Multiphasic analysis of the temporal development of the distal gut microbiota in patients following ileal pouch anal anastomosis

Abstract

Background: The indigenous gut microbiota 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 the gut microbiome in these disorders are typically limited by the fact that sampling of the microbiota generally occurs once disease has become manifest. This limitation could potentially be circumvented by studying patients who undergo total proctocolectomy with ileal pouch anal anastomosis (IPAA) for the definitive treatment of ulcerative colitis. A subset of patients who undergo IPAA develops an inflammatory condition known as pouchitis, which is thought to mirror the pathogenesis of ulcerative colitis. Following the development of the microbiome of the pouch would allow characterization of the microbial community that predates the development of overt disease.

Results: We monitored the development of the pouch microbiota in four patients who underwent IPAA. Mucosal and luminal samples were obtained prior to takedown of the diverting ileostomy and compared to samples obtained 2, 4 and 8 weeks after intestinal continuity had been restored. Through the combined analysis of 16S rRNA-encoding gene amplicons, targeted 16S amplification and microbial cultivation, we observed major changes in structure and function of the pouch microbiota following ileostomy. There is a relative 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 three of the four individuals was quite distinct. In the fourth patient, 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 2 years after IPAA.

Conclusions: The microbiota that inhabit the ileal-anal pouch of patients who undergo IPAA for treatment of ulcerative colitis demonstrate significant structural and functional changes related to the restoration of fecal flow. Our preliminary results suggest once the pouch has assumed the physiologic role previously played by 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 or the reinitiation of the pathogenic cascade that results in intestinal inflammation.

Figure 1

Anatomy of ileal pouch anal anastomosis (IPAA). Patients who undergo a two-stage IPAA procedure initially undergo a total colectomy with the construction of the ileal pouch, which is anastomosed to the rectum. Diversion of the fecal stream occurs through an ileostomy. Study subjects were initially sampled at this stage (visit 1) with specimens harvested from the diverted pouch. In the second stage of the IPAA procedure, the diverting ileostomy is taken down and continuity is restored to the ileum, restoring the flow of intestinal contents to the ileal pouch. The subsequent samples were obtained from the ileal pouch, which was accessed via the rectum (visits 2, 3 and 4). Control samples were obtained from healthy individuals who had the same anatomy as the IPAA subjects prior to the first stage of the procedure.

Figure 2

Shift in mucosal microbiota of the pouch after takedown of diverting ileostomy in patients who had undergone ileal pouch anal anastomosis visualized by principle coordinates analysis of the Unifrac metric based on V3-5 16S rRNA-encoding gene amplicons. The initial time point, prior to takedown of the ileostomy, is indicated by the larger symbols and subsequent time points at the end of the arrows. For comparison, colon mucosal biopsies from healthy individuals are included on the ordination.

Figure 3

Taxonomic classification of V3-5 16S rRNA-encoding gene amplicon sequences from patients who had undergone ileal pouch anal anastomosis. The relative abundance of major phylotypes, classified to the level of family and grouped by phylum, is indicated. Time point 1 is prior to ileostomy takedown, and time points 2, 3 and 4 are 2, 4 and 8 weeks after takedown.

Figure 4

Change in diversity of the mucosal microbial communities inhabiting the ileal/anal pouch of patients after ileostomy takedown. The Shannon diversity index was calculated for each community based on OTUs defined at sequence divergence ≥ 0.03.

Figure 5

Changes in the direct counts and viable cell density from pouch mucosal samples. Brush samples obtained during endoscopy were loaded onto a counting chamber for direct cell counts (A). Error bars were standard errors of three counts per sample. Anaerobic and aerobic cultivation on complex medium was performed. The number of colony-forming units (CFU) on day 5 was used to calculate viable cell density (CFU/ml) (B). Error bars were standard errors of CFU number on triplicate agar plates from independent serial dilutions. For comparison, colon mucosal brushes/biopsies from healthy individuals (control) are included in the figure.

Figure 6

Analysis of 16S rRNA-encoding gene data for butyrate-producing communities. Panel A shows the cumulative result of obtained candidates associated with butyryl-CoA:acetate CoA-transferase (but; grey bar) and butyrate kinase (buk; white stripped bar). Below (B) individual compositions of communities are given. Results are corrected for multiple 16S rRNA-encoding gene copy numbers for individual bacteria. ? = butyrate production was shown for one strain of Subdoligranulum sp. as it is unclear whether all members of this genus have the potential to synthesize butyrate.