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:

• Strain differences among E. coli O157:H7 isolates;
• Types of lettuce and phases of the plant's growth cycle;
• Environmental stress (heat or water) and soil fertility;
• Level of contamination;
• Insect damage to lettuce leaf tissue;
• Physical location of contamination (abaxial or lower leaf surface versus adaxial or upper leaf surface);
• Contribution of soluble organic matter (simulating materials in which E. coli O157 can be found); and
• 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. Close This Area

» Addresses Fresh Express Produce Safety Initiative research priority area 1.

Based on the following hypotheses, the study includes five objectives: E. coli 0157:H7 colonizes different tissues of the spinach plant, and achieves different titers, depending on the site and method of entry. Phytopathogenic bacteria enter plants through natural openings, wounds, or deposition by insects. Depending on the mode of entry, bacteria encounter different plant micro-environments and develop different interactions with the host.

• Objective 1: Assess whether a green fluorescent protein-tagged E. coli 0157:H7, introduced into different plant tissues, colonizes and moves through intercellular spaces, the vascular system, or both, using confocal and fluorescence microscopy of roots, stems and leaves. Spinach cultivars will be inoculated by root drench, leaf surface contamination, needle injection, vacuum infiltration, and an insect vector. Plate counts and quantitative (Q-PCR) will be used to assess bacterial titers reached after internalization.

Spinach cultivar and plant age at the time of inoculation affect susceptibility to E. coli O157:H7 internalization, colonization, and titer. Different plant cultivars often differ in susceptibility to bacterial invasion.

• Objective 2: Test susceptibility to E. coli 0157:H7 for a variety of spinach cultivars. Plant tissues (mature and developing leaves and roots) will be assessed by quantitative polymerase chain reaction (Q-PCR) and culturing. The effect of plant age on colonization will be tested using 1, 2, 3, and 4 week-old plants.

Insect species that are known phytopathogen vectors inhabit livestock pasture areas in close proximity to leafy greens production areas.

• Objective 3: Survey cattle pastures and nearby lettuce/spinach growing areas for insects. Insect sticky traps will be placed in fencerows bordering pastured cattle at three to four locations in Southern California and the Salinas Valley. Sweep samples will be collected from each pasture and insects identified. Pasture grasses will be sampled and tested for E. coli O157:H7.

Insects common to livestock and field production areas (muscid flies, cabbage loopers, leafhoppers, and aphids) are capable of acquiring E. coli from contaminated livestock sources.

• Objective 4a: Confirm that houseflies acquire bacteria. Flies will be exposed to E. coli by caging with open agar cultures, dishes of E. coli suspension, fecal material containing E. coli, or potted grasses sprayed with 105 cells/ml bacteria, and flies will be held in mesh cages and tested for E. coli by polymerase chain reaction (PCR) methodology;
• Objective 4b: Test a butterfly model for its capacity to drink and acquire from above sources. Cabbage looper adults will be enclosed with E. coli-contaminated water or fresh fecal material and then tested along with liquid inoculation sources by PCR and cell plating;
• Objective 4c: Test phytopathogen vectors for their capacity to acquire pathogens from the above sources and from traditional artificial feeding sachets. Circulifer tenellus (a vector of spinach virus), and Acyrthosiphon pisum or Myzus persicae (aphid vectors of plant viruses) will be caged with E. coli contaminated water, fecal material, and plant material, or placed in feeding sachets containing E. coli and insects will be tested after 1, 2 or 3 weeks by PCR and culturing.

Flies, butterflies, leafhoppers, and aphids are competent vectors of E. coli transmitted to leafy greens.

• Objective 5a: Determine dispersal flight distances of flies and cabbage looper butterflies marked with fluorescent powder, released in cattle production area, and recaptured at distances of 0.8 km, 1.6 km, and 2.4 km using fly-bait traps and sugar-water traps set in concentric circles around the release point;
• Objective 5b: Assess propensity of contaminated flies, butterflies to alight on and contaminate greens. Houseflies or butterflies exposed to E. coli will be released into a chamber containing lettuce or spinach plants, and plants will be tested for E. coli by PCR and cell plating, at 1, 3 and 5 days;
• Objective 5c: Evaluate E. coli vector competence of leafhoppers and aphids; C. tenellus, A. pisum, M. persicae and Bemesia tabaci will acquire E. coli from feeding sachets and caged with lettuce or spinach and plants will be tested for E. coli by PCR or culturing. If any experimental transmissions are successful, transmission of E. coli O157:H7 by those insect species will be tested. Close This Area

» Addresses Fresh Express Produce Safety Initiative research priority areas 1 and 3.

Environmental, ultrastructural, molecular, and bactericidal testing approaches will be applied in this project. Environmental studies will assess the role of temperature, pH, incubation time, gas requirements, plant age, and bacterial determinants on the colonization of leafy greens. Ultrastructural research will examine the structures produced by the bacteria that may be involved in bacterial adherence and colonization, such as pili and flagella. Molecular testing will focus on the introduction of specific bacterial effectors into the leaf to determine if they have a role in this phenomenon. Bactericidal testing will be used to assess various bactericidal agents that are non-toxic to humans to determine a more effective method of eliminating this organism from fresh produce.

Preliminary results indicate that E. coli O157:H7 can colonize the surface of spinach leaves using structures resembling flagella and can gain access to the internal leaf structure using the stomata. Colonization of the stomata may be the mechanism by which the E. coli can survive the industrial decontamination process. Further testing will focus on (1) identifying the pathogen’s mechanism for manipulating the stomata, which aids internal colonization; (2) identifying the specific structures that play a role in leaf surface adherence; and (3) developing a method of sterilization that can be applied at the industrial level to eliminate E. coli O157:H7 from produce. Close This Area

» Addresses Fresh Express Produce Safety Initiative research priority areas 1 and 2.

Growth of E. coli O157:H7 inoculated onto lettuce or spinach will be evaluated according to these three objectives:

• For inoculated E. coli O157:H7 on spinach, characterize growth kinetics and evaluate the effects of variety, age of plant, growing conditions, fertility management, growth rate, and day length.
• For inoculated E. coli O157:H7 on washed and bagged romaine lettuce, characterize growth kinetics under different pre-inoculation bacterial growth conditions and stresses.
• For inoculated E. coli O157:H7 on washed and bagged romaine lettuce and spinach, characterize growth kinetics at different holding temperatures, both static and fluctuating.
Romaine lettuce and spinach will be inoculated with different levels of E. coli O157:H7 and held at static temperatures or under conditions where the temperature is shifted for various periods of time from refrigerated to mildly abusive temperatures that permit growth of E. coli O157:H7.

Three varieties of spinach will be grown to three different stages of maturity, at different nitrogen fertilization rates, and using different amounts of supplemental light.

E. coli O157:H7 inoculum will be prepared by several approaches: on solid or liquid medium; in minimal medium or tryptic soy broth; at minimal, moderate, and optimum temperatures; in different inoculum carriers (irrigation water, MilliQ water, 0.1% peptone, and dilute compost); to early or late stationary phase; and held for different times between preparation of inoculum and inoculation.

Results of this study will include the identification of factors under controlled greenhouse conditions that affect leaf structure, leaf breakage, cotyledon dehiscence or senescence, and other aspects of plant morphology relevant to food safety. Data on culture conditions determined to have the greatest impact on the fate of E. coli O157:H7 inoculated onto lettuce will be applicable to methods development and inoculation studies. Data on E. coli O157:H7 survival and growth at constant and fluctuating storage temperatures generated by these experiments will greatly expand the available information relevant to leafy greens. Close This Area

» Addresses Fresh Express Produce Safety Initiative research priority area 4.

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. Close This Area

» Addresses Fresh Express Produce Safety Initiative research priority area 4.

Key environmental factors related to the regrowth and survival of three foodborne pathogens in compost are evaluated in this study. Data from this project can be applied to the design of composting practices in California, the identification of environmental factors conducive to pathogen regrowth, and to the prediction of pathogen inactivation during on-farm composting.

The specific objectives are to:

• Identify the optimal conditions for E. coli O157:H7, Salmonella spp., and L. monocytogenes regrowth in composts under field application; The most desirable environmental conditions for inactivation, such as heavy rain/overhead sprinkling/flooding, nutrient sources (types of compost), and warm temperatures, will be evaluated for pathogen regrowth in compost. A cocktail of three strains for each pathogen will be heat-shocked and then inoculated into the compost with low initial populations. The inoculated compost will be maintained inside a greenhouse for four weeks and sampled periodically for pathogen analysis. Different varieties of compost produced in California will be used for studying pathogen regrowth.


• Determine the fate of heat-adapted avirulent pathogens during composting on the farm; Composting practices in California will be followed and simulated, using on-farm composting of vegetables with dairy manure and ingredients used in California (which can also be found in South Carolina.) The green wastes from either summer or fall organic crops will be collected from Clemson University’s Calhoun Field Laboratory farm. Duplicate compost heaps will be set up on site. The survival of compost-adapted and heat-adapted avirulent strains of E. coli O157:H7, Salmonella spp., and L. innocua will be determined at three locations (surface, center, and bottom) of composting heaps. Field studies will be conducted during summer months (25-35°C) and winter months (5-15°C) to determine the influence of outdoor temperatures and precipitation on the fate of pathogens.


• Study the impact of initial heat resistance of pathogens on their survival during composting. Three strains for each pathogen will be heat-shocked at 45°C prior to the inoculation of compost mixture separately. Cultures without heat-shock treatment will be used as control. The inoculated compost held in a Tyvek® pouch will be kept in an incubator at a range of temperatures (45, 50, 55 and 60°C) to simulate the conditions inside compost heaps during thermophilic composting. At certain intervals, samples will be taken out and analyzed for surviving bacterial counts. Mathematic models based on these data will be developed to predict the thermal inactivation of pathogens during composting.

» Addresses Fresh Express Produce Safety Initiative research priority area 5. Close This Area

The project’s specific objectives are to:

• Determine the transfer coefficients for the numbers of E. coli O157:H7 transferred from dip-inoculated, wet head lettuce and wet spinach to equipment surfaces during conveyance on a conveyor belt, dewatering by shaking/centrifugation, and mechanical shredding using commercial processing equipment.
• Determine the extent of E. coli internalization during processing of head lettuce and spinach; and
• Develop a mathematical risk model for E. coli O157:H7 cross-contamination of head lettuce and spinach during processing, using the model to identify candidate risk mitigation strategies.

Fresh-cut, pre-washed baby spinach and intact heads of California head lettuce (Iceberg variety) will be surface-inoculated with an avirulent strain of E. coli O157:H7 containing the green fluorescent protein (gfp) plasmid by dipping so as to contain approximately 105 CFU/g or cm2. After brief draining to simulate product exiting the flume tank, numbers of E. coli transferred to equipment surfaces will be assessed during conveying, dewatering and shredding. Selected lettuce and spinach samples containing the highest numbers of gfp-labeled E. coli O157:H7 will be examined for both presence and numbers of internalized cells. For quantification, samples will be surface-sterilized by immersion in 1% silver nitrate, rinsed, ground, and then plated on an appropriate medium. Selected samples will also be examined for internalized cells of gfp-labeled E. coli O157:H7 using Confocal Scanning Laser Microscopy. The results will be compared to those from Objective 1 to determine percent and extent of internalization. After determining the transfer coefficients for the different transfer scenarios, the data will be subjected to our existing bacterial transfer model. As the E. coli transfer coefficients are collected, the information will be plotted to determine the relationship between type of contact surface and the rate of bacterial transfer. By examining the plotted experimental results, several mathematical models will be proposed. Software dedicated to model development, such as TableCurve®2D and 3D, will be used to identify alternative mathematical algorithms. In all objectives, transfer of E. coli from an inoculated to an uninoculated surface and vice versa will be assessed using statistically-based models.

A quantitative risk assessment model will be developed for assessing the risk of California-grown leafy greens for consumers, and include mitigation strategies, in accordance with the Codex Alimentarius Commission’s scheme: exposure assessment, hazard characterization, risk characterization and sensitivity analysis.

It is anticipated that conveyance on a conveyor belt, dewatering by shaking or centrifugation, and mechanical shredding will prove to be important multi-directional transfer points for E. coli O157:H7 dissemination, due to incoming contamination on the leafy greens, low cleanability of some areas of the equipment surface, and potential for E. coli O157:H7 to penetrate into cut surfaces of leafy greens during shredding. Transfer coefficients generated from these data can be modeled and incorporated into risk assessments to enhance the safety of leafy greens for consumers. Close This Area

» Addresses Fresh Express Produce Safety Initiative research priority areas 1, 2, and 3.

The project will apply this novel approach to evaluate the following hypotheses: (1) that E. coli O157:H7 strains isolated from produce-associated outbreaks are better able to be internalized into plants than are strains isolated from beef-associated outbreaks or non-pathogenic E. coli strains; (2) that E. coli O157:H7 requires an intact stress response to survive in plants; and (3) that established E. coli O157:H7 virulence factors are heightened, or up-regulated, during growth on leafy greens compared with growth in ground beef.

Specific objectives of the project include:

• Development of strains of E. coli O157:H7 and non-pathogenic E. coli that contain the gfp gene inserted into the bacterial chromosome, using newly developed transformation methods based on transposons as vectors. These strains will have improved bacterial fitness over plasmid gfp strains and will not require a selective agent for detection, enabling accurate assessment of E. coli O157:H7 survival and growth.
• Determination of the survival and growth of gfp-labeled wild type versus stress response gene (rpoS)¬-deficient E. coli O157:H7 populations in internalized plant tissues in different spinach and lettuce varieties. Enumeration and microscopic analysis of E. coli O157:H7 from internalized tissues will reveal the possibility and extent of internalization in plant tissues at specific points during the growing process.
• Determination of differences in the expression of virulence factors in E. coli O157:H7 grown on leafy greens compared with E. coli O157:H7 grown in ground beef. Virulence factors such as ler (positive regulator of type III secretion), eae (outer membrane adhesin intimin), espA (type III secretion filament), ehxA (enterohemolysin), ihaA (adherence factor), rfbE (O157 antigen), and stxII (shiga toxin 2) will be evaluated by real time reverse transcriptase (RT) polymerase chain reaction (PCR) methods. A ratio between virulence factors and housekeeping genes will be determined, and the ratio for E. coli O157:H7 grown on lettuce will be compared to the ratio for E. coli O157:H7 grown on ground beef. This comparison will yield insights into the potential of E. coli O157:H7 to cause human illness through the consumption of contaminated produce. Close This Area

» Addresses Fresh Express Produce Safety Initiative research priority area 1.

Ozone is a potent sanitizer that is well-suited for use with fresh produce. Results of this project may provide the fresh produce industry with a critical decontamination step that can be incorporated into exiting production and processing lines, potentially reducing the health risks associated with sporadic contamination of fresh produce with E. coli O157:H7.

The preliminary experiments will involve the use of an elaborate ozone treatment vessel located in the investigators’ biosafety level-2 facility. Processing variables to be tested include: vacuum level; vessel pressure after ozone introduction; ozone concentration; treatment time; average temperature; and product loading. Preliminary studies will be conducted on diffusion of ozone into various locations within the treatment chamber. A sampling manifold will be installed to draw gas samples from multiple locations when different product loads are tested. Product/vessel volume ratio will be optimized to make sure all leaves are exposed to the sanitizer. If needed, a slow tumbling of the product within the vessel during the treatment will be applied. In all cases, E. coli O157:H7 will be counted (or detected, if the bacterial population is uncountable) in produce before and after treatment, as well as during storage.

This study will identify treatment conditions sufficient to eliminate E. coli O157:H7 in fresh produce, at realistic contamination levels, while maintaining product quality and integrity. It will also optimize an ozonation process for scale-up and incorporation into pre-packaging treatments of fresh produce.

» Addresses Fresh Express Produce Safety Initiative research priority area 2. Close This Area

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