Goals: Although previous studies support that Listeria monocytogenes, Salmonella and Escherichia coli can persist in the food processing plant environment, risk factors for pathogen persistence (i.e., plant practices or pathogen characteristics) have not yet been fully elucidated and there is a clear need to identify risk factors for persistence and develop mitigation strategies to control pathogen persistence. Through the use of combined testing and molecular subtyping, we will identify persistent L. monocytogenes, Salmonella and E. coli O157:H7 strains to be characterized by phenotypic assays in order to probe phenotypes that may contribute to persistence. <P>
Objectives/Expected Outcomes: <OL> <LI> Conduct longitudinal studies in fresh meat, RTE meat and smoked seafood processing plants to detect key foodborne pathogens in the plant environment and associated food products. Upon completion of Objective I, we will have tested approximately 6,300 samples from at least 15 different food processing plants to isolate and detect L. monocytogenes, Salmonella and E. coli O157:H7. In addition, we will have collected data regarding physical plant characteristics, production and process characteristics along with cleaning and sanitation practices. <LI> Subtype isolates from Obj. I to identify persistent strains, elucidate persistent strain transmission patterns and identify risk factors for persistence. Upon completion of this objective, we will have characterized L. monocytogenes, Salmonella, and E. coli O157:H7 isolates from food products and their associated processing environments from Obj. I by PFGE typing. Patterns from PFGE typing will be deposited into PathogenTracker enabling plants to monitor contamination patterns. Identification of persistent strains and their associated harborage sites, surveillance of contamination patterns and analyses to identify risk factors for pathogen persistence will be used to improve general understanding of pathogen persistence and to develop mitigation strategies. <LI> Compare the ability of persistent strains and transient strains representing each target pathogen to adhere to food contact surfaces and resist sanitizers. Upon completion of this objective, we will have characterized the phenotypes (i.e., sanitizer resistance and adherence to food contact surfaces) for representative persistent and transient strains belonging to each target pathogen from the environments of fresh beef, RTE meat and RTE seafood plants. <LI> Extend knowledge regarding persistence of pathogens in food processing plants, including risk factors for persistence and mitigation strategies to control persistent strains. After completion of this objective, we anticipate that we will have (i) conducted at least three one-day "Train-the-trainer" and industry workshops and (ii) held at least one Webinar series for processors. We anticipate that these outreach activities will at least reach 300 to 400 food safety professionals in the U.S. In addition, we will have developed and distributed training materials on pathogen persistence to a variety of outreach and extension personnel through various channels.
NON-TECHNICAL SUMMARY: More than 1,000 of 1,800 deaths caused by known foodborne pathogens can be attributed to Escherichia coli O157, Listeria monocytogenes, and Salmonella. "DNA fingerprinting" or molecular subtyping differentiates isolates beyond the species and serotype level and molecular subtyping is routinely to monitor foodborne pathogen transmission dynamics. Recent multistate outbreaks of foodborne illness attributed to cross-contamination of foods by Listeria monocytogenes or Salmonella strains that persisted in the food processing plant environment for extended periods highlight the continued need for research and training on L. monocytogenes persistence and knowledge gaps regarding Salmonella and Escherichia coli O157 persistence. The overall goal of this project is to integrate applied research and outreach to augment knowledge regarding pathogen persistence. Combined field studies and molecular subtyping will be performed to identify L. monocytogenes, Salmonella and E. coli O157:H7 strains that persist in the food processing plant environment to probe risk factors for persistence and phenotypes that may contribute to persistence. In order to accomplish this overall goal, we will (i) conduct longitudinal studies in fresh meat, RTE meat and smoked seafood processing plants to detect L. monocytogenes, Salmonella and E. coli O157 in the plant environment and associated food products, (ii) subtype isolates to identify persistent strains, elucidate persistent strain transmission patterns and identify risk factors for persistence, (iii) compare the ability of persistent and transient strains representing each target pathogen to adhere to food contact surfaces and resist sanitizers and (iv) extend knowledge regarding persistence of pathogens in food processing plants, including risk factors for persistence and mitigation strategies to control persistence. Knowledge gained will be disseminated to food processors and trainers through a series of outreach activities designed to provide fundamental knowledge regarding pathogen persistence, identification of persistent strains and monitoring transmission patterns, risk factors for persistence and mitigation strategies to control persistence.
APPROACH: Objective I. Researchers at Colorado State University will enroll at least five fresh meat and five RTE meat plants, while researchers at Cornell University will enroll at least five smoked seafood plants in the study. A standardized questionnaire will be administered to collect data on plant physical characteristics, production and process practices and sanitation procedures. Routine sample collections will be performed at each plant on a bi-monthly basis during the first two years of this project. During each sample collection, at least 25 environmental sponge samples will be collected from non-food contact surfaces, at least five environmental sponge samples will be collected from food contact surfaces and five food product samples will be collected. Packaged finished RTE food product samples will be collected from RTE meat and smoked seafood processing plants, while trim and/or fresh ground beef samples will be collected from fresh beef operations. All samples will be microbiologically analyzed to isolate and detect L. monocytogenes, Salmonella, and E. coli O157:H7. Objective II. We will perform pulsed field gel electrophoresis (PFGE) typing of L. monocytogenes, Salmonella and E. coli O157:H7 isolates following standard PFGE protocols developed by the Centers for Disease Control and Prevention (CDC) and deposit PFGE patterns and associated epidemiological information in the publicly available WWW PathogenTracker database. We will also perform statistical analysis to initially identify risk factors for pathogen persistence by probing associations between data collected trough questionnaires and contamination events. Objective III. We will characterize adherence and sanitizer resistance of persistent and transient strains representing each target pathogen on various food contact surfaces, including (i) stainless steel surfaces, (ii) cutting boards and (ii) conveyor belt materials. Food contact surfaces with adherent pathogen populations will be submerged or situated to float in different substrates (i.e., fresh decontamination runoff fluids and fabrication/grinding runoff fluids) and will be stored under different temperatures (i.e., 4oC and 15oC). The effectiveness of sanitizers (e.g., acidified sodium chlorite, sodium hypochlorite, quaternary ammonium compound, peroxyacetic acid, peracetic acid/octanoic acid mixture, cetylpyridinium chloride, and Virkon S) at maximum approved concentrations and other levels for inactivation or sub-lethal injury. Objective IV. Methods to accomplish this objective include (i) regular communication of testing and molecular subtyping results to each plant and annual in-plant trainings, (ii) develop of short one-to-two page fact-sheets on pathogen persistence in the food processing plant environment, (iii) conduct a web-based seminar series to provide information on pathogen persistence to processors and (iv) develop and deliver workshops targeted towards food safety professionals in industry and trainers to provide information on pathogen persistence and mitigation strategies. A project WWW webpage will be developed to make all training materials accessible to food processors, trainers and other stakeholders.