Transmission of Escherichia coli 0157:H7
from Contaminated Manure and Irrigation Water
to Lettuce Plant Tissue and Its Subsequent Internalization

This article Ethan B. Solomon, Sima Yaron, and Karl R. Matthews, Department of Food Science, Rutgers University, New Brunswick, New Jersey, appeared in “Applied and Environmental Microbiology,” January 2002, p. 397-400, Vol. 68, No. 108901.

In recent years, Escherichia coli 0157:H7 has been isolated with increasing frequency from fresh produce, including bean sprouts, cantaloupes, apples, and leaf lettuce. The mechanisms by which the pathogen is introduced into the lettuce plant are not fully understood; however, one hypothesis states that the plant becomes contaminated when grown in fields fertilized with improperly treated manure.

Epidemiological data indicate that E. coli 0157:H7 may be present in up to 8.3 percent of dairy and beef cattle, and that it is shed asymptomatically in the feces. Current manure-handling guidelines suggest a composting period before application of the manure to a field as fertilizer. Research has demonstrated the long-term survival of E. coli 0157:H7 in manure held under a variety of conditions, so even strict adherence to the guideline may result in the application of manure containing culturable E. coli 0157:H7 to production fields.

E. coli 0157:H7 may also be introduced are through flood irrigation with water contaminated with cattle feces, or contact with contaminated surface runoff. A number of recent E. coli 0157:H7 outbreaks have been linked to contaminated water; furthermore, studies have demonstrated the ability of the pathogen to survive for extended periods in water. Cattle in an adjacent field were implicated as the source of E. coli 0157:H7 during a multi-state outbreak associated with the consumption of mesclun lettuce in 1996. The authors speculated that contaminated water was used to irrigate the lettuce fields.

Lettuce production practices commonly include a rinse step, in which the leaves are sanitized using tap water containing 100 to 200 ppm of free chlorine. This level of chlorine has been shown to be only marginally effective at reducing the level of E. coli 0157:H7 on lettuce tissue surfaces. The ineffectiveness of chlorine and other surface-sanitizing agents is likely dependent on whether the target organisms are readily accessible. Cells of E. coli 0157:H7 were shown to penetrate into the stomata and junction zones of cut lettuce leaves, becoming entrapped 20 to 100 µm below the surface of the cut edge. Cells entrapped at subsurface locations were protected from sanitation with chlorine.

Previous studies have not provided a direct link for contamination of lettuce in the field through fertilization with E. coli 0157:H7-contaminated manure or irrigation with contaminated water. Moreover, the sites of association, surface or subsurface, of the pathogen following in-field contamination have not been delineated. We investigated whether E. coli 0157:H7 associated with contaminated manure or irrigation water can be transported from the root system into the edible portion, putatively by the plant vascular system. In this study, we demonstrated the transmission of E. coli 0157:H7 to lettuce plants from contaminated manure incorporated into the soil. Furthermore, the contamination of lettuce through flood irrigation with contaminated water was demonstrated. E. coli 0157:H7 expressing green fluorescent protein (GFP) was used to facilitate detection of the target organism in association with lettuce tissue.

Application of E. coli 0157:H7-contaminated manure to the production field or irrigation with E. coli 0157:H7-contaminated water may result in contamination of the crop in the field. Studies have indicated that E. coli can survive for extended periods in manure and water. We have demonstrated that lettuce grown in soil containing contaminated manure, or irrigated with contaminated water, results in contamination of the edible portion of the lettuce plant. Moreover, the results suggest that edible portions of a plant can become contaminated without direct exposure to a pathogen, but rather through transport of the pathogen into the plant by the root system.

We recognize that the levels of E. coli 0157:H7 used in this study are far greater than what may be found on an agricultural field; however, numbers of bacteria were used that could be readily detected by the assays used in the present study. Under natural conditions, even a low level of contamination could present a significant human health risk, since the infective dose of E. coli 0157:H7 is less than 1,000 cells.

Research suggests that surface sanitizing of lettuce is not an effective method to eliminate all E. coli 0157:H7 cells. The inaccessibility of a large number of organisms, as a consequence of their subsurface location, is perhaps the reason for the lack of effectiveness of surface-sanitizing treatments. The impacts of on-farm practices which may result in E. coli 0157:H7 becoming associated with lettuce, or for that matter other crops, have not been sufficiently explored.

The results of this study emphasize the need for exercising extreme care when using composted manure as a source of crop nutrients. Never use undecomposed manure on vegetable crops.