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Applied and Environmental Microbiology, January 2007, p. 133-147, Vol. 73, No. 1
0099-2240/07/$08.00+0     doi:10.1128/AEM.01453-06

A Single-Nucleotide-Polymorphism-Based Multilocus Genotyping Assay for Subtyping Lineage I Isolates of Listeria monocytogenes{triangledown},{dagger}

Thomas F. Ducey,1 Brent Page,1 Thomas Usgaard,1 Monica K. Borucki,2 Kitty Pupedis,3 and Todd J. Ward1*

Microbial Genomics and Bioprocessing Research Unit, Agricultural Research Service, United States Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604,1 Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, California 94550,2 Microbial Outbreaks and Special Projects Laboratory, Food Safety and Inspection Service, United States Department of Agriculture, Athens, Georgia 306053

Received 23 June 2006/ Accepted 27 October 2006

Listeria monocytogenes is a facultative intracellular pathogen responsible for food-borne disease with high mortality rates in humans and is the leading microbiological cause of food recalls. Lineage I isolates of L. monocytogenes are a 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 for L. monocytogenes lineage I isolates based on nucleotide variation identified by sequencing 23,251 bp of DNA from 22 genes distributed across seven genomic regions in 65 L. monocytogenes isolates. This single-well assay of 60 allele-specific probes captured 100% of the haplotype information contained in approximately 1.5 Mb of comparative DNA sequence and was used to reproducibly type a total of 241 lineage I isolates. The MLGT assay provided high discriminatory power (Simpson's index value, 0.91), uniquely identified isolates from the eight listeriosis outbreaks examined, and differentiated serotypes 1/2b and 4b as well as epidemic clone I (ECI), ECIa, and ECII. In addition, the assay included probes for a previously characterized truncation mutation in inlA, providing for the identification of a specific virulence-attenuated subtype. These results demonstrate that MLGT represents a significant new tool for use in pathogen surveillance, outbreak detection, risk assessment, population analyses, and epidemiological investigations.

* Corresponding author. Mailing address: Microbial Genomics and Bioprocessing Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, IL 61604. Phone: (309) 681-6394. Fax: (309) 681-6672. E-mail: wardtj{at}ncaur.usda.gov.

{triangledown} Published ahead of print on 3 November 2006.

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

Applied and Environmental Microbiology, January 2007, p. 133-147, Vol. 73, No. 1
0099-2240/07/$08.00+0     doi:10.1128/AEM.01453-06




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