Epiphytic, Attached, and Internal <i>Escherichia coli</i><scp>O157</scp>:<scp>H7</scp> 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 &lt; 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 &lt; 0.001). While EDL933 had lower measures of Strongly Attached + Internal cells compared to All and Epiphytic fractions ( p &lt; 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 &lt; 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.