Natural atypical Listeria innocua strains with Listeria monocytogenes pathogenicity island 1 genes

Abstract

Identification of bona fide Listeria isolates into the six species of the genus normally requires only a few tests. Aberrant isolates do occur, but even then only one or two extra confirmatory tests are generally needed for identification to species level. We have discovered a hemolytic-positive, rhamnose and xylose fermentation-negative Listeria strain with surprising recalcitrance to identification to the species level due to contradictory results in standard confirmatory tests. The issue had to be resolved by using total DNA-DNA hybridization testing and then confirmed by further specific PCR-based tests including a Listeria microarray assay. The results show that this isolate is indeed a novel one. Its discovery provides the first fully documented instance of a hemolytic Listeria innocua strain. This species, by definition, is typically nonhemolytic. The L. innocua isolate contains all the members of the PrfA-regulated virulence gene cluster (Listeria pathogenicity island 1) of L. monocytogenes. It is avirulent in the mouse pathogenicity test. Avirulence is likely at least partly due to the absence of the L. monocytogenes-specific allele of iap, as well as the absence of inlA, inlB, inlC, and daaA. At least two of the virulence cluster genes, hly and plcA, which encode the L. monocytogenes hemolysin (listeriolysin O) and inositol-specific phospholipase C, respectively, are phenotypically expressed in this L. innocua strain. The detection by PCR assays of specific L. innocua genes (lin0198, lin0372, lin0419, lin0558, lin1068, lin1073, lin1074, lin2454, and lin2693) and noncoding intergenic regions (lin0454-lin0455 and nadA-lin2134) in the strain is consistent with its L. innocua DNA-DNA hybridization identity. Additional distinctly different hemolytic L. innocua strains were also studied.

FIG. 1.

(a) Dendrogram showing the phylogenetic relationship among the six Listeria species and the atypical hemolytic L. innocua strains based on nucleotide sequence data of the partial 16S rRNA gene. This tree was constructed by the maximum parsimony method in the MEGA 2.1 package. The bootstrap values presented at corresponding branches were evaluated from 500 replications. GenBank accession numbers are indicated for each strain used in creating this dendrogram. (b) Dendrogram showing the phylogenetic relationship among the six Listeria species and the atypical hemolytic L. innocua strains based on nucleotide sequence data of the large 16S-23S rRNA IGS region. This tree was constructed by the maximum parsimony method in the MEGA 2.1 package. The bootstrap values presented at corresponding branches were evaluated from 500 replications. GenBank accession numbers are indicated for each strain used in creating this dendrogram.

FIG. 2.

Alignment of prs-prfA intergenic regions of the atypical hemolytic L. innocua strains with putative insertion junctions. prs and prfA stop codons are boxed (for details see the text). Asterisks indicate the precise halfway points between the sequence numbers.

FIG. 3.

Genetic relationships between the atypical hemolytic L. innocua strains and other Listeria species. The dendrogram is based on invasion-associated protein p60 (iap) gene sequence analysis, and it was constructed by the maximum parsimony method in the MEGA 2.1 package. The bootstrap values presented at corresponding branches were evaluated from 500 replications. GenBank accession numbers are indicated for each strain used in this analysis. To more clearly show the evolutionary relationships occurring among members of the Listeria species, the species subbranches of the phylogenetic tree were clustered.