Fluoroquinolone resistance in Streptococcus dysgalactiae subsp. equisimilis and evidence for a shared global gene pool with Streptococcus pyogenes

Abstract

Quinolone resistance is an emerging problem in Streptococcus pyogenes, and recombination with Streptococcus dysgalactiae DNA has been implicated as a frequent mechanism leading to resistance. We have characterized a collection of S. dysgalactiae subsp. equisimilis isolates responsible for infections in humans (n = 314) and found a high proportion of levofloxacin-resistant isolates (12%). Resistance was associated with multiple emm types and genetic lineages, as determined by pulsed-field gel electrophoretic profiling. Since we could not find evidence for a role of efflux pumps in resistance, we sequenced the quinolone resistance-determining regions of the gyrA and parC genes of representative resistant and susceptible isolates. We found much greater diversity among the parC genes (19 alleles) than among the gyrA genes (5 alleles). While single mutations in either GyrA or ParC were sufficient to raise the MIC so that the strains were classified as intermediately resistant, higher-level resistance was associated with mutations in both GyrA and ParC. Evidence for recombination with S. pyogenes DNA was found in some parC alleles, but this was not exclusively associated with resistance. Our data support the existence of a common reservoir of genes conferring quinolone resistance shared between S. dysgalactiae subsp. equisimilis and S. pyogenes, while no recombination with the animal pathogen S. dysgalactiae subsp. dysgalactiae could be found.

FIG. 1.

Neighbor-joining tree for the parC QRDR. The Streptococcus agalactiae type strain NCTC 8181 parC sequence (GenBank accession number AB101464) was used to root the tree. If the percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (1,000 replicates) was greater than 75%, the values are shown next to the branches. The tree is drawn to scale, with the branch lengths being in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed by the Kimura two-parameter method and are in the units of the number of base substitutions per site. Sp, Streptococcus pyogenes; Sde, Streptococcus dysgalactiae subsp. equisimilis; Sdd, Streptococcus dysgalactiae subsp. dysgalactiae. Sets of numbers and letters identify the GenBank entries whose sequences are identical to the sequences of the alleles found in our collection or the sequences of S. pyogenes used to construct the tree.

FIG. 2.

Neighbor-joining tree for the gyrA QRDR. The Streptococcus agalactiae type strain NCTC 8181 gyrA sequence (GenBank accession number AB101448) was used to root the tree. For details on the methods used to construct the tree, see the legend to Fig. 1. Sp, Streptococcus pyogenes; Sde, Streptococcus dysgalactiae subsp. equisimilis; Sdd, Streptococcus dysgalactiae subsp. dysgalactiae. Sets of numbers and letters identify the GenBank entries whose sequences are identical to the sequences of the alleles found in our collection or the sequences of S. pyogenes used to construct the tree.

FIG. 3.

Diagram of the QRDRs of gyrA and parC in isolates showing recombination events. The box on the left represents the gyrA QRDR, while that on the right represents the parC QRDR. White boxes represent regions that are characteristic of S. dysgalactiae subsp. equisimilis, while black boxes represent regions characteristic of S. pyogenes. Each allele combination represents the sequences resulting from the putative recombination events indicated in Fig. 1 (T₁ to T₃). Recombination breakpoints were determined by the maximum χ² method implemented in RDP3 software, refined by visual inspection of the sequences (Table 3).