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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\u2009\u00b1\u20090.15\u00a0nm, polydispersity index (PDI) of 0.114\u2009\u00b1\u20090.008, and zeta potential of \u2212\u20092.73\u2009\u00b1\u20090.12 mV. TNE demonstrated consistent antimicrobial activity with MIC values of 15\u00a0mg\/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.\n                  <\/jats:p>","DOI":"10.1038\/s41598-025-29481-6","type":"journal-article","created":{"date-parts":[[2025,11,22]],"date-time":"2025-11-22T19:33:28Z","timestamp":1763840008000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Development and optimization of thymol-loaded nanoemulsions for enhanced antimicrobial activity against methicillin-resistant Staphylococcus aureus"],"prefix":"10.1038","volume":"15","author":[{"given":"Lingyong","family":"Song","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weize","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhizhong","family":"Hu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yangbo","family":"Lv","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jincang","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Feng","family":"Cheng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chunping","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,11,22]]},"reference":[{"key":"29481_CR1","doi-asserted-by":"publisher","first-page":"214","DOI":"10.15585\/mmwr.mm6809e1","volume":"68","author":"AP Kourtis","year":"2019","unstructured":"Kourtis, A. 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