Inferred metagenomic comparison of mucosal and fecal microbiota from individuals undergoing routine screening colonoscopy reveals similar differences observed during active inflammation

Abstract

The mucosal microbiota lives in close proximity with the intestinal epithelium and may interact more directly with the host immune system than the luminal/fecal bacteria. The availability of nutrients in the mucus layer of the epithelium is also very different from the gut lumen environment. Inferred metagenomic analysis for microbial function of the mucosal microbiota is possible by PICRUSt. We recently found that by using this approach, actively inflamed tissue of ulcerative colitis (UC) patients have mucosal communities enriched for genes involved in lipid and amino acid metabolism, and reduced for carbohydrate and nucleotide metabolism. Here, we find that the same bacterial taxa (e.g. Acinetobacter) and predicted microbial pathways enriched in actively inflamed colitis tissue are also enriched in the mucosa of subjects undergoing routine screening colonoscopies, when compared with paired samples of luminal/fecal bacteria. These results suggest that the mucosa of healthy individuals may be a reservoir of aerotolerant microbial communities expanded during colitis.

Keywords: acinetobacter; colitis; inflammation; microbiome; mucosa; proteobacteria.

Figures

Figure 1.

(A) The luminal microbiome (represented by fecal samples) has higher microbial diversity than the mucosal microbiome (represented by biopsy samples) as determined by paired t-test. ***P < 0.0005. (B) PCoA plot of unweighted UniFrac distance, showing that the bacterial communities of the stool samples (blue dots) were distinct from those in the biopsy samples (red dots). (C) Relative abundance of taxonomic groups averaging across stool and biopsy samples, with the 10 most abundant phyla being represented. (D) Average UniFrac distance calculated between the luminal and mucosal microbial communities was greater than the average UniFrac distance calculated for different microbial communities between different individuals. (E) Bacterial taxa identified to be differentially abundant in the mucosal and luminal microbiome by LEfSe, at a logarithmic LDA threshold score of 2.0.

Figure 2.

(A) Bacterial taxa that were significantly enriched in the mucosa of participants undergoing routine screening colonoscopy and were previously identified to be differentially enriched in mucosa of patients with colitis. (B) Bacterial taxa that were significantly enriched in the luminal microbiome of participants undergoing routine screening colonoscopy and were previously identified to be reduced in the mucosa of inflamed tissue of UC patients relative to uninflamed normal mucosa. Paired t-tests: *P < 0.05, ***P < 0.0005.

Figure 3.

(A) Supervised analysis with LEfSe (Log LDA > 2.00) showed functional pathways that were differentially enriched in the mucosal microbiome and the luminal microbiome. (B) Paired analyses of stool and biopsy samples from the same individuals found lipid and amino acid metabolism pathways to be enriched in the mucosal microbiome, while carbohydrate and nucleotide metabolism pathways, as well as genetic information processing pathways to be more abundant on the luminal microbiome. Paired t-tests: **P < 0.005, ***P < 0.0005.

Figure 4.

Spearman correlation analysis of the differences in relative abundance values of bacterial taxa from both data sets. The values along the B-S axis represented the differences in relative abundance of bacteria between biopsy and stool samples, while the values along the I-N axis represented the differences in relative abundance of bacteria between inflamed and normal biopsy tissues. Each data point is representative of a single bacteria taxa. The correlation analysis was performed at both the phylum (A) and genus (B) levels, with the data points for the phyla Proteobacteria and Firmicutes, as well as the genus Acinetobacter being highlighted. The same analysis was repeated with relative abundance values of predicted metabolic pathways (C). (D) The enrichment of Acinetobacter genus in mucosal biopsy was validated by performing qPCR on biopsy and stool samples. Mann-Whitney test: ***P < 0.0005.

Figure 5.

(A) In a steady-state, the mucosal microbial communities are kept in check by the luminal bacteria, intestinal immune response and barrier function. An oxygen gradient exists within the intestinal environment, with the mucosal interface being largely aerobic and the intestinal lumen largely anaerobic. (B) During colitis, the intestinal barrier breaks down, increasing the oxygen content within the intestinal lumen and leading to the expansion of aerotolerant mucosal microbial communities, which spill over into the intestinal lumen and translocate across the intestinal epithelial barrier surface to trigger a strong anti-bacterial response.