Genetic markers unique to Listeria monocytogenes serotype 4b differentiate epidemic clone II (hot dog outbreak strains) from other lineages

Abstract

A small number of closely related strains of Listeria monocytogenes serotype 4b, designated epidemic clone I (ECI), have been implicated in numerous outbreaks of food-borne listeriosis described during the past two decades in Europe and North America. In 1998 to 1999, a multistate outbreak traced to contaminated hot dogs involved a different strain type of serotype 4b, with genetic fingerprints rarely encountered before. In spite of the profound economic and public health impact of this outbreak, the implicated bacteria (designated epidemic clone II [ECII]) have remained poorly characterized genetically, and nucleotide sequences specific for these strains have not been reported. Using genome sequence information, PCR, and Southern blots, we identified DNA fragments which appeared to be either absent or markedly divergent in the hot dog outbreak strains but conserved among other serotype 4b strains. PCR with primers derived from these fragments as well as Southern blots with the amplicons as probes readily differentiated ECII from other serotype 4b strains. The serotype 4b-specific region harboring these fragments was adjacent to inlA, which encodes a well-characterized virulence determinant. The findings suggest that ECII strains have undergone divergence in portions of a serotype-specific region that is conserved in other serotype 4b strains. Although the mechanisms that drive this divergence remain to be identified, DNA-based tools from this region can facilitate the detection and further characterization of strains belonging to this lineage.

FIG. 1.

Differentiation of the hot dog outbreak strains (ECII) from other strains of L. monocytogenes serotype 4b by PCR with primers derived from 4bSF7. Lanes: M, 100-bp molecular size marker XIV (Roche); 1 to 8, hot dog outbreak strains; 9 to 18, other serotype 4b strains from the categories shown in Table 1. PCR was done as described in Materials and Methods.

FIG. 2.

Southern blot of EcoRI-digested chromosomal DNAs from the hot dog outbreak strains (ECII) and other representative strains of L. monocytogenes, with 4bSF7 (A) and 4bSF18 (B) as probes. Lanes: M, digoxigenin-labeled λ HindIII digest, used as molecular size markers (fragment sizes are [from top to bottom] 23, 9.4, 6.5, 4.3, 2.3, and 2.0 kb); 1 to 8, hot dog outbreak strains; 9 to 14 and 18, other serotype 4b strains from the categories shown in Table 1; 15, L. monocytogenes G2228 (serotype 1/2a); 16, L. monocytogenes G3978 (serotype 1/2b); 17, L. innocua 101A. Southern blots were done as described in Materials and Methods.

FIG. 3.

Conservation of ORFs in the genomic region harboring 4bSF7 and 4bSF18 between ECII and other serotype 4b strains. DNA probes were generated with the primers listed in Table 2 and used in hybridizations as described in Materials and Methods. + and − indicate the presence and absence of a hybridization signal, respectively. L. monocytogenes strains of serotype 4d yielded results identical to those of other 4b strains. ORFs which lacked detectable homology in ECII strains (on the basis of the Southern blot data) are indicated in black, and serotype 4b-specific ORFs conserved in ECII strains are indicated in gray. The putative ORFs 1373 and 1378 (111 and 90 bp, respectively) were too short for adequate probe construction, and their conservation in ECII strains has not been determined. Arrows indicate the direction of transcription. IGS, intergenic regions. Note that inlA (ORFs 1358), inlB (ORF 1355), and ORF 1376 are much longer than the others, as detailed in Table 3, and are not drawn to scale.

FIG. 4.

Genomic organization in the genomic region harboring 4bSF7 and 4bSF18, and genomic counterparts of the region in the sequenced genomes of L. monocytogenes EGD (serotype 1/2a) and L. innocua. Artemis was used to visualize the location and direction of transcription of the putative ORFs. Black arrows indicate serotype 4b-specific ORFs not detected in ECII strains; gray arrows indicate ORFs that are serotype 4b specific (ORFs 1376 and 1377 are also conserved between serotype 4b strains and L. innocua) and conserved in ECII strains. Diagonal lines indicate ORFs conserved between different genomes. The putative ORFs 1373 and 1378 (111 and 90 bp, respectively) were too short for adequate probe construction, and their conservation in ECII strains has not been determined. Note that inlA (ORFs 1358 and 433), inlB (ORF 1355), and ORF 1376 are much longer than the others, as detailed in Table 3, and are not drawn to scale.