Pathogenic bacteria that become internalized in leaf tissues are protected from the antimicrobial effects of surface treatments. Ionizing radiation is known to penetrate food tissues, but the efficacy of the process against internalized bacteria is unknown. Leaves of Romaine lettuce and baby spinach were cut into pieces, submerged in a cocktail mixture of three isolates of Escherichia coli O157:H7, and subjected to a vacuum perfusion process to force the bacterial cells into the intercellular spaces in the leaves. Scanning electron microscopy was used to evaluate the efficacy of the perfusion process. The inoculated leaves were then treated with a 3-min water wash, a 3-min wash with a sodium hypochlorite sanitizing solution (300 or 600 ppm), or various doses of ionizing radiation (0.25 to 1.5 kGy). Leaves were stomached to recover the internalized pathogen cells, which were enumerated. The vacuum perfusion effectively forced bacteria into the leaf vasculature and apoplast, as confirmed by scanning electron microscopy. For spinach leaf pieces, neither the water nor the sodium hypochlorite washes resulted in significant reductions of E. coli O157:H7 cells relative to the untreated control. For Romaine lettuce leaf pieces, 300 and 600 ppm sodium hypochlorite each resulted in less than 1-log reduction; water wash was ineffective. Ionizing radiation, in contrast, significantly reduced the pathogen population, with 4-log (Romaine lettuce) or 3-log (spinach) reductions at the highest dose tested. In Romaine leaves, the reduction was dose dependent across the range of doses tested, with a D10-value (the amount of irradiation necessary to reduce the population by 1 log unit) of 0.39 kGy. In spinach leaves, the pathogen had a biphasic response, with a D10-value of 0.27 kGy in the range of 0 to 0.75 kGy but only slight additional reductions from 0.75 to 1.5 kGy. In this study, ionizing radiation but not chemical sanitizers effectively reduced viable E. coli O157:H7 cells internalized in leafy green vegetables, but the response of the pathogen to irradiation was more complex in spinach leaves than in Romaine lettuce leaves.
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