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.

FIG. 1.

UPGMA representation of genetic relationships between 52 L. monocytogenes isolates and one L. innocua isolate (INN) based on hybridization data derived from 130 bimodally distributed microarray probes. Phylogenetic divisions are indicated as D1a and D1b (Division I) and D2 (Division II). Isolates B339S and B345S (in boldface) were tested for processing and analysis reproducibility in two separate experiments. Isolates with the same AscI and ApaI PFGE restriction patterns are shown in the same color. Nine isolates were subtyped by PFGE, MLST, and ribotyping and are labeled with a diamond symbol. Ten isolates from the following four different epidemics were tested for subtype grouping: the 1981 Halifax epidemic (HA) (isolates F495E and F496E), the 1994 Illinois epidemic (IL) (isolates B507E and B508B), the 1998 multistate frankfurter-associated epidemic (HD) (isolates F470E, F581E, and F584E), and the 1988 to 1990 United Kingdom epidemic (UK) (isolates F497E, F498E, and F586E).