Multilocus Genotyping Assays for Single Nucleotide Polymorphism-Based Subtyping of Listeria monocytogenes Isolates
Date
2008-12
Authors
Ward, Todd J.
Ducey, Thomas F.
Usgaard, Thomas
Dunn, Katherine A.
Bielawski, Joseph P.
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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 ( 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.
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Citation
Ward, Todd J., Thomas F. Ducey, Thomas Usgaard, Katherine A. Dunn, et al. 2008. "Multilocus Genotyping Assays for Single Nucleotide Polymorphism-Based Subtyping of Listeria
monocytogenes Isolates." Applied and Environmental Microbiology 74(24): 7629-7642.