Use of GFP and LUX to Track Pathogen Contamination, Growth, and Inactivation on Produce Contaminated Via Manure/Water (Farm to Fork)

NON-TECHNICAL SUMMARY: Fresh produce is often contaminated with pathogenic bacteria. The purpose of this study is to identify the pathways for serious contamination events in fresh produce and to test decontamination methods. <P>

APPROACH: Objective 1. Soil, water and manures will be inoculated with different levels of Salmonella spp., E. coli O157:H7, Shigella spp., and L. monocytogenes lux-gfp constructs, and these will be used to challenge cantaloupe, apple, tomatoes and lettuce, respectively. The presence of the lux-gfp systems provides a novel method to carefully track two factors: bacterial presence (both number and location) and in situ activity. This approach allows us to directly track the presence and location of bacteria on the plant surface. <P>Objective 2. We will study the factors involved in bacterial stability on various plant surfaces (in situ) after loading and establishment of initial colonies. We will extend the work to track bacterial survival and growth inside the plant, both in healthy plant tissue, particularly sprouts, and in introduced niches, such as tears and cuts. As above, we will take advantage of the lux-gfp systems to follow the growth and movement of bacteria in the plants. We will also directly assess the ability of Salmonella spp. to utilize plant material as a nutrient and carbon source. <P>Objective 3. Data that we obtain concerning loading rates and niche contamination of the produce will allow us to use both artificial contamination analyses and downstream treatment of the same samples used in Objective 1 and 2 analyses. The same techniques used in the primary studies will be applied to Objective 3; however, the addition of treatment steps and an assessment of handling procedures will be added. <P>Objective 4. Our general approach for outreach-based education can be defined in 6 key steps: 1. Obtain research results and information 2. Develop outreach curriculum and teacher\\\'s kit 3. Establish a website for outreach-based materials 4. Develop a train-the-trainer program for potential deliverers of the program 5. Increase the visibility with science-based symposia 6. Review outreach curriculum, makes needed changes, and translate to Spanish Several extension-based audiences have been identified that could benefit from an outreach education program. Our primary audiences will include Extension Educators and the Food Industry; however, farmers, transportation/distribution outlets, and retailers would also be targeted. <P>Objective 5. The general content of the educational-based materials will be similar to the Extension-based materials, but, the structure will vary considerably. Our primary focus will be for undergraduate and graduate level education for students in food science, horticulture, environmental sciences, microbiology and related fields. Learning sessions will be developed with the following format to aid in undergraduate and graduate education: 1. Learning Session Objectives 2. Essential Terms 3. Core Content Material 4. Learning Session Summary 5. Learning Activities/Laboratory Exercises 6. Discussion Questions 7. Knowledge Impact Questions 8. References, Readings, and Websites The curriculum will be developed for both undergraduate and graduate level students. Innovative teaching methods will be selected and several methods of course distribution possibilities will be considered (including web based learning).
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PROGRESS: 2003/09 TO 2008/09 <BR>
OUTPUTS: A series of education modules, in the form of PowerPoint presentations with instructor notes, were developed as part of the outreach effort of this project. Instructional modules focused on five main criteria that included: Food Safety and Quality for Fruit and Vegetables, Soil Contamination and Control, Manure Contamination and Control, Water Contamination and Control, and Plant Contamination and Control. The PowerPoint presentations are available, in both English and Spanish, to be downloaded from the Purdue University Department of Food Science website at www.foodsci.purdue.edu . A 1-day train-the trainer program was held in Indiana for Extension Educators, State Health Department Officials, and the Food and Drug Administration so that a State training core could be developed. In turn, these participants plan to teach the program to industry stakeholders (growing operations, and manufacturers). These modules and activities represent the most significant outputs and dissemination activities produced during the duration of the funded project. <BR>PARTICIPANTS: Another important outcome of this project was the graduation of several graduate students working directly on this project, including Andrew Curtis (MS; currently at Gallo Wines), Leigh Farris (PhD; works in a small genetics lab), and YanYun Chen (PhD; soon to join Kellog Cereal). The theses titles are listed in the publications section. Two other graduate students contributed to the work; one is now employed by Heinz foods (Elisa Halim, MS), and the other continues to work on new produce safety projects that came out of this project (Amanda Deering, PhD student). In addition to the Purdue graduate students, several visiting undergraduates from Latin America and HBCUs also participated. These students include Shannon Coleman and Jamie Kendrick from Alabama A&M, and Eileen Enid Duarte Gomez and Axel Ramiro Morales Calel from the Zamorano Pan-American School of Agriculture in Honduras. All the undergraduates have finished their degrees and both Jamie and Eileen are enrolled in graduate school. Mussie Habteselassie was also an integral part of the research project. As a postdoctoral researcher he directed many aspects of the work, and helped to prepare the posters and manuscripts. Mussie has moved on to a Faculty position at the University of Georgia. <BR>TARGET AUDIENCES: As described in the Outputs section, a Produce Safety module was produced, and a 1-day train-the trainer program was held in Indiana for Extension Educators, State Health Department Officials, and the Food and Drug Administration so that a State training core could be developed. In turn, these participants plan to teach the program to industry stakeholders (growing operations, and manufacturers). <BR><BR>

IMPACT: 2003/09 TO 2008/09<BR>
One objective of this research was to use bioluminescence for assessing the survival and growth of E. coli 0157:H7 and S. poona on plant material. First, bioluminescent strains of each were constructed and applied to carbon utilization assays. Cells were assayed for increased luminescence over time in media containing glucose, pectin, or apple, cantaloupe, tomato, or alfalfa extracts. The results showed species dependent activity on the various carbon sources. Other assays used extracts from green and red tomatoes, and despite the abundance of free sugar in ripe tomato, the bacterial cells showed equivalent activity on both extracts. Another analysis looked at bacterial spread on alfalfa plants sprouted in sterile agar in test tubes. Emerging plants were contaminated with the bioluminescent strains at various points along the surface and allowed to grow. Bioluminescence was tracked, and the results showed that the bacterial cells spread to all surfaces of the plant, including the leaves during plant germination and growth. A third objective of this study was to understand the factors controlling the transmission of E. coli from soil and irrigation water to produce. Radish and lettuce were grown in soil inoculated with E. coli through uniform distribution in the soil, via manure, or via irrigation water. Irrigation treatment after seeding was the most effective vehicle of transmission as it moistened the seeds, creating maximum contact with E. coli, although the other treatments also resulted in contamination. The highest number of E. coli was associated with the rhizosphere, due to increased availability of carbon from exudates. The internalization of O157:H7 was shown in lettuce, as the attachment and entry of O157:H7-lux along the cut edges of lettuce leaves was demonstrated. The appearance of O157:H7-lux throughout the leaf pieces raises concern about lettuce processing, as it appears to expose multiple sites of pathogen entrance into the plant/leaf vascular system. Exposure of cut leaves to an aqueous suspension containing O157:H7 may result in total colonization of a leaf vascular system, and bagged salad greens have been identified as the vehicle for the transmission of E. coli O157:H7. Cantaloupe contamination experiments showed an increase in bacterial activity in scored tissue, especially at depths of 1-3 cm, compared to intact surface contamination, indicating that S. Poona may get water and nutrients from the mesocarp, whereas availability of water and nutrients is lacking on intact rind. S. Poona was detected up to 14 days in the scored tissue. Similar experiments with tomato showed the same effect: damage leads to serious contamination. In addition, experiments with scored tomatoes showed that the human pathogens are able to survive and remain active in both ripe and green fruit, despite the paucity of free sugars in the latter. Thus, once a fruit is contaminated at any stage of development, it may remain contaminated. All of these data have been presented at meeting and has been or will be published.