Escherichia coli O157:H7 Biofilm Formation on Romaine Lettuce and Spinach Leaf Surfaces Reduces Efficacy of Irradiation and Sodium Hypochlorite Washes

Abstract: Escherichia coli O157:H7 contamination of leafy green vegetables is an ongoing concern for consumers. Biofilm-associated pathogens are relatively resistant to chemical treatments, but little is known about their response to irradiation. Leaves of Romaine lettuce and baby spinach were dip inoculated with E. coli O157:H7 and stored at 4 °C for various times (0, 24, 48, 72 h) to allow biofilms to form. After each time, leaves were treated with either a 3-min wash with a sodium hypochlorite solution (0, 300, or 600 ppm) or increasing doses of irradiation (0, 0.25, 0.5, 0.75, or 1 kGy). Viable bacteria were recovered and enumerated. Chlorine washes were generally only moderately effective, and resulted in maximal reductions of 1.3 log CFU/g for baby spinach and 1.8 log CFU/g for Romaine. Increasing time in storage prior to chemical treatment had no effect on spinach, and had an inconsistent effect on 600 ppm applied to Romaine. Allowing time for formation of biofilm-like aggregations reduced the efficacy of irradiation. D₁₀ values (the dose required for a 1 log reduction) significantly increased with increasing storage time, up to 48 h postinoculation. From 0 h of storage, D₁₀ increased from 0.19 kGy to a maximum of 0.40 to 0.43 kGy for Romaine and 0.52 to 0.54 kGy for spinach. SEM showed developing biofilms on both types of leaves during storage. Bacterial colonization of the stomata was extensive on spinach, but not on Romaine. These results indicate that the protection of bacteria on the leaf surface by biofilm formation and stomatal colonization can reduce the antimicrobial efficacy of irradiation on leafy green vegetables. Practical Application: Before incorporating irradiation into the overall GMP/GHP chain, a packer or processor of leafy green vegetables must determine at what stage of processing and shipping the irradiation should take place. As a penetrating process, irradiation is best applied as a postpackaging intervention. Time in refrigerated storage between packaging and processing may alter the antimicrobial efficacy of irradiation. Irradiation on a commercial scale should include efforts to minimize the time delay between final packaging and irradiation of leafy vegetables.