Misoprostol protects mice against severe Clostridium difficile infection and promotes recovery of the gut microbiota after antibiotic perturbation

Abstract

Clostridium difficile infection (CDI) is one of the most common nosocomial infections worldwide and an urgent public health threat. Epidemiological and experimental studies have demonstrated an association between nonsteroidal anti-inflammatory drug (NSAID) exposure and enhanced susceptibility to, and severity of, CDI. NSAIDs target cyclooxygenase enzymes and inhibit the production of prostaglandins (PGs), but the therapeutic potential of exogenous introduction of PGs for the treatment of CDI has not been explored. In this study, we report that treatment with the FDA-approved stable PGE₁ analogue, misoprostol, protects mice against C. difficile-associated mortality, intestinal pathology, and CDI-mediated intestinal permeability. Furthermore, we report that the effect of misoprostol on the gastrointestinal tract contributes to increased recovery of the gut microbiota following antibiotic perturbation. Together, these data implicate PGs as an important host-factor associated with recovery to C. difficile-associated disease and demonstrate the potential for misoprostol in the treatment of CDI. Further studies to explore the safety and efficacy of misoprostol treatment of CDI in humans is needed.

Keywords: Clinical pharmacology; Colitis; Microbiome; NSAIDs; Prostaglandins.

Fig. 1:. Experimental design.

(a) Female C57BL/6J mice were exposed to cefoperazone ad libitum in drinking water for days −7 to −2 prior to inoculation with 1 × 10⁶ spores of the C. difficile strain M7404 by gavage on day 0. Following infection mice were administered intraperitoneal injections of misoprostol once daily for 7 days. (b) Mice were exposed to cefoperazone as in (a) then treated with misoprostol (20 μg) daily by intraperitoneal injection through 10 days. Feces were collected for microbiota studies.

Fig. 2:. Misoprostol protects animals against severe CDI and decreases intestinal permeability.

Female C57BL/6 mice were treated with cefoperazone for 5 days followed by 2 days of recovery in regular drinking water and then challenged with 1 × 10⁶ spores of strain M7404. (a) Mice received misoprostol by intraperitoneal (i.p.) injection daily starting on the day of inoculation. 60 mg i.p. daily showed the same results as 20 mg (not shown). N = 5 mice per group. * P <0.05 compared to uninfected control by Log-rank (Mantel-Cox) test. (b.) Mice were treated with cefoperazone for 5 days followed by 2 days of recovery in regular drinking water and then challenged with 1 × 10⁴ spores of strain M7404. Mice received misoprostol by i.p. injection (or vehicle) daily and stools were scored for severity of diarrhea on a 4 point scale (1 -- normal, 2 -soft stool/discolored, 3 -- wet stained tail/mucous, 4 -- liquid/no stool). N = 5 mice per group. **P <0.01, ***P < 0.001 by ANOVA followed by Tukey’s multiple comparisons test. (c) To assess intestinal permeability mice were infected with 1×10⁴ spores of M7404 and given misoprostol 20 mg/mouse by IP injection 30 min before C. difficile inoculation, 24 h later and at the time of FITC-dextran treatment. 2 d post infection mice were gavaged with FITC-dextran or vehicle control, then euthanized 4 h later and concentrations of FITC-dextran in plasma were determined. N = 5 mice per group. *P < 0.05 by ANOVA followed by Tukey’s multiple comparisons test.

Fig. 3:. Misoprostol treatment promotes recovery of the gut microbiota following antibiotic exposure.

(a) Shannon diversity index for cefoperazone treated (black) or cefoperazone + Misoprostol (purple) treated mice. (b) Non-metric multidimensional scaling (NMDS) ordination showing β-diversity as measured by Yue and Clayton’s measure of dissimilarity (θ_YC) on day 10. Significance between baseline (grey) and cefoperazone- treated (black) or cefoperazone + Misoprostol (grey) samples measured using analysis of molecular variance (AMOVA) (P<0.001).

Fig 4.. Gut microbiota dynamics following antibiotic and misoprostol treatment

(a) NMDS ordination showing the dynamics of recovery (baseline, day 0, day 1, day 4, day 5, and day 10) following antibiotic treatment and subsequent introduction of PBS (grey scale) or Misoprostol (purple scale).

Fig 5.. Rebound of taxa following antibiotic treatment following introduction of misoprostol

(a) Dynamics of recovery of differentially abundant taxa over a 10-day time course. B represents baseline microbiota pre-treatment. Significantly altered taxa were identified using the linear discriminant analysis (LDA) effect size (LEfSe) algorithm.