Summary of Awards to Date

Comparison of surrogate E. coli survival and epidemiology in the phyllosphere of diverse leafy green crops.


Apr. 1, 2009 - Mar. 31, 2010

Award Number


Amount Awarded



Trevor Suslow, Ph.D.
University of California, Davis


Steven Koike, Richard Smith, Michael Cahn, Ph.D.


The validation and application of surrogates, safe substitutes for pathogens, to study critical aspects of contamination, spread of contaminants during handling, open-environment ecological studies of survival during production, and controls and interventions during processing has had immeasurable value with many food borne pathogens Specific isolates of nonpathogenic E. coli that have been selected for environmental, soil, and leaf surface fitness on lettuce and spinach will be further qualified as surrogates for E. coli O157:H7. These will be made available to the research community to facilitate broad controlled environment and field-based experiments simulating pathogen dispersal, survival, and growth potential on lettuce and leafy greens. Up to fourteen different types and varieties of leafy greens including leaf lettuce, spinach, endive, and mini-greens used in Spring Mix salads will be tested in two seasonal plantings for their comparative capacity to support growth of surrogate E. coli. A separate field trial will investigate the affect of nitrogen application levels on surrogate E. coli survival and growth. By conducting these tests, the database that supports the establishment of industry standards for microbiological quality will be significantly expanded. In separate but related efforts, a regional team of Cooperative Extension Advisors and a campus-based Extension Specialist will conduct detailed microbiological grid-analysis of grower/handler fields, identified as positive for foodborne pathogens, to help identify risk factors and localize natural contamination events. These findings will be widely shared to improve collective food safety management and the best use of rapid pathogen detection screening by the leafy greens industry.

Technical Abstract

The overarching goal of this project is to further validate nonpathogenic surrogates for Escherichia coli O157:H7, and by extension non-O157 enterohaemorrhagic E. coli (EHEC), on diverse leafy greens in an open environment as models to better understand the survival, growth, dispersal, and transference of the pathogenic forms under relevant field conditions. The combined integrated objectives are to 1) Compare the foliar survival and growth of specific
isolates of nonpathogenic E. coli to non-toxigenic (attenuated) E. coli O157:H7 on diverse types and cultivars of lettuce and leafy greens during late spring and late summer conditions.; 2) Conduct microbiological grid-analysis of grower/handler fields, identified as positive for EHEC, to localize natural contamination events in relation to the presence of presumptive indicators.; and 3) Assess the survival of attenuated E. coli O157:H7 on field grown lettuce produced under different nitrogen fertilizer levels. For field trials with antibiotic-resistance-marked inoculum, a dose of log 4 CFU/ml will be spray-inoculated onto the foliage of established lettuce and leafy greens during late spring and late-summer plantings to span the seasonal association of historical risk of outbreak by EHEC on product produced in the CA Central Coast region. Periodic sampling of plants and recovery by direct enumeration on selective/differential agar media and by selective enrichment will be conducted using established protocols from prior field research. At least three sampling dates are proposed for each of three inoculated field trials. Molecular
confirmation, either Pulsed Field Gel Electrophoresis (generic E. coli) or real-time PCR (attenuated E. coli O157:H7), will be conducted on representative colonies recovered from sampling timepoints. In events of suspected natural contamination, we plan to extend our opportunity to understand field distribution of pathogens in relation to current fecal indicators, as well as biological and ecological aspects of microbial contamination. We will coordinate with local produce companies that would alert our Monterey County-based UC Cooperative Extension team when a presumptive pathogen contamination possibility has been identified. We anticipate conducting in-depth examination of a maximum of 3 fields targeting the late summer (August through October) period. In rapid response to notification, a 60-point sampling grid based on the field configuration would be established. Pertinent on-site risk factors will be evaluated. Standardized sampling regimes will be used to determine whether confirmation of contamination is possible as well as to characterize the distribution of E. coli and other indicators. We anticipate that this risk assessment data will be directly applicable to reassessments of microbiological standards and metrics for the leafy greens industry. We expect that this data will be most pertinent to setting practical expectations for irrigation and foliar applied water standards and testing regimes. Furthermore, the result of a rapid local response to presumptively contaminated lots will potentially expand the public knowledge base for the distribution and sources of seasonal contamination. A further benefit, we believe, will be an improved science-basis for continuing dialogue on setting realistic risk expectations with public health regulatory and enforcement agencies.