Abstract: Background: Carvacrol, a monoterpenoid phenol abundant in oregano and thyme essential oils, has shown remarkable antimicrobial and efflux pump inhibitory properties. This study aimed to assess both the in silico binding interactions and the in vitro efflux pump inhibition potential of carvacrol against Staphylococcus aureus isolates from bovine mastitis. Methods: Molecular docking was performed using AutoDock Vina to predict the binding of carvacrol to the NorA efflux pump protein (PDB ID: 3WDO). Three clinical S. aureus isolates obtained from bovine mastitis and a reference strain (S. aureus ATCC 25923) were used for in vitro validation. Carvacrol (≥98% purity, Sigma-Aldrich) was tested for its minimum inhibitory concentration (MIC) using the broth microdilution method according to CLSI guidelines, and its efflux inhibition activity was evaluated by ethidium bromide accumulation assay. Results: Docking analysis revealed strong binding affinity of carvacrol to the NorA efflux pump, with a predicted binding energy of -7.1 kcal·mol⁻¹, mainly stabilized through hydrophobic and hydrogen-bond interactions. In vitro, carvacrol exhibited MIC values ranging from 128-256 µg·mL⁻¹. Ethidium bromide accumulation assays showed a significant increase in intracellular fluorescence (2.4-fold compared to control), indicating inhibition of efflux activity. Conclusion: Carvacrol demonstrated strong in silico interaction with the NorA efflux pump and effective in vitro inhibition of efflux-mediated resistance in S. aureus mastitis isolates. These findings support the potential of carvacrol as a natural efflux pump inhibitor that could enhance antibiotic efficacy against multidrug-resistant S. aureus.
