Synergistic effects of luteolin and Lactobacillus rhamnosus on methicillin-resistant Staphylococcus aureus biofilm inhibition: in vitro and in silico molecular deployment studies.

Methicillin-resistant Staphylococcus aureus (MRSA) remains one of the leading causes of healthcare-associated infections due to its remarkable biofilm-forming ability and multidrug resistance, highlighting the urgent need for alternative antimicrobial strategies. The present study investigated the antibacterial, antibiofilm, antivirulence, and cytotoxic effects of luteolin alone and in combination with Lactobacillus rhamnosus bacterial suspension natant against MRSA. Antibacterial activity was evaluated using minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), disk diffusion, and fractional inhibitory concentration (FIC) assays. Biofilm inhibition was quantified using the crystal violet method, while the expression levels of biofilm-associated genes (icaA, icaB, icaC, icaD, clfA, clfB, fnbA, and fnbB) were determined by quantitative real-time PCR. Molecular docking analysis was performed to investigate the interaction between luteolin and S. aureus Sortase A (SrtA), and the cytotoxic potential of the treatments was evaluated in human dermal fibroblast (HDF) cells using the WST-8 assay. Luteolin exhibited MIC and MBC values of 128 and 256 µg/mL, respectively, whereas L. rhamnosus bacterial suspension natant also demonstrated antibacterial activity against MRSA. Combination treatment significantly increased the inhibition zone diameter and produced a synergistic interaction with an FIC index of 0.50. Moreover, the combination inhibited biofilm formation by approximately 75%, markedly exceeding the inhibitory effects of either treatment alone. Gene expression analysis revealed dose-dependent modulation of several biofilm-associated genes, particularly suppression of fnbA, suggesting interference with bacterial adhesion mechanisms. Molecular docking demonstrated favorable binding of luteolin to the active site of Sortase A with a predicted binding energy of - 9.220 kcal/mol, supporting its potential antivirulence mechanism. No significant cytotoxicity was observed in HDF cells within the tested concentration range. Collectively, these results demonstrate that luteolin combined with L. rhamnosus bacterial suspension natant represents a promising natural therapeutic strategy for controlling MRSA infections by simultaneously targeting bacterial growth, biofilm formation, and virulence while maintaining acceptable biocompatibility.