Progress 09/01/10 to 08/31/13
Outputs
Target Audience: Target audiences include other academic researchers as well as industry and government agencies that use molecular subtyping methods for foodborne pathogens or that use pathogen strains or data to assure food safety through research and/or development efforts. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project has provided training and professional development opportunities for a number of graduate students and post-doctoral fellows that have been fully or partially supported by this project. One of the post-docs that were supported by the project has recently taken on a faculty position at North Dakota State University and continues food safety related research; this project thus has made critical contributions to the training of future food safety professionals in the US. This project also supported meeting attendance by supported students, providing further professional development opportunities for them. How have the results been disseminated to communities of interest? In addition to the peer-reviewed publications reported, the www-based FoodMicrobeTracker database is publicly available at www.foodmicrobetracker.com; this database has received more than 95,000 hits since April 2002. Isolates available from this database have also been distributed to interested parties with appropriate qualifications, for additional research. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This project has completed the development of the FoodMicrobeTracker database (www.foodmicrobetracker.com), which contains isolation data as well as comprehensive characterization data for foodborne pathogen isolates as well as isolates representing other food associated microbes. Overall, this database contains entries for more than 50,000 bacterial isolates, including >11,000 L. monocytogenes and >8,500 Salmonella. Data from the various specific studies conducted under this grants have been curated through this database and are available for use by other researchers, government agencies worldwide as well as others. This database can be used to more rapidly detect disease outbreaks and identify outbreak sources, therefore reducing human foodborne illness cases and outbreaks in the US. Obj. 1 (Perform longitudinal and retrospective studies on L. monocytogenes transmission at retail, including molecular characterization of L. monocytogenes isolates). We conducted a crosssectional study to characterize the prevalence, distribution, and subtype diversity of L. monocytogenes in 120 New York State retail deli establishments. Analysis of these data along with previously reported data for 121 predominantly larger retail establishments in New York State identified establishment size, geographic location, and inspection history as significant predictors of L. monocytogenes presence and prevalence. The odds of an establishment being L. monocytogenes positive were approximately twice as high for large establishments, establishments located in New York City, or establishments with poor inspection history (as compared with establishments without these attributes), even though correlation between location and inspection history complicated interpretation of results. Within an establishment, L. monocytogenes was significantly more prevalent on nonfood contact surfaces than on food contact surfaces; prevalence was particularly high for floors and in floor drains, sinks, the dairy case, and milk crates. These data have been published and widely communicated and have been used by industry to develop and implement control strategies for Listeria monocytogenes at retail. Obj. 2 (Develop, validate, and compare rapid molecular methods for Salmonella serotyping and develop data on transmission and virulence differences among Salmonella serotypes). More than 2,500 Salmonella serotypes have been identified using the Kauffman-White immunologic classification scheme, which is based on somatic (O) and flagellar (H) antigens. We specifically evaluated the ability of four molecular methods (PFGE, rep-PCR, ribotyping, and MLST) to predict serotypes for a set of 46 isolates, which were identified to represent the top 40 reported Salmonella from human and non-human sources reported by the Centers for Disease Control and World Health Organization. MLST was most reliable and able to accurately predict serotypes for 42/46 isolates representing the top 40 serotypes. PFGE, ribotyping, and rep-PCR were able to accurately predict 35/46, 34/46 and 30/46 serotypes, respectively. Across methods, serotypes 4,5,12;i;-, Typhimurium, and Typhimurium var. 5- were frequently not classified correctly, consistent with their close phylogenetic relationship. We also integrated a number of available data sources to develop and validate a PCR-based O-antigen screen with sequencing of internal fliC (H1 antigen) and fljB (H2 antigen) fragments to characterize Salmonella isolates to the serotype level. PCR and sequence based serotyping correctly identified 42/46 common serotypes. As molecular based sequencing was as reliable as existing subtyping methods evaluated, we continued to test this approach against a selection of 70 less common Salmonella serotypes and were able to accurately predict 62/70 Salmonella serotypes. These efforts provide an initial comparison of the ability to identify Salmonella serotypes using different molecular approaches. These data provide critical new knowledge that allows industry, academia, and government agencies to make decisions on molecular methods to use for Salmonella serotyping. Obj. 3. (Continue comprehensive collection as well as molecular and phenotypic characterization of human, food, and animal isolates of Salmonella). We have continued to expand coverage of Salmonella in the Food MicrobeTracker database, which now includes >8,500 Salmonella isolates. A number of these isolates have been characterized by full genome sequencing and sequence data are available and linked in Food MicrobeTracker. Genome analysis work in our laboratories has revealed a islet encoding the Cytolethal distending toxin B (CdtB) in the genomes of 54 nontyphoidal Salmonella. We also evaluated the contributions of this toxin to host cell damage. Our data suggest that cdtB plays a role in the pathogenesis of cytotoxicity associated with infection by Salmonella belonging to Clade B serovars and this inidciates that these isolates may cause disease and sequelae distinct from those caused by other Salmonella serovars that do not carry cdtB. As a consequence of this work, we have provided access to full genome sequence data to researchers as well as public health agencies (e.g., the New York State Department of Health) and have facilitated development and implementation of full genome sequencing based subtyping methods for foodborne pathogens by public health agencies. Completion of this aim thus has facilitated considerable changes that are leading to the use of new and improved technologies for subtyping of bacterial pathogens. Obj. 4. (Continue comprehensive collection as well as molecular and phenotypic characterization of human, food, and animal isolates of L. monocytogenes). We have also continued to expand coverage of Listeria monocytogenes in the Food MicrobeTracker database, which now includes >8,500 Listeria monocytogenes isolates. Isolates from Obj. 1 have also been added to this database and have been characterized by PFGE, a DNA fingerprinting method that is routinely used for outbreak detection and source tracking. In addition, unusual Listeria isolates have been characterized by both DNA-based methods and phenotypic methods; these data have identified a number of isolates representing unusual and previously unrecognized Listeria (e.g., Listeria rocourtiae, hemolytic Listeria innocua). Completion of this aim and associated expansion of the Listeria coverage in the FoodMicrobeTracker database provide considerable new resources that facilitate detection and characterization of Listeria isolates by both industry and government agencies. Obj. 5. (Enhance and maintain the PathogenTracker database, including development of improved data analysis tools and user interfaces, and distribution of strains and isolates). We have also completed a re-design of the PathogenTracker database, which has now been re-named Food Microbe Tracker; this redesign has included implementation of a number of new functionalities. Briefly, Food Microbe Tracker is a public Web-based database that allows archiving and exchange of a variety of molecular subtype data that can be cross-referenced with isolate source data, genetic data, and phenotypic characteristics. Data can be queried with a variety of search criteria, including DNA sequences and banding pattern data (e.g., PFGE type). Food Microbe Tracker allows the deposition of data on any bacterial genus and species. This database facilitates source tracking and outbreak detection, improve discovery of emerging subtypes, and increase our understanding of transmission and ecology of these microbes. Continued addition of subtyping, genetic or phenotypic data for a variety of microbial species will broaden the database and facilitate large-scale studies on the diversity of food-associated microbes.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Shi, C., P. Singh, M. L. Ranieri, M. Wiedmann, and A. I. Moreno Switt. 2013. Molecular methods for serovar determination of Salmonella. Critical Reviews in Microbiology [Nov 14, 2013; Epub ahead of print].
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Ranieri, M., C. Shi, A. Moreno Switt, Henk den Bakker, and M. Wiedmann. 2013. Comparison of typing methods with a new procedure based on sequence characterization for Salmonella serovar prediction. 51:1786-1797
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Progress 09/01/11 to 08/31/12
Outputs
OUTPUTS: During the reporting time frame we have made considerable progress in further development, comparison and validation of molecular methods for serotyping and subtyping of Salmonella. Serotyping is an important characterization tool for Salmonella. More than 2,500 Salmonella serotypes have been identified using the Kauffman-White immunologic classification scheme, which is based on somatic (O) and flagellar (H) antigens, including 46 currently recognized O serogroups, which are encoded in the rfb gene cluster. We specifically evaluated the ability of four molecular methods (PFGE, rep-PCR, ribotyping, and MLST) to predict serotypes for a set of 46 isolates, which were identified to represent the top 40 reported Salmonella from human and non-human sources reported by the Centers for Disease Control and World Health Organization. MLST was most reliable and able to accurately predict serotypes for 42/46 isolates representing the top 40 serotypes. PFGE, ribotyping, and rep-PCR were able to accurately predict 35/46, 34/46 and 30/46 serotypes, respectively. Across methods, serotypes 4,5,12;i;-, Typhimurium, and Typhimurium var. 5- were frequently not classified correctly, consistent with their close phylogenetic relationship. We also integrated a number of available data sources to develop and validate a PCR-based O-antigen screen with sequencing of internal fliC (H1 antigen) and fljB (H2 antigen) fragments to characterize Salmonella isolates to the serotype level. PCR and sequence based serotyping correctly identified 42/46 common serotypes. As molecular based sequencing was as reliable as existing subtyping methods evaluated in this study, we continued to test this approach against a selection of 70 less common Salmonella serotypes and were able to accurately predict 62/70 Salmonella serotypes. These efforts provide an initial comparison of the ability to identify Salmonella serotypes using (i) different molecular methods that predict serotypes based on banding patterns or phylogenetic relationships and (ii) a combined PCR and sequencing based approach that directly targets O and H antigen encoding genes. As recent work in our laboratories has revealed a islet encoding the Cytolethal distending toxin B (CdtB) within the genomes of 54 nontyphoidal Salmonella, we evaluated the contributions of this toxin to host cell damage, using isolates representing different cdtB-positive Clade B serovars. The data suggest that cdtB plays a role in the pathogenesis of cytotoxicity associated with infection by Salmonella belonging to Clade B serovars and suggests that these isolates may cause disease and sequelae distinct from those caused by other Salmonella serovars that do not carry cdtB. In addition, we have also completed whole genome sequencing and analysis of a number of additional Salmonella isolates, including to support outbreak investigations by New York State Department of Health. We have also completed a re-design of the PathogenTracker database, which has now been re-named Food Microbe Tracker; this redesign has included implementation of a number of new functionalities. PARTICIPANTS: Individuals who worked on this project during the reporting period include Martin Wiedmann, Project Director and Kendra Nightingale (project co-director at Texas Tech University) as well as postdoctoral fellows Teresa Bergholz (performed work on phenotypic characterization of Listeria), Henk den Bakker, Renato Orsi, Andrea Moreno Switt (performed molecular characterization of Salmonella including work on molecular serotyping of Salmonella) and Gina Ryan (performed collection and characterization of additional pathogen isolates added to Food Microbe Tracker). In addition, graduate students Travis Chapin (performed collection and characterization of Salmonella and Listeria), Tom Denes, Silin Tang (both performed characterization of Listeria monocytogenes), and Lorraine Rodriguez (performed characterization of Salmonella) and technical staff Sherry Roof, Emily Wright, and Steven Warchocki (performed characterization of Salmonella and Listeria and addition of data to Food Microbe Tracker) participated in this project. TARGET AUDIENCES: Target audiences include other academic researchers as well as industry and government agencies that use molecular subtyping methods for foodborne pathogens or that use pathogen strains or data to assure food safety through research and/or development efforts. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Major outcomes of this project generated over the last project year include an improved knowledge of the performance of different methods that can predict or identify Salmonella serotypes. These data facilitate selection of appropriate methods by industry. This information is important for both the food industry and public health as it will facilitate replacement of traditional serological serotyping methods, which rely on antisera that are difficult and costly to maintain and that are only available through a relatively small number of laboratories. Our work on molecular and phenotypic characterization of Salmonella isolates has lead to an important change in knowledge, as our data provide new knowledge that Salmonella enterica subsp. enterica isolates represent distinct clonal groups that differ in the presence/absence of a specific virulence gene, which affects how these different Salmonella interact with host cells. This knowledge may lead to future efforts to differentially assess the human health risk associated with different Salmonella serotypes, which will ultimately improve food safety. Another major outcome of this project (representing both a change in actions and a change in conditions) is the continued development of whole genome sequencing-based subtyping methods for foodborne pathogens, with a specific focus on Salmonella, including the transfer of these methodologies to the New York State Department of Health laboratories. Through collaborations with our group, the New York State Department of Health laboratories are continuing to successfully implement expanded whole genome sequencing efforts as a routine approach for subtyping of Salmonella isolates; these methods are facilitating detection of foodborne disease outbreaks that may have previously gone un-recognized. These approaches thus will likely facilitate improved food safety and ultimately reduce human foodborne disease cases; it is feasible that these approaches will have a similar profound impact on improved food safety as other molecular subtyping ("DNA fingerprinting") methods had when they were first introduced. These efforts are also supported through joint work with NCBI that focuses in the development of publicly available pipelines that can be used for repaid analysis of whole genome sequencing data, including by public health agencies (a "change in conditions").
Publications
- Den Bakker, H.C., AI Moreno Switt, G Govoni, CA Cummings, ML Ranieri, L Degoricija, K Hoelzer, L.D. Rodriguez-Rivera, S. Brown, E. Bolchacova, M.R. Furtado and M. Wiedmann. 2011. Genome sequencing reveals diversification of virulence factor content and possible host adaptation in distinct subpopulations of Salmonella enterica. BMC Genomics 12(1):425
- Moreno Switt, A. I., H. C. den Bakker, C. A. Cummings, L. D. Rodriguez-Rivera, G. Govoni, M. L Ranieri, L. Degoricija, S. Brown; K. Hoelzer; J. E. Peters, E. Bolchacova; Manohar R Furtado; and M. Wiedmann. 2012. Identification and Characterization of Novel Salmonella Mobile Elements Involved in the Dissemination of Pathogenic Genes. PLoS ONE 7(7): e41247
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: Despite growing concerns about cross-contamination of ready-to-eat foods with Listeria monocytogenes, our knowledge about the ecology and transmission of L. monocytogenes in retail establishments has remained limited. We conducted a crosssectional study to characterize the prevalence, distribution, and subtype diversity of L. monocytogenes in 120 New York State retail deli establishments that were hypothesized to present an increased risk for environmental L. monocytogenes contamination (i.e., small establishments and establishments with a history of failed New York State Agriculture and Markets inspections). Analysis of these data along with previously reported data for 121 predominantly larger retail establishments in New York State identified establishment size, geographic location, and inspection history as significant predictors of L. monocytogenes presence and prevalence. The odds of an establishment being L. monocytogenes positive were approximately twice as high for large establishments, establishments located in New York City, or establishments with poor inspection history (as compared with establishments without these attributes), even though correlation between location and inspection history complicated interpretation of results. Within an establishment, L. monocytogenes was significantly more prevalent on nonfood contact surfaces than on food contact surfaces; prevalence was particularly high for floors and in floor drains, sinks, the dairy case, and milk crates. L. monocytogenes subtype diversity differed between sites, with lineage I isolates significantly associated with nonfood contact surfaces and lineage II isolates significantly associated with food contact surfaces. Isolates belonging to the same ribotype were often found dispersed across multiple sites within an operation. Serotyping is an important characterization tool for Salmonella. More than 2,500 Salmonella serotypes have been identified using the Kauffman-White immunologic classification scheme, which is based on somatic (O) and flagellar (H) antigens, including 46 currently recognized O serogroups, which are encoded in the rfb gene cluster. Currently, rfb gene clusters of more common Salmonella serotypes are available. To expand our knowledge of the rfb locus and to support DNA-based approaches for serotyping, we used whole genome sequencing to analyze the rfb region of 12 less common human disease associated Salmonella serotypes. rfb clusters ranged in size from 8,012 to 23,893 bp and harbored 9 to 15 putative genes. GC content of the rfb clusters ranged from 34% to 43%, which is well below the genome average for Salmonella, suggesting that recent transfer from different bacterial species may contribute to O-antigen diversity. Within Salmonella serogroups or across serogroups sharing a common antigenic factor, there was a high degree of similarity. Comparisons among serogroups revealed considerably less homology in gene content. Overall, rfb cluster analysis will expand our knowledge of serogroup diversity and provide data for the development of molecular based serotyping methods. PARTICIPANTS: Individuals who worked on this project during the reporting period include Martin Wiedmann, Project Director and Kendra Nightingale (project co-director at Colorado State University). TARGET AUDIENCES: Target audiences include other academic researchers as well as industry and government agencies that use molecular subtyping methods for foodborne pathogens. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Major outcomes of this project generated over the last project year include an improved understanding of Listeria monocytogenes transmission in retail. Our data in particular have identified key sites in retail operations that show high prevalence. Our publications on L. monocytogenes at retail had considerable impact by alerting the retail industry to potential issues with L. monocytogenes and providing initial data that has facilitated further efforts to develop science-based control strategies for L. monocytogenes at retail. Overall, at least in part due to our efforts, we have seen a considerable change in how the retail industry addresses L. monocytogenes contamination with an increasing number of pro-active programs in place to address this potential issue. In addition, our project created new knowledge on Salmonella O antigen diversity, which will further facilitate the development of more robust molecular-based serotyping approaches for Salmonella. This information is important for both the food industry and public health as traditional serological serotyping relies on antisera that are difficult and costly to maintain with only a relatively small number of laboratories having serotyping capabilities. Development of molecular serotyping approaches will facilitate serotyping by a broader range of laboratories with more rapid turn around time.
Publications
- Hoelzer, K., B. D. Sauders, M. D. Sanchez, P. T. Olsen, M. M. Pickett, K. J. Mangione, D. H. Rice, J. Corby, S. Stich, E. D. Fortes, S. E. Roof, Y. T. Grohn, M. Wiedmann, and H. F. Oliver. 2011. Prevalence, distribution, and diversity of Listeria monocytogenes in retail environments, focusing on small establishments and establishments with a history of failed inspections. J. Food Prot. 74: 1083-1095
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