The Effect of Atmospheric Cold Plasma on Bacterial Stress Responses and Virulence Using \u3ci\u3e Listeria monocytogenes\u3c/i\u3e Knockout Mutants

Listeria monocytogenes is an opportunistic intracellular pathogen commonly associated with serious 16 infections and multiple food-borne outbreaks. In this study, we investigated the influence of 17 atmospheric cold plasma (80 kV, 50 Hz) on L. monocytogenes (EGD-e) and its knockout mutants of 18 sigB, rsbR, prfA, gadD and lmo0799 genes at different treatment time intervals. Further, to ascertain if 19 sub-lethal environmental stress conditions could influence L. monocytogenes survival and growth 20 responses, atmospheric cold plasma (ACP) resistance was evaluated for the cultures exposed to cold 21 (4°C) or acid (pH 4) stress for 1 h. The results demonstrate that both wild-type and knockout mutants 22 were similarly affected after 1 min exposure to ACP (p \u3e 0.05), with a difference in response noted 23 only after 3 min of treatment. While all L. monocytogenes strains exposed to acid/cold stress were 24 hypersensitive to ACP treatment and were significantly reduced or inactivated within 1 min of 25 treatment (p \u3c 0.05). The results indicate sigB and prfA are important for general stress resistance and 26 biofilm respectively, loss of these two genes significantly reduced bacterial resistance to ACP 27 treatment. In addition, exposure to sub-lethal 1min ACP increased the gene expression of stress 28 associated genes. SigB showed the highest gene expression, increasing by 15.60 fold, followed by 29 gadD2 (7.19) and lmo0799 (8.6) after 1 min exposure. Overall, an increase in gene expression was 30 seen in all stress associated genes analyzed both at 1 min treatment; while long treatment time reduced 31 the gene expression and some cases down-regulated prfA and gadD3 gene expression. By comparing 32 the response of mutants under ACP exposure to key processing parameters, the experimental results 33 presented here provide a baseline for understanding the bacterial genetic response and resistance to 34 cold plasma stress and offers promising insights for optimizing ACP applications