# Resistance of \(L\). _monocytogenes_ and \(S\). Typhimurium
## 3 Results and Discussion
### Influence of the Biofilm Age on the CAP Efficacy Following 10 min of Treatment  
In this section, the log-reductions obtained following 10 min of CAP treatment of the different biofilm ages (i.e., 1, 2, 3, 5, and 7 day(s) old) will be compared for both pathogenic species. The log-reductions have been calculated based on the cell densities of the untreated biofilms and the remaining cell densities following 10 min of CAP treatment. Results have been presented in Figure 4a and Figure 4b for \(L\). _monocytogenes_ and \(S\). Typhimurium, respectively. To the best of the authors' knowledge, only [PERSON] et al. [16] studied the effect of the biofilm age on the CAP inactivation efficacy for (\(S\). Typhimurium, \(L\). _monocytogenes_, and _Escherichia coli_) biofilms before. In that research, it could be concluded that biofilms become more resistant towards CAP treatment as the biofilm ages. Nevertheless, in contrary to the currently presented research, these biofilms were grown on real food products (small pieces of lettuce) and not on abiotic (food) contact surfaces. In addition, only two biofilm ages were compared, i.e., 24 and 48 h old biofilms, which are still two relatively young biofilm ages. Due to the novelty of the presented research, observations within the following paragraphs will be explained based on general biofilm characteristics and/or biofilm inactivation studies with (conventional) antimicrobial agents, although the inactivation mechanism differs. Figure 4: Log-reductions (log(CFU/cm\({}^{2}\))) obtained on general and selective medium for (**a**) \(L\). _monocytogenes_ and (**b**) \(S\). Typhimurium biofilms treated for 10 min with Cold Atmospheric Plasma (CAP) at optimal inactivation conditions (i.e., DBD, 0.0 (\(v\)/\(v\)) % oxygen, 21.88 V input voltage). Bars bearing different letters (no capital or small letters in common for the general and the selective medium, respectively) are significantly different (\(p\leq 0.05\)). Based on the results that are presented in Figure 4a, it can be concluded that significantly lower log-reductions are obtained for the seven days old _L. monocytogenes_ biofilms, and this for both the general and the selective medium. This means that the CAP inactivation efficacy is significantly lower for the seven days old biofilms or, in other words, that the _L. monocytogenes_ biofilm-associated cells become more resistant towards CAP inactivation as the biofilm ages. For the 10 days old biofilms, however, the log-reductions are again similar to those obtained for the 1, 2, and 3 day(s) biofilms, meaning that the resistance towards CAP treatment decreases again. For the _S._ Typhimurium biofilms (Figure 4b), similar observations can be made, i.e., following CAP treatment of the 1, 2, and 3 day(s) old biofilms, the obtained log-reductions do not significantly decrease (compared to the one-day old reference biofilms), meaning that the biofilm resistance does not increase up until this biofilm age. However, following 2 days of incubation, it should be mentioned that the obtained log-reductions are significantly higher than the ones obtained for the 3 days old biofilms. As the _S._ Typhimurium biofilm ages even more, i.e., following 7 days of incubation, the log-reductions following 10 min of CAP treatment decrease compared to the one-day old (reference) biofilms, although only significantly for the general medium. This means that the 7 days old biofilms become more resistant towards CAP treatment. Nevertheless, as for _L. monocytogenes_, the CAP resistance decreases again for the 10 days old biofilms, i.e., log-reductions obtained for the latter biofilm age are similar to the ones obtained for the one-day old (reference) biofilms. The higher resistance of the seven days old _L. monocytogenes_ biofilms can be explained based on the difference in biomass between this biofilm age and the one-day old (reference biofilms), i.e., the OD of the 7 days old biofilms is much higher than the one of the reference biofilms due to a higher amount of produced EPS (Section 3.1).