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The specific project objectives will extend current work on the influences of fertility management of spinach for packaged salads on attachment, and colonization of E. coli O157:H7 and initiate novel research on the development of microbial communities under agronomic practices intended to optimize crop yield and quality. The core hypothesis is that non-pathogenic microbial communities limit the persistence and colonization of human pathogens introduced to soil, but the limitation will depend on the specific agronomic practices and seasonal influences. A corollary hypothesis is then that directed shifts in microbial community abundance and composition will be an effective intervention for food safety risk reduction. The focus of the anticipated outcomes addresses the competitiveness of E. coli O157:H7, and its potential for survival and growth in soil between and within crop cycles of spinach. Research will be conducted to determine the influence of nitrogen fertilization on microbial communities within different cropping conditions (conventional versus organic) of spinach and develop a community fingerprint (taxonomic information) of common bacteria that develop in these systems in relation to E. coli O157:H7 persistence and abundance in soil. The primary long-term outcomes will result in broader science-based guidance and practical interventions in setting sound food safety policies, standards, and criteria for persistence of E. coli O157:H7 introduced to cropped soil.
NON-TECHNICAL SUMMARY: Food borne illnesses associated with consumption of leafy greens have increased noticeably in the last ten years with more than 20 outbreaks involving E. coli O157:H7 on lettuce, mixed leafy greens, and spinach. The consumption of spinach alone, over the last decade, has significantly increased from 1.6 pounds per person in the 1990's to over 2.4 pounds per person in 2005. That spinach consumption has increased, and therefore the proportional risk of sporadic illness (if levels of pathogen contamination have remained constant) is not surprising. This project addresses the persistence of enterohaemorrhagic E. coli on fresh spinach in the preharvest environment. It specifically focuses on the potential for survival and growth of E. coli O157:H7in a fluctuating and potentially competitive soil environment. The specific objectives will evaluate the influences of fertility management on the plant in relation to microbial community development and colonization of E. coli O157:H7 of under these agronomic practices. Comparisons of applied nitrogen concentration or nitrogen-form, in particular, are anticipated to reveal food safety risk factors of fresh spinach and other leafy greens. Fertility inputs effect changes in leaf morphology, potentially elevating risk due directly to changes in pathogen survival, attachment, growth, and internalization. These factors are directly involved in setting standards and `metrics' for replant timing and pre-harvest intervals that are especially critical for spinach and other mechanically harvested mini-greens and spring-mix, commodities. <P>APPROACH: A combined program of model studies with regionally relevant E. coli O157:H7 isolates in challenge inoculation studies within greenhouse, controlled experimental farm facilities, and commercial plantings will be conducted. The approaches used will determine whether excess nitrogen fertilization, regardless of cropping system will shift microbial community composition in a manner that elevates the relative risk of the survival of human pathogens in the soil, transference to edible plant structures, and plant surface or sub-surface colonization. The experimental approach will involve basic plant culture for spinach under different fertility management schemes. For the selected fields, each plot will be organized in a split-plot design with nitrogen and fumigation treatments as the fixed variables (4 treatments per variable). Nitrogen applications will range from 50-200 ppm total N2 (nitrate to ammonia ratio of 75:25). Leaf area and thickness for leaves will be determined for different treatments. Nitrate, ammonia and total sugars and sugar ratio will be compared across treatments and plantings. Leaf structure will be analyzed using light microscopy at 4 and 10X magnification for cross sections of the leaves of 150um. Leaf upper and lower epidermis will be also evaluated for the determination of cracks and stomatal incidence associated with each cropping system. Electrolyte leakage will be carried out as an indicator of tissue integrity. Leaf fragility will be analyzed using a texture analyzer (TA.XTiT instrument). Standard microbiology will be used to introduce and recover attenuated (non-toxigenic) isolates of E. coli O157:H7 from soilless mix in hydroponic systems and soil, rhizosphere, shoots, and irrigation run-off in field settings. In addition, where populations fall below standard detection limits, sensitive concentration and enrichment protocols will be used in combination with detection by semi-quantitative real-time PCR. Within this project we will characterize microbial communities, the relationships between environmental properties, nitrogen fertilization, presence of specific pathogens, and microbial properties using canonical correspondence analysis. Total bacterial community DNA will be extracted from soil core samples in duplicate following a humic acid clean-up step. The pure DNA will be amplified by Polymerase chain reaction using primers, denoted 968F and 1401R, which span the region between nucleotides 968 and 1401 of the 16S rRNA gene. This region includes the variable regions V6 through V8 of E. coli. These amplified small subunit rDNA will be further purified. Phospholipid fatty acid (PLFA) biomarkers is a well established technique for rapid screening of microbial communities in which membrane lipids extracted from organisms provide a fingerprint of the community. PLFA will be extracted from soil core samples obtained from each replicate microcosm and split into polar and non-polar lipid fractions. Terminal restriction fragment length polymorphism (TRLFP) and denaturing gradient gel electrophoresis (DGGE) are DNA-based techniques that will be used for amplification and separation of target sequences for analysis.