据（27%），而年龄匹配的对照者中有7例（10%）。这种差异具有统计学意义（P = 0.016）。总体而言，与对照相比，CRC患者粪便样本中更常检测到ETBF携带，但不同DNA制备方法和检测方法的不同结果表明，敏感性差可能会限制粪便样本中ETBF的分子检测。
