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Comparative Study
. 2011 Jan 10:11:7.
doi: 10.1186/1471-2180-11-7.

High-throughput clone library analysis of the mucosa-associated microbiota reveals dysbiosis and differences between inflamed and non-inflamed regions of the intestine in inflammatory bowel disease

Alan W Walker  1 , Jeremy D Sanderson, Carol Churcher, Gareth C Parkes, Barry N Hudspith, Neil Rayment, Jonathan Brostoff, Julian Parkhill, Gordon Dougan, Liljana Petrovska
Affiliations
Comparative Study

High-throughput clone library analysis of the mucosa-associated microbiota reveals dysbiosis and differences between inflamed and non-inflamed regions of the intestine in inflammatory bowel disease

Alan W Walker et al. BMC Microbiol. .

Abstract

Background: The gut microbiota is thought to play a key role in the development of the inflammatory bowel diseases Crohn's disease (CD) and ulcerative colitis (UC). Shifts in the composition of resident bacteria have been postulated to drive the chronic inflammation seen in both diseases (the "dysbiosis" hypothesis). We therefore specifically sought to compare the mucosa-associated microbiota from both inflamed and non-inflamed sites of the colon in CD and UC patients to that from non-IBD controls and to detect disease-specific profiles.

Results: Paired mucosal biopsies of inflamed and non-inflamed intestinal tissue from 6 CD (n = 12) and 6 UC (n = 12) patients were compared to biopsies from 5 healthy controls (n = 5) by in-depth sequencing of over 10,000 near full-length bacterial 16S rRNA genes. The results indicate that mucosal microbial diversity is reduced in IBD, particularly in CD, and that the species composition is disturbed. Firmicutes were reduced in IBD samples and there were concurrent increases in Bacteroidetes, and in CD only, Enterobacteriaceae. There were also significant differences in microbial community structure between inflamed and non-inflamed mucosal sites. However, these differences varied greatly between individuals, meaning there was no obvious bacterial signature that was positively associated with the inflamed gut.

Conclusions: These results may support the hypothesis that the overall dysbiosis observed in inflammatory bowel disease patients relative to non-IBD controls might to some extent be a result of the disturbed gut environment rather than the direct cause of disease. Nonetheless, the observed shifts in microbiota composition may be important factors in disease maintenance and severity.

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Figures

Figure 1
Figure 1
qPCR analysis of total bacterial load in mucosal biopsy samples. Figures are mean results for each patient cohort. Error bars denote standard deviation from the mean. Total bacterial load was significantly lower in the inflamed CD biopsies than the UC inflamed biopsies.
Figure 2
Figure 2
Compositional analysis of 16S rRNA gene clone libraries. Phylum-level classification of bacterial phylotypes in CD, UC and non-IBD control patients showing significant reduction in the proportion of Firmicutes sequences in UC samples relative to non-IBD controls (*a) and disruption in Firmicutes to Bacteroidetes ratio in IBD patients relative to non-IBD controls (*b).
Figure 3
Figure 3
Measures of bacterial diversity in the mucosal biopsies. 3A) Rarefaction analysis showing number of phylotypes observed with increasing sequencing effort across all patient cohorts. Data points show the observed diversity after each individual biopsy sample was incorporated into the analysis. Colour-coded errors bars show 95% confidence intervals for each patient cohort. Note that, as each patient is incorporated into the analysis, the gap between the number of phylotypes observed in non-IBD patients compared to IBD patients grows larger. The reduction in species richness appeared to be particularly significant in CD patients. Number of sequences per sample: Non-IBD controls = 252-489, CD Inflamed = 248-342, CD Non-inflamed = 287-445, UC Inflamed = 267-469, UC Non-inflamed = 286-499. 3B) Mean Shannon diversity indices (SDI) calculated from the individual biopsies for each sample type. Significantly reduced SDI compared to non-IBD control samples are indicated by * (p = < 0.05). Error bars indicate standard deviation from the mean.
Figure 4
Figure 4
Cluster dendrogram generated using the Jaccard coefficient, illustrating relationship between bacterial species membership and biopsy type across all samples included in the study. Crohn's disease patients are indicated by numbers CD1-CD6. Ulcerative colitis patients are indicated by UC1-UC6. Samples marked with "I" are from inflamed intestinal regions, those marked with "N" are from non-inflamed regions. Non-IBD control samples are indicated with N1-N5. Adjacent bar charts show the Family level classification (as determined by the RDP classifier) for each of the sequences per sample. Families coloured in yellow/brown belong to the Firmicutes phylum, blue = Bacteroidetes, pink = Actinobacteria, green = Proteobacteria, black = all other sequences not belonging to the specified Families.
Figure 5
Figure 5
Principal coordinates analysis of variation between the bacterial communities present in all biopsy samples. Each data point represents an individual sample. Blue circles denote non-IBD control samples, red squares are Crohn's disease samples, green triangles are ulcerative colitis samples. Numbers indicate the donor the samples were obtained from. The paired, inflamed and non-inflamed, biopsy samples from each donor can be seen to cluster together. Figure was calculated using unweighted Fast UniFrac [39].
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