Neutral plasma-activated solution reverses polymyxin to inhibit polymyxin-resistant <i>Acinetobacter baumannii</i> by promoting the release of ROS and destroying the outer membrane <i>in vitro</i>

<i>Acinetobacter baumannii</i> is a prominent pathogen linked to ventilator-associated pneumonia (VAP) and has demonstrated widespread multidrug resistance globally. While the exploration of synergistic antibiotic combinations is increasingly viewed as an innovative treatment strategy, the antibacterial potential of plasma activated water (PAW) in neutralizing formulations remains underdeveloped. This study investigates the synergistic interactions between neutral plasma activated water (NPAW) and polymyxin B against multidrug-resistant strains of <i>A. baumannii</i>. We evaluated the antibacterial activity of the combination of NPAW and polymyxin B against polymyxin-resistant <i>A. baumannii</i> strains both <i>in vitro</i> and <i>in vivo</i>, further exploring the underlying mechanisms of synergy. Through checkerboard assay and time-kill studies, we demonstrated that the combination of NPAW and polymyxin B exhibited a synergistic effect against polymyxin-resistant <i>A. baumannii</i>. In the mouse pneumonia model, we confirmed that the combined treatment significantly reduced bacterial colonization in the lungs. Mechanistic studies indicated that NPAW enhances the bactericidal activity of Polymyxin B by promoting the release of reactive oxygen species (ROS). When used together, NPAW and polymyxin B decreased the production of intracellular ATP and membrane potential and compromised outer membrane integrity. In conclusion, the synergistic interactions between these agents may enable the use of lower concentrations of polymyxin B in treating <i>A. baumannii</i> infections, thereby minimizing dose-dependent side effects and providing a novel therapeutic option for managing these infections.IMPORTANCEPolymyxin-resistant <i>Acinetobacter baumannii</i> poses a global threat as last-line therapies fail. We demonstrate that neutral plasma activated water (NPAW), a reactive oxygen species-rich non-antibiotic agent, synergizes with polymyxin B to overcome resistance. Mechanistically, NPAW disrupts membrane integrity, depletes ATP, and amplifies oxidative stress, enhancing polymyxin B's bactericidal activity and reducing lung bacterial burdens in mice. This synergy enables lower polymyxin B doses, a critical advance for treating ventilator-associated pneumonia.