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The overall goal of the project is to investigate methods to detect and control foodborne pathogens Salmonella spp., Escherichia coli O157:H7, Listeria monocytogenes, and Campylobacter jejuni and to understand key parameters that allow their persistence in the environment and resistance to decontamination or control procedures. The recombinant bioluminescent pathogens will provide insight into factors affecting survival, growth, contamination, and activity of natural food-borne pathogens throughout production, processing and packaging. Bioluminescent bacteria and recombinant bacteriophage will be used to evaluate detection and control strategies for the target foodborne pathogens. UV and E-beam assisted methods for immobilizing bacteriophage will be optimized for detection and control of bacteria in food packages. <P>Specific Objectives include:<OL> <LI> Construct pathogen-specific recombinant bacteriophage to identify the infected host and develop large scale production strategies for the resultant phage<LI> Investigate factors affecting survival and control of foodborne bacteria in fresh produce, minimally-processed foods, or processing equipment <LI> Evaluate methods to immobilize bacteriophage (or other antimicrobials) for detection and control of pathogens.</ol>
Expected Outputs from this project include: recombinant bacteriophage for detection of foodborne pathogens, methods to produce and purify large numbers of phage, experiments to identify factors affecting survival and control of bacteria in fresh produce, minimally-processed foods, or processing equipment, methods to immobilize phage onto packaging materials, and experiments to determine the activity of immobilized phage.
NON-TECHNICAL SUMMARY: Foodborne pathogens are a serious concern in many fresh and minimally-processed foods. Several approaches are possible for addressing this concern. In this project, one approach is the development of rapid methods to detect or control pathogens in the food product. Another approach is to develop methods to track the survival and growth of bacteria throughout the production, harvest, processing, and retail sales to understand those factors affecting the bacterial populations when product reaches the consumer. This project will pursue the development of genetically modified viruses called bacteriophage, for detecting and controlling pathogens on foods, processing equipment, or in the food package. As a result of this project, better knowledge of the factors that affect survival and control of foodborne bacteria on surfaces of produce should be gained. Bacteria-specific viruses immobilized onto food package surfaces will be evaluated for effectiveness in detecting and controlling bacteria.
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APPROACH: Obj.1: This research will generate and purify phages designed to selectively detect pathogenic bacteria. Initial work will focus on generation of E. coli O157:H7 specific bacteriophage utilizing the bacteriophage phi-V10. The phage will initially be modified by inserting cobA into the phages genome using the lambda red mediated homologous recombination system described by others. Expression of the cobA results in the production of red fluorescent pigment which can be observed in visible light. Insertion of the entire luxCDABE will also be attempted. Large quantities of phage are typically grown in its specific host which in this case is E. coli O157:H7. This is problematic for two reasons: first, growing hundreds of liters of E. coli O157:H7 poses a significant risk and would require substantially more monitoring than existing bacterial-based biotechnology protocols; second, if a single O157:H7 used in production is found in the commercialized phage assay, it would be catastrophic to the commercialization. To avoid this potential liability issue we have isolated a phi-V10 lysogen of a non pathogenic E. coli Top10 strain and showed that it can infect E. coli O157:H7 after genetic modification. We plan to increase the yield from the lysogens by manipulating the expression of the repressor, GP40, which we have shown to be capable of repressing the lytic cycle. Because the lysis genes will have been deleted from reporter phage, the lysis genes will have to be expressed from the inducible promoter as well. It is necessary to produce a purified phage preparation free of cell debris which could interfere with immobilization. Purification will be done by column chromatography using a gradient HPLC system. Purification experiments will focus on improving efficiency of this method. Obj. 2: In this effort, foodborne pathogens including E. coli O157:H7, Salmonella enteritidis, S. cholerasuis , S. typhimurium, and L. monocytogenes. The energy dependence of the lux system allows utilization of bioluminescence to monitor gene general cell physiology. These bioluminescent strains will be used to examine bioavailable carbon and overall environmental conditions influencing cell metabolism associated with fresh produce, minimally processed foods and contact surfaces. In situ monitoring of bioluminescence will be used to evaluate antimicrobial treatments for decontamination of fresh produce. Obj. 3: Selected recombinant bacteriophage will be immobilized onto various substrates using both UV and E-beam curable monomers. The minimum monomer layer thickness needed for proper phage attachment and minimum dissolution will be evaluated. Various polymer supports including polyethylene (HDPE, LDPE) and polypropylene will be examined. The activity of the phage on the polymer surface will be measured along with possible dissolution in buffered wash solution using a plaque assay. Successfully immobilized, active phage coupons will be tested for their effectiveness in detecting or controlling bacteria in food packages. For example, immobilized phage for E. coli O157:H7, could be placed in a package with bagged lettuce previously inoculated with the bacteria.