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Frontiers IN

Cold Atmospheric Plasma (CAP) is a promising novel method for biofilm inactivation as log-reduction values up to 4.0 log₁₀ (CFU/cm²) have been reported. Nevertheless, as the efficacy of CAP itself is not sufficient for complete inactivation of mature biofilms, the hurdle technology could be applied in order to obtain higher combined efficacies. In this study, CAP treatment was combined with a mild hydrogen peroxide (H₂O₂) treatment for disinfection of 1 and 7 day(s) old Listeria monocytogenes and Salmonella Typhimurium biofilms. Three different treatment sequences were investigated in order to determine the most effective treatment sequence, i.e., (i) first CAP, then H₂O₂, (ii) first H₂O₂, then CAP, and (iii) a simultaneous treatment of CAP and H₂O₂. Removal of the biofilm, induction of sub-lethal injury, and H₂O₂ breakdown due to the presence of catalase within the biofilms were investigated in order to comment on their possible contribution to the combined inactivation efficacy. Results indicated that the preferred treatment sequence was dependent on the biofilm forming species, biofilm age, and applied H₂O₂ concentration [0.05 or 0.20% (v/v)]. At the lowest H₂O₂ concentration, the highest log-reductions were generally observed if the CAP treatment was preceded by the H₂O₂ treatment, while at the highest H₂O₂ concentration, the opposite sequence (first CAP, then H₂O₂) proved to be more effective. Induction of sub-lethal injury contributed to the combined bactericidal effect, while the presence of catalase within the biofilms resulted in an increased resistance. In addition, high log-reductions were partially the result of biofilm removal. The highest overall log-reductions [i.e., up to 5.42 ± 0.33 log₁₀ (CFU/cm²)] were obtained at the highest H₂O₂ concentration if CAP treatment was followed by H₂O₂ treatment. As this resulted in almost complete inactivation of the L. monocytogenes and S. Typhimurium biofilms, the combined treatment of CAP and H₂O₂ proved to be a promising method for disinfection of abiotic surfaces.