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<oL> <LI> Investigate the population dynamics of Escherichia coli O157:H7 on plant roots grown under various irrigation water salinity regimes in the field and growth chamber. <LI>Investigate persistence of E. coli O157:H7 in soil, irrigation water, and rhizophere under different environmental conditions. <LI>Utilize fluorescence-activated cell sorter (FACS) technologies to determine genes that are specifically expressed by E. coli O157:H7 cells on plant surfaces. <LI> Construct knock-out mutations in E. coli O157:H7 genes that are specifically expressed in the root and test their involvement in survival and interactions with indigenous microflora. <LI> Develop predictive models for E. coli O157:H7 persistence/survival in soil and on plants grown under field conditions.
NON-TECHNICAL SUMMARY: Organic amendments and waste water frequently contain high levels of salinity, and can be a major factor in altering the root systems of plants to facilitate the entry and transport of bacteria into plant tissues. There has been no research to date that has revealed a "kill" step that both reliably destroys pathogenic contamination and retains a level of quality acceptable to consumers. Consequently, contamination by the pathogen must be prevented at all steps of the supply chain, and the first step to accomplish this begins in the field. The most likely sources of E. coli O157:H7 in the production (growing) environment include some of the following: irrigation water quality and manure (feces) associated with cattle and to some extend workers and wildlife. A major objective of this study will be the acquisition of knowledge on risk factors that affect the growth and spread of E. coli O157:H7 on field crops and that can be used to model the likelihood for spread of the pathogen on crop plants. Our studies will focus in particular on identification of genes that are used by E. coli O157:H7 during colonization and growth on plants, and which therefore may influence survival and spread of the pathogen within the field. The data that we will obtain from these studies will then be used to develop models for prediction of how E. coli O157:H7 is influenced by different environmental factors, agricultural practices, and interactions with the indigenous microflora.
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APPROACH: We will use fluorescence microscopy methods to quantitatively study the population dynamics of enteric bacteria on plant surfaces that will show the interactions of O157:H7 strains with indigenous microorganisms and the location of this pathogen in particular microsites and high-throughput fluorescence-activated cell sorter (FACS) to identify genes up-regulated by E. coli O157:H7 cells on plant. We will also use specific real time PCR probes to quantitatively monitor survival/persistence of E. coli in soil, rhizosphere, and on leaf surfaces. Specific evaluations of persistence of E. coli O157:H7 in the lab, growth chamber, and lysimeters at different irrigation water qualities, salinity and other environmental conditions will be determined. Finally, we will model the interaction between environmental factors, population changes, and genes that are expressed during growth of bacterial pathogens in the rhizosphere and on leaf surfaces.