Development and Implementation of Genomic-based Approaches for Molecular Subtyping and Serotyping of L. Monocytogenes and Salmonella

NON-TECHNICAL SUMMARY: Recent estimates project that 76 million cases of gastrointestinal foodborne illnesses occur in the US on an annual basis, resulting in at least 325,000 hospitalizations and 5,000 deaths. L. monocytogenes causes about 2,500 human foodborne listeriosis cases and 500 deaths annually, while Salmonella causes an estimated 1.4 million cases of foodborne disease annually in the US, including approximately 550 deaths. Thus, these two foodborne pathogens have a significant negative impact on human health and well being in the US. This project will develop improved molecular characterization and subtyping methods to further enhance our understanding of the transmission and ecology of Salmonella and Listeria monocytogenes and to more accurately detect and identify foodborne disease outbreaks and outbreaks sources. Strain collections, subtyping and characterization methods, and protocols will be made broadly available to facilitate application of the methodologies developed.

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APPROACH: The FSRC program will be administered by faculty and staff in the Department of Food Science at Cornell. They will administer the funds and conduct research as specified and involve faculty at other universities and organizations as formal or informal collaborators. The program will interface with and draw on the existing 'Cornell Food and Water Safety Program' (see http://www.foodscience.cornell.edu/fws/fws.htm), which includes more than 30 faculty members at different colleges with research, teaching, and extension efforts and interests in food and water safety. We will use the following specific approaches to accomplish the objectives described above: (1) Analyze at least 5 Salmonella genome sequences using evolutionary approaches and construct and evaluate appropriate Salmonella mutants using tissue culture and other assays to probe the importance of strain variation on virulence. Validate strain-specific patterns of positive selection using sequencing of target genes in a diversity set of approximately 30 isolates. (2) Assemble large collections representing the overall diversity of L. monocytogenes and Salmonella serotypes for characterization by diverse subtyping methods including single nucleotide polymorphism-based molecular serotyping methodologies. (3) Obtain human, animal, and food associated Salmonella isolates. Characterize isolates by multilocus sequence typing and other molecular subtyping methods and analyze data to probe ecology and transmission of different Salmonella subtypes along the food system. (4) Obtain human, animal, and food associated L. monocytogenes isolates. Characterize isolates by multilocus sequence typing and other molecular subtyping methods and analyze data to probe ecology and transmission of different L. monocytogenes subtypes along the food system. (5) Develop PathogenTracker interface to provide user-specific search functions. Identify data sets and publications that use isolates already in PathogenTracker and add appropriate links and data information to PathogenTracker. Distribute isolates in our collection to qualified researchers in industry, academia, and government agencies.

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PROGRESS: 2007/08 TO 2008/07<BR>
OUTPUTS: The overall goal of the New York Food Safety Research Consortium is to conduct and coordinate food safety research that provides critical new knowledge on foodborne pathogens and leads to the development of new and innovative food safety tools and intervention strategies. Over the last project year, we have made major progress on two specific projects briefly detailed below. As previous research has shown that L. monocytogenes isolates can be grouped into three genetic lineages, which seem to differ in their ability and likelihood to cause human disease, we have tested the hypothesis that L. monocytogenes lineages may exhibit different stress related phenotypes. To test this hypothesis, isolates representing the three L. monocytogenes lineages were assessed for their ability to grow under salt and cold stress. Specifically, changes in cell density over time in brain heart infusion (BHI) with and without 6% NaCl at 7 and 37 C were quantified by plate counts and growth rates determined using the Baranyi model. In BHI at 37 C without additional NaCl, the growth rates were similar between lineages. However, in BHI with 6% NaCl at 37 C, the average specific growth rate for lineage II strains was 0.23 (SD= 0.03) log CFU/mL/h, which was significantly slower than the growth rates for lineage III strains and lineage I strains. Under these conditions, lineage III strains also had a significantly greater growth rate compared to lineage I isolates. The lag phase duration time upon transfer to BHI with 6% NaCl at 37 C was significantly greater for the lineage III strains compared to the lineage I and II strains. This data, along with previously reported phenotypic stress resistance data suggest that the genetic lineages of L. monocytogenes may be adapted to thrive under different environmental conditions. We are now in process to determine whether there are specific associations between these stress resistance phenotypes and L. monocytogenes SNPs. We have also performed a comprehensive subtype characterization of molecular subtypes (and specifically multilocus sequence typing data and SNP data) of L. monocytogenes strains associated with human listeriosis outbreaks in Europe and North America since the 1980s. The outbreak isolates characterized represented 8 distinct sequence types. Except for one sequence type (the sequence type found in isolates of the 1981 Nova Scotia coleslaw outbreak), all sequence types were also found among L. monocytogenes isolates obtained over the last 10 years from various sources in new York state. Our data thus show that L. monocytogenes strains responsible for human listeriosis outbreaks occur in a wide variety of habitats in urban, farm and in some cases natural environments. Based on these observations we propose that the majority of outbreaks are caused by strains of L. monocytogenes that are widely distributed in the general environment. <BR>
PARTICIPANTS: Individuals who worked on project include Martin Wiedmann, Project Director; Teresa Bergholz, Postdoctoral fellow (her role includes project supervision and phenotypic characterization of different Listeria subtypes); Patrick McGann, Postdoctoral fellow (his role included subtype characterization of L. monocytogenes isolates); Yesim Soyer, Graduate Research Assistant (her role included performance of genome Salmonella analyses), and Mary Palmer, Graduate Research Assistant (her role included phenotypic characterization of L. monocytogenes isolates). Partner organizations and collaborators include (i) New York State Department of Health, (ii) New York State Department of Agriculture and Markets, and (iii) New York State Animal Health Diagnostic center. All of these organizations provide bacterial isolates for molecular subtyping and inclusion on our databases. <BR>
TARGET AUDIENCES: Target audiences include other academic researchers as well as industry and government agencies that use molecular subtyping methods for foodborne pathogens. <BR>
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IMPACT: 2007/08 TO 2008/07<BR>

Major outcomes of this project generated over the last project year include an improved understanding of the phenotypic diversity of L. monocytogenes as well as the availability of new molecular subtype data sets for outbreak associated Listeria monocytogenes isolates. These data will aid in the rapid identification of outbreak associated L. monocytogenes strains among isolates obtained during food, environmental, or human disease surveillance. Inclusion of these data in our comprehensive subtype database that is supported by this project (i.e., the PathogenTracker database; www.pathogentracker.net) will also provide users (including industry, government agencies, and academics) with improved molecular subtype datasets that can be used to rapidly characterize foodborne pathogen isolates. Overall, our project continues to make major contributions to the further development and dissemination of molecular subtyping ("DNA fingerprinting") tools for foodborne pathogens, including the training of individuals with expertise in molecular characterization of foodborne pathogens. The knowledge generated through this project, in combination with the human resources generated, will thus contribute to an improved ability to detect and track human foodborne disease outbreaks and identify the sources of disease outbreaks and sporadic disease cases. Changes leading to improved application of molecular methods across various areas of the food industry are also facilitated through different workshops and industry presentations that assure broad awareness of the data and resources generated through this project.

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PROGRESS: 2006/08/01 TO 2007/07/31<BR>

OUTPUTS: The overall goal of the New York Food Safety Research Consortium (FSRC), which includes this specific project, is to conduct and coordinate food safety research that provides critical new knowledge on foodborne pathogens and leads to the development of new and innovative food safety tools and intervention strategies. Specifically, this project supports (i) further expansion and maintenance of a comprehensive foodborne pathogen subtype database (www.pathogentracker.net) with a particular focus on L. monocytogenes and Salmonella; (ii) analysis of Salmonella full genome sequences to develop improved subtyping methods for this important foodborne pathogen; and (iii) development of single nucleotide polymorphism-based molecular serotyping and subtyping methods for L. monocytogenes and Salmonella, to provide improved tools for source tracking and source attribution. To date, we have continued to expand the PathogenTracker database (www.pathogentracker.net), which now includes data for more than 6,300 Listeria monocytogenes and more than 1,200 Salmonella isolates. In addition, more than 70 references are linked to isolate data and information in this database. We have also completed initial analyses of five Salmonella genomes (representing serotypes Choleraesuis, Paratyphi, Typhi [2 isolates], and Typhimurium) to identify Salmonella genes that are under positive selection and/or genes that show evidence for recombination. Overall, we identified a total of 127 and 41 genes as showing evidence of positive selection using p-value 0.05 significance level and 0.2 cut off q-value, respectively. These rapidly evolving genes may contribute to adaptation of Salmonella to specific niches and hosts and knowledge of these genes may thus allow for a better understanding of Salmonella transmission patterns associated with different subtypes and serotypes. We are currently in the progress of confirming these findings on a larger set of Salmonella isolates. We have also further enhanced and expanded our database of DNA sequences for the Listeria gene sigB. DNA sequencing of this gene allows for rapid and economic subtyping as well as species classification of Listeria. This SNP-based subtyping has also been used to track Listeria contamination patterns in food processing plants to help understand sources and transmission of this foodborne pathogen in order to allow their control and elimination.<BR>
PARTICIPANTS: Individuals who worked on project include Martin Wiedmann, Project Director; Teresa Bergholz, Postdoctoral fellow (her role includes project supervision and phenotypic characterization of different Listeria subtypes); Patrick McGann, Postdoctoral fellow (his role included subtype characterization of L. monocytogenes isolates); Yesim Soyer, Graduate Research Assistant (her role included performance of genome Salmonella analyses), and Mary Palmer, Graduate Research Assistant (her role included phenotypic characterization of L. monocytogenes isolates). Partner organizations and collaborators include (i)New York State Department of Health, (ii) New York State Department of Agriculture and Markets, and (iii) New York State Animal Health Diagnostic center. All of these organizations provide bacterial isolates for molecular subtyping and inclusion on our databases.<BR>
TARGET AUDIENCES: Target audiences include other academic researchers as well as industry and government agencies that use molecular subtyping methods for foodborne pathogens.
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IMPACT: 2006/08/01 TO 2007/07/31<BR>

Outcomes of this project include an improved availability of molecular subtype data for the foodborne pathogens Listeria monocytogenes and Salmonella. These data are critical to allow for a better understanding of transmission routes and sources of foodborne pathogen, representing knowledge that is needed to reduce foodborne illnesses in the US. In addition, this project has developed improved economical subtyping methods for the genus Listeria that can rapidly differentiate potentially human disease causing bacteria from non-pathogenic Listeria species, thus allowing for improved detection and characterization of foodborne pathogens. Improved subtyping methods for Salmonella will also allow for better source tracking for Salmonella outbreaks and contamination events. For example, our extensive Salmonella subtype databases have started to show patterns which indicate that certain subtypes of common Salmonella serotypes may be associated with specific animal source populations. For example certain Salmonella Newport subtypes appear to be associated with cattle. These data will ultimately help both industry and government agencies to more rapidly identify the most likely sources of Salmonella outbreaks and contamination events.