Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases

Abstract

The two primary human inflammatory bowel diseases, Crohn's disease (CD) and ulcerative colitis (UC), are idiopathic relapsing disorders characterized by chronic inflammation of the intestinal tract. Although several lines of reasoning suggest that gastrointestinal (GI) microbes influence inflammatory bowel disease (IBD) pathogenesis, the types of microbes involved have not been adequately described. Here we report the results of a culture-independent rRNA sequence analysis of GI tissue samples obtained from CD and UC patients, as well as non-IBD controls. Specimens were obtained through surgery from a variety of intestinal sites and included both pathologically normal and abnormal states. Our results provide comprehensive molecular-based analysis of the microbiota of the human small intestine. Comparison of clone libraries reveals statistically significant differences between the microbiotas of CD and UC patients and those of non-IBD controls. Significantly, our results indicate that a subset of CD and UC samples contained abnormal GI microbiotas, characterized by depletion of commensal bacteria, notably members of the phyla Firmicutes and Bacteroidetes. Patient stratification by GI microbiota provides further evidence that CD represents a spectrum of disease states and suggests that treatment of some forms of IBD may be facilitated by redress of the detected microbiological imbalances.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Phylogenetic distribution of SSU rRNA sequences. The diagrammatic phylogenetic tree presents a summary of the rRNA sequences isolated in this study. The number of taxa in a clade is designated either on its wedge or parenthetically to its name. Phyla are named to the left of the tree and lower taxonomic levels to the right. Wedge widths represent the taxa with the longest (top bar) and shortest (bottom bar) distances within the clade. The scale bar represents base changes per site.

Fig. 2.

Phylum-level comparison of disease state. Bars shading depicts percentages of cloned sequences in all samples of a particular category that belong to one of the predominant phylum of Bacteria identified in the sample set (Firmicutes are subdivided into Bacillus, Lachnospiraceae, and miscellaneous groups). Phyla of lower abundance were omitted, so bars do not sum to 100%. NI, non-IBD control samples; Con-Sub, Control subset; and IBD-Sub, IBD subset are explained in the text.

Fig. 3.

PCA; 97% OTUs were encoded as presence/absence data for each sample, and the corresponding data matrix was subjected to PCA. Each circle, which is representative of a single sample and shaded according to disease status, is plotted along the first two principal component axes. Dashed lines denote nominal sample clusters, the Control subset and IBD subset.

Fig. 4.

Q-PCR analysis of predominant phylogenetic groups. Selected representatives of the Control- and IBD-subset clusters (see text for details) were assayed by Q-PCR for total bacteria, Lachnospiraceae, Bacteroidetes, and Enterobacteriaceae. Experiments were performed in triplicate. Data are presented as log10-transformed SSU rDNA gene copy numbers, as interpolated from standard curves of plasmid controls. Error bars represent standard deviations of replicate experiments. NI, non-IBD controls. Control- and IBD-subset are explained in the text. The results of Student's t tests are indicated as *, P < 0.05; **, P < 0.01; and ***, P < 0.001. t tests compared the results of Control- and IBD-subset samples for a particular disease state and bacterial group (e.g., Bacteroidetes/CD/Control subset vs. Bacteroidetes/CD/IBD subset). Data for specific groups were normalized to total bacteria.

Fig. 5.

Microbial diversity of the human intestine. OTU richness was estimated for a data set of sequences obtained in this study and those of Eckburg et al. (44) and Ley et al. (32). Sequences were clustered into OTUs and averaged collector's curves simulated by 1,000 random replicate samplings of the OTUs at sample sizes ranging from 1,000 to 45,000 sequences (total sequences, 45,172). The graph shows double reciprocal plots of mean Sobs−1 vs. sequences−1 for 99%, 97%, and 95% OTUs. The Y intercepts of the graphs were extrapolated by linear regression of Sobs−1 and sequences−1, from which expected OTU richness (i.e., Smax) was inferred. The dashed lines extending from each graph approximate the extrapolations.