Reduction in diversity of the colonic mucosa associated bacterial microflora in patients with active inflammatory bowel disease

Abstract

Background and aims: The intestinal bacterial microflora plays an important role in the aetiology of inflammatory bowel disease (IBD). As most of the colonic bacteria cannot be identified by culture techniques, genomic technology can be used for analysis of the composition of the microflora.

Patients and methods: The mucosa associated colonic microflora of 57 patients with active inflammatory bowel disease and 46 controls was investigated using 16S rDNA based single strand conformation polymorphism (SSCP) fingerprint, cloning experiments, and real time polymerase chain reaction (PCR).

Results: Full length sequencing of 1019 clones from 16S rDNA libraries (n = 3) revealed an overall bacterial diversity of 83 non-redundant sequences-among them, only 49 known bacterial species. Molecular epidemiology of the composition of the colonic microflora was investigated by SSCP. Diversity of the microflora in Crohn's disease was reduced to 50% compared with controls (21.7 v 50.4; p<0.0001) and to 30% in ulcerative colitis (17.2 v 50.4; p<0.0001). The reduction in diversity in inflammatory bowel disease was due to loss of normal anaerobic bacteria such as Bacteroides species, Eubacterium species, and Lactobacillus species, as revealed by direct sequencing of variable bands and confirmed by real time PCR. Bacterial diversity in the Crohn's group showed no association with CARD15/NOD2 status.

Conclusions: Mucosal inflammation in inflammatory bowel disease is associated with loss of normal anaerobic bacteria. This effect is independent of NOD2/CARD15 status of patients.

Figure 1

Dendrogram and normalised single strand conformation polymorphism profiles obtained from a cluster analysis of 10 randomly assigned profiles from controls, Crohn’s disease (CD), and ulcerative colitis (UC) patients. Patients are marked with bars in different grey scales, as indicated in the legend. Separation of controls and disease groups is clearly visible, while CD and UC patients show mixed clusters. For cluster analysis, Pearson’s correlation and the unweighted pair group method with arithmetic mean algorithm were applied.

Figure 2

Band numbers and bacterial diversity obtained from single strand conformation polymorphism profiles of inflammatory bowel disease patients (Crohn’s disease (CD) and ulcerative colitis (UC)), non-inflammatory controls (controls−), and inflammatory controls (controls+). For each group, the mean numbers of bands (SD) are shown. Diversity of the controls was significantly higher compared with the disease groups. The weighted diversity scores (SD) according to Shannon and Weaver showed the same pattern. Results for comparison of both non-inflammatory and inflammatory controls and CD/UC were statistically significant: ****p<0.0001.

Figure 3

Bacterial diversity, indicated as number of bands versus medication. Four groups were compared: no medication, medication all (including single patients with combined medication), 5-aminosalicyclic acid (5-ASA), and corticosteroids. For each group, the mean (SD) numbers of bands are shown. Differences were not statistically significant.

Figure 4

Phylogenetic tree of the operational taxonomic units (OTUs) identified in the single strand conformation polymorphism (SSCP) profiles by the Band Matching algorithm. Fragments were identified by NCBI blast search (BLAST, http://www.ncbi.nlm.nih.gov/). The corresponding representative OTUs from the clone libraries (highlighted in grey) were inserted as benchmarks for phylogenetic orientation. The phylogenetic tree was built from sequence data of the SSCP profiles and the corresponding region (V4 and V5) of the sequences of the clone libraries with the ARB software package (http://www.arb-home.de/), using the parsimony algorithm and the Jukes-Cantor correction. Three clusters are defined by phylogenetic relationships: Bacteroides/Prevotella group (cluster 1), Firmicutes phylum (cluster 2), and Enterobacteriaceae family (cluster 3).

Figure 5

Reduction of Bacteroidetes and Enterobacteriaceae, as determined by real time polymerase chain reaction in patients with Crohn’s disease (CD) and ulcerative colitis (UC). All results were statistically significant (***p = 0.003, ****<p<0.0001).

Figure 6

Comparison of the bacterial profiles of 10 patients with active Crohn’s disease (CD) before and after a six week follow up. Bacterial diversity is indicated as number of bands (SD) and as diversity score (SD). Biodiversity was lower after six weeks but the results were not significant (p = 0.054).

Figure 7

Correlation of bacterial diversity and CARD15/NOD2 status of patients with Crohn’s disease (CD). Six of the 26 patients (30%) tested CARD15/NOD2 positive (detection of at least one of the three major single nucleotide polymorphisms—SNP8, SNP12, and SNP13). Diversity, indicated as number of bands and as weighted diversity score, was not significantly different in the CARD15/NOD2 positive group compared with wild-type patients (p = 0.063 and p = 0.11, respectively).