2007 Annual Report 1a.Objectives (from AD-416) The overall objective for this project is to produce an evolutionary framework for understanding the ecology, virulence and epidemiology of Listeria, Clostridium, and Enterococcus that will contribute to the development and implementation of effective control strategies for these species. The specific objectives are: . 1)Characterize lineage evolution, population structure and demographic history of species within the genera Listeria, Clostridium, and Enterococcus;. 2)Determine patterns of selective constraint throughout the genomes of species within the genera Listeria, Clostridium, and Enterococcus; and. 3)Develop molecular subtyping methods for L. monocytogenes and C. perfringens. 1b.Approach (from AD-416) Multilocus DNA sequence datasets will be developed for diverse collections of Listeria, Clostridum and Enterococcus from food, veterinary, clinical and environmental sources. These data will be combined with data from complete genome sequences and analyzed in order to provide an evolutionary framework for understanding the ecology, virulence, and epidemiology of pathogenic and toxigenic species of Listeria, Clostridium, and Enterococcus. In addition, results of the molecular evolutionary analyses will be used to determine if there is an association between a specific adaptive variant and subtypes most frequently associated with food borne illness in humans, providing a scientific basis for subtype-specific risk assessments, regulatory policies, and intervention strategies that provide maximum protection to consumers while limiting the number and size of product recalls. Finally, the evolutionary framework and information on the distribution and adaptive significance of genetic variation within the pathogens examined will be used to develop DNA sequence-based subtyping technologies that will enhance pathogen surveillance, outbreak detection, epidemiological investigation, and source-tracking efforts. 3.Progress Report A manuscript was published describing the first single-nucleotide-polymorphism-based (SNP-based) subtyping system for use in L. monocytogenes risk assessment and outbreak investigation. 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. For example, we used this assay to determine the prevalence of epidemic-associated and virulence-attenuated strains of L. monocytogenes in ready-to-eat meat products. In addition, we completed a large-scale SNP discovery project to identify strain-specific variation among L. monocytogenes commonly associated with contaminated food and animal illness. This project involved collecting over 1.8 million bases of deoxyribonucleic acid (DNA) sequence data in a comparative analysis of 126 L. monocytogenes isolates. SNP variation identified in this project was used to develop and validate multilocus genotyping assays, producing a comprehensive panel of SNP-based assays for L. monocytogenes subtyping. Publication of these data and other technology transfer activities have led to the establishment of collaborative projects with the U.S. Department of Agriculture (USDA) Food Safety Inspection Service (FSIS) to determine subtype prevalence in a large variety of ready-to eat food products and to develop customized subtyping assays that will inform their threat-based risk assessment programs. Additional collaborations have been developed with the Centers for Disease Control and Prevention (CDC) and the North Carolina State Public Health Laboratory to further validate the subtyping technologies we developed and to perform comparative analysis of a variety of molecular subtyping technologies for L. monocytogenes. Finally, we completed comparative genomic analyses of a gene family that includes key virulence factors for L. monocytogenes. As a result of this research, we have characterized the mechanisms leading to diversification in these virulence-associated genes and a paper is being prepared that describes the results of this study. Analysis of nucleotide sequence data from virulence genes of Eneterococus faecium and E. faecalis have been collected and analyzed. These data indicate a lack of genetic variation at these loci. In consultation with National Program staff, we have decided to discontinue further research into Enterococcus. 4.Accomplishments Subtyping of Listeria monocytogenes. As a result of limitations in current subtyping capabilities, the CDC PulseNet Task Force has called for the development and validation of DNA sequence-based methods for subtyping L. monocytogenes as part of outbreak detection and epidemiological investigations. In addition, the recent L. monocytogenes risk assessment completed by the Food and Drug Administration (FDA), FSIS, and CDC identified significant gaps in knowledge regarding subtype prevalence and cited as critical the need for methods to determine the virulence potential of individual strains as FSIS transitions to a risk-based monitoring program. In response to this need for next generation subtyping technologies, we completed the development, validation and publication of the first single-nucleotide-polymorphism-based subtyping assay for the group of L. monocytogenes strains that are most commonly associated with human illness. We demonstrated that the multi-locus genotyping (MLGT) assay provided high discriminatory power and differentiated groups relevant to epidemiological investigations. In addition, our assay provided for the identification of a specific virulence-attenuated subtype, and was used to document the frequency of epidemic and virulence-attenuated subtypes in ready-to-eat meat products. The potential impact of this accomplishment is demonstrated through the establishment of collaborative projects with regulatory and public health agencies aimed at using the technology we have developed to determine subtype prevalence in a large variety of ready-to eat food products and to develop customized subtyping assays that will inform threat-based risk assessment programs. This accomplishment directly addresses National Program 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, 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.Technology Transfer Number of non-peer reviewed presentations and proceedings 7 Review Publications Leverentz, B., Conway, W.S., Janisiewicz, W.J., Kurtzman, C.P., Abadias, M., Camp, M.J. 2006. Biocontrol of foodborne pathogens on fresh-cut apples using naturally occurring bacterial and yeast antagonists. Applied and Environmental Microbiology. 72:1135-1140. Ducey, T.F., Page, B., Usgaard, T.R., Borucki, M.K., Pupedis, K., Ward, T.J. 2007. A single-nucleotide-polymorphism-based multilocus genotyping assay for subtyping lineage I isolates of Listeria monocytogenes. Applied and Environmental Microbiology. 73(1):133-147.