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 h 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 h, 98.02% at 4 h, 99.90% at 6 h, and nearly complete eradication (99.99%) by 8 h. 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.IMPORTANCEThis study unveils a novel, multi-targeted strategy against methicillin-resistant Staphylococcus aureus (MRSA) using the cell-free supernatant (CFS) of Bifidobacterium longum FB1-4. Integrating multi-omics analyses, we systematically demonstrate that FB1-4 CFS acts synergistically to disrupt MRSA's antibiotic tolerance, biofilm formation, virulence (via the SaeR/S system), and core metabolism. This multifaceted mechanism circumvents the resistance risk of single-target antibiotics. FB1-4 CFS also shows significant efficacy in both food matrices and an in vivo model, highlighting its dual potential as a natural food preservative and a therapeutic candidate to combat drug-resistant infections.