Assessment of four Poly Antibiotic-resistant Bacterial Strains of Bacillusclausii (TIL 19T, TIL 21C, TIL 28S, TIL 30R) and their Probiotic Attributes: Implicationsfor Safety and Therapeutic Viability

Abstract

Objective: This study evaluated the probiotic potential and safety of four poly-antibiotic-resistant strains of
Bacillus clausii (TIL 19T, TIL 21C, TIL 28S, and TIL 30R), a spore-forming bacterium known for supporting
gastrointestinal health. Results: The strains demonstrated several desirable probiotic characteristics, including
tolerance to gastrointestinal conditions such as bile salts, lysozyme, and intestinal transit stress, along with strong
auto-aggregation and co-aggregation abilities. Acid, bile, transit, and lysozyme tolerance assays demonstrated
strong gastrointestinal survival abilities in all the four strains. TIL 19T and TIL 30R displayed high survival rates in
lysozyme-rich environments, and TIL 28S and TIL 30R showed strong co-aggregation with E. coli, indicating potential
pathogen inhibition. The strains also adhered effectively to intestinal cell models, produced digestive enzymes and
exopolysaccharides, reduced cholesterol levels, and exhibited antimicrobial properties. Safety assessments further
supported their probiotic candidacy. The strains were heat resistant, capable of moderate biofilm formation, and did
not produce harmful compounds such as biogenic amines, or DNase. Cytotoxicity testing on Vero cells revealed
high cell viability, suggesting a favourable safety profile. Conclusion: Collectively, these findings indicate that the
investigated Bacillus clausii (Shouchella clausii) strains possess strong probiotic and therapeutic potential, making
them promising candidates for future applications in functional foods and gastrointestinal health promotion.