Two strains of <i>Bacillus velezensis</i> : potent probiotics against <i>Escherichia coli</i> K99-induced diarrhea with high safety in mice

<i>Escherichia coli</i> K99 is a primary causative agent of diarrhea in young animals, leading to significant health and economic losses. With growing concerns over antibiotic resistance, probiotics-particularly <i>Bacillus</i> species-are gaining attention as effective alternatives for disease prevention and health promotion in livestock. In this study, 39 <i>Bacillus</i> strains were isolated from fresh cattle feces. Preliminary screening focused on biofilm formation, hemolytic activity, and antibacterial effects against <i>E. coli</i> K99. Promising candidates were further evaluated for <i>in vitro</i> probiotic characteristics and safety, including tolerance to acidic and bile salt conditions, heat resistance, hydrophobicity, enzymatic activity, and the presence of virulence and antibiotic resistance genes. <i>In vivo</i> assessments involved acute and subchronic toxicity tests and protective efficacy evaluations in a mouse model challenged with <i>E. coli</i> K99. Four strains-T36, B102, B116, and B150-demonstrated strong biofilm-forming ability, non-hemolytic properties, and notable antimicrobial activity. Among them, B102 and B116 showed excellent probiotic traits and safety profiles <i>in vitro</i>, lacking gelatinase and lecithinase activity, and were free of detectable virulence or resistance genes. <i>In vivo</i>, oral administration of B102 and B116 at high doses caused no acute or subchronic toxicity in mice. Notably, these strains improved the survival rate of <i>E. coli</i> K99-infected mice to 50% and 30%, respectively, compared to 0% in the untreated group. In conclusion, strains B102 and B116 exhibit strong probiotic potential and safety, offering promising alternatives to antibiotics for the prevention and control of <i>E. coli</i> K99 infections in animals. Their application may contribute to sustainable livestock health management.IMPORTANCE<i>Escherichia coli</i> K99-induced diarrhea causes severe economic losses in livestock farming, with limited safe control options due to antibiotic resistance. This study identifies two <i>Bacillus velezensis</i> strains (B102 and B116) that effectively inhibit <i>E. coli</i> K99, enhance survival rates in infected mice, and exhibit strong safety profiles. Their tolerance to gastrointestinal stress and lack of virulence or resistance genes make them ideal antibiotic alternatives. These strains could promote sustainable livestock health by reducing antibiotic use, mitigating resistance risks, and improving disease control, benefiting both agricultural productivity and public health.