Disruption of Shiga-toxigenic Escherichia coli Biofilms In Vitro and on Food Contact Surfaces Using Bacteriophages

Introduction: Shiga toxin-producing Escherichia coli (STEC) are a significant public health concern. Their biofilms on food and food-contact surfaces pose a significant challenge to the food industry. These biofilms can be very difficult to penetrate, making conventional control methods insufficient. Targeted use of bacteriophages that can disrupt these biofilms could reduce this problem in the food industry.

Purpose: This study evaluates the efficacy of bacteriophages against STEC biofilms in vitro and on food contact surfaces.

Methods: Bacteriophages (n=52), isolated from cattle operations, showing lytic activity towards STEC strains were tested for biofilm-inhibiting capabilities in vitro and on food contact surfaces. Phage treatments (eight log PFU/ml) were prepared in phosphate buffered saline (PBS). Biofilm-forming STEC strains (O157, O26, O45, O103, O111, O121, O145), were used to form biofilms in microtiter plates (seven log CFU/ml), stainless steel (SS), or high density polyethylene (HDPE) coupons (nine log₁₀ CFU/ml). Following biofilm formation, plates/coupons were treated with phage treatments or used as PBS-controls. For in vitro evaluation, microtiter plates were incubated six hours at 37°C. Changes in bacterial absorbance (A₅₉₅) were observed at zero, three, and six hours. Coupons were incubated at 37°C for 16 h and STEC survival was determined by plating onto tryptic soy agar. Data was analyzed using one-way ANOVA (P<0.05).

Results: Bacteriophages showed significant reduction (P<0.05) in STEC biofilms compared to the positive control in vitro and on food contact surfaces. A reduction in absorbance (2.262 nm at 0 h to 0.808 nm at 6 h) was observed in phage-treated wells compared to control wells, indicating STEC biofilm disruption. Of 52 phages, 62% showed STEC biofilm disruption in vitro. On SS and HDPE coupons, phage treatments showed 1.8 to 5.8 log CFU/cm² reduction in STEC populations, compared to the control.

Significance: Bacteriophages specific for STECs, with high lytic activity and ability to reduce biofilm formation, could be used as biological control agents in the food industry.