2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Infection of humans by food borne pathogens is a significant cause of morbidity and mortality worldwide. As summarized in a Scientific Status Summary prepared in 2004 by the Institute of Food Technologists, each year in the United States alone there are an estimated 76 million cases of food borne illness that result in some 325,000 hospitalizations and 5,000 deaths, with attendant losses ranging from about $6 to $35 billion dollars in medical costs and lost productivity. The food borne bacterial pathogens, Salmonella, Campylobacter, Listeria monocytogenes, and Escherichia coli O157:H7 are the most significant based on the number of cases caused by these agents and/or the severity of the ensuing disease. In the aftermath of the catastrophic events of 11 September 2001 and the "anthrax-by-mail attack" shortly thereafter, food microbiologists must also address breaches to the security of our Nation's food supply that may arise due to a terroristic addition of threat agents to foods. One of the most likely bacterial bioterrorism agents is Bacillus anthracis. Thus, a primary focus of this proposal is the characterization and control of L. monocytogenes (Gram-positive bacterium), Salmonella spp. and E. coli O157:H7 (Gram-negative bacteria), and B. anthracis (Gram-positive, spore-forming bacterium) in higher risk foods such as milk, ready-to-eat (RTE) meats and poultry, and Hispanic-style cheese as these pairings pose a significant and immediate threat to food safety and security. Pathogenic microbes exhibit a variety of uncharacterized responses to foods, the handling of foods before processing, the sequence of food processing steps, and the conditions under which foods are processed and stored. Determining the ecological niches, persistence, and physiological responses that are initiated by pathogens under the conditions particular to foods and food processing conditions is an essential prerequisite for the development of directed detection and intervention methods for human pathogens in foods. In light of existing and pending regulatory policies and the paucity of available literature, research is needed to better quantify the association and fate of select food borne pathogens, notably Listeria monocytogenes, and select bioterror threat agents, notably Bacillus anthracis, in higher risk foods such as ready-to eat, comminuted, tenderized,/injected, and/or fermented meat and poultry products, Hispanic-style cheese, and/or fluid milk, and to validate methods to detect and control these agents across a given food system. At present, the identity, nature, and number of genes and proteins essential for pathogens to survive in food are largely unknown. This project will provide information allowing us to identify both regulatory and specific elements, at the gene and protein levels, that are responsible for conferring growth or survival advantages to pathogens in foods, food environments, and under conditions associated with food processing or storage. Ultimately we will use the information on these survival processes to inhibit, control, or otherwise destroy pathogens associated with foods to better protect consumers against foodborne diseases. The overall goal of this research is to reduce the occurrence, risk, and severity of illness associated with consumption of foods contaminated with pathogenic microorganisms. This project will focus on the following three main objectives aimed at increasing our understanding of pathogen persistence in foods and in turn developing and evaluating effective interventions to enhance the safety and security of our Nation's food supply: i) elucidate the ecology (persistence, predominance, behavior, community analysis) of pathogens in various food matrices; specifically focus on foods considered high risk by the stakeholder regulatory agencies (FSIS and FDA), for example ready-to-eat foods, or foods with a short shelf life, ii) develop and validate intervention strategies used either alone or in combination with other processes for pathogen control, and iii) elucidate/define (including at the molecular level) the pathogens physiological responses to various intervention strategies and processes. Examine the influence of the inherent food macro and micro-environments. The proposed research specifically addresses the goals of National Program 108, Food Safety. More specifically, this project focuses on genomic/proteomic analyses of pathogens at points throughout food processing to provide the data necessary for characterizing, monitoring, and controlling undesirable bacteria under sections 1.2.3 Production and Processing Ecology; 1.2.5 Omics; 1.2.8 Pathogenicity: and 1.2.9 Food Security. The research addresses Agency Performance Measure 3.1.2: Develop and transfer to Federal agencies and the private sector systems that rapidly and accurately detect, identify, and differentiate the most critical and economically important food borne microbial pathogens. Target: Develop practices and/or products that reduce post-harvest contamination of major animal- and plant-derived food products. 2.List by year the currently approved milestones (indicators of research progress) FY 2006 Recover L. monocytogenes and other pathogens from dairy farms, cheese plants, and retail. Recover Salmonella and L. monocytogenes from poultry processing facilities. Initiate studies for control of L. monocytogenes in hams, frankfurters, and turkey breasts. Initiate studies on use of HPP to control B. anthracis. Map pathogen distribution on poultry. Initiate studies on gene and protein expression profiles of L. monocytogenes FY 2007 Complete recovery of L. monocytogenes and other pathogens from dairy farms, plants, and retail in Mexico and Brazil. Complete studies on control of L. monocytogenes in hams, frankfurters, and turkey breasts. Initiate studies on cooking turkey breast and steaks to control L. monocytogenes and E. coli O157:H7. Evaluate pathogen persistence during poultry processing. Complete studies on gene and protein expression profiles of L. monocytogenes FY2008 Conduct molecular subtyping of pathogens recovered from dairy and poultry environments/products. Complete studies on cooking turkey breast and steaks to control L. monocytogenes and E. coli O157:H7. Complete experiments using microfiltration to remove B. anthracis from fluid milk. Complete pathogen mapping studies. Complete studies to identify genes and proteins from profiling data related to survival of L. monocytogenes. Initiate construction and profiling of mutants of L. monocytogenes. FY2009 Complete molecular subtyping of pathogens recovered from dairy and poultry environments/products. Evaluate select strains for survival in relevant foods Complete experiments using HPP to control B. anthracis in milk and meats. Complete studies on pathogen persistence related to poultry processing. Complete experiments to generate and characterize specific mutations in genes/proteins of interest. FY2010 Finish experiments, transfer technology, make recommendations to enhance safety of Hispanic-type cheese and RTE and raw poultry products. Upgrade software for ERRC Process Simulator to include mass and energy balances for lethality of pathogens in foods. Complete studies to evaluate parental and mutant strains directly in foods. 4a.List the single most significant research accomplishment during FY 2006. This project recently completed the OSQR NP108 review process. See the report for 1935-42000-052-00D. 4b.List other significant research accomplishment(s), if any. This project recently completed the OSQR NP108 review process. See the report for 1935-42000-052-00D. 4c.List significant activities that support special target populations. This project recently completed the OSQR NP108 review process. See the report for 1935-42000-052-00D. 4d.Progress report. 1935-41420-012-01S: This report serves to document research conducted under a Specific Cooperative Agreement between ARS and the University of Wisconsin. Genomic analysis of Listeria monocytogenes has revealed a large number of surface proteins which might participate in adherence or invasion of mammalian cells. We investigated the role of the prominent listerial surface protein, p60, and several serotype 4b-specific surface antigens associated with teichoic acid glycosylation, in the pathogenesis of gastrointestinal listeriosis. At the time we initiated these experiments, there was little or no information as to whether these virulence-related determinants contribute to food contamination and foodborne illness. Serotype 4b-specific surface antigens associated with the teichoic acid of the cell wall were identified by immunological and genetic approaches. Mutants deficient in these surface antigens revealed that they are essential for invasion of mammalian cells in culture. We investigated several of these mutants for their virulence in a mouse model of gastrointestinal listeriosis and for their ability to invade and multiply within Caco-2 human intestinal epithelial cells. In particular, we noted that two different mutants of gtcA (which places glucose or galactose residues on the teichoic acid of L. monocytogenes serotype 4b) were significantly less virulent than their respective wild type parent strains when inoculated into the g.i. tract of mice. We also found that the mutant strains were less able to invade Caco-2 cells in vitro, and multiplied to a lower final number of listerial cells than did the wild type parent strain of L. monocytogenes. As a result of these promising results, we will focus on how gtcA influences the virulence of L. monocytogenes in the gastrointestinal tract. This will be done by generating mutants of the gtcA gene in serotype 4b strains isolated from food products and patients in the 1998-99 and 2002 outbreaks of listeriosis, that were attributable to contamination of ready-to-eat hot dogs and deli turkey lunch meat, respectively. The influence of gtcA expression on adherence and growth of the L. monocytogenes strains on deli turkey meat and other RTE meats will be assessed. These mutant strains, and their parental wild type strains, will then be assessed for virulence in the A/J mouse model of gastrointestinal listeriosis and for their ability to invade intestinal epithelial cells (Caco-2) and hepatocytes (TIB73 cells) in vitro. The in vitro cell line studies will also provide a means of assessing what mechanisms and mammalian cell targets are involved in the attachment and invasion of relevant cell types by gtcA-expressing L. monocytogenes. These experiments will include the use of selective inhibitors (e.g. monosaccharides, RGD peptides, and other inhibitors of surface proteins likely to be invoked in bacterial cell attachment), to assess receptor-ligand pairs that facilitate attachment and internalization of L. monocytogenes. Other approaches will include the use of confocal microscopy to identify the particular intracellular niche occupied by the listerial cells, and the use of microarrays to identify signaling pathways that are regulated by exposure to gtcA positive and negative strains of L. monocytogenes. The fate of the infected cells (e.g. apoptosis, cytokine release) will be assessed. 1935-41420-012-04G: This report serves to document research conducted under a General Assistance Type Agreement between ARS and the University of Wisconsin. The long-term goal of this project is to better understand bacterial and host factors that influence the virulence of L. monocytogenes in the gastrointestinal (g.i.) tract. In previous studies we showed that the L. monocytogenes wild type parent strain F2365 is relatively virulent for A/J mice in a model of gastrointestinal listeriosis. Mice inoculated with 10*5 to 10*6 CFU develop significant systemic infection. Of 14 transposon mutants of the Crp/Fnr global regulatory genes that were examined, one mutant (prfA) was essentially avirulent in terms of its ability to cause systemic infection of the spleen and liver, even following intragastric inoculation with as many as 10*9 CFU. This observation is consistent with previous reports that prfA regulates a number of virulence genes that are important for L. monocytogenes following parenteral inoculation into mice. Curiously, this prfA mutant survives well in the gastrointestinal tract (i.e. cecum), and was occasionally recovered from the bloodstream and gall bladder. This observation is significant, because it provides evidence that the bacterial factors that influence the survival of L. monocytogenes in the g.i. tract are not necessarily the same as those that regulate its ability to cause invasive infection. We have also evaluated the virulence of a 5-strain cocktail of L. monocytogenes, similar to what is used in many food microbiology studies, in our mouse model. By comparing the recovery of L. monocytogenes from the spleens and livers of mice inoculated i.g. with the 5 strain cocktail to mice inoculated with comparable numbers of the individual strains that make up the cocktail, we evaluated the relative virulence of these strains. Our results suggest that much of the virulence can be attributed to strains Scott A and 101M (both serotype 4b); some of the other strains yielded much lower numbers of listeriae from the spleen and liver following inoculation into the g.i. tracts of mice. We also evaluated a different cocktail of L. monocytogenes strains, obtained from disease outbreaks, to see if these provide a more robust model for assessing virulence in vivo. Here, too, we found that one or two of the strains were largely responsible for the severity of the infection. Thus, we have not yet found evidence that a combination of L. monocytogenes strains results in a more severe infection than the individual strains used to prepare that combination. 1935-41420-012-06S: This report serves to document research conducted under a Specific Cooperative Agreement between ARS and Drexel University. Hispanic-style cheese has been associated with both cases and outbreaks of listeriosis. As such, we compared the growth/viability of Listeria monocytogenes in vacuum-packaged slices of commercially-prepared Queso Blanco during storage at temperatures ranging from 5 to 25C. Results revealed that this type of cheese provides a suitable environment for growth of the pathogen at both refrigerated and abuse temperatures. Although there were subtle differences in the duration of the lag phase and growth rate at the 5 temperatures tested, the average maximum population density for all temperatures was about 8.3 log(10) per gram. These findings will be used to estimate growth of listeriae in higher risk foods such as Hispanic-style cheese and to develop appropriate interventions to reduce the potential risk of human illness. 5.Describe the major accomplishments to date and their predicted or actual impact. This project recently completed the NP 108 OSQR Review Process and was certified. See the report for 1935-42000-052-00D. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? This project recently completed the NP 108 OSQR Review Process and was certified. See the report for 1935-42000-052-00D. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). This project recently completed the NP 108 OSQR Review Process and was certified. See the report for 1935-42000-052-00D.