Persistence and internalization of Listeria monocytogenes in romaine lettuce, Lactuca sativa var. longifolia

Listeria monocytogenes, has been implicated in a number of outbreaks involving fresh produce. While no L. monocytogenes outbreaks have been linked to romaine lettuce, the number of lettuce recalls specific to L. monocytogenes is increasing. Understanding the potential of persistence and internalization of L. monocytogenes on and within romaine lettuce will aid in determining food safety risk. Persistence of three L. monocytogenes strains on three romaine lettuce cultivars was assessed independently by inoculating seeds in 25 ml of 8 log CFU/ml for 30 minutes. Seeds were grown on two soil types (i.e. standard potting mix, Indiana top soil) or sterile soft-top agar for up to 60 days. Average CFU/g of L. monocytogenesretained on seeds or persisting on growing plants was calculated from a total of 5 replicates per harvest day. Plants grown on sterile soft-top agar maintained between 4.4 to 7.8 log CFU/g L. monocytogenes after a 60 day period, while pathogen levels dropped below the limit of detection (2 log CFU/g) by Day 18 in 75% Indiana top soil, and by Day 45 in commercial potting mix. This suggests that soil microflora may impede pathogen persistence. L. monocytogenes strain differences and the presence of a clay coating on seeds were not factors that affected persistence. Cultivar differences, however, potentially influenced L. monocytogenes growth and survival. For internalization studies, seeds were inoculated with a L. monocytogenes strain constitutively expressing green fluorescent protein (GFP). Three plants were fixed, paraffin embedded, and sectioned; localization was studied using standard immunohistochemistry techniques. A total of 539 L. monocytogenes cells were internalized in all major tissue types of the hypocotyl with the majority localizing in the pith followed by cortex, xylem, phloem and epidermis. The presence of the bacterium in the plant vasculature indicates its potential to be transported throughout the plant system and reside within edible tissue. The significance of these findings is that romaine lettuce can support growth and internalization of L. monocytogenes, which could serve as a vehicle for L. monocytogenestransmission to consumers. Additionally, seeds from 16 cultivars of romaine lettuce and one cultivar of radicchio were examined as a potential source of contamination in a 2014 commercial recall of bagged salad products. Only a single seed, out of 1,700 tested, was found to be contaminated using PCR based methods on isolates recovered. These data indicate that while it is possible that the seed could be a source of potential L. monocytogenescontamination, it is unlikely that the seeds of the tested cultivars were the source of bagged salad recalls