Elucidation of Molecular Mechanisms of Foodborne Pathogen Inactivation by Cold Plasma through RNA-Seq Analysis

Introduction: As an emerging technology, cold atmospheric plasma offers a dry, non-thermal, rapid decontamination process with minimal damage to food products, therefore overcoming many of the limitations of current food decontamination methods.

Purpose: Although the bactericidal effects of cold plasma are well documented, the molecular mechanisms and modes of action underlining such effects are not well understood. The purpose of this study was to elucidate the molecular response of Salmonella Enteritidis to cold plasma treatment through transcriptomic analysis by RNA-Seq.

Methods: Culture suspensions of Salmonella Enteritidis were spot inoculated (100 µl in 20-25 spots) onto 10 sterile cover slips (10⁸ cells/cover slip, 10⁶ -10⁷ cells/spot) and treated with cold plasma for 2 min at 1 cm from actuators. Three treated and untreated (control) samples (10 cover slips per sample) were washed by vortexing in sterile peptone and the resulting wash fluid was used for total RNA isolation, cell enumeration, and evaluation of bacterial inactivation. rRNA-depleted libraries of isolated RNA were sequenced by Illumina HiSeq and transcriptomic differences between control and treated samples were evaluated using the web-based Activesite Comparative Expression Viewer (Cofactor Genomics).

Results: Among 375 differentially expressed genes (treated and control) with fold changes greater than 2.00 (P < 0.05), 101 were moderately up-regulated (fold changes between 2.10 to 5.19) after plasma treatment. Approximately 50% of the up-regulated genes were associated with known Salmonella responses to macrophage infection and constituents of the Salmonella pathogenicity island 2 (SPI2), indicating possible oxidative stress responses similar to those experienced in the macrophage environment. A majority of genes down-regulated after plasma treatment were associated with nutrient uptake and desiccation stress, indicating a possible shift in cellular response to plasma treatment from desiccation stress (experienced by the control samples) to oxidative stress (experienced by the treated samples).

Significance: These results confirm the key role of oxidative stress in the inactivation of bacterial pathogens by cold plasma treatment. Cold plasma actuator designs can thus be optimized to maximize reactive oxygen and nitrogen species production for effective food decontamination applications.