Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/27/2006
Publication Date: 1/1/2007
Citation: Ducey, T.F., Page, B., Usgaard, T.R., Borucki, M.K., Pupedis, K., Ward, T.J. 2007. A single-nucleotide-polymorphism-based multilocus genotyping assay for subtyping lineage I isolates of Listeria monocytogenes. Applied and Environmental Microbiology. 73(1):133-147. Interpretive Summary: Listeria monocytogenes is a food-borne bacterium that places a serious burden on human health and the agricultural economy. Listeria monocytogenes is responsible for over one-quarter of foodborne disease-related deaths linked to known pathogens, and L. monocytogenes contamination has been the leading cause of food recalls due to biological concerns in recent years. Discrimination of strains below the species level (subtyping) is critical to epidemiological investigations, threat-based risk assessment, and the development of effective disease control strategies that provide maximum protection to consumers while limiting the number and size of product recalls. Currently used subtyping methods are labor intensive, time consuming, and can not be used to determine the virulence potential of L. monocytogenes strains. To address these issues, we developed the first high-throughput subtyping test based on DNA sequence differences between L. monocytogenes strains. This test provides for the rapid and accurate identification of strains. In addition, the assay also can be used for risk assessment, and was designed to look at specific mutations that result in strains with a reduced ability to cause infection. Preliminary surveys of subtype prevalence indicate that such strains are common in food products. These data confirm that we have developed a technology that will serve as a multifunctional and important tool in the prevention and analysis of L. monocytogenes infections.
Technical Abstract: Listeria monocytogenes is a facultative intracellular pathogen responsible for food-borne disease with high mortality rates in humans and other animals. L. monocytogenes is also the leading microbiological cause of food recalls. Lineage 1 isolates of L. monocytogenes are of particular public health concern because they are responsible for most sporadic cases of listeriosis and the vast majority of epidemic outbreaks. Rapid, reproducible and sensitive methods for differentiating pathogens below the species level are required for effective pathogen control programs, and the CDC PulseNet Task Force has called for the development and validation of DNA sequence-based methods for subtyping food-borne pathogens. Therefore, we developed a multilocus genotyping (MLGT) assay based on nucleotide variation identified from 22 genes distributed across seven genomic regions in 65 L. monocytogenes isolates. This single-well assay of 60 allele-specific probes provided high discriminatory power (Simpson’s index = 0.91) and epidemiologically relevant, phylogenetically informative, subtype data. The MLGT assay uniquely identified isolates from the eight listeriosis outbreaks examined, and differentiated serotypes 1/2b and 4b as well as epidemic clones 1, 1a and 2. In addition, the assay included probes for a previously characterized truncation mutation in inlA, providing for the identification of a specific virulence-attenuated subtype. All 240 isolates examined were reproducibly typed with the MLGT assay, which captured 100% of the haplotype information identified in analyses of over 23,000 nucleotides of DNA sequence. These results demonstrate that MLGT represents a significant new tool for use in pathogen surveillance, outbreak detection, risk assessment, population analyses and epidemiological investigations.