Drug repurposing of Nifuratel against methicillin-resistant Staphylococcus aureus through proton motive force disruption.

Introduction: The diminishing efficacy of conventional antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) necessitates novel therapeutic strategies. Drug repurposing represents a promising approach. This study investigates the antibacterial potential of Nifuratel, a repurposed agent, against MRSA. Methods: In vitro antibacterial activity was assessed against type strains and clinical isolates via minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. The propensity for resistance development was evaluated. Sub-MIC effects on key virulence phenotypes-biofilm formation, hemolysis, auto-aggregation, and surface spreading-were examined. The mechanism of action was investigated using transmission electron microscopy, fluorescence probes, and molecular dynamics simulations. Efficacy and biocompatibility were evaluated in vivo using murine abscess and wound infection models, with assessments of bacterial load, inflammation, wound healing, hemolysis, and organ toxicity. Results: Nifuratel exhibited potent bactericidal activity with MICs of 2-8 µg/mL and MBCs of 8-16 µg/mL, and a low propensity for resistance development. At sub-MIC concentrations, it significantly suppressed MRSA virulence phenotypes. Mechanistic studies revealed that Nifuratel disrupts the proton motive force by dissipating both the transmembrane potential and proton gradient, without causing direct membrane damage. In vivo, Nifuratel treatment significantly reduced bacterial loads, attenuated inflammation, and promoted wound healing comparably to fusidic acid. The compound demonstrated excellent biocompatibility with minimal hemolysis and no observed organ toxicity. Discussion: These results identify Nifuratel as a promising repurposed antimicrobial agent against MRSA. Its dual capability to exert direct bactericidal activity by disrupting PMF and attenuate key virulence factors, combined with a favorable resistance profile and biocompatibility in vivo, supports its potential for further therapeutic development.