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United States Department of Agriculture

Agricultural Research Service

Research Project: INTERVENTION TECHNOLOGIES FOR ENHANCING THE SAFETY AND SECURITY OF FRESH AND MINIMALLY PROCESSED PRODUCE AND SOLID PLANT-DERIVED FOODS

Location: Food Safety and Intervention Technologies

Title: Escherichia coli O157:H7 biofilm formation and internalization on lettuce and spinach leaf surfaces reduces efficacy of irradiation and sodium hypochlorite washes

Authors
item Niemira, Brendan
item Cooke, Peter

Submitted to: Journal of Food Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 19, 2010
Publication Date: May 8, 2010
Citation: Niemira, B.A., Cooke, P.H. 2010. Escherichia coli O157:H7 biofilm formation and internalization on lettuce and spinach leaf surfaces reduces efficacy of irradiation and sodium hypochlorite washes. Journal of Food Science. 75(5):M270-M277.

Interpretive Summary: Escherichia coli O157:H7 contamination of leafy green vegetables is an ongoing concern for consumers. Pathogens which live in tightly knit communities called biofilms, or pathogens which are living inside plant tissues are relatively resistant to chemical treatments. However, little is known about the response of these protected pathogens to irradiation. Leaves of Romaine lettuce and baby spinach were dip inoculated in a cocktail of three strains of E. coli O157:H7 and stored at refrigeration temperatures for various times (0, 24, 48, 72h) to allow biofilms to form. After each time, the leaves were treated with either a 3-minute wash with a chlorine-based sanitizing solution (0, 300 or 600ppm) or increasing doses of irradiation (0, 0.25, 0.5, 0.75 or 1.0 kGy). Viable bacteria were recovered and enumerated. Chlorine washes were generally only moderately effective, and resulted in maximal reductions of 95% for baby spinach and 98% for Romaine. Increasing time in storage prior to chemical treatment had no effect on spinach, but reduced the efficacy of the 600ppm wash applied to Romaine. For both types of leaves, allowing time for biofilm formation reduced the efficacy of irradiation. At the highest dose tested, 1kGy, irradiation treatment immediately after storage resulted in 99.999% reductions. After 72 h in refrigerated storage, the same treatment of 1kGy gave reductions of 99.4%. Scanning electron microscopy was used to visualize the developing biofilms on both types of leaves during storage. Bacterial were seen to proliferate inside the stomata on spinach leaves, but not on lettuce leaves. These results indicate that the protection of bacteria due to the specific nature of the ways that they attach and grow on the leaf surface, including biofilm formation and internalization, can reduce the efficacy of irradiation in eliminating pathogens from leafy green vegetables. Therefore, the time between packaging and irradiation should be kept to a minimum, to reduce the potential of this type of development.

Technical Abstract: Escherichia coli O157:H7 contamination of leafy green vegetables is an ongoing concern for consumers. Biofilm-associated and internalized pathogens are relatively resistant to chemical treatments, but little is known about the response of these protected pathogens to irradiation. Leaves of Romaine lettuce and baby spinach were dip inoculated in a cocktail of three strains of E. coli O157:H7 and stored at 4C for various times (0, 24, 48, 72h) to allow biofilms to form. After each time, the leaves were treated with either a 3-minute wash with a sodium hypochlorite solution (0, 300 or 600ppm) or increasing doses of irradiation (0, 0.25, 0.5, 0.75 or 1.0 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, but reduced the efficacy of 600ppm applied to Romaine. For both types of leaves, allowing time for biofilm formation reduced the efficacy of irradiation. D10 values (the dose required for a 1 log reduction) significantly increased with increasing storage time, up to 48h post-inoculation. At 0h of storage, D10 was 0.19kGy for both vegetables; a dose of 1kGy yielded log reductions of 5.07-5.51 log cfu/g. D10 increased to 0.40-0.43 kGy for Romaine and 0.52-0.54 kGy for spinach, with a dose of 1kGy yielding pathogen reductions of 2.20 and 2.19 log cfu/g, respectively. SEM was used to visualize the developing biofilms on both types of leaves during storage. Bacterial internalization by means of the stomata was extensive on spinach leaves, but not on lettuce leaves. Bacterial cells were seen to proliferate within these internal spaces. These results indicate that the protection of bacterial due to the specific nature of the phytoplane microbial ecology, including biofilm formation and internalization, can reduce the efficacy of irradiation in eliminating pathogens from leafy green vegetables.

Last Modified: 9/23/2014
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