Phenotypic and Molecular Studies to Facilitate Improved Control of L. Monocytogenes and Salmonella Transmission

NON-TECHNICAL SUMMARY: A study published in 1999 estimated that 76 million cases of gastrointestinal food-borne illnesses occur in the US on an annual basis, resulting in at least 325,000 hospitalizations and 5,000 deaths. While subsequent data indicated that the incidence of human listeriosis and selected other food-borne disease decreased since these initial estimates, 2008 data indicate no further declines in food-borne illness frequencies. Thus, food-borne illnesses and their medical sequelae continue to have a significant negative impact on human health and well being. Food recalls due the presence of food-borne pathogens are not uncommon and have significant negative economic consequences for the food industry. To track and control sources of food-borne pathogens, agricultural and food industries have a critical need for access to advanced molecular and genetics based tools, including future employees trained in the use of these tools. The overall goal of this project is to assure access to these advanced tools, by the food and meat industries to aid in their efforts to control food-borne pathogens. Thus, one of the goals of this project is the further development and expansion of a WWW-based publicly available DNA sub-type database for microorganisms causing food-borne illnesses. Further goals of this project include applications of this database to (i) help detect food-borne disease outbreaks and outbreak sources, (ii) develop improved "DNA fingerprinting" methods for food-borne pathogens, (iii) develop a better under-standing of the transmission, and evolution of food-borne pathogens, (iv) to define specific bacterial sub-types that differ in their ability to cause food-borne disease, and (v) provide scientific information that can be used to enhance risk assessments for food-borne illnesses. L. monocytogenes causes about 2,500 human food-borne listeriosis cases and 500 deaths annually in the US and has been commonly found in many different environments. This food-borne pathogen thus represents a considerable concern, not only due to its ability to cause severe human disease, but also because contamination of Ready-To-Eat food products with this organism is not uncommon. Recalls due to the presence of L. monocytogenes are thus not uncommon and both, human listeriosis cases and costly recalls, place a significant financial and emotional toll in the US population and national economy. Salmonella causes an estimated 1.4 million cases of food-borne disease annually in the US, including approximately 550 deaths. L. monocytogenes and Salmonella combined thus cause approx. 1,050 food-borne deaths annually in the US, out of an estimated total 1,800 deaths due to known food-borne pathogens. Significant efforts in this project are thus focused on the development of an improved understanding of farm-to-table food-borne transmission and ecology of L. monocytogenes and Salmonella, including the development and application of better tools to control and minimize contamination with these bacteria throughout the food system.
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APPROACH: <BR> (1) Isolate and characterize Listeria and Salmonella phages and develop an initial collection of wild-type lytic phages for these pathogens: Samples for isolation of lytic phages infecting L. monocytogenes and Salmonella will be collected from different farms throughout New York State. Feed and manure samples will be used for phage isolation using both a direct isolation protocol and a phage enrichment protocol. Phage characterization will be performed using (i) host range determinations, (ii) electron microcopy, and (iii) phage genome restriction enzyme analysis. In addition, phage genomes will be sequenced for selected phage isolates. <BR> <BR> (2) Perform full genome sequencing, using new rapid sequencing methods, and virulence studies of selected Salmonella strains and serotypes: We anticipate that we will perform full genome sequencing for (i) about 4 - 6 Salmonella isolates that are not included among the isolates sequenced to date, focusing on strains that appear to be characterized by unique virulence and epidemiological features (e.g., underrepresentation among human clinical cases as compared to prevalence among foods, unique virulence characteristics) and (ii) approx.5 - 10 genomes of epidemiologically related and unrelated isolates with identical sub-types as determined by other sub-typing methods (e.g., MLST; PFGE). Selection of Salmonella strains with evidence for unique virulence or transmission characteristics will be accomplished using a combination of epidemiological and sub-type data as well as tissue culture pathogenicity data. <BR> <BR> (3) Continue comprehensive collection as well as molecular and phenotypic characterization of human, food, and animal isolates of Salmonella: Human Salmonella isolates will be obtained through collaborations with the NYS Department of Health (NYSDOH). Farm animal isolates will be obtained from the routine patient submissions to the NYS Animal Health Diagnostic Center (AHDC) at Cornell. Selected isolates will be characterized by MLST, PFGE, and by molecular serotyping schemes.<BR> <BR> (4) Continue comprehensive collection as well as molecular and phenotypic characterization of human, food, and animal isolates of L. monocytogenes. Human isolates will be obtained through collaborations with the NYS Department of Health (NYSDOH), while animal isolates will be obtained from the routine patient submissions to the NYS Animal Health Diagnostic Center (AHDC) at Cornell. Selected isolates will be characterized by ribotyping, PFGE, and DNA sequencing-based sub-typing methods.<BR> <BR> (5) Enhance and maintain the Pathogen Tracker database, including development of improved data analysis tools and user interfaces, and distribution of strains and isolates, including phage isolates.