Stressor exposure disrupts commensal microbial populations in the intestines and leads to increased colonization by Citrobacter rodentium

Abstract

The gastrointestinal tract is colonized by an enormous array of microbes that are known to have many beneficial effects on the host. Previous studies have indicated that stressor exposure can disrupt the stability of the intestinal microbiota, but the extent of these changes, as well as the effects on enteric infection, has not been well characterized. In order to examine the ability of stressors to induce changes in the gut microbiota, we exposed mice to a prolonged restraint stressor and then characterized microbial populations in the intestines using both traditional culture techniques and bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). Exposure to the stressor led to an overgrowth of facultatively anaerobic microbiota while at the same time significantly reducing microbial richness and diversity in the ceca of stressed mice. Some of these effects could be explained by a stressor-induced reduction in the relative abundance of bacteria in the family Porphyromonadaceae. To determine whether these alterations would lead to increased pathogen colonization, stressed mice, as well as nonstressed controls, were challenged orally with the enteric murine pathogen Citrobacter rodentium. Exposure to the restraint stressor led to a significant increase in C. rodentium colonization over that in nonstressed control mice. The increased colonization was associated with increased tumor necrosis factor alpha (TNF-alpha) gene expression in colonic tissue. Together, these data demonstrate that a prolonged stressor can significantly change the composition of the intestinal microbiota and suggest that this disruption of the microbiota increases susceptibility to an enteric pathogen.

FIG. 1.

Exposure to prolonged restraint significantly changed the levels of bacteria that could be cultured from the distal small intestine, cecum, and large intestine. The data are the means + standard errors, with panel A showing levels of total (i.e., both Gram-positive and Gram-negative) aerobic and facultatively anaerobic bacteria and panel B showing Gram-negative aerobes and facultative anaerobes. n = 3 preexperiment animals; n = 9 per group at days 1, 3, 5, and 7. *, P < 0.05 versus FWD levels on the same culture day. In this experiment, nonstressed home cage controls were used to set preexperiment values.

FIG. 2.

Exposure to prolonged restraint significantly changes community structure at the genus level. The double dendrogram describes the top 15 genera detected among the FWD, RST, and HCC samples. The heat map indicates the relative percentage of the given genera within each sample ID with a color legend and scale provided. The distance of the samples based upon weighted pair linkage and Manhattan distance methods with no scaling is provided at the top of the figure along with a distance score. The bacterial genera and the associated clustering are provided along the y axis, and their associated distance scores are indicated. n = 3 per group on days 1, 3, 5, and 7; n = 8 HCC controls.

FIG. 3.

Exposure to prolonged restraint significantly changes community structure at the family level. The double dendrogram describes the top 10 classes detected among the FWD, RST, and HCC samples. The heat map indicates the relative percentage of the given classes within each sample ID with a color legend and scale provided. The distance of the samples based upon weighted pair linkage and Manhattan distance methods with no scaling is provided at the top of the figure along with a distance score. The bacterial classes and the associated clustering are provided along the y axis, and their associated distance scores are indicated. n = 3 per group on days 1, 3, 5, and 7; n = 8 HCCs.

FIG. 4.

Rarefaction analysis of microbial communities on different sampling days. The number of phylotypes, identified with >95% similarity, is plotted for FWD controls (open circles) and the RST mice (closed inverted triangles) after 1, 3, 5, and 7 exposures. The data indicate that, over time, the number of phylotypes in the FWD control mice slightly increased, with the number of phylotypes in the RST mice decreasing with repeated exposure to RST. n = 3 per group at each time point; n = 4 to calculate baseline values. RST mice had a decreased number of phylotypes across the sampling days [F(1, 15) = 4.40, P < 0.05], with the largest differences occurring on days 5 and 7 (*, P < 0.05). In this experiment, nonstressed HCC mice were used to set baseline values.

FIG. 5.

Exposure to prolonged restraint prior to oral challenge with C. rodentium significantly increased the number of C. rodentium bacteria shed in the stool. Mice were exposed to prolonged restraint on 7 consecutive nights prior to oral challenge with 3 × 10⁸ to 5 × 10⁸ CFU of C. rodentium. The C. rodentium bacteria were then cultured from the stool during the first 21 days of infection. The data are the means ± standard errors of 10 to 16 mice per group. *, P < 0.05 versus both FWD and HCC at the specified time point.

FIG. 6.

Exposure to prolonged restraint prior to oral challenge with C. rodentium significantly altered colonic gene expression. Mice were exposed to prolonged restraint on 7 consecutive nights prior to oral challenge with 3 × 10⁸ to 5 × 10⁸ CFU of C. rodentium. Mice were euthanized on the indicated days postinfection, and gene expression for TNF-α (A), iNOS (B), IL-1β (C), and IL-10 (D) in colonic tissue was determined. The data are the means ± standard errors of the fold increase in gene expression over the FWD controls on day 1 postinfection. n = 6 mice per group on day 1 postchallenge; n = 3 mice per group on days 6, 12, and 24 postchallenge. The asterisk indicates a main effect of RST having higher gene expression than HCC and FWD across all days of infection (P < 0.05). **, P < 0.05 versus HCC and FWD controls at the same time point; †, marginally significant main effect of RST having higher gene expression than HCC and FWD across all days of infection (P = 0.07).

FIG. 7.

Colonic histopathology is increased in mice exposed to prolonged restraint prior to C. rodentium challenge. The data are the means ± standard errors of the cumulative histologic colitis score. n = 6 per group on day 1 postchallenge; n = 3 per group on days 6, 12, and 24 postchallenge. *, P < 0.05 versus HCC and FWD controls on day 6 postchallenge.

FIG. 8.

Prolonged restraint had little effect on measures of mucosal immunity. (A) Colonic IgA levels were unaffected by restraint exposure prior to challenge. n = 3 per group prechallenge; n = 5 per group on days 1, 6, 12, and 24 postchallenge. (B) Gene expression for mouse β-defensin 1 was similar in RST mice and in HCCl mice, with FWD control mice having lower gene expression on day 6 postchallenge (*, P < 0.05). n = 5 per group prechallenge; n = 3 per group on days 1, 6, 12, and 24 postchallenge. (C) TNF-α levels were suppressed in RST mice compared to HCC mice on days 12 and 24 postchallenge (*, P < 0.05 versus HCC mice). n = 3 per group prechallenge; n = 5 per group on days 1, 6, 12, and 24 postchallenge. In all cases the data are the means ± standard errors.