Foodborne Pathogens and Fresh Produce: Diagnostics, Colonization, and Controls

Non-Technical Summary: Human pathogen contamination of fresh produce has become a growing cause of outbreaks of foodborne illness in the United States including Oklahoma. Contamination of fresh produce by foodborne human pathogens can happen in the field (pre-harvest), or at down-stream nodes such as processing or packaging plants, distribution systems, and storage and marketing sites. Research that addresses critical issues throughout the fresh produce supply chain, such as rapid and accurate diagnostic methods, interactions between the human pathogens, host plants, and associated microflora, and practical prevention strategies will enhance significantly our ability to control the contamination of fresh produce by foodborne human pathogens. This project focuses on the development of rapid and accurate detection and differentiation methods for Salmonella, studying the survival and colonization of the pathogen on fresh produce (cantaloupe), and development of biological control agents against foodborne human pathogens on fresh produce. Results of this study will 1) increase our diagnostic capacity in detecting foodborne human pathogens either for daily monitoring or outbreak/forensic investigations; 2) provide a basic understanding of the mechanisms underlying the colonization of Salmonella on cantaloupe which is the foundation for the development of control strategies; 3) produce biological control agents that can be used against foodborne human pathogens on fresh produce. <P> Approach: The quantitative PCR for sensitive and selective detection and quantification of viable Salmonella in fresh produce will be achieved by treatment of samples with propidium monazide (PMA) prior to DNA extraction to render the DNA from non-viable cells incompatible with RT-PCR amplification. Subtyping methods, such as Multiple Loci Variable Number of Tandem Repeats Analysis (MLVA) and Single-nucleotide polymorphism (SNP), will be explored to differentiate Salmonella isolates that cannot be achieved by PFGE. For the characterization of the survival and growth of Salmonella on preharvest cantaloupes as influenced by the presence of plant pathogen E. tracheriphila, green house-grown cantaloupes will be inoculated with either or both pathogens that have been tagged with different fluorescent proteins, and the colonization/interaction among these pathogens will be monitored by convention cultural, molecular (PCR), and microscopic (scanning electron microscopy, SEM; and confocal laser scanning microscopy, CLSM) assays. To develop biological control agents against foodborne human pathogens on fresh produce, potential isolates, primarily bacteria, will be isolated from fresh produce (iceberg lettuce, spinach, tomatoes, cilantro, parsley, and jalapeno peppers) and screened for antagonism against E. coli O157:H7, Salmonella, and Shigella. Selected isolates will be characterized and evaluated on fresh produce by mimicking commercial practices.