This project examines factors affecting the uptake and internalization of E. coli O157:H7 in lettuce plants. Growth and survival of the pathogen will be evaluated according to several key variables, including pathogen strain, produce type, growth cycle phase, environmental factors, and mechanical processing. The results will improve our understanding of the factors that influence E. coli O157:H7 growth and survival in and on produce, enabling the development of enhanced risk mitigation strategies.
It is anticipated that E. coli O157:H7 is internalized through the roots of seedlings or older plants when the plants are subjected to stressful conditions (such as excessive heat or restricted water/nutrients), although this route of internalization may result in the pathogens either not surviving or failing to be transported up through the plants' vascular system to their edible tissues.
Internalization may occur more readily through the leaf surface, depending on the physical location of E. coli O157:H7 on the leaves, availability of nutrients, and insect-related mechanical damage to the leaf. Contaminated cutting blades used to harvest the lettuce could also serve as a tool to expose internal tissues to E. coli O157:H7, although treatment with a chlorinated water rinse soon after the contamination may reduce pathogen levels.
In this study, the following factors will be evaluated systematically for their impact on the extent of E. coli O157:H7 internalization, colonization, survival, and growth in and on lettuce plants:
<li>Strain differences among E. coli O157:H7 isolates;
<li>Types of lettuce and phases of the plant's growth cycle;
<li>Environmental stress (heat or water) and soil fertility;
<li>Level of contamination;
<li>Insect damage to lettuce leaf tissue;
<li>Physical location of contamination (abaxial or lower leaf surface versus adaxial or upper leaf surface);
<li>Contribution of soluble organic matter (simulating materials in which E. coli O157 can be found); and
<li>Coring by an E. coli O157:H7-contaminated knife.
All experiments will be conducted in environmentally controlled growth chambers at the Georgia Envirotron facility at the University of Georgia. Lettuce seeds will first be germinated in trays containing horticultural vermiculite. One week after germination, healthy seedlings will be transplanted into pots containing either 1:5 dairy manure:soil (leaf surface contamination studies) or sifted sandy loam soil (root contamination studies) obtained from horticultural fields on the Tifton Campus at the University of Georgia. The pots will be illuminated (14 hours per day) in growth chambers held at 23 C during the day and 7 C at night.
All E. coli O157:H7 strains used in the project will be labeled with the green-fluorescent protein (gfp) and will either be suspended in a carrier solution or manure filtrate (approximately 104 or 106 CFU/ml) for leaf surface contamination studies or incorporated into the soil (approximately 103 or 106 CFU/g) for root contamination studies. Exposure of lettuce plants to contaminated soil will occur either initially at seedling transplantation or 30 days after seedling transplantation. In all leaf surface contamination studies, the pathogen mixture will be applied as drops to either the abaxial or adaxial leaf surfaces at one of three different times (7, 30 and 60 days) following seedling transplantation.
Studies addressing the potential for insects to act as contributing factors in the contamination of lettuce tissues with E. coli O157:H7 will involve two scenarios: pathogen contamination occurring 24 hours before insect infestation and immediately following insect infestation.
For all studies, triplicate samples will be obtained following contamination at selected intervals up to and including harvest. Plants will be carefully removed and dissected into root and aerial tissue portions. Enumeration will be conducted for both surface and internalized E. coli O157:H7 in lettuce root and aerial tissue samples. In addition, soil samples beneath and in contact with plant roots will be obtained for enumeration of E. coli O157:H7 in root contamination studies. When pathogens cannot be detected by direct plating, a selective enrichment method will be used and representative colonies will be confirmed by the E. coli O157:H7 latex agglutination test. At selected sampling times, plant tissues will also be obtained for confocal and epifluorescent microscopic analyses.
To simulate contamination of iceberg head lettuce through mechanical damage incurred during harvest, uncored head lettuce will be cored with a field corer that has been exposed to E. coli O157:H7-contaminated soil (102 or 105 CFU/g). Following coring, the lettuce heads will be rinsed with chlorinated water (0, 100, or 200 ppm) and examined for E. coli O157:H7 through enumeration and enrichment culture.