Selective Discrimination of Listeria monocytogenes Epidemic Strains by a Mixed-Genome DNA Microarray Compared to Discrimination by Pulsed-Field Gel Electrophoresis, Ribotyping, and Multilocus Sequence Typing

Abstract

Listeria monocytogenes can cause serious illness in humans, and subsequent epidemiological investigation requires molecular characterization to allow the identification of specific isolates. L. monocytogenes is usually characterized by serotyping and is subtyped by using pulsed-field gel electrophoresis (PFGE) or ribotyping. DNA microarrays provide an alternative means to resolve genetic differences among isolates, and unlike PFGE and ribotyping, microarrays can be used to identify specific genes associated with strains of interest. Twenty strains of L. monocytogenes representing six serovars were used to generate a shotgun library, and subsequently a 629-probe microarray was constructed by using features that included only potentially polymorphic gene probe sequences. Fifty-two strains of L. monocytogenes were genotyped by using the condensed array, including strains associated with five major listeriosis epidemics. Cluster analysis of the microarray data grouped strains according to phylogenetic lineage and serotype. Most epidemiologically linked strains were grouped together, and subtyping resolution was the same as that with PFGE (using AscI and ApaI) and better than that with multilocus sequence typing (using six housekeeping genes) and ribotyping. Additionally, a majority of epidemic strains were grouped together within phylogenetic Division I. This epidemic cluster was clearly distinct from the two other Division I clusters, which encompassed primarily sporadic and environmental strains. Discriminant function analysis allowed identification of 22 probes from the mixed-genome array that distinguish serotypes and subtypes, including several potential markers that were distinct for the epidemic cluster. Many of the subtype-specific genes encode proteins that likely confer survival advantages in the environment and/or host.

Pulsed-Field Gel Electrophoresis (PFGE) Analysis of Temporally Matched Listeria monocytogenes Isolates from Human Clinical Cases, Foods, Ruminant Farms, and Urban and Natural Environments Reveals Source-Associated as Well as Widely Distributed PFGE Types

“…Rapid, discriminatory, and standardized molecular subtyping methods are critical for effective food-borne disease surveillance and outbreak investigations. While automated ribotyping provides superior standardization and speed compared to many other molecular subtyping methods for bacterial isolates (58), not surprisingly and consistent with the findings of a number of previous studies (1,5,24,32,56), two-enzyme PFGE using the standard PulseNet protocol provides significantly higher subtype discrimination than EcoRI ribotyping. While automated ribotyping is thus suitable for population-based studies or subtype characterization of large numbers of isolates (1), the discriminatory power of PFGE clearly is critical for human disease outbreak investigations.…”

Suspension Microarray with Dendrimer Signal Amplification Allows Direct and High-Throughput Subtyping of Listeria monocytogenes from Genomic DNA

“…Future experiments will include design and testing of additional probes to improve L. monocytogenes suspension array subtyping resolution, including the addition of phylogeneticdivision-and species-specific probes. Variable regions of the L. monocytogenes genome have already been identified using planar-microarray analysis (1,3,5). This information can be readily used for the design of additional suspension array probes to allow development of a rapid and high-resolution subtyping assay for L. monocytogenes isolates.…”

“…The most recent examples of this are multilocus sequence subtyping (12,17) and microarray genomic analysis (1,5,26). Recent studies have shown that a relatively simple genotyping microarray has subtyping resolution comparable to that of pulsed-field gel electrophoresis (AscI and ApaI digestion) and superior to that of multilocus sequence subtyping (six housekeeping genes) and ribotyping (3). L. monocytogenes subtyping using DNA microarrays is also genetically informative because it can identify the genes or alleles that characterize subtypes.…”

Presencia de Listeria monocytogenes en alimentos listos para el consumo, procedentes de plazas de mercado y delicatessen de supermercados de cadena, Bogotá, D.C, 2002-2008

“…Con la clasificación alimentaria expuesta anteriormente, el criterio establecido en los Estados Unidos para aceptar como inocuos los alimentos listos para el consumo en los que puede crecer L. monocytogenes, es de cero tolerancia (10,16,47,48,65,68,69). Europa acepta niveles hasta de 100 unidades formadoras de colonias (UFC) para algunos alimentos que no estén destinados a poblaciones vulnerables (47).…”