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

Abstract

Plasma, a unique state of matter with properties similar to those of ionized gas, is an effective biological disinfectant. However, the mechanism through which nonthermal or "cold" plasma inactivates microbes on surfaces is poorly understood, due in part to challenges associated with processing and analyzing live cells on surfaces rather than in aqueous solution. Here, we employ membrane adsorption techniques to visualize the cellular effects of plasma on representative clinical isolates of drug-resistant microbes. Through direct fluorescent imaging, we demonstrate that plasma rapidly inactivates planktonic cultures, with >5 log(10) kill in 30 s by damaging the cell surface in a time-dependent manner, resulting in a loss of membrane integrity, leakage of intracellular components (nucleic acid, protein, ATP), and ultimately focal dissolution of the cell surface with longer exposure time. This occurred with similar kinetic rates among methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Candida albicans. We observed no correlative evidence that plasma induced widespread genomic damage or oxidative protein modification prior to the onset of membrane damage. Consistent with the notion that plasma is superficial, plasma-mediated sterilization was dramatically reduced when microbial cells were enveloped in aqueous buffer prior to treatment. These results support the use of nonthermal plasmas for disinfecting multidrug-resistant microbes in environmental settings and substantiate ongoing clinical applications for plasma devices.