Association of gut microbiota with post-operative clinical course in Crohn's disease

Abstract

Background: The gut microbiome is altered in Crohn's disease. Although individual taxa have been correlated with post-operative clinical course, global trends in microbial diversity have not been described in this context.

Methods: We collected mucosal biopsies from the terminal ileum and ascending colon during surgery and post-operative colonoscopy in 6 Crohn's patients undergoing ileocolic resection (and 40 additional Crohn's and healthy control patients undergoing either surgery or colonoscopy). Using next-generation sequencing technology, we profiled the gut microbiota in order to identify changes associated with remission or recurrence of inflammation.

Results: We performed 16S ribosomal profiling using 101 base-pair single-end sequencing on the Illumina GAIIx platform with deep coverage, at an average depth of 1.3 million high quality reads per sample. At the time of surgery, Crohn's patients who would remain in remission were more similar to controls and more species-rich than Crohn's patients with subsequent recurrence. Patients remaining in remission also exhibited greater stability of the microbiota through time.

Conclusions: These observations permitted an association of gut microbial profiles with probability of recurrence in this limited single-center study. These results suggest that profiling the gut microbiota may be useful in guiding treatment of Crohn's patients undergoing surgery.

Figure 1

Inter-individual variability of the gut microbiota is greater in Crohn’s disease than healthy controls. (A) Principal coordinates analysis (PCoA) plot based on weighted UniFrac distances between biopsies from Crohn’s and non-IBD patients. Biopsies in Crohn’s patients were taken of inflamed and healthy-appearing mucosa. Biopsies did not cluster by inflammation or location. (B) Average pairwise weighted UniFrac distances (±s.e.m.) among subsets of biopsies. Asterisks indicate significant differences: * P<0.05; ** P<0.005 (Student’s t-test with 1,000 Monte Carlo simulations). Phylum-level classifications of a single (C) ileal or (D) colonic sample from each patient’s initial procedure within this study. The Rutgeerts score at post-operative colonoscopy is denoted below surgical samples from Crohn’s patients. Phyla are sorted from top to bottom in overall decreasing prevalence.

Figure 2

Crohn’s patients with recurrence have significant dysbiosis at the time of surgery relative to control patients. (A) PCoA plot of weighted UniFrac distances between all surgical biopsies and non-IBD colonoscopic biopsies, with Crohn’s surgical samples (red) sized according to Rutgeerts score at post-operative colonoscopy. A Rutgeerts score of 2 or more indicates recurrence; less than 2, remission. (B) Average weighted UniFrac distances (±s.e.m.) between Crohn’s surgical samples and surgical controls were significantly greater in recurrence. The asterisk indicates a significant difference: * P<0.05. (C) Heat map showing probability of remission or recurrence based on weighted UniFrac distance to a single surgical control biopsy, binned in increments of 0.1. Each row adds up to 1. Based on a single biopsy per Crohn’s patient, differences in UniFrac distance distributions between remission and recurrence were not consistently significant, as demonstrated by (D) random selection of a single surgical biopsy from each of the six Crohn’s patients and determination of UniFrac distance to a randomly selected control biopsy (repeated 10,000 times), and (E) examination of all 486 combinations of pairwise comparisons between each surgical biopsy from the six Crohn’s patients and the centroid in ordination space of control biopsies. Dark gray shaded bars indicate a P-value of less than 0.05.

Figure 3

Recurrence is associated with instability through time. (A) Average weighted UniFrac distances (±s.e.m.) between surgical and post-operative colonoscopy biopsies for an individual. The asterisks indicate a significant difference: ** P<0.005. (B) Heat map showing probability of remission or recurrence based on a single pairwise weighted UniFrac distance at surgery and post-operative colonoscopy, binned in increments of 0.1. Each row adds up to 1. (C) Phylum-level classifications of biopsies acquired at surgery (“surg”) and post-operative colonoscopy (“colo”) suggest greater stability in remission. Location (i = ileum; c = colon) and presence of inflammation at the biopsy site are indicated.