Sub-inhibitory concentrations of fosfomycin enhance Staphylococcus aureus biofilm formation by a sarA-dependent mechanism.

Staphylococcus aureus readily forms biofilms, which contribute to antimicrobial resistance and the persistence of chronic infections. This study investigates the effects of sub-inhibitory concentrations of fosfomycin on S. aureus biofilm formation and elucidates the underlying molecular mechanisms. Using crystal violet staining and confocal laser scanning microscopy, we demonstrated that fosfomycin at 1 µg/mL significantly enhanced biofilm biomass by 1.82- to 4.27-fold and led to denser biofilm structures. Adhesion assays further revealed that fosfomycin significantly promoted the initial attachment of S. aureus to solid surfaces, a critical early step in biofilm development. Phenotypic analyses showed increased production of polysaccharide intercellular adhesin, enhanced bacterial aggregation, and accelerated autolysis, resulting in elevated extracellular DNA release. Enzymatic disruption experiments indicated that, in addition to PIA and eDNA, proteins also play an important role in fosfomycin-enhanced biofilm formation. RT-qPCR revealed significant upregulation of key biofilm-associated genes, including icaA, icaB, fnbA, fnbB, emp, cidA, and the global regulator sarA. Notably, deletion of sarA abolished fosfomycin-induced promotion of both adhesion and biofilm formation, while complementation restored the phenotype, confirming a sarA-dependent mechanism underlying the fosfomycin-mediated enhancement of biofilm formation. These findings suggest that sub-inhibitory concentrations of fosfomycin promote S. aureus biofilm formation via sarA-mediated regulation, involving increased PIA synthesis, eDNA release, and protein-dependent matrix components. This mechanism may contribute to treatment failure and the development of persistent, antibiotic-resistant infections.IMPORTANCEBiofilm formation is a major factor in the persistence and antibiotic resistance of Staphylococcus aureus infections. Although fosfomycin is increasingly used to treat multidrug-resistant bacterial infections, its sub-inhibitory effects on biofilm formation have not been fully elucidated. Our study reveals that low-dose fosfomycin can significantly enhance S. aureus biofilm formation through a sarA-dependent mechanism. This finding raises concerns about the potential risks of sub-optimal dosing and highlights the need for careful evaluation of treatment strategies to avoid promoting persistent infections and resistance.