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Applied and Environmental Microbiology, December 2008, p. 7629-7642, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.01127-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Multilocus Genotyping Assays for Single Nucleotide Polymorphism-Based Subtyping of Listeria monocytogenes Isolates{triangledown} ,{dagger}

Todd J. Ward,1* Thomas F. Ducey,1,{ddagger} Thomas Usgaard,1 Katherine A. Dunn,2 and Joseph P. Bielawski2

Microbial Genomics and Bioprocessing Research Unit, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604,1 Department of Biology, Dalhousie University, Halifax, Nova Scotia B3H 4J1, Canada2

Received 19 May 2008/ Accepted 9 October 2008

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 (SNPs) 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 the second assay combined probes for lineage III strains with probes for strains representing a recently characterized fourth evolutionary lineage (IV) of L. monocytogenes. These 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 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 associated with attenuated virulence in cell culture and animal models. These mutations were restricted to serogroup 1/2 isolates, which may explain the overrepresentation of serotype 4b isolates in human listeriosis cases.

* Corresponding author. Mailing address: Microbial Genomics and Bioprocessing Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, USDA, Peoria, IL 61604. Phone: (309) 681-6394. Fax: (309) 681-6672. E-mail: todd.ward{at}ars.usda.gov

{triangledown} Published ahead of print on 17 October 2008.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Current address: Coastal Plains Soil, Water and Plant Research Center, Agricultural Research Service, USDA, 2611 W. Lucas Street, Florence, SC 29501.

Applied and Environmental Microbiology, December 2008, p. 7629-7642, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.01127-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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