The efflux pump QacA mediates the transition to vancomycin heteroresistance in sequence type 5 methicillin-resistant...

The heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) phenotype in methicillin-resistant S. aureus (MRSA) is a major driver of vancomycin treatment failure. Within the prevalent ST5-MRSA lineage, the efflux pump gene qacA has emerged as a key factor in the development of hVISA. However, the underlying mechanism remains elusive, as vancomycin is not a typical efflux substrate. In this study, we demonstrated that qacA triggered the vancomycin-susceptible S. aureus (VSSA)-to-hVISA phenotypic conversion independently of direct efflux, cell wall thickening, or biofilm formation. Membrane fluidity assays and fluorescence recovery after photobleaching (FRAP) analysis confirmed that QacA expression significantly increased membrane fluidity and accelerated lateral diffusion rates. Correspondingly, confocal microscopy assays revealed that vancomycin probe binding to cells was reduced in the qacA-carrying strains. Lipidomic profiling further demonstrated that qacA induced membrane lipid reprogramming, characterized by an enrichment of diunsaturated glycerophospholipids. Furthermore, exogenous supplementation with long-chain unsaturated fatty acids exerted potent synergistic bactericidal effects with vancomycin. In conclusion, our study showed that qacA drives the VSSA-to-hVISA transition through a distinct lipid reprogramming. This process forms a hyper-fluid membrane "entropy barrier" that disrupts vancomycin binding to Lipid II, rather than relying on direct drug transport. These findings challenge the traditional understanding of antiseptic resistance determinants and highlight the bacterial membrane lipidome as a critical, yet overlooked, target for potentiating vancomycin activity.