Disease-Specific Enteric Microbiome Dysbiosis in Inflammatory Bowel Disease

Abstract

Inflammatory Bowel disease (IBD) is traditionally divided into Crohn's disease (CD) and ulcerative colitis (UC). UC is a relapsing non-transmural inflammatory disease that is restricted to the colon and is characterized by flare-ups of bloody diarrhea. CD is a chronic, segmental localized granulomatous disease that can affect any part of the entire gastrointestinal tract. Ileo-anal pouch is a procedure restoring functionality of the rectum after a colectomy. IBD is a multifactorial disease and flares of IBD are probably triggered by changes in the intestinal microbiota followed by an abnormal immune response. In this study, we aim to analyze the intestinal bacterial diversity in IBD patients during various stages of disease compared with healthy controls. Permission for human experiments and recruitment of participants was obtained from the Ethic Committee for Copenhagen County hospitals (Permission no. KA-03019, Permission no. KA-20060159). Stools from 26 healthy controls, 42 CD, 38 UC and 18 pouch patients were collected. Stool DNA extraction was performed using Qiagen, DNA mini stool kit Denmark. DGGE-PCR amplifying the V2-V3 region of 16S-rDNA gene of the bacteria was amplified by universal primers HDA1 and HDA2. Analysis of DGGE was performed blinded using BioNumerics version 7.5. After normalization, a DGGE gel band matching was performed. The similarities between profiles were calculated with a ranked Pearson correlation coefficient based on the band matching results using band intensities. Simpson's index of diversity and Pielou's species evenness were calculated. Based on the Shannon Diversity Index, UC patients had lower species diversity and bacterial evenness in comparison to healthy persons, p < 0.05. However, only CD and disease pouch patients had lower species diversity compared to those with inactive disease and healthy controls. Well-functioning pouch patients had decreased species evenness in comparison to diseased pouch patients and control group. During the active disease stage in CD and pouch, the patients have a low bacterial diversity in their gut when compared to the inactive stage. In UC patients, a generally low diversity was observed at all stages of the disease compared to healthy controls.

Keywords: crohn's disease; dysbiosis; ileo-anal pouch; inflammatory bowel disease; intestinal microbiome; pielou index; shannon diversity index; ulcerative colitis.

Figure 1

(A) Shannon Diversity Index for patients with active (UCA) and inactive (UCI) Ulcerative colitis vs. healthy person/Control group shows patients group (UCI and UCA) have a significantly decreased bacterial abundance in comparison to controls group, p < 0.05^*(B) Pielou index shows significantly decreased bacterial evenness in the patient groups (UCI and UCA) vs. controls, p < 0.05.

Figure 2

(A) Cluster analysis of control group and patient groups with active (UCA in red)/inactive (UCI in blue) ulcerative colitis. (B) LDA shows that controls in green are mostly gathered in the red circle.

Figure 3

(A) Shannon Diversity Index for CD patients with active (CDA) and inactive disease (CDI) vs. controls show CDA have significantly decreased bacterial abundance in comparison to CDI and controls, p > 0.05*. (B) Pielou index shows no significant differences in species evenness in patient groups vs. controls (p < 0.083).

Figure 4

(A) Cluster Analysis of controls and patient groups with active (CDA, in red) and inactive (CDI, in blue) Crohn's disease shows the group clustering in colors (B) LDA shows control in green are mostly separated in the red ring, from CDA in red and CDI in blue.

Figure 5

(A) Shannon Diversity Index of pouch patient groups with active (PA) and inactive disease (PI) and controls shows PA has significant decreased bacterial diversity index in comparison to the PI and control group, p < 0.05. The differences were not significant in PI, p-value = 0.06. (B) Species evenness (Pielou index) was lower in the patient groups compared to the healthy controls, however these differences were only significant in PI, p < 0.05.

Figure 6

(A) Cluster analysis for pouch patients with active (PA, in red) and inactive (PI, in blue) disease shows controls (green) clustered mostly together. (B) LDA graph shows most of controls in green are separated from PI in blue and PA in red.