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The overall goal of the New York Food Safety Research Consortium (FSRC) is to conduct and coordinate food safety research, which provides critical new knowledge on foodborne pathogens and which leads to the development of new foods and innovative food safety intervention strategies for application by the food industry. We propose to use CSREES special grant funds supporting the FSRC to (i) further expand and maintain a comprehensive foodborne pathogen subtype database (www.pathogentracker.net) and (ii) to expand our efforts to characterize L. monocytogenes and Listeria spp. isolates to improve our understanding as to the ecology and evolution of the foodborne pathogen L. monocytogenes. The recent description of hemolytic Listeria strains, which appear to group into the species L. innocua based on most phenotypic and genetic characteristics, further supports the need for a comprehensive phylogenetic and virulence characterization of the genus Listeria to define the foodborne virulence potential of different phylogenetic groups within the genus Listeria. <P>
The overall project aims will be achieved through the following specific objectives: <OL> <LI> Develop a comprehensive collection of human, food, and animal isolates of L. monocytogenes and characterize all isolates by different molecular subtyping data. At the completion of this project we will have developed a publicly accessible database and collection of human, food, and animal isolates from New York State covering a continuous 9 year period.<LI>Apply molecular subtyping methods to characterize at least 200 Listeria spp. isolates collected from various environments in New York State to develop an understanding of the ecology and evolution of the genus Listeria. <LI>Characterize the ability of selected Listeria and L. monocytogenes isolates to (i) cause infections in an oral guinea pig infection model: (ii) form biofilms under different environmental conditions, and (iii) to survive in the presence of protozoan organisms.<LI> Use isolates from our extensive strain collection to develop new rapid detection and subtyping methods for Listeria spp. and L. monocytogenes based on Ion Mobility Spectrometry.
NON-TECHNICAL SUMMARY: Listeria monocytogenes is a foodborne pathogen that causes an estimated 2,500 human disease cases, including 500 deaths annually. Improved control and prevention of this disease requires an in-depth understanding of the ecology and transmission of this organism. This project will ultimately provide an improved understanding of the environmental survival and virulence characteristics of evolutionary distinct groups of the species Listeria. This knowledge will not only aid in the definition of the particular Listeria subtypes that provide a public health hazard, but will also provide an understanding of the specific factors which contribute to the environmental persistence of Listeria.
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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) Obtain human, food, and animal isolates from state health departments and other sources. Characterize isolates by automated ribotyping and multilocus sequence typing. 2) Characterize 200 Listeria spp. isolates by multilocus sequence typing and determine Listeria phylogeny using the Neighbor Joining Method. 3) Characterize selected L. monocytogenes and Listeria spp. isolates for guinea pig virulence, biofilm formation, and survival in co-culture with Tetrahymena. 4) Modify existing Ion Mobility Spectrometry (IMS) equipment to allow temperature stepping. Use the equipment developed to test its ability to specifically detect and subtype L. monocytogenese and Listeria spp.
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PROGRESS: 2003/05 TO 2006/04 <BR>
Through this project we have been able to expand the PathogenTracker database (www.pathogentarcker.net) to include more than 5,000 L. monocytogenes isolates. Subtype data collected through this project have contributed to the detection of multiple human listeriosis outbreaks and provided critical data for large scale analyses of associations between different L. monocytogenes subtypes and different sources. Specifically, data analyses indicated that (i) L. monocytogenes from different sources show a high level of diversity, (ii) even though populations overlap, L. monocytogenes subtypes differ significantly in their associations with different environments, and (iii) a higher proportion of isolates from environmental sources, as compared to isolates from human clinical cases, classify into L. monocytogenes lineage II, supporting classification of this lineage as an environmentally adapted subgroup. Further use of our L. monocytogenes subtype database showed that (i) while human listeriosis cases are caused by a diversity of EcoRI ribotypes, specific lineage I epidemic clones cause a large number of human listeriosis cases; (ii) a considerable number of human listeriosis cases represent statistically significant temporal clusters, including widely distributed and region specific clusters caused by various ribotypes; (iii) L. monocytogenes lineages and EcoRI ribotypes do not appear to differ in their likelihood of causing different clinical manifestations or mortality. In order to develop rapid molecular methods for speciation and subtyping of Listeria spp., we characterized 101 Listeria isolates obtained from urban and pristine environments by sequencing of two genes (gap and sigB). Phylogenetic analyses revealed four well supported gap and sigB clusters as well as four main sigB clusters, which showed close concordance with Listeria species identification by API, such that each cluster could be assigned a specific Listeria species. sigB sequencing provided more distinct subtypes than gap sequencing and was thus chosen as the subtyping method for subsequent characterization of an additional >500 Listeria isolates. Analyses of these data showed that L. seeligeri and L. welshimeri were significantly associated with pristine environments, while L. monocytogenes and L. innocua were significantly associated with urban environments, indicating that Listeria spp. differ in their ecology. These findings have important implications for the use of Listeria as an indictor for the presence of L. monocytogenes. We have also completed experiments using L. monocytogenes isolates from our collection to evaluate their ability to f to attach to innate surfaces. Our data suggest that L. monocytogenes does not form true biofilms but is able to rapidly (i.e., in less than 5 min) and efficiently attach to surfaces and form mixed biofilms with P. fluorescens. Interestingly, sigma B does not seem to be required for single species surface attachment or multispecies biofilm formation but is essential for long-term survival. Our data suggest that the universal biofilm model may not be appropriate for the Listeria surface attachment community.
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IMPACT: 2003/05 TO 2006/04<BR>
This project has allowed us to further expand and maintain a comprehensive foodborne pathogen subtype database (www.pathogentracker.net) including a large collection of L. monocytogenes isolates available for the broad food safety research community. Specifically, isolates available through this database have been distributed to a large number of researchers in academia, government agencies, and private industry. Characterization of large number of isolates included in our database by various methods (e.g., animal virulence assays, DNA sequencing-based subtyping) also has provided information on the ecology and foodborne transmission characteristics of different L. monocytogenes and Listeria subtypes, which will aid in the development of prevention and tracking strategies targeting those subtypes most likely to cause human disease. While isolates available through this database have also been used in a number of specific research projects that are often supported by funding from other sources, this project specifically provided an infrastructure for data and isolate sharing and collection of large isolates and data sets for analyses, thus maximizing benefits of various research projects for other researchers, industry, and government agencies. Finally, subtype data available through the PathogenTracker database have been critical for the detection of multiple human listeriosis outbreaks, thus directly contributing to a reduced foodborne disease burden in the US.