Molecular characterization of the bacteria adherent to human colorectal mucosa

Abstract

Aims: To study large intestinal mucosal bacterial communities by Denaturing Gradient Gel Electrophoresis (DGGE) profiling and sequencing of 16S rRNA gene polymerase chain reaction (PCR) products amplified from DNA extracted from colorectal biopsies taken from healthy individuals. The specific aims were to determine how similar the mucosa‐associated bacterial communities are within and between individuals and also to characterize the phylogenetic origin of isolated DGGE bands. Methods and Results: Human colorectal biopsies were taken at routine colonoscopy from 33 patients with normal looking mucosa. The DNA was extracted directly from single biopsies and the bacterial 16S rDNA PCR amplified. The PCR products were profiled using DGGE to generate a fingerprint of the dominant members of the bacterial community associated with the biopsy. The reproducibility of this method was high (>98%). Washed and unwashed biopsies gave similar DGGE banding patterns (Median Similarity Coefficient – MSC 96%, InterQuartile Range – IQR 3·0%, n = 5). Adjacent biopsies sampled from the same patient using different forceps gave similar DGGE profiles (MSC 94%, n = 2). Two colorectal biopsies sampled at locations 2–5 cm apart, from each of 18 patients, resulted in very similar profiles (MSC 100%, IQR 2·8%). Biopsies sampled from different locations within the large intestine of the same patient also gave similar DGGE profiles (MSC 98% IQR 3·3%n = 6). Although all patients (n = 33) gave different DGGE profiles, some similarity (c. 34%) was observed between profiles obtained from 15 patients arbitrarily selected. 35 DGGE bands were excised and sequenced. Many were found to be most closely related to uncultured bacterial sequence entries in the Genbank database. Others belonged to typical gut bacterial genera including Bacteroides, Ruminococcus, Faecalibacterium and Clostridium. Conclusions: Bacterial communities adherent to colorectal mucosa within a normal patient show little variation; in contrast, mucosal bacterial communities sampled from different patients with normal colorectal mucosa show a high degree of variation. Significance and Impact of the Study: This research demonstrates that DGGE profiling of 16S rRNA gene PCR products amplified from DNA extracted directly from mucosal samples offers fresh insight into the bacterial communities that are adherent to colorectal mucosa. These findings are important with respect to further studies on the gastrointestinal tract in health and disease.