# Resistance of \(L\). _monocytogenes_ and \(S\). Typhimurium
###### Abstract
Due to all of these characteristics, biofilm-associated cells are already very resistant towards the inactivation methods that are currently applied in an industrial (food processing) environment. These methods involve the use of (hot) water and antimicrobial agents, in combination with a mechanical action, such as the use of brushes [6]. In addition, many studies proved that the resistance of biofilms towards these methods further increases as the biofilm ages [12; 13; 14; 15]. This phenomenon can be explained by the fact that a mature biofilm is denser and more complex when compared to a younger one, limiting the penetration of antimicrobial agents [15]. Also, microorganisms in a biofilm can be less susceptible to antimicrobial agents due to their altered physiological state, which might be more prominent as the biofilm ages [14]. To ensure food safety, new biofilm inactivation methods should be investigated and implemented in the existing cleaning processes. One of the promising technologies for biofilm inactivation is the use of Cold Atmospheric Plasma (CAP), since log-reductions up to 4-log CFU/sample have been obtained for _L. monocytogenes_ and _S._ Typhimurium biofilms [16; 17]. Plasma can be defined as a (partially) ionized gas, consisting of ions (positive and negative), photons, free electrons, and activated neutral species (excited and radical) [18; 19]. CAP is a specific type of plasma, which can be created by using an electric discharge in a gas, at room temperature and at atmospheric pressure. The use of CAP as inactivation technology for (food) contact surfaces has many advantages when compared to the currently applied methods, i.e., (i) it is fast, (ii) it can be performed at a low temperature, (iii) the plasma components fade out immediately after treatment, (iv) it is environmentally friendly, (v) it has a low running cost, and (vi) cells can be inactivated by multiple inactivation mechanisms [18; 19; 20; 21]. The latter has an additional advantage since it is thought that the broad range of active agents, mechanisms of action, and target sites, would decrease the likelihood that (biofilm-associated) cells are resistant towards this treatment [21]. However, despite the promising biofilm inactivation efficacy of CAP reported in literature, further investigation is still required to examine whether the efficacy would decrease with an increased biofilm age. If biofilms grown on (food) contact surfaces are not (completely) inactivated by means of CAP or other (traditional) inactivation methods, they might become more mature and more resistant towards CAP treatment. Consequently, the chance of (cross) contamination of food products would increase, implying higher food safety risks. For this reason, it is necessary to investigate the influence of the biofilm age on the CAP inactivation efficacy. In addition, the induction of sub-lethally injured cells following CAP treatment of biofilms with different ages should be investigated as well. Sub-lethal injury of (biofilm-associated) cells can be the result of an inefficient treatment, which was only able to damage the cells without killing them. This phenomenon has been reported with other (traditional) inactivation methods (such as a mild heat treatment) and it is important to take into account since sub-lethally injured cells might recover if they re-encounter optimal growth conditions [22; 23; 24]. Therefore, the goal of this research was to examine the influence of the biofilm age on (i) the total biofilm mass of the untreated biofilms, (ii) the cell density of the untreated biofilms, (iii) the CAP efficacy for biofilm inactivation, and (iv) the CAP inactivation kinetics. _L. monocytogenes_ (Gram-positive) and \(S\). Typhimurium (Gram-negative) biofilms with different ages (i.e., 1, 2, 3, 7, and 10 day(s)) were developed and treated with a previously determined combination of optimal plasma characteristics [17].