International Life Sciences Institute North America Listeria monocytogenes strain collection: development of standard Listeria monocytogenes strain sets for research and validation studies.

Research and development efforts on bacterial foodborne pathogens, including the development of novel detection and subtyping methods, as well as validation studies for intervention strategies can greatly be enhanced through the availability and use of standardized strain collections. These types of strain collections are available for some foodborne pathogens, such as Salmonella and Escherichia coli. We have developed a standard Listeria monocytogenes strain collection that has not been previously available. The strain collection includes (i) a diversity set of 25 isolates chosen to represent a genetically diverse set of L. monocytogenes isolates as well as a single hemolytic Listeria innocua strain and (ii) an outbreak set, which includes 21 human and food isolates from nine major human listeriosis outbreaks that occurred between 1981 and 2002. The diversity set represents all three genetic L. monocytogenes lineages (I, n = 9; II, n = 9; and III, n = 6) as well as nine different serotypes. Molecular subtyping by EcoRI automated ribotyping and pulsed-field gel electrophoresis (PFGE) with AscI and ApaI separated the 25 isolates in the diversity set into 23 ribotypes and 25 PFGE types, confirming that this isolate set represents considerable genetic diversity. Molecular subtyping of isolates in the outbreak set confirmed that human and food isolates were identical by ribotype and PFGE, except for human and food isolates for two outbreaks, which displayed related but distinct PFGE patterns. Subtype and source data for all isolates in this strain collection are available on the Internet and are linked to the PathogenTracker database (www.pathogentracker.com), which allows the addition of new, relevant information on these isolates, including links to publications that have used isolates from this collection. We have thus developed a core L. monocytogenes strain collection, which will provide a resource for L. monocytogenes research and development efforts with centralized Internet-based data curation and integration.

Food microbe tracker: a web-based tool for storage and comparison of food-associated microbes.

Large amounts of molecular subtyping information are generated by the private sector, academia, and government agencies. However, use of subtype data is limited by a lack of effective data storage and sharing mechanisms that allow comparison of subtype data from multiple sources. Currently available subtype databases are generally limited in scope to a few data types (e.g., MLST. net) or are not publicly available (e.g., PulseNet). We describe the development and initial implementation of Food Microbe Tracker, 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., ribotype or pulsed-field gel electrophoresis type). Food Microbe Tracker allows the deposition of data on any bacterial genus and species, bacteriophages, and other viruses. The bacterial genera and species that currently have the most entries in this database are Listeria monocytogenes, Salmonella, Streptococcus spp., Pseudomonas spp., Bacillus spp., and Paenibacillus spp., with over 40,000 isolates. The combination of pathogen and spoilage microorganism data in the database will facilitate 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.

Pulsed-field gel electrophoresis (PFGE) analysis of temporally matched Listeria monocytogenes isolates from human clinical cases, foods, ruminant farms, and urban and natural environments reveals source-associated as well as widely distributed PFGE types.

A total of 495 temporally and geographically matched Listeria monocytogenes isolates from human clinical cases, foods, ruminant farms, and urban and natural environments were used to investigate L. monocytogenes pulsed-field gel electrophoresis (PFGE) type diversity. Two-enzyme (AscI and ApaI) PFGE discriminated 310 PFGE types and exhibited higher overall discriminatory power (Simpson's index of discrimination [D] = 0.995) than either EcoRI ribotyping (D = 0.950) or AscI or ApaI single-enzyme PFGE (D = 0.992 for both). Seven PFGE types showed significant associations with specific sources, including one and four PFGE types, respectively, associated with human clinical cases and foods. Spatial analysis of 13 PFGE types occurring >5 times showed that two PFGE types were specific to a single processing facility each, where they appear to have persisted over time. Nine PFGE types were geographically widespread and occurred among isolates from multiple sources. For example, a PFGE type that matched isolates from listeriosis outbreaks in Los Angeles and Switzerland occurred among isolates from farms (n = 7), human clinical cases (n = 4), environmental sources (n = 3), and foods (n = 1). Our data indicate that (i) PFGE is highly discriminatory for the subtyping of L. monocytogenes, (ii) some L. monocytogenes PFGE types are associated with specific sources, and (iii) some L. monocytogenes PFGE types are widely distributed and appear to be stable and pandemic. Large PFGE type databases representing isolates from different sources are thus needed to appropriately interpret subtype data in epidemiological investigations and to identify common as well as source-specific PFGE types.