Synergistic Antimicrobial Effects of Baicalin Combined with Kanamycin Against MRSA: Underlying Mechanisms and Diminished Colonization on Lettuce

<b>Background:</b> The treatment of methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) infections is extremely challenging due to its antibiotic resistance, and the combination of plant active ingredients with antibiotics represents a potential strategy to address this issue. <b>Methods:</b> We determined the combinatorial relationship between baicalin (BA) and kanamycin (KM) using the checkerboard dilution method. The antibacterial activity of the baicalin-kanamycin (BA/KM) combination was evaluated through growth curve determination assays and scanning electron microscopy (SEM). The effects of the BA/KM combination on the cell membrane and cell wall of MRSA were analyzed using reactive oxygen species (ROS) detection assays, intracellular protein leakage experiments, alkaline phosphatase (AKP) activity assays, laser scanning confocal microscopy (LSCM) observations, and molecular docking simulations. The antibiofilm activity and related mechanisms of the BA/KM combination were elucidated via crystal violet staining, MTT assay, phenol-sulfuric acid method, congo red staining, staphyloxanthin determination assays, and quantitative real-time polymerase chain reaction (qPCR). The safety of the BA/KM combination was assessed through hemolytic activity analysis, and its anti-MRSA efficacy was evaluated on lettuce. <b>Results:</b> BA/KM combination showed a synergistic antibacterial effect on MRSA USA300. Mechanistic studies revealed that BA may interact with amino acid residues of peptidoglycan synthetase PBP2a to hinder peptidoglycan synthesis, thereby facilitating KM penetration through the cell wall. Subsequently, BA binds to amino acid residues of the membrane transporter NorA, leading to disruption of cell membrane homeostasis and enhancing KM's ability to induce intracellular ROS accumulation in MRSA. Furthermore, the BA/KM combination reduced MRSA biofilm formation by 77.85% and decreased the metabolic activity of biofilm cells by 42.93% through inhibiting the synthesis of biofilm components EPS and PIA. Additionally, this combination suppressed the synthesis of staphyloxanthin and downregulated the expression of <i>agrA</i> and <i>agrC</i> genes. When 1/8 MIC BA was combined with 1/4 MIC KM, the count of MRSA on lettuce surfaces was reduced by 0.88 log CFU/cm², an effect comparable to that of 0.2% (<i>v</i>/<i>v</i>) hydrogen peroxide. <b>Conclusions:</b> According to these findings, the BA/KM combination may offer a promising option for enhancing antibacterial efficacy through synergism, reducing antibiotic usage concentrations, and limiting MRSA transmission in fresh agricultural products.