Flagella mediate attachment of enterotoxigenic Escherichia coli to fresh salad leaves

P>Enterotoxigenic Escherichia coli (ETEC) causes child and travelers' diarrhea and is presumed to be water- and food-borne. Sporadic outbreaks were traced to consumption of contaminated fresh produce, particularly salad leaves as lettuce and parsley. Importantly, the mechanism by which ETEC binds salad leaves is not known. In this study we investigated the ability of clinical ETEC isolates to adhere to Eruca vesicaria (commonly known as rocket). Towards this end we inoculated pieces of cut E. vesicaria leaves with clinical ETEC isolates grown in Luria broth at 20 degrees C, conditions that are not permissive for expression of the plasmid-encoded colonization factors and hence mimic the actual transmission pathways of ETEC through intake of contaminated food. We found that ETEC strains bind E. vesicaria at various efficiencies. Examination of representative strains by scanning electron microscopy revealed that they adhere to the E. vesicaria surface in a diffuse pattern by extended filaments resembling flagella. Using the prototype ETEC strain H10407 we found that it also binds to lettuce, basil and spinach leaves. Binding of H10407 was dependent on flagella as a fliC mutant attached to leaves at a much lower efficiency. Interestingly, under the employed environmental conditions EtpA, which forms a flagellar tip structure, and colonization factor I are dispensable for leaf attachment. The results show that ETEC can bind specifically to salad leaves, which might represent an important, yet less recognized, source of infection.