The Clinical Drug Ebselen Attenuates Inflammation and Promotes Microbiome Recovery in Mice after Antibiotic Treatment for CDI

Abstract

Clostridium difficile infection (CDI) is an enteric bacterial disease that is increasing in prevalence worldwide. C. difficile capitalizes on gut inflammation and microbiome dysbiosis to establish infection, with symptoms ranging from watery diarrhea to toxic megacolon. We reported that the safe-in-human clinical drug ebselen (ClinicalTrials.gov: NCT03013400, NCT01452607, NCT00762671, and NCT02603081) has biochemical, cell-based, and in vivo efficacy against the toxins of C. difficile. Here, we show that ebselen treatment reduces recurrence rates and decreases colitis in a hamster model of relapsing CDI. Furthermore, ebselen treatment does not alter microbiome diversity and promotes recovery back to that of healthy controls after antibiotic-induced dysbiosis in healthy and C. difficile-infected mice. This increased microbiome recovery upon ebselen treatment correlates with a decrease in host-derived inflammatory markers, suggesting that the anti-inflammatory properties of ebselen, combined with its anti-toxin function, help to mitigate the major clinical challenges of CDI, including recurrence, microbial dysbiosis, and colitis.

Graphical abstract

Figure 1

Ebselen Reduces Inflammation and Recurrence in a Hamster Model of Relapsing CDI (A) Schematic of hamster models. Top: hamster model of CDI. Golden Syrian hamsters were randomly divided into four treatment groups: vehicle 1 (0.9% NaCl; n = 5), vehicle 2 (6.7% DMSO, 1% Tween 80 in PBS; n = 4), ebselen (100 mg/kg; n = 10), or vancomycin (10 mg/kg; n = 10). Dysbiosis was induced with clindamycin on day −1 prior to challenge with BI/NAP1 027 ribotype C. difficile (BAA-1805) spores on day 0. Vancomycin was dosed twice daily via oral gavage starting on day 0 for 5 days. Ebselen was dosed twice daily via oral gavage starting on day 0 for 10 days. Hamsters were observed for 28 days post-infection. Bottom: hamster model of relapsing CDI. Hamsters were randomly divided into four treatment groups of vehicle (n = 10), ebselen (100 mg/kg; n = 10), vancomycin (10 mg/kg; n = 10), or vancomycin and ebselen (10 mg/kg and 100 mg/kg respectively; n = 10). Dysbiosis was induced with clindamycin on day −1 prior to challenge with BI/NAP1 027 ribotype C. difficile (VA20) spores on day 0. Vancomycin was dosed once daily via oral gavage starting on day 0 for 5 days. Ebselen was dosed twice daily via oral gavage starting on day 0 for the duration of the study. Hamsters were observed for 21 days post-infection. (B) A Kaplan-Meier curve of a hamster model of CDI. (C) Representative histological images for the hamster model of CDI. The cecum and colon were harvested from animals at the time of humane sacrifice, death, or on day 28 for surviving animals. Samples were formalin fixed and paraffin embedded for H&E staining and histopathology. (D) Histopathology quantification from cecum and colon samples from hamsters from (B) treated with vehicle (0.9% NaCl; n = 8), vancomycin (n = 20), vehicle (6.7% DMSO, 1% Tween 80 in PBS; n = 10), or ebselen (n = 20). Slides were scored blinded by a board-certified veterinary pathologist for epithelial damage, vascular congestion and hemorrhage, and inflammation, with statistical analysis by unpaired t test. ∗p < 0.05. (E) Kaplan-Meier curve for relapse hamster model. Hazard ratio of co-treatment with ebselen and vancomycin to vancomycin: 0.537 (95% CI 0.1595–1.808) measured by a Mantel-Haenszel test. Results are presented from single experiments for each model.

Figure 2

Ebselen Does Not Alter the GI Microbiome (A) Conventional Swiss Webster mice were randomly divided into four groups and treated with vehicle (6.7% DMSO, 1% Tween 80 in PBS; n = 4), ebselen (100 mg/kg; n = 4), vancomycin (100 mg/kg; n = 4), or vancomycin and ebselen (100 mg/kg and 100 mg/kg, respectively; n = 4). Animals were dosed once daily via oral gavage for five treatments starting on day 0 after sampling. Fecal samples were collected daily for the 5-day experiment. (B) A principal-component analysis (PCA) plot of weighted UniFrac distances between microbiota of individual mice on day 5. Daily fecal samples were analyzed for GI microbial diversity via 16S rRNA amplicon analysis. (C) Weighted UniFrac distances between microbiota at day 5. Quantification is shown for intra-group measurements (side-by-side spheres, all intra-group comparisons NS; n = 14 for each) and inter-group comparisons (indicated by spheres below; n = 16 for each). Statistics were measured via one-way ANOVA with multiple comparisons; ∗∗∗∗p < 0.0001. (D) Alpha diversity of GI microbial amplicon sequence variants (ASVs) measured over the treatment period via the Shannon diversity index (n = 4 for each treatment at each time point except vehicle at day 3 where n = 3). (E) Statistical analysis of microbial diversity shown in (D): plots of Shannon diversity index for combined data for samples representing the treatment period (days 1–5; n = 20 per condition per day except for vehicle where n = 19 for all time points). Significance was determined by the Kruskal-Wallis test with the Dunn’s multiple comparison test. NS, non-significant; ∗∗∗∗p < 0.0001. Results are presented for a single animal experiment.

Figure 3

Ebselen Promotes Microbiome Recovery after Antibiotic Treatment (A) Schematic of experiment. Conventional Swiss Webster mice were randomly divided into four groups treated with one of four treatments: vehicle (6.7% DMSO, 1% Tween80 in PBS; n = 6), ebselen (100 mg/kg; n = 6), vancomycin (100 mg/kg; n = 6), or vancomycin and ebselen (100 mg/kg and 100 mg/kg, respectively; n = 6). Treatments were dosed once daily via oral gavage for five treatments (treatment phase) starting on day 0 after sampling. (B) Fecal samples (n = 4 for vehicle; n = 6 for all others) were analyzed for GI microbial diversity via 16S rRNA amplicon analysis. Alpha diversity of GI microbial ASVs was measured over the treatment and recovery phases via Shannon diversity index. (C) Statistical analysis of microbial diversity shown in (B) at day 28 (n = 4 for vehicle and n = 6 for all others): statistical analysis by one-way ANOVA. ∗∗p < 0.01 where indicated, all other comparisons were non-significant (NS). (D) Weighted UniFrac distances between vehicle/vancomycin (n = 24), vehicle/ebselen and vancomycin (n = 24), and vehicle/vehicle (n = 6) measured at day 8. Colored spheres indicate groups from which distances were measured; statistical analysis via one-way ANOVA, ∗p < 0.05. Results are presented for a single animal experiment.

Figure 4

Ebselen Attenuates Host-Derived Inflammation and Promotes Microbiome Recovery in a Mouse Model of CDI (A) Schematic of a mouse model of CDI. Dysbiosis was induced in conventional Swiss Webster mice with an antibiotic cocktail in drinking water (kanamycin 0.4 mg/mL, gentamicin 0.035 mg/mL, colistin 850 U/mL, metronidazole [0.215 mg/mL], and vancomycin [0.045 mg/mL]) for 3 days, beginning 6 days before inoculation (gray box, pre-treatment). Mice were switched to regular water for 2 days and then treated with 1 mg of clindamycin via oral gavage on day −1. On day 0, mice were randomly divided into an uninfected control group (n = 5) or four treatment groups (n = 7 for each) treated with one of four treatments: vehicle (6.7% DMSO, 1% Tween80 in PBS), ebselen (100 mg/kg), vancomycin (100 mg/kg), or vancomycin and ebselen (100 mg/kg and 100 mg/kg, respectively). Mice were orally challenged with approximately 10⁸ CFUs of C. difficile strain 630 (treatment groups) or PBS control. Treatments were dosed once daily via oral gavage for five treatments (acute infection and treatment phase) starting on day 0 after sampling. Fecal samples were collected for 28 days post-infection. (B) PCA plot of weighted UniFrac distances of GI microbiota at day 28. Fecal samples from each animal were analyzed for GI microbial diversity via 16S rRNA amplicon analysis. (C) Weighted UniFrac distances between vehicle/vancomycin (n = 42), vehicle/ebselen and vancomycin (n = 42), vancomycin/ebslen and vancomycin (n = 42), and intra groups (n = 21 each) at day 28. Colored spheres indicate groups from which distances were measured. Analysis of comparison between weighted UniFrac distances were made via one-way ANOVA, ∗∗p < 0.01. (D) Fecal lipocalin measured via ELISA for four treatment groups (n = 7 for each group and time point except uninfected where n = 4), measured during acute infection and treatment phase (days 2 and 5), and recovery phase (days 17, 24, and 28). Alternating gray and white rectangles delineate different days. Statistical analysis between treatment groups at each day was via one-way ANOVA with Tukey’s multiple comparisons. NS, non-significant, ∗p < 0.05, ∗∗p < 0.01. Results are shown for a single animal experiment.