Epiphytic, Attached, and Internal Escherichia coli O157:H7 Subpopulations Associating With Romaine Lettuce Are Strain-Dependent and Affected by Relative Humidity and Pre- and Postharvest Plant State

Abstract

Romaine lettuce is susceptible to Escherichia coli O157:H7 contamination. We evaluated strain and pre- and postharvest lettuce product differences in E. coli O157:H7 subpopulation distribution on romaine lettuce at two relative humidity (RH) levels. Plants of romaine lettuce cultivar 'Carlsbad' harvested and processed 'Carlsbad' leaves, and store-bought ready-to-eat romaine lettuce were inoculated with E. coli O157:H7 reference strain EDL933 and romaine lettuce outbreak strain 2705C. Using four processing methods, we determined pathogen cell fractions representing All (entire population), Epiphytic (loosely attached cells), Strongly Attached + Internal (excluding loosely attached cells), and Internal (excluding epiphytic cells) subpopulations. Preharvest, 80% RH favored subpopulations in each cell fraction, compared to 40% RH (p < 0.01 for both strains) and yielded 92%–100% internalization incidence of E. coli O157:H7, compared to 50%–57% at 40% RH. Levels of internal EDL933 cells were also 1.1 log higher than 2705C cells from plants kept at 80% RH (p < 0.001). While EDL933 had lower measures of Strongly Attached + Internal cells compared to All and Epiphytic fractions (p < 0.01), 2705C yielded no difference. Taken together, data suggest that the lettuce outbreak strain had a higher propensity for strong attachment to leaves and EDL933 internalized more successfully. Moreover, the Strongly Attached + Internal fractions of both strains were lower on preharvest 'Carlsbad' compared to 'Carlsbad' processed leaves (p < 0.01), suggesting that E. coli O157:H7 attached less strongly to preharvest plants than postharvest cut and stored leaves of the same variety. Our study uncovers important factors influencing cultivar- and strain-specific differences in association and internalization of enteric pathogens on leafy greens.