Establishment of incubation conditions to optimize the in vitro formation of mature Listeria monocytogenes biofilms on food-contact surfaces

Most microbial populations in food industry environments grow as adhered communities with subsequent biofilm formation on different food-contact surfaces, causing possible cross-contaminations to food products and posing a relevant risk to public health. The aim of this study was to develop a laboratory scale model to optimally form Listeria monocytogenes biofilms in their mature stage on stainless steel surfaces. The results showed that the best initial incubation period is 48 h when compared with 24 h and 72 h. The maximum growth for biofilm formation in the in vitro conditions tested was obtained after a week of incubation with different series of washing and nutrient renewal, with a total cell amount of 7.25 log CFU cm−2 and a cell survival rate of 94.47%. Significant differences were obtained (P < 0.05) when 48 h as the total incubation period was compared with 48 h + 24 h, 48 h + 48 h and 48 h + 24 h + 24 h. These conditions were also compared with each other and no statistical differences were found (P = 0.5392, P = 0.5542, P = 0.9965, respectively). When the same conditions were compared with the one-week incubation, significant differences were obtained (P < 0.001) in all the cases except for the condition of 48 h + 24 h + 24 h + 72 h + 24 h (P = 0.1654), but they presented a lower count and cell survival percentage. The incubation period, the series of washes and the renewal of nutrients directly influences the extracellular matrix production, demonstrating that a one-week incubation period is the most suitable condition for producing the proteins and carbohydrates that give the biofilm system consistency and robustness. The effect of the conditioned protein layer demonstrated an 8-13% greater biofilm formation of the strains CECT 5366, 5672 and 5873 on the polystyrene surfaces, but no significant differences were found in microbial counts (P > 0.05). The proposed in vitro model to form biofilms in their mature stage is completely crucial to understanding how to reproduce and eliminate them not only in vitro, but also in real conditions.