Enzymatic Inactivation of Pathogenic and Nonpathogenic Bacteria in Biofilms in Combination with Chlorine

Abstract

ABSTRACT 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.