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Listeria monocytogenes  is an opportunistic intracellular pathogen commonly associated with serious infections and multiple food-borne outbreaks. In this study, we investigated the influence of atmospheric cold plasma (80 kV, 50 Hz) on  L. monocytogenes  (EGD-e) and its knockout mutants of  sigB ,  rsbR ,  prfA ,  gadD , and  lmo0799  genes at different treatment time intervals. Further, to ascertain if sub-lethal environmental stress conditions could influence  L. monocytogenes  survival and growth responses, atmospheric cold plasma (ACP) resistance was evaluated for the cultures exposed to cold (4°C) or acid (pH 4) stress for 1 h. The results demonstrate that both wild-type and knockout mutants were similarly affected after 1 min exposure to ACP ( p  > 0.05), with a difference in response noted only after 3 min of treatment. While all  L. monocytogenes  strains exposed to acid/cold stress were hypersensitive to ACP treatment and were significantly reduced or inactivated within 1 min of treatment ( p  < 0.05). The results indicate  sigB  and  prfA  are important for general stress resistance and biofilm, respectively, loss of these two genes significantly reduced bacterial resistance to ACP treatment. In addition, exposure to sub-lethal 1min ACP increased the gene expression of stress associated genes.  SigB  showed the highest gene expression, increasing by 15.60 fold, followed by  gadD2  (7.19) and  lmo0799  (8.6) after 1 min exposure. Overall, an increase in gene expression was seen in all stress associated genes analyzed both at 1 min treatment; while long treatment time reduced the gene expression and some cases down-regulated  prfA  and  gadD3  gene expression. By comparing the response of mutants under ACP exposure to key processing parameters, the experimental results presented here provide a baseline for understanding the bacterial genetic response and resistance to cold plasma stress and offers promising insights for optimizing ACP applications.

The Effect of Atmospheric Cold Plasma on Bacterial Stress Responses and Virulence Using Listeria monocytogenes Knockout Mutants