Internalization and Fate of Escherichia coli O157:H7 in Leafy Green Phyllosphere Tissue Using Various Spray Conditions

In the past decade, leafy greens have been implicated in several outbreaks of foodborne illness, and research has focused on contamination during preharvest operations. Concerns have been raised that internalization of pathogens into the edible tissue occurs where postharvest chemical interventions would be ineffective. This study was initiated to measure the degree and fate of Escherichia coli O157:H7 internalized in the phyllosphere tissue of leafy greens when spray conditions, inoculum level, and type of leafy green were varied. Two spraying treatments were applied: (i) spraying individual spinach or lettuce leaves on plants once with a high dose (7 to 8 log CFU/ml) of E. coli O157:H7 and (ii) spraying spinach, lettuce, or parsley plants repeatedly (once per minute) with a low dose (2.7 to 4.2 log CFU/ml) of E. coli O157:H7 over a 10- to 20-min period. With the high-dose spray protocol, no significant differences in the prevalence of internalization occurred between Shiga toxin-negative E. coli O157:H7 isolates and virulent isolates (P > 0.05), implying that the Shiga toxin virulence factors did not influence internalization or the subsequent fate of those populations under these test conditions. Significantly greater internalization of E. coli O157:H7 occurred in spinach leaves compared with lettuce leaves when leaves were sprayed once with the high-dose inoculum (P < 0.05), whereas internalization was not observed in lettuce leaves but continued to be observed in spinach and parsley leaves following repeated spraying of the low-dose inoculum. Based on these results, it is surmised that a moisture film was generated when spraying was repeated and this film assisted in the mobilization of pathogen cells to plant apertures, such as stomata. E. coli O157:H7 cells that were internalized into spinach tissue using a low-dose repeat-spray protocol were temporary residents because they were not detected 2 days later, suggesting that plant-microbe interactions may be responsible.