Listeria monocytogenes internalizes in Romaine Lettuce grown in greenhouse conditions

Authors: SHENOY, ARCHANA - Purdue University; OLIVER, HALEY - Purdue University; DEERING, AMANDA - Purdue University

Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/22/2016
Publication Date: 3/1/2017
Citation: Shenoy, A.G., Oliver, H.F., Deering, A.J. 2017. Listeria monocytogenes internalizes in Romaine Lettuce grown in greenhouse conditions. Journal of Food Protection. 80(4):573–581.

Interpretive Summary: Harmful bacteria have been found in fresh produce and have caused foodborne illness outbreaks and deaths. In order to improve the handling and minimize the risk associated with consumption of fresh produce, it is important to document where harmful bacteria reside on or in plants, and also how long harmful bacteria may persist on the produce. Scientists from the Purdue University Center for Food Safety Engineering determined the persistence and internalization potential of one harmful bacterium, Listeria monocytogenes, in romaine lettuce. Lettuce seeds were contaminated with L. monocytogenes cells and grown in laboratory and standard greenhouse conditions. In laboratory conditions, high levels of L. monocytogenes persisted on romaine lettuce plants grown on a synthetic medium; however, the harmful bacteria were found in lower numbers on plants grown in soil and greenhouse conditions, indicating that competition with the natural soil microbes reduces the presence of harmful bacteria in produce. Harmful bacteria cells were located in all major tissue types (pith, cortex, xylem, phloem, and epidermis), indicating that the harmful bacteria can be transported throughout the plant into the edible plant tissue. If the bacteria are located on the inside of the lettuce, they could not be washed off. This may contribute to recent outbreaks associated with romaine lettuce, and suggest that it is better to prevent contamination than to try to wash harmful organisms from produce.

Technical Abstract: Listeria monocytogenes has been implicated in a number of outbreaks involving fresh produce, including an outbreak in 2016 resulting from contaminated packaged salads. The persistence and internalization potential of L. monocytogenes in romaine lettuce was evaluated, and the persistence of two L. monocytogenes strains was assessed on three romaine lettuce cultivars. Seeds were germinated, and plants grown in three soil types (i.e., standard potting mix, autoclaved potting mix, and top soil) and sterile soft-top agar for up to 21 days. Average CFU per gram of L.monocytogenes on seeds and plants was calculated from five replicates per harvest day. Up to 8.2 log CFU/g L. monocytogenes persisted on romaine lettuce plants (Braveheart cultivar) grown in soft-top agar, while those grown in commercial potting mix (initial soil aerobic plate count of 4.0 3 104 CFU/g) had a final concentration of 5.4 log CFU/g, and autoclaved commercial potting mix had a final concentration of 3.8 6 0.2 log CFU/g after a 21-day period. Pathogen levels dropped below the limit of detection (2 log CFU/g) by day 18 in 75% topsoil (initial soil aerobic plate count of 4.03101 CFU/g); this did not occur in sterile media. Although L. monocytogenes strain differences and presence of a clay coating on seeds did not affect persistence, differences were observed in L. monocytogenes growth and survival among cultivars. To assess internalization, seeds were inoculated with L. monocytogenes expressing green fluorescent protein. Three plants were fixed, paraffin embedded, and sectioned; localization was studied by using standard immunohistochemistry techniques. A total of 539 internalized L. monocytogenes cells were visualized among three 20-day seedlings. L. monocytogenes cells were located in all major tissue types (pith followed by cortex, xylem, phloem, and epidermis). The presence of L.monocytogenes in the plant vasculature suggests potential for transport throughout the plant into edible tissue.