Determination of the combined effect of grape seed extract and cold atmospheric plasma on foodborne pathogens and their environmental stress knockout mutants

The study aimed to explore the antimicrobial efficacy of grape seed extract (GSE) and cold atmospheric plasma (CAP) individually or in combination against <i>L. monocytogenes</i> and <i>E. coli</i> wild type (WT) and their isogenic mutants in environmental stress genes. More specifically, we examined the effects of 1% (wt/vol) GSE, 4 min of CAP treatment, and their combined effect on <i>L. monocytogenes</i> 10403S WT and its isogenic mutants Δ<i>sigB</i>, Δ<i>gadD1</i>, Δ<i>gadD2</i>, Δ<i>gadD3,</i> as well as <i>E. coli</i> K12 and its isogenic mutants Δ<i>rpoS</i>, Δ<i>oxyR</i>, and Δ<i>dnaK</i>. In addition, the sequence of the combined treatments was tested. A synergistic effect was achieved for all <i>L. monocytogenes</i> strains when exposure to GSE was followed by CAP treatment. However, the same effect was observed against <i>E. coli</i> strains, only for the reversed treatment sequence. Additionally, <i>L. monocytogenes</i> Δ<i>sigB</i> was more sensitive to the individual GSE and the combined GSE/CAP treatment, whereas Δ<i>gadD2</i> was more sensitive to CAP<i>,</i> as compared to the rest of the mutants under study. Individual GSE exposure was unable to inhibit <i>E. coli</i> strains, and individual CAP treatment resulted in higher inactivation of <i>E. coli</i> in comparison to <i>L. monocytogenes</i> with the strain Δ<i>rpoS</i> appearing the most sensitive among all studied strains. Our findings provide a step toward a better understanding of the mechanisms playing a role in the tolerance/sensitivity of our model Gram-positive and Gram-negative bacteria toward GSE, CAP, and their combination. Therefore, our results contribute to the development of more effective and targeted antimicrobial strategies for sustainable decontamination.IMPORTANCEAlternative approaches to conventional sterilization are gaining interest from the food industry, driven by (i) the consumer demand for minimally processed products and (ii) the need for sustainable, environmentally friendly processing interventions. However, as such alternative approaches are milder than conventional heat sterilization, bacterial pathogens might not be entirely killed by them, which means that they could survive and grow, causing food contamination and health hazards. In this manuscript, we performed a systematic study of the impact of antimicrobials derived from fruit industry waste (grape seed extract) and cold atmospheric plasma on the inactivation/killing as well as the damage of bacterial pathogens and their genetically modified counterparts, for genes linked to the response to environmental stress. Our work provides insights into genes that could be responsible for the bacterial capability to resist/survive those novel treatments, therefore, contributing to the development of more effective and targeted antimicrobial strategies for sustainable decontamination.