Submitted to: Applied and Environmental Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/10/2008
Publication Date: 12/1/2008
Citation: Ward, T.J., Ducey, T.F., Usgaard, T.R., Dunn, K.A., Bielawski, J.P. 2008. Multilocus Genotyping Assays for SNP-based Subtyping of Listeria monocytogenes. Applied and Environmental Microbiology. 74(24):7629-7642. 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 a modular set of high-throughput subtyping tests based on DNA sequence differences between L. monocytogenes strains. These tests address the need for comprehensive subtyping of all L. monocytogenes isolates. In addition, these tests provide information that is critical to risk assessment and evaluation of the public health threat posed by individual strains. We used these tests to demonstrate that a substantial fraction of L. monocytogenes isolates from food products and processing facilities surveyed by the Food Safety and Inspection Service have reduced abilities to cause invasive illness in humans. These results demonstrate that we have developed a technology that will serve as a multifunctional and important tool in the prevention of L. monocytogenes infections and detection of disease outbreaks.
Technical Abstract: Listeria monocytogenes is responsible for serious invasive illness associated with consumption of contaminated food, and places a significant burden on public health and the agricultural economy. We recently developed a multilocus genotyping (MLGT) assay for high-throughput subtype determination of L. monocytogenes lineage I isolates based on interrogation of single nucleotide polymorphisms via multiplexed primer extension reactions. Here we report the development and validation of two additional MLGT assays that address the need for comprehensive, DNA sequence-based subtyping of L. monocytogenes. The first of these novel MLGT assays targeted variation segregating within lineage II, while we combined probes for lineage III strains with probes for strains representing a novel evolutionary lineage (IV) defined on the basis of phylogenetic analysis. These lineage-specific assays were based on nucleotide variation identified in > 3.8 Mb of comparative DNA sequence and consisted of 115 total probes that differentiated 93% of the 100 haplotypes defined by the multilocus sequence data. MLGT reproducibly typed the 173 isolates used in SNP discovery, and the 10,448 genotypes derived from MLGT analysis of these isolates were consistent with DNA sequence data. Application of the MLGT assays to assess subtype prevalence among isolates from ready-to-eat (RTE) foods and food-processing facilities indicated a low frequency (6.3%) of epidemic clone subtypes and a substantial population of isolates (> 30%) harboring mutations in inlA that are associated with attenuated virulence.