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Mutations in toll-like receptors that mediate bacterial recognition by the mammalian innate immune system have the potential to substantially alter the composition of an individual's microbiota. Here we tested this hypothesis by comparing the intestinal microbiota of toll-like receptor 2-deficient mice, both young and middle aged, with that of wild-type mice of the same genetic background, housed together under specific pathogen-free conditions. Bacterial DNA was extracted from mouse caecal tissue samples, amplified using universal bacterial 16S ribosomal RNA gene primers, and cloned into a plasmid vector. Insert-containing colonies were picked for high-throughput sequencing, and sequence data were analysed yielding species-level phylogenetic data. Clone libraries were compared by phylogenetic composition analysis using UniFrac. While pairwise differences in phylogenetic population structure between mutant and wild-type mice were not statistically significant, anosim analysis did demonstrate a significant difference between toll-like receptor 2-deficient mice and their wild-type counterparts. The difference observed was probably due to a high level of colonization of the toll-like receptor 2-deficient mice by two distinct Helicobacter phylotypes that were totally absent from wild-type mice. Principal coordinate analysis clustering indicated that age is a weaker determinate than genotype and maternal heritage in the mouse caecal microbiota. The findings suggest that although mutations in toll-like receptors may cause increased susceptibility to specific opportunistic bacteria, they do not dramatically alter the phylogenetic structure of microbiota., (© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd.)

A leading hypothesis for the role of bacteria in inflammatory bowel diseases is that an imbalance in normal gut flora is a prerequisite for inflammation. Testing this hypothesis requires comparisons between the microbiota compositions of ulcerative colitis and Crohn's disease patients and those of healthy individuals. In this study, we obtained biopsy samples from patients with Crohn's disease and ulcerative colitis and from healthy controls. Bacterial DNA was extracted from the tissue samples, amplified using universal bacterial 16S rRNA gene primers, and cloned into a plasmid vector. Insert-containing colonies were picked for high-throughput sequencing, and sequence data were analyzed, yielding species-level phylogenetic data. The clone libraries yielded 3,305 sequenced clones, representing 151 operational taxonomical units. There was no significant difference between floras from inflamed and healthy tissues from within the same individual. Proteobacteria were significantly (P = 0.0007) increased in Crohn's disease patients, as were Bacteroidetes (P < 0.0001), while Clostridia were decreased in that group (P < 0.0001) in comparison with the healthy and ulcerative colitis groups, which displayed no significant differences. Thus, the bacterial flora composition of Crohn's patients appears to be significantly altered from that of healthy controls, unlike that of ulcerative colitis patients. Imbalance in flora in Crohn's disease is probably not sufficient to cause inflammation, since microbiotas from inflamed and noninflamed tissues were of similar compositions within the same individual.