Incidence of naturally internalized bacteria in lettuce leaves

Lettuce is the fresh leafy vegetable most frequently involved in foodborne disease outbreaks. Human bacterial pathogens may be experimentally internalized into lettuce plants, but the occurrence of natural microflora inside lettuce leaves has not been elucidated. To characterize the endophytic microorganism residing in commercial lettuce leaves, two separate studies were conducted. First, a total of 30 and 25 heads of romaine and red leaf lettuce, respectively, served as the source of individual leaves which were surface sterilized, stomached, enriched in BHI broth for 24 h and plated onto BHI agar for non-selective isolation of internalized microorganism. In a separate survey, 80 heads of each of the two types of lettuce were similarly processed, except that GN broth and MacConkey agar (MCA) were used for isolation of Gram negative bacteria. Thirty-eight out of 100 leaves were positive for internalized microorganisms, and Bacillus, Pseudomonas and Pantoea were the genera most frequently found in both types of lettuce. Members of the genus Erwinia were isolated from romaine lettuce only. In the second study, 21 and 60% of romaine and red leaf lettuce heads, respectively, had internalized bacteria capable of growing on MCA. Among the Gram negative strains, Pseudomonas and Pantoea genera were most frequently isolated. Enterobacter isolates were obtained from three red leaf samples. In summary, spore-forming bacteria and traditional epiphytic bacterial genera were frequently detected in surface-sterilized commercial lettuce leaves. Despite the common occurrence of internalized bacteria, only Enterobacter was related to Escherichia coli O157:H7 and Salmonella.