The structures of the colonic mucosa-associated and luminal microbial communities are distinct and differentially affected by a prolonged murine stressor

Abstract

The commensal microbiota of the human gastrointestinal tract live in a largely stable community structure, assisting in host physiological and immunological functions. Changes to this structure can be injurious to the health of the host, a concept termed dysbiosis. Psychological stress is a factor that has been implicated in causing dysbiosis, and studies performed by our lab have shown that restraint stress can indeed shift the cecal microbiota structure as well as increase the severity of a colonic infection caused by Citrobacter rodentium. However, this study, like many others, have focused on fecal contents when examining the effect of dysbiosis-causing stimuli (e.g. psychological stress) upon the microbiota. Since the mucosa-associated microbiota have unique properties and functions that can act upon the host, it is important to understand how stressor exposure might affect this niche of bacteria. To begin to understand whether chronic restraint stress changes the mucosa-associated and/or luminal microbiota mice underwent 7 16-hour cycles of restraint stress, and the microbiota of both colonic tissue and fecal contents were analyzed by sequencing using next-gen bacterial tag-encoded FLX amplicon technology (bTEFAP) pyrosequencing. Both control and stress groups had significantly different mucosa-associated and luminal microbiota communities, highlighting the importance of focusing gastrointestinal community structure analysis by microbial niche. Furthermore, restraint stress was able to disrupt both the mucosa-associated and luminally-associated colonic microbiota by shifting the relative abundances of multiple groups of bacteria. Among these changes, there was a significant reduction in the immunomodulatory commensal genus Lactobacillus associated with colonic mucosa. The relative abundance of Lactobacillus spp. was not affected in the lumen. These results indicate that stressor-exposure can have distinct effects upon the colonic microbiota situated at the mucosal epithelium in comparison to the luminal-associated microbiota.

Keywords: Psychological stress; inflammation; microbiota; pyrosequencing.

Figure 1.

The mucosa-associated and luminal-associated microbiota communities are significantly different from each other. Alpha diversity measurements,including Shannon Diversity, Chao1, and equitability were calculated using QIIME and compared. (A) The Shannon Diversity Index was significantly increased in mucosal samples over luminal samples as measured with QIIME and averaged by group. (B) Richness, estimated with Chao1, was unchanged between both compartments. (C) Evenness, using the equitability measurement, was also increased in the mucosal samples over luminal samples. All data in A-C are mean ± SD. All α diversity measurements were analyzed using a parametric T-test on QIIME at a sequence depth of 6177. (D) Luminal samples and mucosal samples clustered independently of one another based upon unweighted Unifrac distances on a Principle Coordinate Analysis using the ANOSIM statistic (P < 0.0005). (E) Major phyla, including Firmicutes and Bacteroidetes, were unchanged when compared between luminal and mucosal-associated samples. (F) There were significant differences in the lesser phyla when compared between mucosa-associated microbiota and luminally-associated microbiota. The relative abundance of Actinobacteria, Deferribacteres, Proteobacteria, and Acidobacteria were all significantly different when compared between both gastrointestinal compartments. Phyla-level relative abundance was compared using Mann-Whitney U non-parametric tests. Mucosal-associated samples are n = 15; luminal-associated samples are n = 14.

Figure 2.

Stressor exposure was not associated with shifts in α diversity in the luminally-associated microbiota. (A) The Shannon Diversity index (SDI), (B) richness (using Chao1) and (C). Evenness (using equitability measurement) was measured for each sample using QIIME and averaged by group. None of the α diversity measurements were affected by stressor exposure. Data are mean ± SD. N = 5 for all groups, with the exception of HCC-Control, which had n = 4. Groups were compared using parametric T-tests on QIIME with a modified Bonferroni correction for multiple comparisons.

Figure 3.

Stressor exposure significantly affects the community structure of the luminally-associated microbiota. (A) Major phyla were unchanged between any group. (B) Among the lesser phyla, Actinobacteria was significantly reduced in RST Stressor mice compared to both HCC Control and FWD Control, while Deferribacteres was significantly reduced in RST Stressor mice compared to HCC Control and FWD Control mice. Abundances were compared using non-parametric Kruskal-Wallis tests, and post-hoc testing was performed with non-parametric Mann-Whitney U tests. (C) Principle coordinate analysis was used to visualize stressor exposure-induced community profile clustering based upon unweighted Unifrac distances. RST Stressor shifted the community structure of the luminally-associated microbiota compared to FWD Control mice (P < 0.01) significantly using the ANOSIM statistic, but not significantly compared to HCC Control mice. FWD and HCC Controls were unchanged compared to each other. Data are from n = 5 for RST Stressor and FWD Control groups, and n = 4 for HCC Control.

Figure 4.

Stressor exposure significantly reduces α diversity in the mucosa-associated microbiota. (A) The Shannon Diversity index (SDI) was measured for each sample and averaged by group. RST Stressor was significantly lower than FWD Control mice (P < 0.05), but was not significantly different than HCC Control SDI values. (B) Richness (by Chao1 measurement) and (C) evenness (by equitability) were then calculated using QIIME and averaged by group. RST Stressor did not affect overall richness, but evenness was reduced in RST Stressor-exposed mice when compared to FWD Control mice (P< 0.05). Data are mean ± SD for n = 5 for all groups. For α diversity, groups were compared using parametric T-tests on QIIME with a modified Bonferroni correction for multiple comparisons.

Figure 5.

Stressor exposure significantly alters the community structure of the mucosa-associated microbiota. (A) Firmicutes and Bacteroidetes were unchanged between any of the groups in the experiment. (B) Upon examining the lesser phyla, Actinobacteria was significantly reduced in the RST Stressor group compared to FWD Control and HCC Control mice. Deferribacteres was significantly increased in RST Stressor mice compared to HCC Control mice, but not FWD Control mice. Phyla abundance data were compared first using non-parametric Kruskal-Wallis test, and Mann-Whitney U tests were used post-hoc. (C) Principle coordinate analysis was used to visualize clustering of similar community profiles. Restraint stressor-exposed mice significantly clustered using the ANOSIM statistic (HCC Control vs. RST Stressor, P < 0.01) (FWD Control vs. RST Stressor, P < 0.01). FWD Control and HCC Control also clustered significantly apart from each other (P < 0.05). Data are for N = 5 for all groups.