Kinetics of biofilm formation by pathogenic and spoilage microorganisms under conditions that mimic the poultry, meat, and egg processing industries

Abstract

Pathogens and spoilage microorganisms can develop multispecies biofilms on food contact surfaces; however, few studies have been focused on evaluated mixed biofilms of these microorganisms. Therefore this study investigated the biofilm development by pathogenic (Bacillus cereus, Escherichia coli, Listeria monocytogenes, and Salmonella enterica Enteritidis and Typhimurium serotypes) and spoilage (Bacillus cereus and Pseudomonas aeruginosa) microorganisms onto stainless-steel (SS) and polypropylene B (PP) coupons; under conditions that mimic the dairy, meat, and egg processing industry. Biofilms were developed in TSB with 10% chicken egg yolk (TSB + EY), TSB with 10% meat extract (TSB + ME) and whole milk (WM) onto SS and PP. Each tube was inoculated with 25 μL of each bacteria and then incubated at 9 or 25 °C, with enumeration at 1, 48, 120, 180 and 240 h. Biofilms were visualized by epifluorescence and scanning electron microscopy (SEM). Biofilm development occurred at different phases, depending on the incubation conditions. In the reversible adhesion, the cell density of each bacteria was between 1.43 and 6.08 Log₁₀ CFU/cm² (p < 0.05). Moreover, significant reductions in bacteria appeared at 9 °C between 1 and 48 h of incubation. Additionally, the constant multiplication of bacteria in the biofilm occurred at 25 °C between 48 and 180 h of incubation, with increments of 2.08 Log₁₀ CFU/cm² to S. Typhimurium. Population establishment was observed between 48 and 180 h and 180-240 h incubation, depending on the environmental conditions (25 and 9 °C, respectively). For example, in TSB + ME at 25 °C, S. Typhimurium, P aeruginosa, and L. monocytogenes showed no statistical differences in the amounts between 48 and 180 h incubation. The dispersion phase was identified for L. monocytogenes and B. cereus at 25 °C. Epifluorescence microscopy and SEM allowed visualizing the bacteria and extracellular polymeric substances at the different biofilm stages. In conclusion, pathogens and spoilage microorganisms developed monospecies with higher cellular densities than multiespecies biofilms. In multispecies biofilms, the time to reach each biofilm phase varied is depending on environmental factors. Cell count decrements of 1.12-2.44 Log₁₀ CFU/cm² occurred at 48 and 240 h and were most notable in the biofilms developed at 9 °C. Additionally, cell density reached by each microorganism was different, P. aeruginosa and Salmonella were the dominant microorganisms in the biofilms while B. cereus showed the lower densities until undetectable levels.

Keywords: Food contact surfaces; Food industry; Multispecies biofilms.