Plasma-Treated Water: Role of Superoxide in Antimicrobial Activity

Abstract

Antimicrobial resistance is a growing global concern, affecting human health, veterinary settings, and food industry. In some regions, the overuse and uncontrolled dispensing of antibiotics have exacerbated this issue, leading to treatment complications and increased resistance rates. Listeria monocytogenes and Enterococcus faecalis are particularly problematic due to their resistance to environmental stresses and antibiotics (Border SO, et al., 2024). While conventional thermal treatments compromise product quality, alternative methods such as bacteriophages, essential oils, and non-thermal technologies offer promising solutions, however, most of them require combination strategies to achieve optimal efficacy. In this study, we investigate four variants of plasma treated water (PTW) generated using a bubbling method with four different airflow rates (PTW-60, PTW-80, PTW-100, PTW-120). Given PTW's well-documented antimicrobial properties, its effectiveness against L. monocytogenes and E. faecalis was evaluated. Additionally, high-performance liquid chromatography (HPLC), electron paramagnetic resonance (EPR) combined with spin-trapping and scavenger assays were performed to identify the reactive oxygen and nitrogen species present in each PTW sample. HPLC analysis revealed that higher airflow during PTW generation resulted in lower concentrations of the reactive species detected (OH•, NO• and NO2•). The intensity of DMPO radical adducts in the EPR spectra for both solvents, PTW and DMSO:PTW (9:1) was correlated with the airflow rates used when generating the PTW (PTW-60 < PTW-80 < PTW-100 < PTW-120). Although DMPO primarily formed DMPO-OH, experiments in 90% DMSO confirmed the presence of a stabilized superoxidederived adduct. EPR analysis performed with added superoxide scavenger SOD showed that the adduct signal was nearly absent, supporting the hypothesis that superoxide plays a key role in PTW's antimicrobial activity. Microbiological assays further confirmed this finding, as increasing SOD concentrations led to a significant reduction in bacterial inactivation (Hu X, et al., 2023). These results underscore the crucial role of superoxide radical in PTW-mediated bacterial inactivation and highlight the importance of reactive species characterization for optimizing PTW-based antimicrobial strategies.