Viridans group streptococci are donors in horizontal transfer of topoisomerase IV genes to Streptococcus pneumoniae

Abstract

A total of 46 ciprofloxacin-resistant (Cip(r)) Streptococcus pneumoniae strains were isolated from 1991 to 2001 at the Hospital of Bellvitge. Five of these strains showed unexpectedly high rates of nucleotide variations in the quinolone resistance-determining regions (QRDRs) of their parC, parE, and gyrA genes. The nucleotide sequence of the full-length parC, parE, and gyrA genes of one of these isolates revealed a mosaic structure compatible with an interspecific recombination origin. Southern blot analysis and nucleotide sequence determinations showed the presence of an ant-like gene in the intergenic parE-parC regions of the S. pneumoniae Cip(r) isolates with high rates of variations in their parE and parC QRDRs. The ant-like gene was absent from typical S. pneumoniae strains, whereas it was present in the intergenic parE-parC regions of the viridans group streptococci (Streptococcus mitis and Streptococcus oralis). These results suggest that the viridans group streptococci are acting as donors in the horizontal transfer of fluoroquinolone resistance genes to S. pneumoniae.

FIG. 1.

Nucleotide sequence variations in the ParC, ParE, and GyrA QRDRs. The nucleotides present at each polymorphic site are shown for strain R6, but for the other strains, only the nucleotides that differ from those in R6 are shown. Changes yielding amino acid substitutions, including those involved in fluoroquinolone resistance, are shown in boldface. Codon numbers are indicated vertically above the sequences. Positions 1, 2, and 3 in the fourth row refer to the first, second, and third nucleotides in the codon, respectively. Strains whose sequences do not differ from that of R6 more than 1% are shaded in gray. SPN, S. pneumoniae; SOR, S. oralis; SMI, S. mitis.

FIG. 2.

UPGMA trees of concatenated atpC and atpA genes, full-length parE genes, and ant genes. Phylogenetic and molecular evolutionary analyses were conducted with the MEGA program (version 2.1) by the UPGMA method. Only bootstrap confidence intervals exceeding 90% are shown. The GenBank accession numbers for each nucleotide sequence are shown in parentheses.

FIG. 3.

Mosaic structures of the gyrA, parC, and parE genes of the indicated strains of S. pneumoniae and VS. The locations of the QRDRs are indicated at the tops of the gyrA and parC sequences. The positions of the active Tyr residues (Y120 in GyrA and Y118 in ParC) that bind to DNA and the Ser residues that are changed in strain 4391 (S81 in GyrA and S79 in ParC) and that are involved in resistance are marked. Blocks showing the percent sequence divergence from the corresponding regions of S. pneumoniae R6 are indicated. White box, region of the sequence that differed by ≤1.5%; gray boxes, regions that differed by more than 1.5% but less than 9%; black boxes, regions that differed by >9%. The strains used were S. pneumoniae (SPN) R6 (GenBank accession number AE008451), S. pneumoniae TIGR4 (GenBank accession number AE007391), S. pneumoniae 4391, S. mitis (SMI) NCTC 12261, and S. oralis (SOR) NCTC 11427.

FIG. 4.

Restriction map of the parE-parC region of S. pneumoniae strains and strains of VS and its genetic organization as deduced from Southern blotting experiments and nucleotide sequence analyses. E, EcoRV; N, NcoI. The parE and parC genes with mosaic structures are indicated by with striped arrows. SOR, S. oralis; SMI, S. mitis.

FIG. 5.

Southern blot hybridization of S. pneumoniae strains and strains of VS with an ant-specific probe. Chromosomal DNAs from the indicated strains were cleaved with EcoRV-NcoI, and the fragments were separated in 0.8% agarose gels. The gels were blotted, and the blot was probed with a biotinylated probe derived from S. pneumoniae 3870 containing positions 26 to 290 of the ant gene. The numbers on the bottom right are molecular sizes (in kilobases).

FIG. 5.

Southern blot hybridization of S. pneumoniae strains and strains of VS with an ant-specific probe. Chromosomal DNAs from the indicated strains were cleaved with EcoRV-NcoI, and the fragments were separated in 0.8% agarose gels. The gels were blotted, and the blot was probed with a biotinylated probe derived from S. pneumoniae 3870 containing positions 26 to 290 of the ant gene. The numbers on the bottom right are molecular sizes (in kilobases).