COMPARATIVE ANALYSIS OF THE MUCOSAL ASSOCIATED MICROBIOTA (MMB) IN CROHN'S DISEASE (CD) and NON‐IBD CONTROL SUBJECTS

Abstract

The cause(s) of CD are not known, but its pathogenesis depends on the interaction of enteric bacteria and food antigen products with the mucosal immune system. Accumulated evidence clearly demonstrates that enteric microbiota influence the ontogeny and behavour of the innate and acquired mucosal immune system. Although there are significant enteric bacteria resident in the lumen of the human colon (up to 1013/g wet faeces), it is microbiota that associate with mucosa of intestinal epithelium (MMB) that are more likely to influence the mucosal immune system, to effect inflammation or tolerance. Aims To analyse the MMB communities in CD vs. ulcerative colitis (UC) and non‐IBD subjects using molecular ecological approach. Methods Total DNA are extracted from washed colonoscopic biopsy samples from macroscopically non‐involved CD (to date, n = 6 ileum and n = 1 colon), ulcerative colitis (n = 1 colon) and non‐IBD (n = 2 terminal ileum, n = 1 colon). Bacterial 16s‐rDNA are amplified by PCR with universal bacterial primers. After cloning, the diversity of bacterial communities are characterized by RFLP pattern analysis (ribotyping). Bacteria are then identified by phylogenetic analysis of DNA sequences of individual RFLP patterns. Results There was significantly more bacterial DNA in CD than non‐IBD with mean total DNA (ng) in amplicons in ileal samples from CD subjects, 39.2, compared with non‐IBD, 4.25 and in CD colon, 24.6, compared with non‐IBD colon, 13.4 ng (Mann–Whitney, P = 0.014). Bacteroides sp. were numerically predominant both in four of seven CD and three of three non‐IBD biopsy samples. five of seven CD biopsies had a striking number of bacterial clones phylogenetically identified to belong to Clostridium cluster XIVa (13–36%) vs. 0–9% in non‐IBD controls and 11% in UC colon. The three CD with low bacteroides and lower Clostridium cluster XIVa (4–11%), had a high frequency of Pseudomonas spp. (19–40%), together with high occurrence of Klebsiella spp., as did one of three non‐IBD controls (36%) and the UC biopsy (11%). Conclusions CD ileum may harbour an abnormal and more abundant resident MMB. Further study is required to describe the heterogeneous MMB in both normal and IBD ileum and colon. One of the XIVa species is extremely mucolytic. A Pseudomonas has recently been implicated in CD. The full 16s‐rDNA sequences of two XIVa species and the Pseudomonas spp. are being obtained, as well as specific PCR primers to screen a larger number of biopsies for the presence of these bacteria.