Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae

Abstract

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.

Pharmacodynamic evaluation of tosufloxacin against Streptococcus pneumoniae in an in vitro model simulating serum concentration

“…If the frequency of resistant subpopulations after treatment with each fluoroquinolone had increased, the susceptibility of the colonies obtained on the plate containing the fluoroquinolone, and gene mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC of representative colonies, were investigated. The QRDRs of gyrA and parC were amplified using the polymerase chain reaction (PCR), following the method of Pan et al 3 DNA sequences were determined with an ABI Prism 310 DNA sequencer (Applied Biosystems, Foster City, CA, USA), and were analyzed with DNASIS sequence analysis programs (ver. 2.1, Hitachi Software Engineering, San Francisco, CA, USA).…”

“…[1][2][3][4] For a long time penicillin was the most effective drug against such infections. However, penicillin-resistant S. pneumoniae was first reported in 1967, followed by the report that the frequency of isolation of penicillin-nonsusceptible strains of S. pneumoniae increased dramatically during the 1990s.…”

Antimicrobial activity of DW286 against Streptococcus pneumoniae

“…Polymerase chain reaction (PCR) was used to amplify the QRDRs in gyrA, gyrB, parC and parE using previously described primer pairs and cycling conditions [12]. Template DNA was prepared using Genomic DNA Isolation Solution (NucleoGen Inc., Siheung, South Korea) following the manufacturer's instructions.…”

Quinolone resistance: Older concepts and newer developments

“…For example, selection in vitro has shown that a first-step mutation in topoisomerase IV confers low-level resistance to ciprofloxacin, levofloxacin and ofloxacin, while low-level resistance to sparfloxacin, moxifloxacin and gatifloxacin is the result of a first-step mutation in DNA gyrase [52][53][54][55]. In the case of clinafloxacin, DNA gyrase and topoisomerase IV are targeted equally [56].…”