Multi-species biofilms of environmental microbiota isolated from fruit packing facilities promoted tolerance of to benzalkonium chloride.

may survive and persist in food processing environments due to formation of complex multi-species biofilms of environmental microbiota that co-exists in these environments. This study aimed to determine the effect of selected environmental microbiota on biofilm formation and tolerance of to benzalkonium chloride in formed biofilms. The studied microbiota included bacterial families previously shown to co-occur with in tree fruit packing facilities, including , and Biofilm formation ability and the effect of formed biofilms on the tolerance of to benzalkonium chloride was measured in single- and multi-family assemblages. Biofilms were grown statically on polystyrene pegs submerged in a R2A broth. Biofilm formation was quantified using a crystal violet assay, spread-plating, confocal laser scanning microscopy, and its composition was assessed using amplicon sequencing. The concentration of in biofilms was determined using the most probable number method. Biofilms were exposed to the sanitizer benzalkonium chloride, and the death kinetics of were quantified using a most probable number method. A total of 8, 8, 6, and 3 strains of , and , respectively, were isolated from the environmental microbiota of tree fruit packing facilities and were used in this study. Biofilms formed by , and all multi-family assemblages had significantly higher concentration of bacteria, as well as compared to biofilms formed by alone. Furthermore, multi-family assemblage biofilms increased the tolerance of to benzalkonium chloride compared to mono-species biofilms and planktonic multi-family assemblages. These findings suggest that control strategies should focus not only on assessing the efficacy of sanitizers against but also against biofilm-forming microorganisms that reside in the food processing built environment, such as or .