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OBJECTIVES: 1) Design attenuated strains from virulent pathogen isolates as well as isolate potential surrogates from animal fecal matter and compare their phenotypic traits (growth, attachment to soil and plants, and susceptibility to environmental and biological stress conditions) in both growth chamber and field studies. <br/>2) Determine the role of microbial soil diversity in human enteric pathogen survival when systems are subjected to different moisture and temperature conditions. <br/>3) Assess the role of plant defenses on the fate of surface and internalized pathogens in leafy greens. <br/>4) Develop a metabolomics protocol for rapid detection (less than 8 h) of human enteric pathogens in plant tissues and other environmental sources by providing enrichment conditions that would create pathogen-specific volatile signatures that could be detected by rapid-GC headspace analyses.
Non-Technical Summary:<br/>
Determining the persistence of human enteric bacterial pathogens in pre-harvest produce field systems has been hampered by the limited number of available surrogates for these pathogens, the lack of tools to monitor pathogen presence rapidly, and the availability of assays that would be indicative of the contribution of biological antagonism to pathogen inactivation. This project will address these weaknesses and information gaps through four main thrusts: construct or isolate non-virulent surrogates to human enteric bacterial pathogens for use in field studies; evaluate the role of biological antagonism to inactivation of pathogens in soil by measuring microbial diversity and their associated activity through molecular and enzymatic assays; determine whether plant defenses are activated in response to pathogen intrusion; and develop a method to detect pathogens on plant tissue within eight hours based on the metabolic volatile compounds generated by the pathogens.
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Approach:<br/>
To address the first objective, potential non-virulent surrogates of human bacterial enteric pathogens will be constructed as well as collected from natural sources for subsequent laboratory comparison to the virulent pathogens for their capacity to attach and to survive on plant tissue and soil particles. Promising surrogates will then be introduced into field systems where leafy greens and other fresh-cut produce items are being grown. To address the second objective, soils exhibiting different microbial diversities will be collected from different regions of the country and inoculated with human bacterial enteric pathogens for determination of the persistence of those pathogens at either 25 degrees or 35 degrees C and in the presence of different moisture conditions (30 or 60% water holding capacity). Several indigenous microbial groups (copiotrophic, oligotrophic, and actinomycete) and enzyme activities (dehydrogenase, phosphatase, urease, and ?-glucosidase) within the soil will also be monitored in these soils to determine whether they are indicative of the microbial diversity and in turn, the capacity of the soil to eliminate enteric pathogens introduced into the system. To address the third objective, leafy greens will be grown to a harvestable size in growth chambers and then sprayed with a low dose inoculum of E. coli O157:H7. Internalized pathogen populations and expression of plant defenses will be monitored initially and on 1 and 2 days post-exposure to determine whether plants actively respond to the invasive agent. To address the fourth objective, the formulation of culture media will be modified to include chemicals that would lead to enhanced growth and the generation of unique metabolic volatile profiles by specific human bacterial enteric pathogens contaminating lettuce tissue initially at low levels (< 10 CFU/g). Differentiation of the pathogens by their volatile profiles would be achieved by sampling with a fast gas chromatography instrument (Znose -Registered sign) the headspace of samples cultured for short periods (< 7 h) in this media recipe and applying pattern recognition statistical programs to the data.