Different Inactivation Mechanisms of Staphylococcus aureus and Escherichia coli in Water by Reactive Oxygen and Nitrogen Species Generated from an Argon Plasma Jet

The atmospheric pressure plasma jet (APPJ) is a promising technology for inactivating waterborne pathogens by generating diverse reactive species under ambient conditions. However, uncertainties regarding the bacterial inactivation mechanisms persist due to varying findings in prior research. This study aimed to clarify the inactivation mechanisms of two representative bacteria, Staphylococcus aureus (S. aureus, Gram-positive) and Escherichia coli (E. coli, Gram-negative), using an argon-based APPJ (Ar-APPJ) system in a controlled medium, primarily deionized water. We identified several reactive oxygen and nitrogen species (RONS), including hydrogen peroxide, peroxynitrous acid/peroxynitrite (ONOOH/ONOO–), hydroxyl radical (•OH), and hydroperoxyl radical/superoxide radical, and evaluated their roles in bacterial inactivation. Inactivation experiments and quantification of suspected RONS revealed that ONOOH was the primary lethal agent for S. aureus, while •OH predominantly inactivated E. coli. Assessment of cell membrane integrity and intracellular RONS levels showed that E. coli, with its thinner cell wall, was more vulnerable to surface damage caused by •OH. In contrast, for S. aureus, with its thicker cell wall, intracellular attack by penetrated ONOOH, being significantly more diffusive than •OH, was more effective, as •OH alone could not induce sufficient surface damage. These findings advance our understanding of bacterial inactivation by the Ar-APPJ and provide valuable insights for designing effective water disinfection strategies utilizing this technology.