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

Plasma activated water (PAW) has gained significant global recognition due to its diverse applications in the biomedical and food preservation industries. The interaction between plasma’s ionized particles, comprising ions, electrons, and neutral atoms, and water molecules results in high concentrations of reactive oxygen species (ROS) and reactive nitrogen species (RNS), making PAW effective across various medical and agricultural fields. Previous research has demonstrated that effectiveness of PAW largely depends on its acidic pH, which generates several nucleophilic oxidants or secondary reactive nitrogen intermediates, such as peroxynitrite, derived from primary ROS and RNS. In one of our earlier studies, we were successful in generation of PAW at a neutral pH, which is more practical for real world applications. In this research, we explored the potential of the neutral pH high strength buffered PAW (hsPAbW) as an antimicrobial agent against ESKAPE pathogens and investigated its mechanism of action. Our findings revealed that hsPAbW effectively reduced bacterial viability during their exponential growth phase. We demonstrated that the ROS in hsPAbW are insufficient to independently kill the bacteria. Instead, the RNS components of hs-PAbW can exert microbicidal effects either independently or through the formation of peroxynitrite intermediates, which lead to nitrotyrosine production. As a result, we eliminated the need for low pH to induce the antimicrobial effects of hsPAbW, making it more practical for real world applications. Finally, our study showed that hs-PAbW is more effective at killing bacteria than high doses (50 microgram/mL) of antibiotics like ceftazidime, ciprofloxacin, and meropenem. Overall, our research highlights the promising potential of neutral pH PAW as a sustainable solution for combating ESKAPE pathogens.