Internalization of E. coli in Apples under Field and Laboratory Conditions

This will be accomplished through the combined use of bacteria expressing the green-fluorescent protein (GFP) and confocal microscopy to observe migration patterns. Antibiotic-resistant bacteria may also be used to observe movement. This aspect will be performed primarily within the laboratory using pathogenic species, with potential follow-up work under field conditions using non-pathogenic species. Provide a scientific basis for Good Agricultural Practices for producers and processors to minimize or prevent the of internalization of microbial pathogens occurring during production, harvesting, storage, handling, and utilization.
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Certain agricultural practices contribute to how bacteria get into fruit such as apples that might be used for the production of apple cider. This project examines how E. coli occurs within the apple on several levels by investigating apple variety, apple development patterns, and the movement of E. coli throughout the apple tissue. This project will provide valuable information in developing effective control procedures in apple production, harvesting, storage, and utilization. The techniques that are developed in this study can be used for the examination of similar phenomena in other fruits and vegetables.
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Field studies will be conducted in typical apple orchards in the three cooperating states (Virginia, New York, and Washington) during two different growing seasons for each state. Typical plots of land will be identified and isolated using wire mesh fencing to control large pests. Analysis will occur under varying environmental conditions in apple fields during times when apples are normally picked from the tree and when fresh apple cider is usually produced. Weather conditions during each study will be recorded. A known amount of the pathogen surrogate, E. coli ATCC 25922 will be applied to the plot. Simulated drop or windfall apples will be analyzed for internalization within the plot of land. The apple varieties to be studied will be the ones that are most commonly used for cider production in each participating state: New York - Red Delicious, Northern Spy, Rhode Island Greening, and Cortland; Virginia - Red Delicious, Golden Delicious, Rome Beauty, and York; Washington - Red Delicious, Golden Delicious, Fuji, and Gala. Apples will be recorded as falling calyx facing up or down. In addition, apples for each variety will be punctured through the skin and placed puncture side down to simulate this type of damage when the apple falls from the tree. Apples from at least two varieties that have natural surface defects such as scald will be placed on the ground, damage side down. Apples will be sampled every other day for ten to fourteen days. Each apple will be removed aseptically from the plot and placed into a sample bag. After the excess dirt has been gently removed, each apple will be cut into four regions: skin, inner core, outer core, and flesh. Each region will be stomached for approximately two minutes and then plated using a spiral plater. Dilutions will be plated onto MacConkey agar supplemented with cycloheximide to control potential mold growth. Data will be correlated with weather conditions, region of production, developmental stage of the apple, apple variety, position relative to the calyx, and apple condition. In order to more definitively trace the movement of enteric pathogens through apple slices, bacteria will be transformed to express the green fluorescent protein. For enhanced analysis, bacteria will be transformed to express either blue or green fluorescence to observe movement from various entry points. For each variety of apple from the three states, blue fluorescent bacteria may be applied to stem area and green fluorescent bacteria will be applied to the calyx opening of the same apple. Likewise artificial puncture areas and naturally damaged areas of the same apple for each variety in the study will be treated with blue and green fluorescent bacteria. Then apples will be sliced, plated as previously outlined, and observed for fluorescence using laser confocal imaging. These studies will be repeated using a simulated flume system and water contaminated with the fluorescent bacteria.
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This research project addresses the issue of the ability of Escherichia coli O157:H7 to internalize and survive in whole apples before and after harvest. Four cultivars of apples, Redfree, Red Delicious, Golden Delicious, and York, were inoculated under field conditions with a surrogate strain of E. coli, Escherichia coli ATCC 25922. The Redfree cultivar was inoculated at the beginning of its growth stage (day 0), and again 30 days later, and sampled for two weeks, until E. coli was not recoverable through microbiological methods after three successive sampling days. Red Delicious, Golden Delicious, and York cultivars were spray inoculated with the surrogate strain two weeks before their anticipated harvest date and sampled every other day until E. coli was not recoverable for three successive sampling days. For each cultivar, the presence of E. coli ATCC 25922 was not detectable after 7 to 9 days. In the laboratory study the Red Delicious, Golden Delicious, Rome, and York cultivars received one of three treatments; unblemished control, bruising, or puncturing. The apples were inoculated by immersion in cold water containing E. coli O157:H7 GFP, incubated for three days then microbiologically analyzed for presence of the bacteria. In all cases, the punctured apples of each cultivar showed the greatest uptake of E. coli O157:H7 GFP. Escherichia coli O157:H7 GFP was visualized in flesh and core sections of untreated, bruised, and punctured apples of all cultivars. The microbe was found in between cells, but not within cells of the apple.
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Internalization of Escherichia coli in whole apples on the tree is not likely, and leads to the conclusion that internalization is a post-harvest problem. Internalization may occur before pressing or processing of apples, leading to an increased risk of infection with E. coli for consumers of apple products that are not properly treated to destroy pathogens. Internalization does occur when apples are immersed in solutions containing the pathogen Escherichia coli O157:H7, and better post harvest controls need to be implemented in order to prevent this in whole apples that are used for cider and juice production.