# Resistance of \(L\). _monocytogenes_ and \(S\). Typhimurium
## 3 Results and Discussion
### Influence of the Biofilm Age on the Inactivation Kinetics
[27] model for the inactivation curves of one and seven day(s) old _L. monocytogenes_ and _S. Typhimurium biofilms. Results for the one-day old biofilms have been presented before in the research of [PERSON] et al. [17]. declines again until a residual percentage of approximately 75% is obtained. Important to mention is that the maximum percentage of sub-lethal injury coincides with the end of the log-linear inactivation phase, suggesting a mechanism of injury accumulation eventually resulting in cell lysis [23; 41]. For the seven days old biofilms, the percentage of sub-lethal injury remains constant (at 0%) until 8 min of CAP treatment, meaning that no sub-lethal injury is induced during the beginning of the treatment. Upon further treatment, the percentage increases until a maximum of approximately 10% is reached following 15 min of CAP treatment. Next, the percentage of sub-lethal injury decreases again until it reaches 0%. For this biofilm age, the maximum percentage of sub-lethal injury does not coincide with the end of the log-linear inactivation phase. However, since the maximum percentage of sub-lethal injury is relatively low, it is more likely that the peak in sub-lethal injury would be the result of experimental variability and not due to the CAP treatment. As for _L. monocytogenes_, several observations can be drawn for the influence of the biofilm age on the \(S\). Typhimurium inactivation kinetics (Figure 5b and Table 1). First of all, differences can be observed between the initial cell density (\(N_{0}\)) of the one and seven day(s) old biofilms, i.e., the cell density decreases as the biofilm ages, although not significantly. This is in contradiction with the results that were obtained in Section 3.1, however, this might be the result of experimental/biological variability or due to the model fit. When the percentages of sub-lethally injured cells prior to treatment are compared, it can be observed that this percentage slightly increases with an increased biofilm age, which might be the result of nutrient limitations and/or waste accumulation [38]. Nevertheless, this small difference could also be the result of experimental variability. When the shapes of the inactivation curves are compared, it can be observed that for both the one and seven day(s) old biofilms, a log-linear inactivation phase is followed by a tail. Thus, for both biofilm ages, a prolonged treatment would not result in higher log-reductions, i.e., a more resistant sub-population is present within the biofilms. In addition, no significant differences can be observed for the inactivation rates (\(k_{max}\)) obtained for both biofilm ages. This was to be expected, since no significant differences between the OD values of the one and seven days old biofilms were observed in Section 3.1 (Figure 2b). Nevertheless, the duration of the log-linear inactivation phase is shorter for the seven days old biofilms, i.e., for this biofilm age, the tail phase already starts following approximately 3 min of CAP treatment. For the one-day old biofilms on the other hand, CAP treatment up to 7.5 min is required to reach this tail phase. The combined effect of (i) no significant differences between the \(k_{max}\) values obtained for both biofilm ages and (ii) the shorter duration of the log-linear inactivation phase for the seven days old \(S\). Typhimurium biofilms results in an increased resistance of the seven days old \(S\). Typhimurium biofilms following 10 min of CAP treatment (Section 3.2). As mentioned before, this increased resistance might be the result of a limited penetration of the CAP species, i.e., they cannot reach the lower layers of the biofilm and/or an altered growth rate of the biofilm-associated cells [40; 15]. Following 30 min of CAP treatment, the residual \(S\). Typhimurium population (\(N_{res}\)) of the seven days old biofilms is similar to the one for the one-day old biofilms, and this for both the general and the selective medium. However, the average values for the one-day old \(S\). Typhimurium biofilms are (slightly) higher.