Reactive nitrogen species (RNS) and its reaction intermediates with reactive oxygen species (ROS) in pH-neutral high-strength plasma-activated water determines the antimicrobial activity against ESKAPE Pathogens

Abstract

Plasma-activated water (PAW) is an emerging antimicrobial strategy, though its efficacy has traditionally depended on acidic pH to generate reactive species. To improve clinical and practical applicability, we developed a neutral pH formulation using buffered water--hs-PAbW--and evaluated its antimicrobial activity and mechanism of action against ESKAPE pathogens. hs-PAbW significantly reduced bacterial viability during the exponential growth phase. While reactive oxygen species (ROS) alone were insufficient for bacterial killing, reactive nitrogen species (RNS), particularly peroxynitrite, mediated strong microbicidal effects via nitrotyrosine formation. These effects occurred independently of acidic conditions, demonstrating that antimicrobial activity is retained at neutral pH. Alongside, hs-PAbW effectively reduced bacterial burden on metal surfaces and exhibited superior killing compared to high doses of standard antibiotics, including ceftazidime, ciprofloxacin, and meropenem in vitro. A non-cytotoxic dose of hs-PAbW also reduced bacterial load in infected wounds and accelerated wound closure in vivo. These findings highlight the dual functionality of hs-PAbW in surface sanitation and wound care, and support its potential as a sustainable, broad-spectrum antimicrobial agent for clinical use.