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. 2018 Jun;3(6):662-669.
doi: 10.1038/s41564-018-0150-6. Epub 2018 Apr 23.

Microbiota-accessible carbohydrates suppress Clostridium difficile infection in a murine model

Andrew J Hryckowian  1 , William Van Treuren  1 , Samuel A Smits  1 , Nicole M Davis  1 , Jackson O Gardner  1 , Donna M Bouley  2 , Justin L Sonnenburg  3
Affiliations

Microbiota-accessible carbohydrates suppress Clostridium difficile infection in a murine model

Andrew J Hryckowian et al. Nat Microbiol. 2018 Jun.

Abstract

Clostridium difficile is an opportunistic diarrhoeal pathogen, and C. difficile infection (CDI) represents a major health care concern, causing an estimated 15,000 deaths per year in the United States alone 1 . Several enteric pathogens, including C. difficile, leverage inflammation and the accompanying microbial dysbiosis to thrive in the distal gut 2 . Although diet is among the most powerful available tools for affecting the health of humans and their relationship with their microbiota, investigation into the effects of diet on CDI has been limited. Here, we show in mice that the consumption of microbiota-accessible carbohydrates (MACs) found in dietary plant polysaccharides has a significant effect on CDI. Specifically, using a model of antibiotic-induced CDI that typically resolves within 12 days of infection, we demonstrate that MAC-deficient diets perpetuate CDI. We show that C. difficile burdens are suppressed through the addition of either a diet containing a complex mixture of MACs or a simplified diet containing inulin as the sole MAC source. We show that switches between these dietary conditions are coincident with changes to microbiota membership, its metabolic output and C. difficile-mediated inflammation. Together, our data demonstrate the outgrowth of MAC-utilizing taxa and the associated end products of MAC metabolism, namely, the short-chain fatty acids acetate, propionate and butyrate, are associated with decreased C. difficile fitness despite increased C. difficile toxin expression in the gut VSports手机版. Our findings, when placed into the context of the known fibre deficiencies of a human Western diet, provide rationale for pursuing MAC-centric dietary strategies as an alternate line of investigation for mitigating CDI. .

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Conflict of interest statement

Competing interests

The authors declare no competing interests.

Figures

Figure 1
Figure 1. Dietary MACs toggle the fitness of Clostridium difficile (Cd) in the gut while engendering distinct microbiota states
Humanized, age matched female Swiss-Webster mice were maintained on a diet containing a complex mixture of MACs (MAC+, n=4 mice) or on diets deficient in MACs (MD1, n=4 mice; and MD2, n=4 mice) starting 8 days pre-infection. See Methods for details of diet compositions. (a) Mice were subsequently gavaged with clindamycin at 1 day before infection with Cd. At 36 days post-infection, mice fed the MD diets were switched to the MAC+ diet. Cd burdens were monitored over time by selective culture, as described in Methods. One of the MD2 fed mice was moribund on D10 and was euthanized. Individual per-sample Cd burdens are plotted and lines represent geometric mean burdens per time point. (b–d) Principal coordinates analysis plots of Weighted UniFrac distances between microbiota samples collected from these mice were prepared and overlaid with log-fold contour plots of Cd burdens, as measured in panel a. Clindamycin affects the composition of the microbiota in all groups of mice from D-1 (annotated with ellipses representing 80% CI) to D0 (dotted lines), resulting in a dysbiotic state permissive to CDI. In each panel, a line is drawn through the centroid of the points for a given experiment day. (b) In mice fed the MAC+ diet, the microbiota returns to resemble the pre-infection state as Cd burdens decrease. (c, d) In mice fed the MD1 and MD2 diets, respectively, CDI remains unresolved until dietary intervention with the MAC+ diet at D36, which shifts the microbiota to resemble that of other MAC+ fed mice as Cd burdens decrease (brown lines). Points are colored by the highlighted treatment group, or alternatively are retained as gray points for reference. See Fig. S4 for further explanation on generation and interpretation of the contoured PCoA (cPCoA) plots shown in panels b–d.
Figure 2
Figure 2. A diet containing inulin as the sole MAC source reduces Cd burdens without increasing microbial diversity
Mice with persistent CDI were subjected to a dietary intervention of the complex MAC+ diet (n=3), a diet containing inulin as the sole MAC source (n=4), or were maintained on the MD1 diet (n=4). See Methods for details of diet compositions. (a) Burdens of Cd were monitored over time by selective culture as described in Methods. Individual per-sample Cd burdens are plotted and lines represent geometric mean burdens per time point. (b) A contoured PCoA (cPCoA) plot of weighted UniFrac distances between microbiomes of these mice was prepared and overlaid with log-fold contour plots of Cd burdens, as measured in panel A. A line is drawn through the centroid of the points for a given experimental day. See Fig. S4 for further explanation on generation and interpretation of cPCoA plots. (c) Alpha diversity of communities was determined longitudinally for the microbiota of these mice by Shannon Diversity Index. Differences in Cd burdens and alpha diversity between dietary conditions was determined by collapsing all time points after the diet shift time point. In the box and whiskers plot shown, the box extends from the 25th to 75th percentiles, with the central line in each box representing the median value of the dataset. The whiskers extend from the smallest to largest values in the datasets. See Fig. S11 for temporal differences in Shannon Diversity Index. Statistical significance was determined by Kruskal Wallis test with Dunn’s multiple comparison test (**** = p<0.0001, see also Table S5).
Figure 3
Figure 3. Acetate, propionate, and butyrate are elevated in the ceca of mice fed MACs, and affect growth and toxin production in C. difficile
(a) The short chain fatty acids acetate, propionate, and butyrate were measured in cecal contents of mice fed the MD1, MAC+, and inulin containing diets (n=8 per dietary condition). See Fig S6 for time points of sacrifice corresponding to measurements). Data points represent per sample concentration of each SCFA and bars represent the mean per-diet SCFA concentration. Statistical significance was assessed by ordinary one-way ANOVA and Tukey’s multiple comparison test. (**, p<0.01; ****, p<0.0001, see also Table S5) (b) Doubling time was calculated for Cd grown in CDMM supplemented with 0, 10, and 30 mM sodium acetate (NaOAc), sodium propionate (NaOPr), sodium butyrate (NaOBu), or sodium chloride (NaCl, sodium matched controls) as described in Methods. For each growth condition, n=12 independent cultures were assessed. (c) TcdB was quantified in supernatants from n=12 independent cultures from each growth condition (corresponding to those analyzed in panel b) as described in Methods. Points represent mean values, error bars represent ±SEM (panels b–c). Statistical significance was assessed for each SCFA and sodium matched controls at each concentration via Mann-Whitney test (**** = two-tailed p<0.0001, see also Table S5).
Figure 4
Figure 4. Inflammation and Cd toxin expression are diet-dependent
Age-matched, female, humanized Swiss-Webster mice were fed the MD1 diet, and underwent experimental CDI as in Fig. 1 or were mock infected with filter sterilized PBS. After 7 days of infection, mice were switched either to the MAC+ or inulin-containing diet. (a) Mice were euthanized at time points specified in Table S4. Histopathology was carried out on proximal colon tissue from these mice as described in the “Histology and histopathological scoring” section of Methods (n=4, n=9, and n=4 for mock-infected mice fed the MD1 diet, the MAC+ diet, and the inulin diet, respectively; and n=13, n=15, and n=5 for infected mice fed the MD1 diet, the MAC+ diet, and the inulin diet, respectively). Points represent the sum histopathological score and lines are drawn at the mean score for each group (see Table S4 for individual parameters scored). Statistical significance between relevant groups was assessed by unpaired T test (*= two-tailed p<0.05; **= two-tailed p<0.01). (b) For infected mice where >5 mg fecal material could be collected (n=14, n=13, and n=13 for mice at 0, 2, and 4 days post MD1 to MAC+ diet shift; n=8, n=9, and n=6 for for mice at 0, 2, and 4 days post MD1 to inulin diet shift; and n=5, n=9, and n=9 at matched time points for mice that were maintained on the MD1 diet), levels of TcdB in the feces were measured and normalized to the burdens of Cd detected. Points represent normalized TcdB abundance for individual fecal samples. Lines are drawn through geometric mean normalized toxin abundances. For mice switched to the MAC+ diet, normalized TcdB abundance increases 14.5-fold from day 0 to day 2 (p<0.0001) and 6.5-fold from day 0 to day 4 (p=0.0066). For mice switched to the inulin diet, normalized TcdB abundance increases 8.0-fold from day 0 to day 2 (p=0.0016) and 6.5-fold from day 0 to day 4 (p=0.0426). Statistical significance between relevant pairs of treatment groups was assessed by Mann-Whitney test (*= two-tailed p<0.05; **= two-tailed p<0.01; ***= two-tailed p<0.001; **** = two-tailed p<0.0001, see also Table S5).
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