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Sirikwan Sangboonruang,1 Natthawat Semakul,2 Mohammad A Obeid,3 Marta Ruano,4 Kuntida Kitidee,5 Usanee Anukool,6,7 Kidsadagon Pringproa,8 Panuwan Chantawannakul,9 Valerie A Ferro,4 Yingmanee Tragoolpua,9 Khajornsak Tragoolpua6,7 1Biotechnology Section, Graduate School, Chiang Mai University, Chiang Mai, Thailand; 2Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand; 3Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, Yarmouk University, Irbid, Jordan; 4Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK; 5Center for Research and Innovation, Faculty of Medical Technology, Mahidol University, Salaya, Nakhon Pathom, Thailand; 6Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand; 7Infectious Diseases Research Unit (IDRU), Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand; 8Department of Veterinary Biosciences and Veterinary Public Health, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, Thailand; 9Division of Microbiology, Department of Biology, Faculty of Sciences, Chiang Mai University, Chiang Mai, ThailandCorrespondence: Khajornsak TragoolpuaDivision of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, 50200, ThailandTel +66 5393 6039Fax +66 5393 6042Email khajornsak.tr@cmu.ac.thBackground: Staphylococcus aureus is an important human pathogen, especially causing skin and soft tissue infections (SSTIs). Over the decades, the infections caused by antibiotic-resistant strains have often become life-threatening. Consequently, exploration and development of competent approaches to combat these serious circumstances are urgently required.Methods: The antibacterial activity of melittin (Mel) on S. aureus, methicillin-resistant S. aureus (MRSA) and clinical isolates of vancomycin-intermediate S. aureus (VISA) was investigated by minimum inhibitory concentration (MIC) and time-killing assays. The localization of Mel on the bacterial cell was visualized by confocal laser scanning microscopy and its effect on the membrane was indicated based on propidium iodide uptake. The non-ionic surfactant vesicle (NISV) or niosome nanocarrier was established for Mel loading (Mel-loaded NISV) by the thin-film hydration method. Physicochemical and in vitro biological properties of Mel-loaded NISVs were characterized. The cellular uptake of Mel-loaded NISVs was evaluated by holotomography analysis. In addition, an ex vivo study was conducted on a porcine ear skin model to assess the permeation ability of Mel-loaded NISVs and their potential to inhibit bacterial skin infection.Results: The effective inhibitory activity of Mel on skin pathogens was demonstrated. Among the tested strains, VISA was most susceptible to Mel. Regarding to its function, Mel targeted the bacterial cell envelope and disrupted cell membrane integrity. Mel-loaded NISVs were successfully fabricated with a nano-size of 120– 200 nm and entrapment efficiency of greater than 90%. Moreover, Mel-loaded NISVs were taken up and accumulated in the intracellular space. Meanwhile, Mel was released and distributed throughout the cytosol and nucleus. Mel-loaded NISVs efficiently inhibited the growth of bacteria, particularly MRSA and VISA. Importantly, they not only penetrated epidermal and dermal skin layers, but also reduced the bacterial growth in infected skin.Conclusion: Mel-loaded NISVs have a great potential to exhibit antibacterial activity. Therapeutic application of Mel-loaded NISVs could be further developed as an alternative platform for the treatment of skin infection via dermal and transdermal delivery.Keywords: melittin, niosome, drug resistance, skin infection, Staphylococcus aureus, dermal and transdermal delivery