Nonthermal Dielectric-Barrier Discharge Plasma-Induced Inactivation Involves Oxidative DNA Damage and Membrane Lipid Peroxidation in Escherichia coli

Abstract

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 ␣-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.Nonthermal (cold) dielectric-barrier discharge (DBD) atmospheric-pressure plasma is widely under investigation for use as an alternative sterilization and disinfection method in the fields of biology and medicine. Most recently, we demonstrated that Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus in both their planktonic form and in biofilms are rapidly inactivated by nonthermal DBD plasma using a floating-electrode technique (11). Complete inactivation of E. coli was seen in less than 120 s when E. coli was present in its planktonic form, and complete inactivation occurred in about 180 s when it was in the biofilm form, making this technique attractive for sterilization processes. E. coli is one of the most common Gram-negative bacterial contaminants responsible for hospital-acquired infections (HAI) and one of the most widely studied organisms in the laboratory and therefore is a good choice to track various oxidative-stress pathways.A DBD plasma-generating probe is an apparatus that generates microsecond-long, high-voltage-pulsed cold plasma between the primary electrode covered with a quartz surface and the surface of the biological sample, which serves as a second electrode. The high-voltage electrode is completely covered with a dielectric barrier, which makes it safe for sterilization applications, and the nature of the applied microsecond pulses do not elevate the surface temperature above 28°C. In the floating-electrode DBD (FE-DBD) plasma setup, the second electrode (biological sample) is not grounded and remains at a floating potential. Discharge ignites when the powered electrode approaches the surface to be treated at a distance (discharge gap) less than about 3 mm, depending on the form, duration, and polarity of the driving voltage, and it is safe to apply...

Nonthermal Atmospheric Plasma Rapidly Disinfects Multidrug-Resistant Microbes by Inducing Cell Surface Damage

“…Using membrane adsorption techniques to mount and stain cells on a supportive surface for epifluorescence microscopy, we observe that brief exposure to plasma (ϳ7 s) elicits a rapid loss of cell membrane integrity (poration), while longer exposures (ϳ30 s) result in visible lesions on the microbial surface (rupture of cell wall or outer lipid membrane). These results agree with data obtained through more indirect forms of imaging (e.g., scanning electron microscopy, atomic force microscopy, and Gram stain) by groups testing similar plasma devices (2,8,13,15,20,26,28) and are consistent with recent evidence that nonthermal plasma induces membrane lipid peroxidation (9). Analogous superficial effects are reported following the treatment of cultured human and mammalian cells with NTP (25).…”

“…6). Nevertheless, given that cells are permeabilized by plasma over time and eventually release intracellular contents, secondary damage to DNA and protein may occur outside the cell or downstream of cell lysis with prolonged plasma exposures (9). Altogether, these data support our initial microscopy findings and confirm that plasma rapidly inactivates cells by permeabilizing the cell surface, resulting in loss of membrane integrity, leakage of intracellular components (ATP, nucleic acid, protein), and ulti- mately focal dissolution of the cell exterior as a function of treatment time.…”

Mechanisms of Inactivation by High-Voltage Atmospheric Cold Plasma Differ for Escherichia coli and Staphylococcus aureus

“…The amount of reactive species generated, including ozone, has been correlated with inactivation efficacy (12,36,(39)(40)(41). Among the reactive species generated during HVACP treatment, ROS contributed as major antimicrobial factors.…”

“…However, the inactivation efficacy can be varied by changing the working gases, which results in different types or amounts of reactive species generated (9)(10)(11). ROS are often identified as the principal effecting species, with a relatively long half-life and strong antimicrobial effects, which are generated in oxygen-containing gases (12).…”

Atomic oxygen patterning from a biomedical needle-plasma source

“…Initial radical formation breaks C-H, C-O and C-C bonds on the polymer surface such as peptidoglycan in bacteria cell walls 41 , lipid layers in animal cell membranes 42 or plastic materials such as polypropolene 27 . RONS flux loss at a treated surface is primarily dependent on the density of available reaction sites and the reaction rate for each species adsorption.…”