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? Species of Listeria, Clostridium, and Enterococcus pose significant threats to the safety and security of the U.S. food supply. Listeria monocytogenes is responsible for approximately one-quarter of food borne disease-related deaths, and is the leading cause of food recalls due to microbiological concerns. Clostridium perfringens is among the most common causes of food borne illness in the U.S., resulting in annual economic losses of nearly 100 million dollars. Enterococcus faecalis and Enterococcus faecium are important industrial probiotic strains that have emerged as serious nosocomial pathogens due to multiple antibiotic resistance and potential for food borne transmission. As such, this set of species has a significant negative impact on public health and the agricultural economy. Other pathogenic food borne species of Clostridium (including C. botulinum and related species) are a significant national security threat due to their capacity to produce highly potent neurotoxins that could be used in bio-weapons. This project has been developed to provide the evolutionary framework and subtype characterization needed for threat-based risk assessments and the development and implementation of more effective intervention strategies and regulatory policies that provide maximum protection to consumers while limiting the number and size of product recalls. In addition, the proposed research will provide molecular tools that enable application of subtype-specific monitoring and threat-based regulatory action, and will also contribute to the public health infrastructure for dealing with these food borne pathogens. A combination of detailed analyses of large sequence data sets from targeted genes as well as novel evolutionary genomic approaches are being used to accomplish these objectives. In addition, single nucleotide polymorphisms (SNP) identified in these comparative sequence data sets are serving as the basis for the development of novel multilocus genotyping (MLGT) assays for pathogen subtype identification and epidemiological investigation. This project directly supports National Program (NP) 108 priority objectives 1.2.1 Detection and Validation, 1.2.5 Omics, 1.2.8 Pathogenicity, and 1.2.9 Food Security; as specified in the 2006-2010 NP-108 Action Plan. In addition, this 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. 2.List by year the currently approved milestones (indicators of research progress) FY2006: - Complete multilocus DNA sequence databases for Listeria and Clostridium. - Develop first-generation MLGT for L. monocytogenes lineage 1. FY2007: - Determine phylogeny, population structure, and historical demography for Listeria and Clostridium. FY2008: - Complete phylogenomic analyses for Listeria. - Complete Enterococcus strain collection and DNA sequence database. - Complete molecular evolutionary analyses of virulence genes for Listeria and Clostridium. - Complete analyses of lineage-specific differences in selective constraint for Listeria and Clostridium. - Develop first-generation MLGT for C. perfringens and L. monocytogenes lineages 2 and 3. FY2009: - Determine phylogeny, population structure, and historical demography for Enterococcus. - Determine genome wide patterns of molecular evolution for Listeria. FY2010: - Complete data collection and phylogenetic analyses of genetic lineages in Listeria and Clostridium. - Complete molecular evolutionary analyses of virulence genes in Enterococcus. - Complete multilocus genotyping analyses for Listeria and Clostridium. 4a.List the single most significant research accomplishment during FY 2006. Genetic diversity and lineage evolution of Clostridum perfringens: A manuscript describing cryptic lineages of Clostridium perfringens was produced and published in GENETICS. This manuscript describes work on the genetic diversity of C. perfringens, an important human and domestic animal bacterial pathogen that causes food poisoning, gastrointestinal necrosis, gangrene, and a multitude of other diseases. The current line of thinking regarding the diversity of C. perfringens is that it is a single evolutionary lineage. However, our research shows that it is actually composed of five distinct lineages that differ genetically as well as in the type of disease that they cause. Our findings carry important ramifications for the diagnosis of C. perfringens disease and for efforts aimed at C. perfringens disease surveillance and source-tracking. Specifically, this information will help public health officials, clinicians, and researchers to better diagnose and track C. perfringens disease. This accomplishment directly addresses the 2006-2010 NP-108 Action Plan Food Safety (animal and plant products) priority objectives 1.2.5 Omics, 1.2.8 Pathogenicity, and 1.2.9 Food Security. In addition, the proposed 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. 4b.List other significant research accomplishment(s), if any. DNA sequence-based subtyping of Listeria monocytogenes: A manuscript describing a single-well MLGT assay for Listeria monocytogenes subtype identification was produced and is currently in peer-review. This test provides for the rapid and accurate identification of L. monocytogenes strains responsible for serious invasive illness in humans and domestic animals. In addition, the assay can be used for threat-based risk assessment, and was designed to look at specific mutations that result in strains with a reduced ability to cause infection. Preliminary surveys of subtype prevalence indicate that such strains are common in food products. These data confirm that we have developed a technology that will serve as a multifunctional and important tool in the prevention and analysis of L. monocytogenes infections. This accomplishment directly addresses the 2006-2010 NP-108 Food Safety (animal and plant products) Action Plan priority objectives 1.2.1 Detection and Validation, 1.2.5 Omics, 1.2.8 Pathogenicty, and 1.2.9 Food Security. In addition, the proposed 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. 4c.List significant activities that support special target populations. None. 4d.Progress report. None. 5.Describe the major accomplishments to date and their predicted or actual impact. This project was split out of 3620-42000-031-00D and was approved in December 2005. FY2006 accomplishments represent the entire life of the project and consist of characterization of lineage evolution, population structure and demographic history of species within Listeria and Clostridium, and the development of molecular subtyping methods for L. monocytogenes and C. perfringens. This work provides the evolutionary framework and subtype characterization needed for threat-based risk assessments and the development and implementation of more effective intervention strategies and regulatory policy. In addition, we have developed molecular tools that enable application of subtype-specific monitoring and threat-based regulatory action, addressing specific needs (pathogen surveillance, outbreak detection, and source tracking) of food producers and public health and regulatory agencies such as Food Safety Inspection Service (FSIS), Food and Drug Administration (FDA), and the U.S. Centers for Disease Control and Prevention (CDC). This accomplishment directly addresses the 2006-2010 NP-108 Food Safety (animal and plant products) Action Plan priority objectives 1.2.1 Detection and Validation, 1.2.5 Omics, 1.2.8 Pathogenicty, and 1.2.9 Food Security. In addition, the proposed 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. 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? The first high-throughput subtyping technology based on DNA sequence differences between L. monocytogenes strains is being made available, through publication, to scientists, regulatory and public health agencies, and the food industry. This technology will likely become available to end-users within a year. 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). Ducey, T. F. A SNP-based Multilocus Genotyping (MLGT) Assay for subtyping Listeria monocytogenes. Symposium on DNA-based serotyping and subtyping of enteric bacteria. Centers for Disease Control and Prevention, Atlanta, GA, March, 2006. Publications are listed on 3620-42000-031-00D, from which this new project was derived in December 2005.