Dual-Species Model Biofilm Consisting of and Typhimurium: Development and Inactivation With Cold Atmospheric Plasma (CAP).

Most environmental biofilms contain a variety of species. These species can establish cooperative and competitive interactions, possibly resulting in an increase or a decrease in antimicrobial resistance. Therefore, results obtained following inactivation of single-species biofilms by means of different technologies (e.g., Cold Atmospheric Plasma, CAP) should be validated for multi-species biofilms. First, a strongly adherent and mature and . Typhimurium dual-species biofilm was developed by altering different incubation conditions, i.e., growth medium, incubation temperature, inoculum ratio of and . Typhimurium cells, and incubation time. Adherence and maturity were quantified by means of optical density measurements and viable plate counts, respectively. Secondly, both the (1 day old) reference biofilm and a more mature 7 days old biofilm were treated for different CAP treatment times (0-30 min). Viable plate counts were again used to determine the (remaining) cell density. For both the biofilm development and inactivation, predictive models were applied to describe the growth/inactivation kinetics. Finally, the kinetics of the [1 and 7 day(s) old] dual-species biofilms were compared with those obtained for the corresponding single-species biofilms. Results implied that a strongly adherent and mature reference dual-species biofilm was obtained following 24 h of incubation at 25°C using 20-fold diluted TSB and an inoculum ratio of 1:1. Main observations regarding CAP inactivation were: (i) the dual-species biofilm age had no influence on the CAP efficacy, although a longer treatment time was required for the oldest biofilm, (ii) for the 1 day old biofilms, CAP treatment became less efficient for . Typhimurium inactivation when this species was part of the dual-species biofilm, while inactivation was not influenced by the biofilm type, and (iii) for the 7 days old biofilms, CAP inactivation of both species became more efficient when they were part of the dual-species biofilms. It can be concluded that the efficacy of the CAP treatment is altered when cells become part of a dual-species biofilm, which is quite important with respect to a possible application of CAP for biofilm inactivation within the food industry.