Discovery of natural atypical nonhemolytic Listeria seeligeri isolates

Abstract

We found seven Listeria isolates, initially identified as isolates with the Xyl(+) Rha(-) biotype of Listeria welshimeri by phenotypic tests, which exhibited discrepant genotypic properties in a well-validated Listeria species identification oligonucleotide microarray. The microarray gives results of these seven isolates being atypical hly-negative L. seeligeri isolates, not L. welshimeri isolates. The aberrant L. seeligeri isolates were d-xylose fermentation positive, l-rhamnose fermentation negative (Xyl(+) Rha(-)), and nonhemolytic on blood agar and in the CAMP test with both Staphylococcus aureus (S(-) reaction) and Rhodococcus equi (R(-) reaction). All genes of the prfA cluster of L. seeligeri, located in the prs-ldh region, including the orfA2, orfD, prfA, orfE, plcA, hly, orfK, mpl, actA, dplcB, plcB, orfH, orfX, orfI, orfP, orfB, and orfA genes, were checked by PCR and direct sequencing for evidence of their presence in the atypical isolates. The prs-prfA cluster-ldh region of the L. seeligeri isolates was approximately threefold shorter due to the loss of orfD, prfA, orfE, plcA, hly, orfK, mpl, actA, dplcB, plcB, orfH, orfX, and orfI. The genetic map order of the cluster genes of all the atypical L. seeligeri isolates was prs-orfA2-orfP-orfB-orfA-ldh, which was comparable to the similar region in L. welshimeri, with the exception of the presence of orfA2. DNA sequencing and phylogenetic analysis of 17 housekeeping genes indicated an L. seeligeri genomic background in all seven of the atypical hly-negative L. seeligeri isolates. Thus, the novel biotype of Xyl(+) Rha(-) Hly(-) L. seeligeri strains can only be distinguished from Xyl(+) Rha(-) L. welshimeri strains genotypically, not phenotypically. In contrast, the Rha(+) Xyl(+) biotype of L. welshimeri would not present an identification issue.

FIG. 1.

Schematic diagram of virulence gene clusters of L. seeligeri strain ATCC 35967, the atypical nonhemolytic L. seeligeri biotype, and L. welshimeri. PCR and sequencing showed that the PrfA-regulated virulence gene cluster was altered in the nonhemolytic L. seeligeri variants by the deletion of 13 genes, namely, orfD-prfA-orfE-plcA-hly-orfK-mpl-actA-dplcB-plcB-orfH-orfX-orfI. Thus, the isolates had the cluster prs-orfA2-(13-gene deletion)-orfP-orfB-orfA-ldh.

FIG. 2.

Dendrograms showing phylogenetic relationships among Listeria species and the atypical nonhemolytic L. seeligeri strains based on complete nucleotide sequence data for the orfA (A), orfB (B), and orfP (C) genes. The trees were constructed by the minimum evolution method in the MEGA 2.1 package. The bootstrap values presented at corresponding branches were evaluated from 1,000 replications. GenBank accession numbers are indicated for each strain used in creating the dendrograms.

FIG.3.

Dendrograms showing phylogenetic relationships among Listeria species and the atypical nonhemolytic L. seeligeri strains based on nucleotide sequence data for the partial 16S rRNA gene (A), the large 16S-23S ITS (B), and the complete iap gene (C). The trees were constructed by the minimum evolution method in the MEGA 2.1 package. The bootstrap values presented at corresponding branches were evaluated from 1,000 replications. GenBank accession numbers are indicated for each strain used in creating the dendrograms.

FIG.3.

Dendrograms showing phylogenetic relationships among Listeria species and the atypical nonhemolytic L. seeligeri strains based on nucleotide sequence data for the partial 16S rRNA gene (A), the large 16S-23S ITS (B), and the complete iap gene (C). The trees were constructed by the minimum evolution method in the MEGA 2.1 package. The bootstrap values presented at corresponding branches were evaluated from 1,000 replications. GenBank accession numbers are indicated for each strain used in creating the dendrograms.