Figure 5.
Protection from C. difficile morbidity and mortality is achieved by prophylactic administration of succinate-producing bacteria and depends on the presence of succinate and Pou2f3-dependent TCs. (A) Experimental diagram of the cefoperazone-based C. difficile infection animal model that includes prophylactic administration of bacterial consortia and subsequent infection with C. difficile. (B and C) (B) Probability of survival and (C) change in weight compared with weight preinfection of C57BL/6 WT mice infected with C. difficile following treatment with a succinate-producing bacterial consortium. Representative of two independent experiments, n = 5 female mice per treatment. Log-rank test was performed to evaluate the difference in survival probability between each two treatments. Benjamini-Hochberg-corrected two-sample t test at different time points were performed to assess differences in weight loss between every two treatments. (D) Estimation of C. difficile toxin A (tcdA) titer per 100 mg of cecal content using RT-qPCR in cefoperazone-treated mice administered with succinate-producing bacterial consortium or PBS and infected with C. difficile, 2 days after infection; n = 10 female mice per treatment. Two-sample t test was performed to assess statistical significance. (E and F) (E) Probability of survival and (F) change in weight compared to weight pre-infection of Pou2f3−/− or Pou2f3+/− mice infected with C. difficile following treatment with a succinate-producing bacterial consortium, the consortium after heat killing, or sterile PBS. Representative of two independent experiments, n = 5 female mice per treatment. Log-rank test was performed to evaluate difference in survival probability between each two treatments. Benjamini-Hochberg-corrected two-sample t test at different time points were performed to assess differences in weight loss between every two treatments. Pou2f3+/− and Pou2f3−/− were littermate cohoused after weaning. (G and H) (G) Probability of survival and (H) change in weight from preinfection of Pou2f3−/− and Pou2f3+/+ mice infected with C. difficile following treatment with sterile PBS. Representative of two independent experiments, n = 5 female mice per treatment. Log-rank test was performed to evaluate difference in survival probability between each two treatments. Benjamini-Hochberg-corrected two-sample t test at different time points were performed to assess differences in weight loss between treatments. (I) Comparison of colonic TC numbers in C57BL/6 WT mice given cefoperazone in the drinking water versus PBS. TC percentage was evaluated via flow cytometry by gating on CD45−Epcam+Siglecf+ cells. Representative of two independent experiments, n = 4 female mice per treatment. Two-sample t test was performed to assess statistical significance. (J) Comparison of TC numbers in the colon and ileum of Pou2f3+/+ and Pou2f3−/− mice administered with succinate producers or PBS immediately prior to C. difficile infection. Representative of two independent experiments, n = 4 female mice per treatment. ANOVA with Tukey post-hoc test was run to determine differences. Statistical significance was estimated at FDR of 0.05. Pou2f3−/− and Pou2f3+/+ mice were co-housed for at least 2 wk for basal microbiome equilibration before antibiotic treatment and administration of bacteria. (K) Targeted metabolomics to evaluate SCFAs and succinate concentration from colonic and ileal content from the mice of panel J. ANOVA with Tukey post-hoc test was run to determine metabolites significantly different across conditions. Statistical significance was estimated at FDR of 0.05. (L and M) (L) Probability of survival and (M) change in weight compared to weight pre-infection of C57BL/6 WT mice infected with C. difficile following treatment with B. thetaiotaomicron versus B. thetaiotaomicron Δfrd infection. Data are representative of two independent experiments repeated twice, n = 10 female mice per treatment. Log-rank test was performed to evaluate difference in survival probability between each two treatments. Benjamini-Hochberg-corrected two-samples t test at different time points was performed to assess differences in weight loss between treatments.

Protection from C . difficile morbidity and mortality is achieved by prophylactic administration of succinate-producing bacteria and depends on the presence of succinate and Pou2f3-dependent TCs . (A) Experimental diagram of the cefoperazone-based C. difficile infection animal model that includes prophylactic administration of bacterial consortia and subsequent infection with C. difficile. (B and C) (B) Probability of survival and (C) change in weight compared with weight preinfection of C57BL/6 WT mice infected with C. difficile following treatment with a succinate-producing bacterial consortium. Representative of two independent experiments, n = 5 female mice per treatment. Log-rank test was performed to evaluate the difference in survival probability between each two treatments. Benjamini-Hochberg-corrected two-sample t test at different time points were performed to assess differences in weight loss between every two treatments. (D) Estimation of C. difficile toxin A (tcdA) titer per 100 mg of cecal content using RT-qPCR in cefoperazone-treated mice administered with succinate-producing bacterial consortium or PBS and infected with C. difficile, 2 days after infection; n = 10 female mice per treatment. Two-sample t test was performed to assess statistical significance. (E and F) (E) Probability of survival and (F) change in weight compared to weight pre-infection of Pou2f3−/− or Pou2f3+/− mice infected with C. difficile following treatment with a succinate-producing bacterial consortium, the consortium after heat killing, or sterile PBS. Representative of two independent experiments, n = 5 female mice per treatment. Log-rank test was performed to evaluate difference in survival probability between each two treatments. Benjamini-Hochberg-corrected two-sample t test at different time points were performed to assess differences in weight loss between every two treatments. Pou2f3+/− and Pou2f3−/− were littermate cohoused after weaning. (G and H) (G) Probability of survival and (H) change in weight from preinfection of Pou2f3−/− and Pou2f3+/+ mice infected with C. difficile following treatment with sterile PBS. Representative of two independent experiments, n = 5 female mice per treatment. Log-rank test was performed to evaluate difference in survival probability between each two treatments. Benjamini-Hochberg-corrected two-sample t test at different time points were performed to assess differences in weight loss between treatments. (I) Comparison of colonic TC numbers in C57BL/6 WT mice given cefoperazone in the drinking water versus PBS. TC percentage was evaluated via flow cytometry by gating on CD45−Epcam+Siglecf+ cells. Representative of two independent experiments, n = 4 female mice per treatment. Two-sample t test was performed to assess statistical significance. (J) Comparison of TC numbers in the colon and ileum of Pou2f3+/+ and Pou2f3−/− mice administered with succinate producers or PBS immediately prior to C. difficile infection. Representative of two independent experiments, n = 4 female mice per treatment. ANOVA with Tukey post-hoc test was run to determine differences. Statistical significance was estimated at FDR of 0.05. Pou2f3−/− and Pou2f3+/+ mice were co-housed for at least 2 wk for basal microbiome equilibration before antibiotic treatment and administration of bacteria. (K) Targeted metabolomics to evaluate SCFAs and succinate concentration from colonic and ileal content from the mice of panel J. ANOVA with Tukey post-hoc test was run to determine metabolites significantly different across conditions. Statistical significance was estimated at FDR of 0.05. (L and M) (L) Probability of survival and (M) change in weight compared to weight pre-infection of C57BL/6 WT mice infected with C. difficile following treatment with B. thetaiotaomicron versus B. thetaiotaomicron Δfrd infection. Data are representative of two independent experiments repeated twice, n = 10 female mice per treatment. Log-rank test was performed to evaluate difference in survival probability between each two treatments. Benjamini-Hochberg-corrected two-samples t test at different time points was performed to assess differences in weight loss between treatments.

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