Development and optimization of thymol-loaded nanoemulsions for enhanced antimicrobial activity against methicillin-resistant Staphylococcus aureus

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) necessitates the development of novel antimicrobial strategies. This study developed and optimized thymol-loaded nanoemulsions (TNE) as a standardized, reproducible alternative to variable essential oil formulations using central composite design. The optimal formulation comprised 4.6% oil phase, 18.5% surfactant, and 2.5:1 surfactant-to-cosurfactant ratio, yielding nanoemulsions with particle size of 25.51 ± 0.15 nm, polydispersity index (PDI) of 0.114 ± 0.008, and zeta potential of - 2.73 ± 0.12 mV. TNE demonstrated consistent antimicrobial activity with MIC values of 15 mg/mL against MRSA and other bacterial strains. Comprehensive mechanistic studies using five complementary assays revealed that TNE exerts antimicrobial effects through multiple membrane-targeting pathways, including membrane permeabilization, depolarization, hydrophobicity alteration, and rigidity modulation. The multi-target mechanism suggests reduced likelihood of resistance development compared to conventional single-target antibiotics. These findings demonstrate that systematically optimized thymol nanoemulsions represent a promising standardized platform for combating multidrug-resistant infections, offering enhanced stability, controlled release characteristics, and reduced resistance potential.