Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae.
Ciprofloxacin-resistant mutants of Streptococcus pneumoniae 7785 were generated by stepwise selection at increasing drug concentrations. Sequence analysis of PCR products from the strains was used to examine the quinolone resistance-determining regions of the GyrA and GyrB proteins of DNA gyrase and the analogous regions of the ParC and ParE subunits of DNA topoisomerase IV. First-step mutants exhibiting low-level resistance had no detectable changes in their topoisomerase quinolone resistance-determining regions, suggesting altered permeation or another novel resistance mechanism. Nine of 10 second-step mutants exhibited an alteration in ParC at Ser-79 to Tyr or Phe or at Ala-84 to Thr. Third- and fourth-step mutants displaying high-level ciprofloxacin resistance were found to have, in addition to the ParC alteration, a change in GyrA at residues equivalent to Escherichia coli GyrA resistance hot spots Ser-83 and Asp-87 or in GyrB at Asp-435 to Asn, equivalent to E. coli Asp-426, part of a highly conserved EGDSA motif in GyrB. No ParE changes were observed. Complementary analysis of two S. pneumoniae clinical isolates displaying low-level resistance to ciprofloxacin revealed a ParC change at Ser-79 to Phe or Arg-95 to Cys but no changes in GyrA, GyrB, or ParE. A highly resistant isolate, in addition to a ParC mutation, had a GyrA alteration at the residue equivalent to E. coli Asp-87. Thus, in both laboratory strains and clinical isolates, ParC mutations preceded those in GyrA, suggesting that topoisomerase IV is a primary topoisomerase target and gyrase is a secondary target for ciprofloxacin in S. pneumoniae.
Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, University of London, United Kingdom.
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Mehtar
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Fisher
D000595:Amino Acid Sequence; D000890:Anti-Infective Agents / Q000494:pharmacology*; D001483:Base Sequence; D002939:Ciprofloxacin / Q000494:pharmacology*; D027101:DNA Topoisomerase IV; D005798:Genes, Bacterial / Q000235:genetics; D006801:Humans; D008173:Lung Diseases, Obstructive / Q000382:microbiology; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D013296:Streptococcus pneumoniae / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D059005:Topoisomerase II Inhibitors
D000890:Anti-Infective Agents; D059005:Topoisomerase II Inhibitors; D002939:Ciprofloxacin; D027101:DNA Topoisomerase IV