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

ABSTRACT 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. IMPORTANCE This 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.