Cold atmospheric plasma interactions with biofilm cells and the extracellular matrix

Abstract

The microbial biofilms represent a significant threat to human health and contribute to substantial costs associated with cleaning and maintenance. Cold plasma, a partially ionized gas, along with plasma-activated water (PAW), has shown potent antimicrobial properties. However, the ideal conditions for generating PAW and the mechanisms by which it exerts its antimicrobial effects remain poorly understood. This study aims to advance PAW as an innovative disinfectant for tackling microbial biofilm contamination. In this work, PAWs were produced using different gases in a plasma bubble spark discharge (BSD) reactor, and their effectiveness in biofilm removal was evaluated under both direct and indirect PAW treatments. The stability of PAWs was assessed through storage experiments. Results revealed that direct PAW treatment was more effective in eradicating biofilms, with further optimization achieved by altering the activated gases used. The physical and chemical properties of various PAWs were systematically optimized, and the underlying antimicrobial mechanisms were explored at both the intracellular and extracellular levels. A key finding was the significant increase in intracellular reactive oxygen species (ROS) levels in Escherichia coli (E. coli) ATCC 25922 biofilms treated with PAW generated with oxygen gas, where superoxide anion radicals (•O2-) were identified as crucial contributors to biofilm inactivation. Confocal microscopy analysis confirmed the removal of the majority of biofilm cells from the surface, leaving only a small fraction of dead cells behind. Additionally, the study revealed that PAW effectively targets both the extracellular matrix and bacterial cells in biofilms from both Gram-negative E. coli UTI 89 and Gram-positive Staphylococcus aureus (S. aureus) NCTC 8325 bacteria. A proteomic analysis further elucidated the response of E. coli and S. aureus biofilms to PAW-induced stress