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<p>Multidrug-resistant (MDR) ESKAPE pathogens pose an escalating threat to global health, necessitating innovative therapeutic platforms capable of overcoming resistance, biofilm formation, and reduced antibiotic efficacy. This study reports the development, characterization, and antimicrobial evaluation of ozonated nanoemulsions (OZNEs) and dual nanomaterials (DNMs) formulated by integrating biogenic silver nanoparticles (AgNPs) with ozonated oil nanoemulsions. OZNEs prepared using castor, olive, and sunflower oils via high-energy microfluidization (20,000 psi) exhibited uniform nanodroplet sizes (82–118&#xa0;nm), low polydispersity (0.21–0.36), acidic pH (2.8–3.5), and excellent kinetic stability. DNMs demonstrated larger integrated droplet diameters (195–320&#xa0;nm) with PDI values of 0.210–0.495, confirming successful nanoparticle–nanodroplet assembly. In vitro assays revealed potent antimicrobial activity of OZNEs against all ESKAPE strains, with MIC values ranging from 1:256 to 1:4096 dilutions, and MBC values confirming complete bactericidal action. Anti-adherence assays showed significant inhibition of <i>S. aureus</i>,<i> A. baumannii</i> and <i>P. aeruginosa</i>. OZNEs reduced bacterial adherence by 45– 87% compared with Cefotaxime and Domex. Biofilm inhibition studies demonstrated that OZNEs achieved 68– 78% biofilm disruption, making it the most effective OZNE formulations. DNMs displayed superior synergistic activity, reducing bacterial viability by 2.5 − 4.8 log units and achieving statistically significant biofilm eradication (<i>P</i> &lt; 0.005) across fifteen ESKAPE strains, including clinical isolates. In vivo therapeutic evaluation using <i>Bombyx mori</i> larvae confirmed high biocompatibility and efficacy of CA-B, OL-B, and SF-B formulations, which restored larval survival to 78 − 92%, outperforming the Cefotaxime control (61%). Behavioral normalization and pigmentation recovery further supported therapeutic potential. Overall, the results demonstrate that ozonated nanoemulsions and their AgNP-integrated dual nanomaterials represent powerful, synergistic antimicrobial platforms capable of overcoming MDR mechanisms, inhibiting biofilm maturation, and outperforming conventional antibiotics. These nanoformulations offer significant translational potential for the development of next-generation therapeutics against MDR ESKAPE pathogens.</p>

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Ozonated Nanoemulsions and Dual Nanomaterials as Potent Antimicrobial Platforms Against Multidrug-Resistant ESKAPE Pathogens: In Vitro and In Vivo Evaluation

  • 期刊论文

  • 发表日期：

    2026年4月14日

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  • Mohd Hashim Khan,
  • Karthikeyan Ramalingam

摘要

Multidrug-resistant (MDR) ESKAPE pathogens pose an escalating threat to global health, necessitating innovative therapeutic platforms capable of overcoming resistance, biofilm formation, and reduced antibiotic efficacy. This study reports the development, characterization, and antimicrobial evaluation of ozonated nanoemulsions (OZNEs) and dual nanomaterials (DNMs) formulated by integrating biogenic silver nanoparticles (AgNPs) with ozonated oil nanoemulsions. OZNEs prepared using castor, olive, and sunflower oils via high-energy microfluidization (20,000 psi) exhibited uniform nanodroplet sizes (82–118 nm), low polydispersity (0.21–0.36), acidic pH (2.8–3.5), and excellent kinetic stability. DNMs demonstrated larger integrated droplet diameters (195–320 nm) with PDI values of 0.210–0.495, confirming successful nanoparticle–nanodroplet assembly. In vitro assays revealed potent antimicrobial activity of OZNEs against all ESKAPE strains, with MIC values ranging from 1:256 to 1:4096 dilutions, and MBC values confirming complete bactericidal action. Anti-adherence assays showed significant inhibition of S. aureus, A. baumannii and P. aeruginosa. OZNEs reduced bacterial adherence by 45– 87% compared with Cefotaxime and Domex. Biofilm inhibition studies demonstrated that OZNEs achieved 68– 78% biofilm disruption, making it the most effective OZNE formulations. DNMs displayed superior synergistic activity, reducing bacterial viability by 2.5 − 4.8 log units and achieving statistically significant biofilm eradication (P < 0.005) across fifteen ESKAPE strains, including clinical isolates. In vivo therapeutic evaluation using Bombyx mori larvae confirmed high biocompatibility and efficacy of CA-B, OL-B, and SF-B formulations, which restored larval survival to 78 − 92%, outperforming the Cefotaxime control (61%). Behavioral normalization and pigmentation recovery further supported therapeutic potential. Overall, the results demonstrate that ozonated nanoemulsions and their AgNP-integrated dual nanomaterials represent powerful, synergistic antimicrobial platforms capable of overcoming MDR mechanisms, inhibiting biofilm maturation, and outperforming conventional antibiotics. These nanoformulations offer significant translational potential for the development of next-generation therapeutics against MDR ESKAPE pathogens.