Genome-resolved, preclinical safety evaluation of infant-derived Clostridium butyricum S-45-5 as a probiotic candidate.

backgroundClostridium butyricum is a butyrate-producing, spore-forming bacterium present in the human gut microbiota and fermented foods that has emerged as a next-generation probiotic candidate. The infant-derived strain C. butyricum S-45-5 exhibits desirable probiotic traits, including acid and bile tolerance, antimicrobial activity against intestinal pathogens and broad-spectrum antiviral effects in vitro and in vivo. However, its strain-specific safety at exposure levels relevant for human use has not been rigorously established. We therefore sought to characterise the safety of C. butyricum S-45-5 using a genome-resolved, microbiology-centred approach complemented by guideline-aligned toxicology.

resultsHybrid long- and short-read sequencing yielded a closed, two-replicon genome for S-45-5. In silico screening against VirulenceFinder, PathogenFinder and ToxFinder detected no virulence- or toxin-associated genes, and ABRicate/MEGARes did not identify acquired antimicrobial resistance (AMR) determinants. A single intact prophage region was observed, none harbouring virulence or AMR genes. In vitro assays showed low cytotoxicity towards Caco-2 cells compared with Escherichia coli O157:H7, a γ-hemolytic (non-hemolytic) phenotype on blood agar, and no detectable D-lactate or bile salt hydrolase activity. E-test MICs indicated susceptibility to six clinically relevant antibiotics; elevated aminoglycoside MICs were consistent with intrinsic low susceptibility of obligate anaerobes and were not associated with acquired AMR genes. In OECD/GLP-compliant studies, S-45-5 was well-tolerated in acute and 90-day oral toxicity studies in rats (no-observed-adverse-effect level, 2,000 mg/kg/day), and genotoxicity tests were negative.

conclusionsGenomic, phenotypic and toxicological data converge to indicate that C. butyricum S-45-5 lacks recognised virulence and acquired AMR determinants, displays a benign in vitro safety profile and does not induce adverse effects or genotoxicity in preclinical models, even at high exposure levels. These findings provide a robust preclinical safety foundation for the development of C. butyricum S-45-5 as a probiotic or food-associated microorganism and may help to inform safety evaluation approaches for other next-generation probiotic candidates.These integrated genomic, phenotypic and OECD/GLP toxicology data support a favourable preclinical safety profile for C. butyricum S-45-5 at high exposure levels, supporting its development as a probiotic or food-associated microorganism.