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 3AE0%, n ¼ 5). Adjacent biopsies sampled from the same patient using different forceps gave similar DGGE profiles (MSC 94%, n ¼ 2). Two colorectal biopsies sampled at locations 2-5 cm apart, from each of 18 patients, resulted in very similar profiles (MSC 100%, IQR 2AE8%). Biopsies sampled from different locations within the large intestine of the same patient also gave similar DGGE profiles (MSC 98% IQR 3AE3% n ¼ 6). Although all patients (n ¼ 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.

Bacteria, phages and pigs: the effects of in-feed antibiotics on the microbiome at different gut locations

“…Many of the other bacteria enriched on the mucosa, such as Campylobacter spp., Weissella spp, and Sutterella spp., are known mucosal colonizers or pathogens and are likely mucosal specialists (On, 1996). These genera may be broadly specific to mucosal surfaces across mammals, having been identified on the mucosal surfaces of multiple host species (On, 1996;Heilig et al, 2002;Green et al, 2006). …”

“…Resident bacteria benefit from nutrient-rich digesta while assisting the host via nutrient digestion, vitamin synthesis, pathogen displacement and immune system maturation (Green et al, 2006;Bird et al, 2010). Factors such as nutrient availability, pH, redox potential and peristalsis strongly influence the composition of bacterial communities in the intestinal tract (Hao and Lee, 2004).…”

Loss of Bacterial Diversity during Antibiotic Treatment of Intubated Patients Colonized with Pseudomonas aeruginosa

“…Highly conserved regions of the 16S rRNA gene enable amplification of this gene from most bacteria with "universal" PCR primers, while variable regions within this gene permit discrimination between bacterial types (16). This approach has been applied successfully to the analysis of environmental (2,20,35,37,42) and human (18,26,28,29,38) bacterial communities and has revealed a much broader bacterial diversity than has traditional culture-based techniques (38). However, to our knowledge, no previous study has examined the extent of bacterial diversity within the lungs of intubated patients by culture-independent methods.…”

Numerical ecology validates a biogeographical distribution and gender-based effect on mucosa-associated bacteria along the human colon

“…Despite these observations, and the technological advances that have been applied to the analysis of gut microbial communities, evidence of a biogeographical pattern within the microbiome of the human large intestine has been elusive to obtain, principally because of the high degree of intersubject variability and constraints in sample number and size. A number of studies have shown a lack of variation in the mucosa-associated microbial communities along the human colon (Zoetendal et al, 2002;Lepage et al, 2005;Bibiloni et al, 2006;Green et al, 2006;Willing et al, 2009) and between diseased and healthy mucosal tissue Vasquez et al, 2007). These studies have usually relied on alphaand beta-diversity measures for the comparative analysis of microbial communities.…”