BACKGROUND: Alterations of the intestinal microbiota have been associated with irritable bowel syndrome (IBS), however the compositional changes in this complex microbial community in IBS is not clear. Additionally, the microbiota in the lumen of the gut is significantly different in composition and diversity from the microbiota associated with the intestinal mucosa (Durban et al., 2010). In this study we expand our previous preliminary report comparing the composition and diversity of the intestinal microbiota within fecal and colonic mucosal niches between patients with Diarrhea-predominant IBS (D-IBS) and healthy controls (HC) on a larger study population with T-RFLP fingerprints using three restriction enzymes.
METHODS: The bacterial 16S ribosomal RNA gene was amplified from fecal and un-prepped colonic mucosal biopsy samples from 16 D-IBS subjects and 21 HC. T-RFLP fingerprints were generated from fecal and colonic mucosal samples by digestion with the restriction enzyme Hha I, and subsequently separated on a capillary sequencer. Further T-RFLP fingerprints were generated for fecal samples using Hae III and Msp I restriction enzymes. TRFLP community fingerprints were compared by multivariate analysis using PRIMER™ v6.
RESULTS: I. T-RFLP fingerprint analysis revealed a significant decrease in the diversity of microbial groups within fecal samples from D-IBS subjects when compared to HC: 1.2 fold decrease with Hha I-generated fingerprints (P = 0.008), and 1.06 fold decrease (P = 0.015) with Hae III-generated fingerprints. The decrease in microbial diversity in D-IBS fecal samples did not reach statistical significance with Msp I-generated fingerprints (1.05 fold, P = 0.296). No difference in diversity was detected between D-IBS and HC in mucosal samples. II. Multivariate analysis of Hha I-generated T-RFLP fingerprints demonstrated distinct microbial communities between luminal versus colonic mucosal niches in HC and D-IBS groups (HC feces vs. HC mucosa, R = 0.41, P = 0.001; D-IBS feces vs. D-IBS mucosa, R = 0.22, P = 0.001). However, no differences in the global composition of the microbial communities were identified in fecal or colonic mucosal samples between D-IBS subjects and HC. III. Comparison of microbial diversity between fecal and colonic mucosal samples revealed a significant 2 fold increase in fecal samples in HC (P = 0.0001) and a 1.7 fold increase in fecal samples in D-IBS patients (P = 0.001).
CONCLUSIONS: We demonstrate a significant decrease in luminal microbial diversity in D-IBS compared to healthy controls. Additionally, the microbiota within the lumen of the intestine displays higher diversity than the colonicassociated microbiota. Our results further support a role for an altered intestinal microbiota in the pathogenesis of IBS and suggest that both luminal and mucosal niches need to be investigated.
