Characterization of a novel strain of Bacillus subtilis and its antimicrobial activity against Clostridium...

Clostridium perfringens (C. perfringens) causes enteric diseases that are increasing threats to both humans and the livestock industry, leading to substantial economic losses globally. In response to restrictions on antibiotic growth promoters in livestock production, probiotics have been widely regarded as a potential alternative to antibiotics for sustainable agriculture. In this study, we isolated and characterized a novel strain of probiotic Bacillus subtilis (B.subtilis) L5 with antimicrobial activity and strong environmental resilience. Our results showed that L5 is tolerant to low pH and high salt environments, adheres to IPEC-J2 cells and modulates host gene expression in vitro. Specifically, L5 treatment increased the expression of cell junction-related genes CLND1 and OCLN1, and anti-apoptosis gene Bcl2 (p < 0.05), while decreasing the expression of inflammation-related genes IL-6 and IL-8, pro-apoptosis gene CASP3 (p < 0.05), suggesting its potential as a probiotic for swine, although efficacy in other livestock species, particularly poultry, remains to be validated. Furthermore, co-culture assays demonstrated that L5 inhibited the proliferation of C. perfringens, reduced the expression of the plc gene in type A C. perfringens and the netB gene in type G C. perfringens (p < 0.05), and alleviated C. perfringens-induced cytotoxicity in IPEC-J2 cells. To better understand the underlying mechanisms and explore the potential applications of L5, metabolomic and whole genome sequencing were performed. Genomic characterization of L5 revealed biosynthetic gene clusters for known antimicrobials (e.g., surfactin, fengycin), while untargeted metabolomics demonstrated that co-culture with C. perfringens induces distinct metabolic shifts in L5, including elevated secretion of putative metabolites potentially responsible for its inhibitory effects and numerous unannotated compounds warranting further investigation. Collectively, these findings underscore the potential of B.subtilis L5 as a viable probiotic candidate with broad-spectrum antimicrobial properties, positioning it as a promising alternative to antimicrobials in sustainable livestock production practices.