Subinhibitory Concentrations of Rifampicin Synergize with Linezolid to Delay Resistance Evolution in Clinical Methicillin-Resistant Staphylococcus Aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen. Long-term clinical use of linezolid readily induces bacterial resistance in MRSA. This study explored resistance evolution and related mechanisms of MRSA to linezolid under subinhibitory concentrations of rifampicin, as well as the antibacterial activity and anti-resistance potential of the combination. Synergistic effects were confirmed via the broth microdilution method, checkerboard method, and time-kill curve assay. The mutant prevention concentration (MPC) was determined to assess suppression of resistant mutant enrichment. We used a 28-day adaptive evolution model and compared resistance dynamics between linezolid monotherapy and its combination with subinhibitory concentrations of rifampicin. We analyzed the growth characteristics, biofilm formation, virulence phenotypes, and resistance-related mutations of induced strains. The combination exerted synergistic or additive effects, reducing the MPC of linezolid, narrowing the mutant selection window, and delaying resistance development. Strains induced by the combination exhibited slower growth, a greater reduction in biofilm formation, and significantly lower hemolytic activity and attenuated in vivo virulence in the Galleria mellonella infection model. Sanger sequencing revealed specific mutations in the 23S rRNA gene and the ribosomal protein gene (rplC). Linezolid combined with rifampicin synergistically suppresses resistant mutant enrichment and delays resistance evolution, providing experimental support for optimizing anti-MRSA therapeutic regimens.