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

Abstract

Atmospheric cold plasma (ACP) is a promising nonthermal technology effective against a wide range of pathogenic microorganisms. Reactive oxygen species (ROS) play a crucial inactivation role when air or other oxygen-containing gases are used. With strong oxidative stress, cells can be damaged by lipid peroxidation, enzyme inactivation, and DNA cleavage. Identification of ROS and an understanding of their role are important for advancing ACP applications for a range of complex microbiological issues. In this study, the inactivation efficacy of in-package high-voltage (80 kV [root mean square]) ACP (HVACP) and the role of intracellular ROS were investigated. Two mechanisms of inactivation were observed in which reactive species were found to either react primarily with the cell envelope or damage intracellular components. Escherichia coli was inactivated mainly by cell leakage and low-level DNA damage. Conversely, Staphylococcus aureus was mainly inactivated by intracellular damage, with significantly higher levels of intracellular ROS observed and little envelope damage. However, for both bacteria studied, increasing treatment time had a positive effect on the intracellular ROS levels generated.

FIG 1

A schematic diagram of the HVACP device. 1, High voltage; 2, Bergoz current; 3, capacitor; 4, high-voltage electrode; 5, ground electrode; 6, oscilloscope; 7, plasma discharge; 8, sealed polypropylene (PP) container; 9, directly exposed; 10, indirectly exposed; 11, emission; 12, fiber optic cable; 13, spectrometer.

FIG 2

Absorbance of HVACP-treated E. coli NCTC 12900 suspension in PBS at 260 nm with different posttreatment storage times. The data points at 0 min of treatment time refer to untreated control stored for 0 h (■), 1 h (◆), and 24 h (▲) in PBS. Treatment times were 1, 3, and 5 min at 80 kV_RMS at the different posttreatment storage times. Solid line, direct exposure; dotted line, indirect exposure.

FIG 3

Absorbance of HVACP-treated S. aureus ATCC 25923 suspension in PBS at 260 nm with different posttreatment storage times. The data points at 0 min of treatment time refer to the untreated control stored for 0 h (■), 1 h (◆), and 24 h (▲) in PBS. Treatment times were 1, 3, and 5 min at 80 kV_RMS at the different posttreatment storage times. Solid line, direct exposure; dotted line, indirect exposure.

FIG 4

Emission spectrum of dielectric barrier discharge atmospheric cold plasma operating in air under atmospheric pressure. (a) Emission spectrum of empty box; (b) emission intensity at 336.65 nm. ■, empty box; ▲, direct exposure; ◆, indirect exposure. A.U., absorbance units.

FIG 5

E. coli NCTC 12900 and S. aureus ATCC 25923 intracellular ROS density assay by DCFH-DA. Treatment times were 1, 3, and 5 min at 80 kV_RMS with 0 h of posttreatment storage. Striped bars, E. coli NCTC 12900; dark shaded bars, S. aureus ATCC 25923. The presence of an asterisk (*) indicates a significant difference at the P ≤ 0.05 level between E. coli and S. aureus. Critical controls were provided as untreated samples with no posttreatment storage. AFU, arbitrary fluorescence units.

FIG 6

E. coli NCTC 12900 and S. aureus ATCC 25923 DNA quantification assay by SYBR green 1. Treatment times were 1, 3, and 5 min at 80 kV_RMS with 24 h of posttreatment storage. Striped bars, E. coli NCTC 12900; dark shaded bars, S. aureus ATCC 25923. The presence of an asterisk (*) indicates a significant difference at the P ≤ 0.05 level between E. coli and S. aureus. Critical controls were provided as untreated samples with no posttreatment storage. AFU, arbitrary fluorescence units.

FIG 7

SEM images of control and treated cells after indirect exposure with plasma at 80 kV_RMS for 1 min following 24 h of posttreatment storage. (a) Untreated S. aureus ATCC 25923; (b) treated S. aureus ATCC 25923; (c) untreated E. coli NCTC 12900; (d) treated E. coli NCTC 12900. Red arrows indicate the significantly different damaging patterns on cell envelopes of E. coli and S. aureus.

FIG 8

Proposed mechanism of action of HVACP with Gram-negative and Gram-positive bacteria. (a to c) Proposed inactivation mechanism of Gram-negative bacteria. (a) Structure of Gram-negative bacteria before treatment, in which the cell envelope consists of a thin layer of peptidoglycan and lipopolysaccharide; (b) ACP-generated ROS attacking both cell envelope and intracellular components, in which the cell envelope is the major target; (c) inactivation mainly caused by cell leakage, with some DNA damage possible. (d to f) Proposed inactivation mechanism of Gram-positive bacteria. (d) Structure of Gram-positive bacteria before treatment, in which the cell envelope consists of a thick rigid layer of peptidoglycan; (e) ACP-generated ROS attacking both cell envelope and intracellular components, in which intracellular materials are the major targets; (f) inactivation mainly caused by intracellular damage (e.g., DNA breakage) but not leakage.