1a. Objectives (from AD-416)
Objective 1. Investigate the mechanism(s) of introduction and transference of Shiga toxigenic E. coli (STEC) and Salmonella to fresh produce during growing, harvest, and postharvest handlings: (1a) Evaluate the growth and survival potential of Enterohemorrhagic E. coli (EHEC) and Salmonella in compost manure applied to leafy green fields and validate cross-contamination and temperature control steps in a Hazard Analysis Critical Control Point (HACCP) system for compost, (1b) Develop a science-based proximity assessment index for airborne bioaerosol emissions for tomato and fresh produce (leafy greens -LG) fields, (1c) Determine the influence of biocontrols and insect amplification and transmission of EHEC and Salmonella in tomato and leafy green crops, and (1d) Evaluate the role of cultivars and fertilization on leafy-green damage during processing and its potential for pathogen contamination. Objective 2. Determine the persistence and survival of pathogens on fresh and fresh-cut leafy green produce: (2a) Determine the persistence of pathogens introduced at different levels on spinach and lettuce plants to verify the adequacy of the California Leafy Greens Marketing Agreement (LGMA), (2b) Identify and evaluate suitable surrogate microorganism for generic E. coli in irrigation water, and (2c) Develop a field-scale-zero valent or slow sand filtration column to inactivate E. coli O157:H12 (surrogate strain) in a field study. Objective 3. Determine the effects of pathogen attachment, biofilm formation, and internalization on the extent of contamination of fresh produce: (3a) Determine the role of various virulence and stress factors on the ability of EHEC to persist on leafy green surfaces, and (3b) Determine the effect of biofilm formation on survival of STEC and Salmonella on fresh produce. Objective 4: Develop effective intervention technologies during processing to inactivate pathogens, and/or to reduce their survival and growth while maintaining produce quality and shelf-life: (4a) Minimizing pathogen transference and infiltration during produce harvesting and post-harvest handling, (4b) Optimizing fresh-cut produce wash system configurations and operations to improve pathogen inactivation and minimize cross-contamination, and (4c) Effect of temperature and packaging atmospheric conditions on survival, growth, and virulence of EHEC.
1b. Approach (from AD-416)
Mechanisms of introduction and transfer of pathogens on fresh produce (lettuce, spinach, tomatoes, leafy greens) at the farm level will be investigated. Growth and survival patterns of avirulent strains of Enterohemorrhagic E. coli (EHEC) and Salmonella will be evaluated in composted manure with different pile size and configuration. Deposition of airborne-microbes onto fresh produce will be evaluated during different times of the year to determine proximity distance between fields and suspected nearby source of contamination. The role of insect vectors in transmission of pathogens to fresh produce will be studied and biocontrols will be used as a potential deterrent to insects. Bacterial analysis will include the use of microbial culture and molecular methods to detect target pathogens in samples. Persistence of EHEC and Salmonella on fresh produce will be determined when these pathogens are introduced at different levels via irrigation water. Clostridium perfringens and coliphages will be evaluated as suitable indicators for fecal contamination of irrigation water. Zero-valent iron (ZVI) columns will be evaluated as an intervention for removing pathogens from irrigation water. The role of specific virulence and stress factors on the ability of EHEC to attach and persist on fresh produce will be determined. Wild-type and curli- and cellulose-deficient strains of EHEC and Salmonella will be evaluated for their attachment and biofilm formation on fresh produce; biofilm formation on foliar surfaces will be determined by confocal laser scanning microscopy (CLSM). Existing and novel new antimicrobial wash treatments which remove biofilm from foliar surfaces will be evaluated. The effect of tomato dump tank management parameters on the probability and extent of Salmonella infiltration will be determined; the infiltration pattern will be determined by CLSM. The effect of the wash-cut sequence on pathogen cross-contamination during cutting and washing of fresh produce will be investigated. To minimize the chlorine degradation of wash water used for fresh produce wash, chlorine stabilizer will be used in wash water to evaluate its effect on chlorine stability of wash water and additional pathogen reduction on fresh produce. Pathogen growth and virulence as impacted by temperature abuse at a retail level and modified atmosphere packaging (MAP) of fresh produce will be evaluated.
3. Progress Report
Persistence of coliphages, a potential indicator for fecal contamination of water, was evaluated on spinach. Coliphages (MS2 and phiX174) were inoculated on to spinach plants and recovery and enumeration techniques for phages were evaluated and optimized. The use of beef extract recovered slightly but significantly more phiX 174 phages than either sterile water or phosphate buffered saline from growing spinach plants after 20 days. When low levels of phage titers were applied to spinach plants, phiX 174 phages declined more rapidly on the surface of spinach plants than MS2 phage; however, at higher phage levels, MS2 phages declined more rapidly than phiX174 phages. The role of curli (a bacteria surface appendage) was investigated relative to its function in bacterial attachment to produce surfaces. The curli deficient mutants didn’t express curli at 20, 26, and 37ºC, whereas wild strain and curli +ve strain developed a clear red phenotype on CR medium at 20 and 27ºC. Curli deficient mutants attached to spinach leaves at significantly lower numbers than the wild strain. Lack of cellulose expression did not affect its attachment to spinach leaves. Confocal microscopy analysis of inoculated spinach revealed that bacteria firmly persisting on leaf surface were surrounded by an extracellular matrix recognized by rabbit anti-curli antibodies.