Bacillus Subtilis THC1I Restores Intestinal Barrier Integrity and Gut Microbiota Balance in a Mouse Model of Antibiotic-Associated Diarrhea.

Antibiotic-associated diarrhea (AAD) remains a common complication of antibiotic therapy. While probiotics show therapeutic potential, the novel strain Bacillus subtilis THC1I has not been previously evaluated for AAD treatment. This study aimed to assess the effects of B. subtilis THC1I on intestinal barrier integrity and gut microbiota dysbiosis in an AAD mouse model. Fifty mice were randomized into five groups (n = 10): control, model, positive control (Enterogermina®, B. clausii), and a B. subtilis THC1I spore suspension at 0.82 × 10⁹ and 1.64 × 10⁹ CFU/kg/day. AAD was induced with gentamycin sulfate and cefradine for five consecutive days. Outcomes included clinical symptoms, hematological and biochemical parameters, colonic macroscopic and histopathological indices, inflammatory cytokines, and gut microbiota analyzed by 16S rRNA sequencing. B. subtilis THC1I significantly improved body weight, water intake, fecal scores, and fecal water content. Treatment restored electrolyte balance (sodium and potassium), reduced white blood cell counts, and decreased relative colon weight and inflammation scores. Histopathological analysis revealed restored epithelial architecture and increased goblet cell density. Pro-inflammatory cytokines (TNF-α, IL-1β) were significantly reduced. B. subtilis THC1I partially improved microbial diversity (Shannon index) and modulated microbiota composition at both phylum and genus levels, decreasing pathogenic bacteria (Proteobacteria phylum: Escherichia-Shigella, Klebsiella, Salmonella; Firmicutes phylum: Clostridioides), and modulating the dysbiotic overgrowth of the beneficial commensal Blautia, while promoting beneficial bacteria (Bacteroidota phylum: Bacteroides, Muribaculaceae; Firmicutes phylum: Lachnospiraceae, Lactobacillus). B. subtilis THC1I demonstrates restorative effects on intestinal barrier damage and gut microbiota dysbiosis in AAD mice, supporting its potential as a therapeutic candidate for clinical application.