Bacterial Mucosa-associated Microbiome in Inflamed and Proximal Noninflamed Ileum of Patients With Crohn's Disease

Abstract

Background: Microbiota is most likely essential in the pathogenesis of Crohn's disease (CD). Fecal diversion after ileocecal resection (ICR) protects against CD recurrence, whereas infusion of fecal content triggers inflammation. After ICR, the majority of patients experience endoscopic recurrence in the neoterminal ileum, and the ileal microbiome is of particular interest. We have assessed the mucosa-associated microbiome in the inflamed and noninflamed ileum in patients with CD.

Methods: Mucosa-associated microbiome was assessed by 16S rRNA sequencing of biopsies sampled 5 and 15 cm orally of the ileocecal valve or ileocolic anastomosis.

Results: Fifty-one CD patients and forty healthy controls (HCs) were included in the study. Twenty CD patients had terminal ileitis, with endoscopic inflammation at 5 cm, normal mucosa at 15 cm, and no history of upper CD involvement. Crohn's disease patients (n = 51) had lower alpha diversity and separated clearly from HC on beta diversity plots. Twenty-three bacterial taxa were differentially represented in CD patients vs HC; among these, Tyzzerella 4 was profoundly overrepresented in CD. The microbiome in the inflamed and proximal noninflamed ileal mucosa did not differ according to alpha diversity or beta diversity. Additionally, no bacterial taxa were differentially represented.

Conclusions: The microbiome is similar in the inflamed and proximal noninflamed ileal mucosa within the same patients. Our results support the concept of CD-specific microbiota alterations and demonstrate that neither ileal sublocation nor endoscopic inflammation influence the mucosa-associated microbiome.

Keywords: Crohn’s disease; microbiome; mucosal microbiota.

FIGURE 1.

Illustration of study design with mucosal pinch biopsy location and number from the study participants. Fifty-one Crohn’s disease patients including 7 CD patients with ileal stenosis (CD-S), 22 CD patients with terminal ileitis (CD-TI) with endoscopic inflamed mucosa at 5-cm location (Rutgeerts score ≥1) and normal endoscopic appearing mucosa at 15-cm location, 10 CD patients with active disease (CD-A) with endoscopic inflamed mucosa at both 5-cm and 15-cm location, and 12 CD patients in remission (CD-R) with endoscopic normal appearing mucosa at both 5-cm and 15-cm location and 40 healthy controls (HC) were included in the cohort. Two mucosal pinch biopsies were collected on each location in the 44 CD patients and HCs, in total 4 mucosal pinch biopsies per study participant. Two mucosal biopsies were collected in the 7 CD-S patients at 5-cm location because of ileal stenosis mucosal pinch biopsies could not be collected at 15-cm location.

FIGURE 2.

Ileal mucosa-associated bacterial microbiome in 51 Crohn’s disease patients and 40 healthy controls, mucosal biopsies sampled 5 and 15 cm proximal from the ileocecal valve or anastomosis, respectively (except for 7 CD patients with stenosis where only 5 cm sample was obtained). In total, 95 biopsy specimens from 51 CD patients and 80 biopsy specimens from 40 HCs. A, Alpha diversity illustrated by Shannon entropy index in CD patients vs HC compared with Wilcoxon test. B, Beta diversity illustrated by Bray-Curtis dissimilarity index on nonmetric multidimensional scaling plot, each sample colored according to phenotype (CD and HC). C, Bacterial taxa significantly differentially represented (adjusted P < 0.05) in patients with CD vs HC, illustrated by log2 fold change.

FIGURE 3.

Ileal mucosa-associated bacterial microbiome at 5-cm and 15-cm location (proximal from the ileocecal valve or anastomosis respectively) in 51 Crohn’s disease patients; biopsies not collected from 7 CD patients at 15-cm location due to ileal stenosis. A, Alpha diversity, illustrated by Shannon entropy index, at 5-cm and 15-cm location compared with Wilcoxon test. B, Beta diversity illustrated by Bray-Curtis dissimilarity index on nonmetric multidimensional scaling plot; each sample colored according to mucosal pinch biopsy location; 5 and 15 cm. C, Beta diversity in 51 CD patients and 40 healthy controls, illustrated by Bray-Curtis dissimilarity index on nonmetric multidimensional scaling plot; each sample colored according to phenotype and location; CD 15 cm, CD 5 cm, HC 15 cm, and HC 5 cm.

FIGURE 4.

Ileal mucosa-associated bacterial microbiome in 20 Crohn’s disease patients with terminal ileitis with endoscopic inflamed mucosa at 5 cm proximal from the ileocecal valve or anastomosis and normal endoscopic appearing mucosa at 15 cm proximal from the ileocecal valve or anastomosis. A, Alpha diversity, illustrated by Shannon entropy index according to location, compared with Wilcoxon test. B, Beta diversity illustrated by Bray-Curtis dissimilarity index on nonmetric multidimensional scaling plot, each sample colored according to mucosal pinch biopsy location; 5 and 15 cm. C, Alpha diversity, illustrated by Shannon entropy index, at 5-cm location in 20 CD-TI patients vs 40 healthy controls compared with Wilcoxon test. D, Beta diversity, illustrated by Bray-Curtis dissimilarity index on nonmetric multidimensional scaling (NMDS) plot, in 5-cm samples from CD-TI patients and HCs; each sample colored according to phenotype; HC and CD-TI.

FIGURE 5.

Ileal mucosa-associated bacterial microbiome in 95 mucosal pinch biopsy specimens (sampled from 5 and 15 cm proximal from the ileocecal valve or anastomosis) from 51 Crohn’s disease patients according to endoscopic inflammation (Rutgeerts score ≥1) and histologic inflammation (GHAS and Robarts score ≥1) at biopsy sample location. A, Alpha diversity, illustrated by Shannon entropy index, according to endoscopic inflammation compared with Wilcoxon test. B, Alpha diversity, illustrated by Shannon entropy index, according to histologic inflammation compared with Wilcoxon test. C, Beta diversity illustrated by Bray-Curtis dissimilarity index on nonmetric multidimensional scaling plot in 95 mucosal pinch biopsies from 51 CD patients and 80 mucosal pinch biopsies from 40 healthy controls; each sample colored according to endoscopic inflammation (yes or no) or HC. D, Beta diversity illustrated by Bray-Curtis dissimilarity index on nonmetric multidimensional scaling plot in 95 mucosal pinch biopsies from 51 CD patients; each sample colored according to histologic inflammation (yes or no).

FIGURE 6.

Ileal mucosa-associated bacterial microbiome composition in 40 healthy controls, 20 Crohn’s disease patients with terminal ileitis and endoscopic inflammation, 12 CD patients in remission and endoscopic normal appearing mucosa, and in 7 CD patients with ileal stenosis. Beta diversity illustrated by Bray-Curtis dissimilarity index on nonmetric multidimensional scaling plot; each biopsy sample colored according to phenotype; HC, CD-TI, CD-R and CD-S. Each study participant represented with 1 mucosal pinch biopsy sampled 5 cm proximal to the ileocecal valve or anastomosis, respectively.