Cold plasma inactivation of internalised bacteria and biofilms for Salmonella enterica serovar Typhimurium, Listeria monocytogenes and Escherichia coli

Microbial biofilms and bacteria internalised in produce tissue may reduce the effectiveness of decontamination methods. In this study, the inactivation efficacy of in-package atmospheric cold plasma (ACP) afterglow was investigated against Salmonella Typhimurium, Listeria monocytogenes and Escherichia coli in the forms of planktonic cultures, biofilms formed on lettuce and associated bacteria internalised in lettuce tissue. Prepared lettuce broth (3%) was inoculated with bacteria resulting in a final concentration of ~7.0log₁₀CFU/ml. For biofilm formation and internalisation, lettuce pieces (5×5cm) were dip-inoculated in bacterial suspension of ~7.0log₁₀CFU/ml for 2h and further incubated for 0, 24 and 48h at either 4°C or room temperature (~22°C) in combination with light/dark photoperiod or at 4°C under dark conditions. Inoculated samples were sealed inside a rigid polypropylene container and indirectly exposed (i.e. placed outside plasma discharge) to a high voltage (80kV_RMS) air ACP with subsequent storage for 24h at 4°C. ACP treatment for 30s reduced planktonic populations of Salmonella, L. monocytogenes and E. coli suspended in lettuce broth to undetectable levels. Depending on storage conditions, bacterial type and age of biofilm, 300s of treatment resulted in reduction of biofilm populations on lettuce by a maximum of 5log₁₀CFU/sample. Scanning electron and confocal laser microscopy pointed to the incidence of bacterial internalisation and biofilm formation, which influenced the inactivation efficacy of ACP. Measured intracellular reactive oxygen species (ROS) revealed that the presence of organic matter in the bacterial suspension might present a protective effect against the action of ROS on bacterial cells. This study demonstrated that high voltage in-package ACP could be a potential technology to overcome bacterial challenges associated with food produce. However, the existence of biofilms and internalised bacteria should be considered for further optimisation of ACP treatment parameters in order to achieve an effective control of the realistic challenges posed by foodborne pathogens.