Factors Influencing Bactericidal Effect of Plasma-Activated Water Against Klebsiella pneumoniae

Plasma activated water (PAW) has demonstrated potent antimicrobial efficacy, which is mainly attributed to the reactive oxygen and nitrogen species (RONS). Our study assessed bactericidal activity of PAW against multidrug-resistant Klebsiella pneumoniae in relation to plasma treatment time, volume of treated water and PAW application time on bacteria samples. The model organism we selected is an important nosocomial pathogen, capable of persistence on various surfaces and difficult to eradicate due to resistance to harsh conditions and antimicrobials. The plasma source was 3-pin jet with needle electrodes inserted inside 3 separate glass tubes and positioned vertically above the water sample. Working gas was argon (2.5 slm), frequency of high-voltage power supply 340 kHz and applied power 14 W. We have treated 150 ml of deionized water for 20 min (PAW1, pH=6.2) and 15 ml for 10 min (PAW2, pH=6.5). Characterization of the PAW chemistry was performed by using colorimetric methods: PAW1 (H2O2=10 ppm, NO3-=34 ppm, NO2-=0 ppm) and PAW2 (H2O2=80 ppm, NO3-=97 ppm, NO2-=41 ppm). An aliquot of 100 μL of the suspension (10E8 cells/ml, K. pneumoniae ATCC BAA-1705) was added to 900 μL of PAW in microtubes, homogenized, and maintained for 15 min and 60 min. Bactericidal effects of PAW were assessed by the standard plate count method and expressed in log CFU/mL. Inactivation activity of PAW against K. pneumoniae increased by reducing the water volume for PAW preparation and by extending the incubation time of the bacteria in PAW. PAW1 reduced K. pneumoniae by ≈1.1 log CFU after 60 min of application time, while no reduction was noted after 15 min exposure. PAW2 applied for 15 min resulted in ≈1.6 log CFU reduction, while 60 min application achieved complete eradication of K. pneumoniae.