Gut microbiota-produced succinate promotes C. difficile infection after antibiotic treatment or motility disturbance

Abstract

Clostridium difficile is a leading cause of antibiotic-associated diarrhea. The mechanisms underlying C. difficile expansion after microbiota disturbance are just emerging. We assessed the gene expression profile of C. difficile within the intestine of gnotobiotic mice to identify genes regulated in response to either dietary or microbiota compositional changes. In the presence of the gut symbiont Bacteroides thetaiotaomicron, C. difficile induces a pathway that metabolizes the microbiota fermentation end-product succinate to butyrate. The low concentration of succinate present in the microbiota of conventional mice is transiently elevated upon antibiotic treatment or chemically induced intestinal motility disturbance, and C. difficile exploits this succinate spike to expand in the perturbed intestine. A C. difficile mutant compromised in succinate utilization is at a competitive disadvantage during these perturbations. Understanding the metabolic mechanisms involved in microbiota-C. difficile interactions may help to identify approaches for the treatment and prevention of C. difficile-associated diseases.

Figure 1. Metabolic strategies employed by C. difficile in response to a human gut commensal

a. C. difficile density in feces at 1 day post-infection of germ free (Cd) or Bt-associated mice (Cd + Bt) and fed standard diet (Std diet) or polysaccharide-deficient diet (Pd diet) (n=4/group). b. C. difficile catabolic pathways with significant differences in gene expression levels in vivo in the presence and absence of Bt. Mice were fed standard diet (Std) or polysaccharide-deficient diet (Pd). Colors indicate the deviation of each gene’s signal above (purple) and below (green) its mean (black) expression value across all sixteen in vivo samples (n=4/group) at 5 days post-infection in cecal contents. Fold change values correspond to gene expression in the Bt-biassociation relative to the monoassociation state. c. Concentration of the short-chain fatty acid butyrate in cecal contents of standard diet-fed mice colonized with Bt, C. difficile and Bt (Cd + Bt), or C. difficile (Cd) at 5 days post-infection (n=4/group).

Figure 2. A C. difficile operon encodes succinate-to-butyrate conversion

a. Growth of wild-type C. difficile (Cd) and CD2344 mutant C. difficile (Cd-CD2344⁻) in minimal media (MM) containing 1% glucose (left), water (center), or 0.1% succinate (right). OD₆₀₀ measurements were taken every 30 minutes (n=3/group). Representative of at least 3 replicates. b. Concentrations of butyrate produced after growth of Cd or Cd-CD2344⁻ in minimal media containing 0.5% glucose (MM + glucose) or 0.1% succinate (MM + succinate) (n=3/group). c. Concentrations of succinate remaining after growth of Cd or Cd-CD2344⁻ in minimal media containing 0.5% glucose (MM + glucose) or 0.1% succinate (MM + succinate) (n=3/group). d. Levels of ¹³C-butyrate produced by Cd or Cd-CD2344⁻ in minimal media containing 0.5% glucose (MM + glucose) or 0.1% ¹³C-succinate (MM + succinate) (n=3/group). e. Levels of ¹³C-succinate remaining after growth of Cd or Cd-CD2344⁻ in minimal media containing 0.5% glucose (MM + glucose) or 0.1% ¹³C-succinate (MM + succinate) (n=3/group).

Figure 3. C. difficile consumes the Bt fermentation product succinate to expand in the intestinal ecosystem

a. C. difficile density in feces at 2 days post-infection of germ-free mice standard diet (Std) or polysaccharide deficient diet (Pd), supplemented with water containing 1% sorbitol (Sor), 1% succinate (Suc) or both 1% sorbitol and 1% succinate (Sor + Suc) (n=5/group). **** indicates statistical significance at an alpha value of <0.05 by one-way ANOVA with Bonferroni multiple comparisons test. b. C. difficile density in feces of germ-free mice consuming polysaccharide-deficient diet and infected with wild-type C. difficile (Cd) or Cd-CD2344⁻ mutant C. difficile (Cd-CD2344⁻) and administered water (−) or water containing 1% sorbitol and 1% succinate (+) at 1 day post-infection (n=5/group). c. C. difficile density in feces of Bt-associated mice infected with wild-type C. difficile (Bt + Cd) or Cd-CD2344⁻ mutant C. difficile (Bt + Cd-CD2344⁻) at day 1 post-infection and under standard dietary conditions (n=5/group). d. Concentration of butyrate in cecal contents of Bt-associated mice infected with wild-type C. difficile (Cd) or Cd-CD2344⁻ mutant C. difficile (Cd-CD2344⁻) at day 3 post-infection and under standard dietary conditions (n=5/group).

Figure 4. C. difficile exploits a spike in succinate availability after microbiota perturbations

a. Concentrations of acetate, butyrate, propionate, lactate and succinate detected in cecal contents of streptomycin-treated conventional mice before treatment (Conv) and 3 days after antibiotic treatment (Abx) (n=5/group). b. DNA of wild-type C. difficile (Cd) versus Cd-CD2344⁻ mutant C. difficile (Cd-CD2344⁻) in feces of conventional mice treated with antibiotics (Abx) or PEG 3 days after infection with a 1:1 mixture of the two strains (n=5/group). *, p<0.05, one-sample t-test vs. a theoretical mean of 1.0. c. C. difficile density in feces of conventional (Conv) or PEG-treated mice infected with wild-type C. difficile at day 3 post-infection (n=5 in Conv and n=4 in PEG). Dotted line represents the detection limit. d. Concentrations of acetate, butyrate, propionate, lactate and succinate detected in cecal contents of PEG-treated conventional mice before treatment (Conv) and 3 days after treatment (PEG) (n=5/group).