Isolation, Characterization, and Probiotic Potential of Bacillus subtilis Strain WW12 from Wild Wolf (Canis lupus) Feces with Protective Effects Against Murine Colitis

Wild wolves (Canis lupus), as the closest wild relatives of domestic dogs, harbor a gut microbiota uniquely adapted to their natural environment, representing a valuable reservoir of host-adapted probiotics. In this study, we isolated Bacillus subtilis strains from wild wolf feces and systematically evaluated their probiotic properties, including stress tolerance, antimicrobial activity, auto-aggregation, surface hydrophobicity, antibiotic susceptibility, and genomic safety, followed by in vivo assessment using a dextran sulfate sodium (DSS)-induced colitis mouse model. Among the isolates, strain WW12 exhibited the most promising probiotic characteristics. It demonstrated high survival rates in simulated gastric (94.3%) and simulated intestinal fluids (85.7%), strong tolerance to high temperatures (65.2% survival at 70°C), and excellent auto-aggregation (78.71%) and co-aggregation with pathogenic bacteria. WW12 showed broad-spectrum antimicrobial activity against Escherichia coli, Staphylococcus aureus and Salmonella Typhimurium , with inhibition zones of 18.3 mm, 19.7 mm and 19.3 mm, respectively. The strain was sensitive to all tested antibiotics, and genomic analysis confirmed the absence of transferable antibiotic resistance genes (ARGs); the only 10 intrinsic ARGs detected were located on the chromosome, indicating a favorable safety profile. In the DSS-induced colitis model, oral administration of WW12 significantly ameliorated disease severity, as evidenced by reduced disease activity index (DAI), restored colon length, improved histopathological scores, and modulation of inflammatory cytokines (decreased IL-1β, IL-6; increased IL-10). Metagenomic analysis revealed that WW12 treatment reshaped the gut microbiota by increasing beneficial bacteria ( Bacteroides fragilis etc. ) and reducing pathogenic taxa ( Clostridioides difficile etc. ). Although not detected in canine feces after oral administration—suggesting possible intestinal mucosal colonization—WW12’s efficacy in the murine model strongly supports its probiotic potential. Transcriptomic analysis further showed that WW12 downregulated the Cytokine-cytokine receptor interaction pathway and the PI3K-Akt signaling pathway. Collectively, these findings establish Bacillus subtilis WW12 as a safe, host-adapted probiotic candidate derived from wild wolves, with significant protective effects against intestinal inflammation, offering a novel natural intervention for inflammatory bowel disease in companion animals.