# Resistance of \(L\). _monocytogenes_ and \(S\). Typhimurium
###### Abstract  
The biofilm mode of growth protects bacterial cells against currently applied disinfection methods for abiotic (food) contact surfaces. Therefore, innovative methods, such as Cold Atmospheric Plasma (CAP), should be investigated for biofilm inactivation. However, more knowledge is required concerning the influence of the biofilm age on the inactivation efficacy in order to comment on a possible application of CAP in the (food) processing industry. \(L\). _monocytogenes_ and \(S\). Typhimurium biofilms with five different ages (i.e., 1, 2, 3, 7, and 10 days) were developed. For the untreated biofilms, the total biofilm mass and the cell density were determined. To investigate the biofilm resistance towards CAP treatment, biofilms with different ages were treated for 10 min and the remaining cell density was determined. Finally, for the one-day old reference biofilms and the most resistant biofilm age, complete inactivation curves were developed to examine the influence of the biofilm age on the inactivation kinetics. For \(L\). _monocytogenes_, an increased biofilm age resulted in (i) an increased biomass, (ii) a decreased cell density prior to CAP treatment, and (iii) an increased resistance towards CAP treatment. For \(S\). Typhimurium, similar results were obtained, except for the biomass, which was here independent of the biofilm age. Listeria monocytogenes; Salmonella Typhimurium; biofilm age; Cold Atmospheric Plasma; plate counts; crystal violet assay +
Footnote †: journal: Applied Sciences  
_Applied_  
_Acticle_  
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exchangers, and corrosion of surfaces [2; 3; 4; 5]. With respect to contamination of food products, it has become clear that pathogenic bacteria, such as _Listeria monocytogenes_, _Salmonella_ spp., _Cannylobacter jejuni_, and _Yersinia enterocolitic_, grow predominantly as biofilms on (a)biotic surfaces rather than as planktonic cells or colonies [6; 7]. In addition, these pathogenic species were mainly responsible for the foodborne illnesses reported in 2015 within the European Union [8]. Based on their notification and (high) fatality rates, two of these species (i.e., S. Typhimurium and _L. monocytogenes_), were selected as model microorganisms to conduct this research. Salmonella (caused by _S._ (Typhimuriumium)) has a high notification rate (21.2/100,000 capita) and a relatively high fatality rate (0.24%). Listeriosis (mainly caused by _L. monocytogenes_) has a relatively low notification rate (0.46/100,000 capita), but a very high fatality rate (17.7%) [8]. The biofilm mode of living protects the cells from environmental stresses, such as shear forces, nutrient limitations, pH changes, disinfectants, and antibiotics [9; 10]. This increased resistance is the result of different biofilm-specific characteristics, such as the production of Extracellular Polymeric Substances (EPS), the presence of less metabolically active cells, and the accumulation of protective enzymes within the matrix [1; 2; 10; 11]. The EPS matrix has several functions, i.e., it (i) prevents the cells from dehydration and starvation, (ii) keeps the cells/biofilm attached to the surface, and, (iii) limits the diffusion of antimicrobial agents into the three-dimensional structure of the matrix [1; 2; 11]. Biofilm-associated cells can also undergo physiological changes, i.e., a subpopulation of the cells can become metabolically dormant with a decreased growth rate and a decreased consumption of nutrients and oxygen [2]. Since antimicrobial agents (and antibiotics) are far more effective towards actively growing cells, this results in an incomplete inactivation of the biofilm population [2]. Finally, the production of antibiotic degrading enzymes (such as \(\beta\)-lactamase) and the subsequent accumulation within the matrix is also one of the possible defence mechanisms of biofilm-associated cells, although this only concerns treatment of health related (medical) issues [2].