Atmospheric Nonthermal Plasma-Treated PBS Inactivates Escherichia coli by Oxidative DNA Damage.

We recently reported that phosphate-buffered saline (PBS) treated with nonthermal dielectric-barrier discharge plasma (plasma) acquires strong antimicrobial properties, but the mechanisms underlying bacterial inactivation were not known. The goal of this study is to understand the cellular responses of Escherichia coli and to investigate the properties of plasma-activated PBS. The plasma-activated PBS induces severe oxidative stress in E. coli cells and reactive-oxygen species scavengers, α-tocopherol and catalase, protect E. coli from cell death. Here we show that the response of E. coli to plasma-activated PBS is regulated by OxyR and SoxyRS regulons, and mediated predominantly through the expression of katG that deactivates plasma-generated oxidants. During compensation of E. coli in the absence of both katG and katE, sodA and sodB are significantly overexpressed in samples exposed to plasma-treated PBS. Microarray analysis found that up-regulation of genes involved in DNA repair, and E. coli expressing recA::lux fusion was extremely sensitive to the SOS response upon exposure to plasma-treated PBS. The cellular changes include rapid loss of E. coli membrane potential and membrane integrity, lipid peroxidation, accumulation of 8-hydroxy-deoxyguinosine (8OHdG), and severe oxidative DNA damage; reveal ultimate DNA disintegration, and cell death. Together, these data suggest that plasma-treated PBS contains hydrogen peroxide and superoxide like reactive species or/and their products which lead to oxidative changes to cell components, and are eventually responsible for cell death.

Control of multi-drug-resistant pathogens with non-thermal-plasma-treated alginate wound dressing.

BACKGROUND: Non-thermal dielectric-barrier discharge plasma (non-thermal plasma) is being investigated for use in wound healing. Alginate gel, already in clinical use, is non-toxic but has no meaningful antimicrobial property. This study reports that a non-thermal-plasma-treated alginate wound dressing has strong antimicrobial properties. METHODS: Alginate gel was treated with non-thermal plasma in room air and inoculated with bacterial pathogens. At 15 min after this, bacterial cell viability was determined by colony assay or 2,3-bis-(2-methoxy-4- nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. The anti-biofilm efficacy of the non-thermal-plasma-treated alginate gel was investigated and the treated gel was tested against vascular endothelial cells for a cytotoxic effect. The proliferation and migration of bacterial cells before and after exposure to the treated gel were investigated with an in vitro wound testing assay. Scanning electron microscopy was used to observe changes in the gel surface associated with exposure to bacterial pathogens. The treated gel was tested against Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus, S. epidermidis, Candida albicans, and C. glabrata as representative pathogens (at 10(6)-10(9) colony-forming units [CFU]/mL), and the thickness of a plasma-treated gel dressing and distance between a glass dielectric-barrier discharge plasma probe and the gel surface were kept constant. RESULTS: Non-thermal-plasma-treated alginate gel exhibited a strong biocidal property and inactivated all of the pathogens included in the study at counts of 10(8) CFU/mL and within 15 sec of treatment. The treated gel inactivated 10(9) CFU/mL of the organisms within 1 min, and 3 min of exposure to the treated gel inactivated pathogens embedded in biofilms. The plasma-treated gel showed no significant cytotoxicity, and endothelial cells exposed to the treated gel proliferated and migrated well across a wound area over a period of time. Dressings made with the treated gel retained their biocidal effects for about a month. Scanning electron microscopy showed no damage to the surfaces of treated gels, but damage to the bacterial pathogens on plasma exposure. CONCLUSION: A non-thermal-plasma-treated alginate gel dressing has the clinical potential to decontaminate wounds, prevent surgical site infection, and promote wound healing.

Nonthermal dielectric-barrier discharge plasma-induced inactivation involves oxidative DNA damage and membrane lipid peroxidation in Escherichia coli.

Oxidative stress leads to membrane lipid peroxidation, which yields products causing variable degrees of detrimental oxidative modifications in cells. Reactive oxygen species (ROS) are the key regulators in this process and induce lipid peroxidation in Escherichia coli. Application of nonthermal (cold) plasma is increasingly used for inactivation of surface contaminants. Recently, we reported a successful application of nonthermal plasma, using a floating-electrode dielectric-barrier discharge (FE-DBD) technique for rapid inactivation of bacterial contaminants in normal atmospheric air (S. G. Joshi et al., Am. J. Infect. Control 38:293-301, 2010). In the present report, we demonstrate that FE-DBD plasma-mediated inactivation involves membrane lipid peroxidation in E. coli. Dose-dependent ROS, such as singlet oxygen and hydrogen peroxide-like species generated during plasma-induced oxidative stress, were responsible for membrane lipid peroxidation, and ROS scavengers, such as α-tocopherol (vitamin E), were able to significantly inhibit the extent of lipid peroxidation and oxidative DNA damage. These findings indicate that this is a major mechanism involved in FE-DBD plasma-mediated inactivation of bacteria.