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. 2017 Oct 11;2(2):121-128.
doi: 10.1109/TRPMS.2017.2761405. eCollection 2018 Mar.

Nontarget Biomolecules Alter Macromolecular Changes Induced by Bactericidal Low-Temperature Plasma

A Privat-Maldonado  1   2   3 , Y Gorbanev  2   4 , D O'Connell  2 , R Vann  2 , V Chechik  4 , M W van der Woude  5   6
Affiliations

Nontarget Biomolecules Alter Macromolecular Changes Induced by Bactericidal Low-Temperature Plasma

A Privat-Maldonado et al. IEEE Trans Radiat Plasma Med Sci. .

Abstract

Low-temperature plasmas (LTPs) have a proven bactericidal activity governed by the generated reactive oxygen and nitrogen species (RONS) that target microbial cell components. However, RONS also interact with biomolecules in the environment. Here we assess the impact of these interactions upon exposure of liquid suspensions with variable organic content to an atmospheric-pressure dielectric barrier discharge plasma jet. Salmonella enterica serovar Typhimurium viability in the suspension was reduced in the absence [e.g., phosphate buffered saline (PBS)], but not in the presence of (high) organic content [Dulbecco's Modified Eagle's Medium (DMEM), DMEM supplemented with foetal calf serum, and Lysogeny Broth]. The reduced viability of LTP-treated bacteria in PBS correlated to a loss of membrane integrity, whereas double-strand DNA breaks could not be detected in treated single cells. The lack of bactericidal activity in solutions with high organic content correlated with a relative decrease of •OH and O3/O2(a1[Formula: see text])/O, and an increase of H2O2 and [Formula: see text] in the plasma-treated solutions. These results indicate that the redox reactions of LTP-generated RONS with nontarget biomolecules resulted in a RONS composition with reduced bactericidal activity. Therefore, the chemical composition of the bacterial environment should be considered in the development of LTP for antimicrobial treatment, and may affect other biomedical applications as well.

Keywords: Bacterial inactivation; low-temperature plasma (LTP); plasma-treated media; reactive oxygen and nitrogen species (RONS).

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Figures

Fig. 1.
Fig. 1.
Schematic of the experimental setup.
Fig. 2.
Fig. 2.
Effect of external organic content on bacterial viability. CFU Log10 reduction of S. Typhimurium in the five solutions exposed to plasma. Data presented as mean ± S.D. ****: formula image.
Fig. 3.
Fig. 3.
Representative PI/SYTO9 dot plots of untreated and LTP-treated S. Typhimurium in the five liquids tested. The gating areas represent dead, intermediate (int.), and live populations.
Fig. 4.
Fig. 4.
Cell sorting of live/dead stained S. Typhimurium suspensions in PBS. (a) Cell sorting identified four populations: 1) livelow; 2) livehigh; 3) intermediate; and 4) dead. (b) Viability of 500 000 events from each population of S. Typhimurium in PBS. Data presented as mean ± S.D. *: formula image.
Fig. 5.
Fig. 5.
dsDNA breaks in LTP-treated S. Typhimurium suspensions. S. Typhimurium suspended in PBS, DMEM, and LB were treated with the AP-DBD plasma jet for 90 s and analyzed with the DDD assay. (a) Fold change in bacterial DNA diffusion in plasma-treated bacteria relative to the mean of untreated bacteria. Each dot represents a single cell; horizontal bars: mean values ± S.D.; ****: formula image. (b) Representative single cells showing the level of DNA fragmentation observed in plasma-treated S. Typhimurium in DMEM suspension. Scale bar 10 mm.
Fig. 6.
Fig. 6.
Detection of RNOS in plasma-treated liquids. (a) Colorimetric assay for the detection of H2O2 by reaction with titanium(IV). Spin-trapped radical adducts of (b) DMPO-OH adduct, (c) TEMPO without added NaN3, and (d) TEMPO with added NaN3. Horizontal bars: mean ± S.D. (e) Azo dye formed by NO2−, assessed by Griess assay. Single measurements for 30- and 60-s treatments and mean values for 90-s treatment (formula image). Length of treatment expressed in seconds.
See this image and copyright information in PMC

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