The antimicrobial efficacy of remote cold atmospheric plasma effluent against single and mixed bacterial biofilms of varying age

Cold atmospheric plasma (CAP) is a minimal food processing technology of increasing interest in the food industry, as it is mild in nature compared to traditional methods (e.g. pasteurisation) and thus can maintain the food’s desirable qualities. However, due to this mild nature, the potential exists for post-treatment microbial survival and/or stress adaptation. Furthermore, biofilm inactivation by CAP is underexplored and mostly studied on specific foods or on plastic/polymer surfaces. Co-culture effects, biofilm age, and innate biofilm-associated resistance could all impact CAP efficacy, while studies on real foods are limited to the food product investigated without accounting for structural complexity. The effect of a Remote and Enclosed CAP device (Fourth State Medicine Ltd) was investigated on Escherichia coli and Listeria innocua grown as planktonic cells and as single or mixed bacterial biofilms of variable age, on a biphasic viscoelastic food model of controlled rheological and structural complexity. Post-CAP viability was assessed by plate counts, cell sublethal injury was quantified using flow cytometry, and biofilms were characterised and assessed using total protein content and microscopy techniques. A greater impact of CAP on planktonic cells was observed at higher air flow rates, where the ReCAP device operates in a mode more favourable to reactive oxygen species than reactive nitrogen species. Although planktonic E. coli was more susceptible to CAP than planktonic L. innocua, the opposite was observed in biofilm form. The efficacy of CAP was reduced with increasing biofilm age. Furthermore, E. coli produced much higher protein content in both single and mixed biofilms than L. innocua. Consequently, greater survival of L. innocua in mixed biofilms was attributed to a protective effect from E. coli. These results show that biofilm susceptibility to CAP is age and bacteria dependent, and that in mixed biofilms bacteria may become less susceptible to CAP. These findings are of significance to the food industry for the development of effective food decontamination methods using CAP.