Escherichia coli O157:H7 is one of the most important pathogens worldwide. In this study, three different kinds of enzymes, DNase I, proteinase K and cellulase were evaluated for inhibitory or degrading activity against E. coli O157:H7 biofilm by targeting extracellular DNA, proteins, and cellulose, respectively. The cell number of biofilms formed under proteinase K resulted in a 2.43 log CFU/cm 2 reduction with an additional synergistic 3.72 log CFU/cm 2 reduction after NaClO post-treatment, while no significant reduction occurred with NaClO treatment alone. It suggests that protein degradation could be a good way to control the biofilm effectively. In preformed biofilms, all enzymes showed a significant reduction of 16.4–36.7% in biofilm matrix in 10-fold diluted media ( p < 0.05). The sequential treatment with proteinase K, cellulase, and NaClO showed a significantly higher synergistic inactivation of 2.83 log CFU/cm 2 compared to 1.58 log CFU/cm 2 in the sequence of cellulase, proteinase K, and NaClO ( p < 0.05). It suggests that the sequence of multiple enzymes can make a significant difference in the susceptibility of biofilms to NaClO. This study indicates that the combination of extracellular polymeric substance-degrading enzymes with NaClO could be useful for the efficient control of E. coli O157:H7 biofilms.

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This study investigated the effects of enzyme application on biofilms of bacterial isolates from a cafeteria kitchen and foodborne pathogens and the susceptibility of Salmonella biofilms to proteinase K combined with chlorine treatment. For four isolates from a cafeteria kitchen (Acinetobacter, Enterobacter, and Kocuria) and six strains of foodborne pathogens (Salmonella enterica, Staphylococcus aureus, and Vibrio parahaemolyticus), the inhibitory effect of enzymes on biofilm formation at 25°C for 24 h or the degradative efficacy of enzymes on 24-h mature biofilm at 37°C for 1 h in tryptic soy broth (TSB) was examined in a polystyrene microtiter plate. The effect of enzymes was also evaluated on a subset of these strains in 20 times diluted TSB (1/20 TSB) at 25°C. The working concentrations of five enzymes were 1 U/100 μL for α-amylase, amyloglucosidase, cellulase, and DNase and 1 milli-Anson unit/100 μL for proteinase K. In addition, 24-h mature Salmonella Typhimurium biofilm on a stainless steel coupon was treated with proteinase K for 1 h at 25°C followed by 20 ppm of chlorine for 1 min at 25°C. The results showed that certain enzymes inhibited biofilm formation by the kitchen-originated bacteria; however, the enzymatic effect was diminished on the mature biofilms. Biofilm formation of V. parahaemolyticus was suppressed by all tested enzymes, whereas the mature biofilm was degraded by α-amylase, DNase I, and proteinase K. Proteinase K was effective in controlling Salmonella biofilms, whereas a strain-dependent variation was observed in S. aureus biofilms. In 1/20 TSB, Enterobacter cancerogenus and Kocuria varians were more susceptible to certain enzymes during biofilm formation than those in TSB, whereas the enzymatic effect was much decreased on 24-h mature biofilms, regardless of nutrient conditions. Furthermore, synergistic inactivation of Salmonella Typhimurium in biofilms was observed in the combined treatment of proteinase K followed by chlorine. Live/Dead assays also revealed a decrease in density and loss of membrane integrity in Salmonella Typhimurium biofilms exposed to the combined treatment. Therefore, certain enzymes can control biofilms of isolates residing in a cafeteria kitchen and foodborne pathogens. This study demonstrates the potential of enzymes for the sanitation of food processing environments and of proteinase K combined with chlorine to control Salmonella biofilms on food contact surfaces.

The complex roles of cell surface modification in the biofilm formation of Campylobacter jejuni, a major cause of worldwide foodborne diarrheal disease, are poorly understood. In a screen of mutants from random transposon mutagenesis, an insertional mutation in the eptC gene (cj0256) resulted in a significant decrease in C. jejuni NCTC11168 biofilm formation (<20%) on major food contact surfaces, such as polystyrene and borosilicate glass, when compared with wild-type cells (p < 0.05). In C. jejuni strain 81-176, the protein encoded by eptC modified cell surface structures, such as lipid A, the inner core of lipooligosaccharide, and the flagellar rod protein (FlgG), by attaching phosphoethanolamine. To assess the role of eptC in C. jejuni NCTC11168, adherence and motility tests were performed. In adhesion assays with glass surfaces, the eptC mutant exhibited a 0.77 log CFU/cm 2 decrease in adherence compared with wild-type cells during the initial 2 h of the assay (p < 0.05). These results support the hypothesis that the modification of cell surface structures by eptC affects the initial adherence in biofilm formation of C. jejuni NCTC11168. In motility tests, the eptC mutant demonstrated reduced motility when compared with wild-type cells, but wild-type cells with the transposon inserted in a gene irrelevant to biofilm formation (cj1111c) also exhibited decreased motility to a similar extent as the eptC mutant. This suggests that although eptC affects motility, it does not significantly affect biofilm formation. This study demonstrates that eptC is essential for initial adherence, and plays a significant role in the biofilm formation of C. jejuni NCTC11168.

Bacterial contamination of food-contact surfaces can be a potential risk factor for food quality and safety. To evaluate the spatial and temporal variations of the potential cross-contamination routes, we conducted a biogeographical assessment of bacteria in a foodservice facility based on the diversity of microflora on each surface. To this end, we performed high-throughput amplicon sequencing of 13 food-contact and non-food contact surfaces in a foodservice facility throughout a year. The results showed that Bacillus, Acinetobacter, Streptophyta, Enterobacter, Pseudomonas, Serratia, Enhydrobacter, Staphylococcus, Paracoccus, and Lysinibacillus were the dominant genera found on the kitchen surfaces of the foodservice facility. Depending on the season, changes in Firmicute/Proteobacteria ratios were observed, and the fan becomes the main source of outdoor air contamination. The microbial flow associated with spoilage was also observed throughout food preparation. Taken together, our results would be a powerful reference to hygiene managers for improvement of food processes.