We investigated the inhibitory potential of combining linezolid with fosfomycin against biofilms formed by vancomycin-resistant Enterococcus faecium (VREfm), aiming to provide a conceptual framework for managing infections associated with VREfm biofilms. This investigation used four clinical VREfm isolates (designated No. 2, No. 4, No. 5, and No. 6) sourced from the First Affiliated Hospital of Anhui Medical University. Synergy between linezolid and fosfomycin was evaluated through the checkerboard assay. Crystal violet staining quantified biofilm biomass suppression, whereas an Alamar Blue cell viability test measured metabolic function. The impact of drug exposure on the expression of genes implicated in biofilm development was probed using quantitative real-time polymerase chain reaction (RT-qPCR). Fractional inhibitory concentration index (FICI) values indicated that the linezolid-fosfomycin pairing acted synergistically against all VREfm isolates tested. Compared with linezolid alone, the dual-drug regimen markedly reduced both biomass accumulation and metabolic function in biofilms, particularly those at the mature stage. Data from RT-qPCR indicated that the combined therapy hindered biofilm development by repressing transcription of cylA, ebpA, and gelE during both early and late biofilm phases. Regarding mature biofilms specifically, the combination additionally downregulated asa1, atlA, and esp. Pairing linezolid with fosfomycin yielded a more robust suppression of VREfm biofilm formation than linezolid administered alone.
