The objective of this study is to determine the ability of E. coli O157:H7 to multiply in the presence of normal background microorganisms on iceberg lettuce and spinach under conditions that mimic actual practices between production and retail sale, including: <ul>
<li>Transportation from harvest field to cooler
<li>Transportation and distribution as <ul>
<li> cored product in a nitrogen atmosphere (lettuce) <li> open 20-pound returnable plastic totes (spinach) <li> finished packaged salads in a low oxygen/high carbon dioxide atmosphere in both common and abusive temperatures.</ul></ul>
By simulating conditions and practices of the fresh-cut industry, this study allows for the evaluation of the fate of E. coli O157:H7 on produce during typical handling practices. The results will provide insight into how E. coli O157:H7 on iceberg lettuce and spinach interacts with naturally-occurring bacteria, which may have competitive or antagonistic influences on the growth of E. coli O157:H7 in these plants. This knowledge can be used to identify handling and packaging routines that reduce the potential for contamination E. coli O157:H7 in fresh produce.
Variations on the way iceberg lettuce and spinach are handled in commercial operations and packaged will be reflected in the sampling plans. Green fluorescent pigment-expressing (GFP) E. coli O157:H7 will be used to trace the organism on inoculated product during handling and storage treatments.
Fresh iceberg lettuce and spinach, harvested from fields in central California, will be forced-air cooled and shipped overnight to the University of Georgia's Department of Food Science and Technology. Field location, proximity of harvest location to cattle operations, rivers, and other wildlife habitats, and temperature during harvest will be recorded. Prior to inoculation, the greens will be removed from the cooler and equilibrated to 25o or 32oC to allow for a comparison of two harvest conditions (one considered desirable and the other considered abusive). For spinach, individual leaves will be sampled. For iceberg lettuce, outer leaves will be removed and the heads cored by hand and washed in chlorinated water as typically done in the field. Exterior leaves and those from the next layer underneath will be sampled to determine the background microflora (aerobic mesophilic and psychrotrophic bacteria, coliforms, yeasts and molds, and lactic acid bacteria).
Multiple areas on the exterior leaves of the iceberg lettuce and leaves in the next layer will be surface-inoculated with 1,000-10,000 GFP-expressing E. coli O157:H7 per cm2. For spinach, individual leaves will be inoculated. After the inoculum dries, a section (approximately 25 cm2) will be removed from the inoculated leaves and the E. coli O157:H7 will be enumerated.
Heads of lettuce inoculated with the GFP-expressing E. coli O157:H7 will be placed in returnable plastic totes, held at 25oC and 32oC, and sampled for GFP-expressing E. coli O157:H7 and background microflora at 0, 5 and 10 hours. These temperatures and times correspond to reasonable conditions and abusive conditions for transporting greens from the field to the cooling facility. After sampling, totes will be placed in the vacuum cooler and chilled to approximately 40oF within 20-40 min. Totes will be held at either 4oC or 12oC and the inoculated heads will be sampled for GFP-expressing E. coli O157:H7 and background microflora at 0, 10, and 72 hours. Sampling after 10 hours represents transit to a fresh-cut operation within the region where the lettuce was harvested, while sampling after 72 hours represents the typical transit from central California to eastern areas like Atlanta. Lettuce will be chop-cut and held in chilled water with or without chlorine (pH adjusted), with agitation to simulate contact with water in fresh-cut operations. After dewatering and bagging, lettuce samples will be held at storage temperatures (desired and abusive temperatures that could occur during distribution of the retail product) and sampled over several days. Baby spinach will be forced air cooled to 4oC and bagged in macro-perforated bags, not chopped or shredded, simulating industry practice.
Naturally-occurring bacteria on the product will be screened for their ability to inhibit the pathogen. Randomly selected isolates from the plates used to enumerate the various types of bacteria, yeasts, and molds present on the lettuce and spinach at the different stages of handling and processing will be tested for any notable competitive or antagonistic effect on E. coli O157:H7.