A Spore-Forming Bacillus Strain FM2 Isolated from Fish Market Runoffs with Remarkable Probiotic Attributes for...

The use of probiotics in aquaculture has emerged as a sustainable strategy for enhancing fish health and reducing the use of antibiotics, thereby serving the cause of One Health. In the present study, we employed a novel, distinctive, rigorous, and multi-tiered plate-based screening methodology to isolate spore-forming probiotic bacterial strains from fish market drain water for aquaculture applications. Based on their non-pathogenicity, potential for extracellular enzyme production, bile salt tolerance, and biofilm formation capabilities, three isolates, FM1, FM2, and FM4, with positive performance for all tested assays, were chosen and comprehensively characterized. Strain FM2, identified based on 16S rRNA gene sequence homology as Bacillus sp., was highly effective, exhibiting high sporulation efficiency (91%) and yield (9.3 × 10⁸ CFU /mL). It also had broad-spectrum antimicrobial activity against eight common fish pathogens, with high biofilm-formation inhibition, and preformed biofilm-disruption potential. The strain further demonstrated excellent adhesive capabilities (97% auto-aggregation at 24 h, significant co-aggregation with pathogens, and high cell surface hydrophobicity), robust antioxidant activity, and notable exopolysaccharide production. It was susceptible to key antibiotics, including gentamicin, imipenem, amikacin, and ciprofloxacin, ensuring biosafety. Its spores can withstand environmental and gastrointestinal stress conditions, such as heat (110 °C), UV light, lysozyme, and bile salts, and survive in simulated gastric and intestinal fluids. Thus, FM2 is a promising, safe, and multi-functional probiotic for aquaculture, capable of improving fish gut health, inhibiting pathogens, and surviving under challenging environmental conditions, and has enormous potential for commercialisation in aquaculture.