Loop ileostomy-mediated fecal stream diversion is associated with microbial dysbiosis

Abstract

Loop ileostomy is an effective procedure to protect downstream intestinal anastomoses. Ileostomy reversal surgery is often performed within 12 months of formation but is associated with substantial morbidity due to severe post-surgical complications. Distal ileum is deprived of enteral nutrition and rendered inactive, often becoming atrophied and fibrotic. This study aimed to investigate the microbial and morphological changes that occur in the defunctioned ileum following loop ileostomy-mediated fecal stream diversion. Functional and defunctioned ileal resection tissue was obtained at the time of loop-ileostomy closure. Intrapatient comparisons, including histological assessment of morphology and epithelial cell proliferation, were performed on paired samples using the functional limb as control. Mucosal-associated microflora was quantified via determination of 16S rRNA gene copy number using qPCR analysis. DGGE with Sanger sequencing and qPCR methods profiled microflora to genus and phylum level, respectively. Reduced villous height and proliferation confirmed atrophy of the defunctioned ileum. DGGE analysis revealed that the microflora within defunctioned ileum is less diverse and convergence between defunctioned microbiota profiles was observed. Candidate Genera, notably Clostridia and Streptococcus, reduced in relative terms in defunctioned ileum. We conclude that Ileostomy-associated nutrient deprivation results in dysbiosis and impaired intestinal renewal in the defunctioned ileum. Altered host-microbial interactions at the mucosal surface likely contribute to the deterioration in homeostasis and thus may underpin numerous postoperative complications. Strategies to sustain the microflora before reanastomosis should be investigated.

Keywords: Loop ileostomy; atrophy; dysbiosis; enteral nutrition; microbiota; morphology; small intestine.

Figures

Figure 1.

Structure of the intestine. (A) Loop ileostomy and (B) following reanastomosis. Block arrows denote presence and direction of luminal contents flow. Tissue located above dashed lines represent areas of intestine removed before reanastomosis and form the specimen acquired for our research.

Figure 2.

Histological analyses of function and defunctioned ileum. (A) Representative H&E stained sections, magnification 4x. (B) Average villous height ± SEM (n = 9, p = 0.0004) (C) Paired villous height.

Figure 3.

Bacterial enumeration of (A) average- and (B) absolute-16S rRNA gene copy numbers per gram of mucosal tissue in functional and defunctioned intestine (n = 27; p = 0.0003).

Figure 4.

(A) Example of denaturation gradient gel electrophoresis (DGGE) profiles. Band classes are depicted using characters a through h and corresponding bands are enclosed. F, functional ileum; D, defunctioned ileum. (B) Microbial diversity in functional and defunctioned ileum expressed as total number of bands in DGGE profiles (n = 11; P = 0.04). (C) Purified and sequenced band classes expressed as a percentage presence in DGGE profiles across all patients. Characters a-h correspond to the band classes highlighted in A. Inclined, corresponding assigned bacterial genus per band class.

Figure 5.

Hierarchical cluster analysis of DGGE profiles represented in graphical form as an UPGMA dendogram. F: functional ileum, D: defunctioned ileum (n = 11).

Figure 6.

Percentage change of phyla abundance in defunctioned ileum relative to functional. Data normalized to universal 16S rDNA primers (Firmicutes (n = 18, p = 0.02), Bacteroiodetes (n = 18, NS), y-Proteobacteria (n = 9, p ≤ 0.05).

Figure 7.

(A) Representative immunofluorescent PCNA labeling (green) to measure proliferation. All nucleated cells, counterstained using Hoechst 33342, are colored blue. Magnification 10x. (B) Average percent proliferating PCNA positive cells/crypt ± SEM (n = 5; p = 0.01). (C) Paired percent PCNA-positive cells per crypt in the functional and defunctioned intestine.