Non- and Medium-Polar Fractions from Trametes villosa Inhibit Staphylococcus aureus Growth, Virulence, and Biofilm Formation

Background: Infections caused by Staphylococcus aureus are a health problem worsened by antibiotic resistance. New drugs, including those inhibiting virulence and resistance mechanisms, are needed. This study aimed to evaluate the anti-growth, anti-virulence, and anti-biofilm activities of Trametes villosa. (2) Methods: Fractions were obtained from the basidiomata of T. villosa. Anti-growth, anti-hemolysis, and anti-biofilm activities were tested against S. aureus strains using resazurin microtiter, blood cell lysis, and crystal violet assays, respectively. Cytotoxicity was evaluated in Vero and HaCaT cells using sulforhodamine B. The active fractions were subjected to GC-MS analysis and molecular docking with S. aureus quorum-sensing receptors. Results: The n-hexane and ethyl acetate (EtOAc) fractions exhibited anti-growth activity against all strains (MIC: 31.2–2000 µg/mL). These fractions also displayed anti-hemolysis (IC50 = 33.8 ± 1.1–53.8 ± 5.1 µg/mL) and anti-biofilm formation activity (IC50 = 106.6 ± 4.8–383.4 ± 31.4 µg/mL), while exhibiting low cytotoxicity in Vero and HaCat. GC-MS analysis revealed that both active fractions mainly contained alkanes, aldehydes, and fatty acids. Molecular docking revealed that isovanillic acid, identified in the EtOAc fraction, exhibited optimal interactions with S. aureus quorum-sensing receptors AgrA and SarA. (4) Conclusions: Our research highlights the potential of T. villosa as a source of bioactive compounds effective against S. aureus.