Bacterial population analysis of human colon and terminal ileum biopsies with 16S rRNA-based fluorescent probes: Commensal bacteria live in suspension and have no direct contact with epithelial cells

Background: The commensal intestinal microflora has important metabolic and perhaps also immune modulatory functions. Evidence has accumulated that the microflora plays a role in the pathogenesis of inflammatory bowel disease. Therefore, there is a growing interest in the intestinal microflora and its interaction with the host. Presumably, this interaction takes place at the mucus layer. In this study, we investigated the microflora that is present at the mucus layer and addressed the following questions. Does a specific mucus-adherent microflora exist? Is there direct contact between commensal bacteria and epithelial cells? Methods: Snap-frozen biopsies were taken of 5 colon regions and of the terminal ileum in 9 subjects with a normal colon. Fecal samples were also collected. Bacteria were detected in cryosections with fluorescent in situ hybridization (FISH) with 16S ribosomal (r)RNA-targeted probes for all bacteria and specific probes for the major representatives of anaerobic microflora (bifidobacteria, Bacteroides, clostridia, atopobia) and aerobic microflora (Enterobacteriaceae, enterococci, streptococci, lactobacilli). Results: With this sensitive technique, bacteria were only observed at the luminal side of the intestinal mucus layer. Very few microcolonies were present at the mucus layer, and the composition of the bacterial microflora present in the feces was similar to that at the mucus layer of the terminal ileum and colon regions. Conclusions: We did not observe direct contact between bacteria and epithelial cells. The equal distribution of bacterial species suggests that intestinal commensal bacteria live in suspension in the lumen and that there is no specific mucus-adherent microflora