Antimicrobial efficacy of Bifidobacterium longum FB1-4 cell-free supernatant against MRSA: Insights into mechanisms and food matrix application

The food safety landscape faces a significant challenge from methicillin-resistant Staphylococcus aureus (MRSA), a virulent and antibiotic-resistant foodborne pathogen. This study investigates the antimicrobial properties and mechanisms of cell-free supernatant (CFS) from Bifidobacterium longum FB1-4 against MRSA. The CFS exhibited robust antibacterial activity, achieving a minimum inhibitory concentration (MIC) of 5.56 mg/ml and maintaining a bacterial elimination rate of over 80 % for 8 hours at 2× MIC, demonstrating superior sustained bactericidal efficacy compared to vancomycin. Antibacterial component analysis revealed that while the initial effect is partly pH-dependent, the activity is largely attributable to heat-stable molecules, retaining 90 % efficacy after high-temperature treatment. In a food matrix, FB1-4 CFS exhibited impressive bactericidal effects against three MRSA strains, leading to elimination rates of 94.64 % at 2 hours, 98.02 % at 4 hours, 99.90 % at 6 hours, and nearly complete eradication (99.99 %) by 8 hours. Furthermore, FB1-4 CFS significantly suppressed all three tested MRSA strains (MRSA-43300, MRSA-337371, and MRSA-361194); P < 0.05) mecA expression, increased antibiotic susceptibility, and diminished biofilm formation by 70 %. Transcriptomic and metabolomic analyses revealed that FB1-4 CFS modulates two-component systems and quorum sensing, leading to reduced virulence factor expression. In vivo studies confirmed a 90 % reduction in MRSA colonization in mouse intestines. These findings provide critical insights into how probiotic metabolites inhibit MRSA, underscoring their potential as natural therapeutic agents in food safety.