Analysis of additional virulence genes and virulence gene regions in Listeria monocytogenes confirms the epidemiologic relevance of multi-virulence-locus sequence typing

Previous molecular subtyping studies have defined four epidemic clones (ECs) of Listeria monocytogenes (ECI, ECII, ECIII, and ECIV). Partial sequences of eight virulence genes were previously shown to be identical within individual ECs of L. monocytogenes. The present study was conducted to determine if the sequences of other virulence genes and virulence gene regions are also conserved within these ECs. Six additional virulence genes - bsh, hly, inlJ, lplAl, pgdA, and srtA - and three additional virulence gene regions of actA, inlA, and inlB were selected based on their role in L. monocytogenes virulence, and intragenic regions of each gene were sequenced. Sequencing was performed on a diverse set of 44 to 48 L. monocytogenes strains. Results demonstrated that the sequenced regions of the nine virulence genes were identical within each of the ECs, and 257 new single nucleotide polymorphism (SNPs) were identilied. ECIII (lineage II) was easily distinguislwble front the other ECs, as 238 SNPs were observed in ECIII due to its significant evolutionary divergence from lineage I. With regard to the other ECs, there were 5 SNPs that represented an informative set, since these SNPs were able to differentiate specific ECs from all either unrelated strains used in this study. This study confirms our previous finding that virulence gene sequences are highly conserved within individual ECs and cuintain stable SNPs that can be used to very accurately differentiate ECs of L. monocytogenes from each other and from other diverse strains.