Microbes in the human gut are thought to play a crucial role in the development and maintenance of the abnormal inflammatory responses that are the hallmark of inflammatory bowel disease. Direct tests of the role of gut microbes in these disorders are limited by the fact that gut microbial sampling generally occurs once disease has manifest. In collaboration with investigators at four other institutions, we have completed a preliminary study that examines the development of the microbial community over time in patients who undergo total proctocolectomy with ileal pouch anal anastomosis (IPAA) for the treatment of ulcerative colitis. A subset of these patients develop an inflammatory condition known as pouchitis, which is thought to mirror the pathogenesis of ulcerative colitis. We characterized the microbial community that predates overt disease by tracking the development of the pouch microbiome.

We monitored the development of the pouch microbiota in four patients who underwent IPAA. Collaborators at the University of Chicago obtained mucosal tissue biopsies and fecal samples prior to take down of the diverting ileostomy and at two, four and eight weeks after intestinal continuity was restored (Figure X). Through the combined analysis of 16S rRNA gene amplicons, targeted functional gene amplification, and microbial cultivation we observed major changes in structure and function of the pouch microbiota following ileostomy. We see an increase in anaerobic microorganisms with the capacity for fermentation of complex carbohydrates, which corresponds to the physical stasis of intestinal contents in the ileal pouch. Compared to the microbiome structure encountered in the colonic mucosa of healthy individuals, the pouch microbial community in 3 of the 4 individuals was quite distinct (Figure Y). In the fourth patient (210), a community that was much like that seen in a healthy colon was established and this patient also had the most benign clinical course of the four patients, without the development of pouchitis two years after IPAA.

We concluded that the pouch microbiota of IPAA patients demonstrates significant structural and functional changes related to the restoration of fecal flow. Our preliminary results suggest that once the pouch replaces the intact colon, the precise structure and function of the pouch microbiome relative to a normal colonic microbiota will determine if there is establishment of a stable, healthy mucosal environment rather than a pathogenic cascade that results in intestinal inflammation.

Going forward, we continue to analyze many more samples, both 16S gene sequences and full metagenomic sequencing. The subject pool includes many more patients, some of whom have developed pouchitis and others who have not, as well as healthy controls. We are developing and improving our methods, for example, determining optimum library insert size for Illumina sequencing, reducing the amount of contaminating host DNA in biopsy samples, and estimating accuracy, rather than simply quality, of Illumina and 454 data.