Genetic Analyses of Mutations Contributing to Fluoroquinolone Resistance in Clinical Isolates of Streptococcus pneumoniae

Abstract

Twenty-one clinical isolates of Streptococcus pneumoniae showing reduced susceptibility or resistance to fluoroquinolones were characterized by serotype, antimicrobial susceptibility, and genetic analyses of the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE. Five strains were resistant to three or more classes of antimicrobial agents. In susceptibility profiles for gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin, ofloxacin, sparfloxacin, and trovafloxacin, 14 isolates had intermediate- or high-level resistance to all fluoroquinolones tested except gemifloxacin (no breakpoints assigned). Fluoroquinolone resistance was not associated with serotype or with resistance to other antimicrobial agents. Mutations in the QRDRs of these isolates were more heterogeneous than those previously reported for mutants selected in vitro. Eight isolates had amino acid changes at sites other than ParC/S79 and GyrA/S81; several strains contained mutations in gyrB, parE, or both loci. Contributions to fluoroquinolone resistance by individual amino acid changes, including GyrB/E474K, ParE/E474K, and ParC/A63T, were confirmed by genetic transformation of S. pneumoniae R6. Mutations in gyrB were important for resistance to gatifloxacin but not moxifloxacin, and mutation of gyrA was associated with resistance to moxifloxacin but not gatifloxacin, suggesting differences in the drug-target interactions of the two 8-methoxyquinolones. The positions of amino acid changes within the four genes affected resistance more than did the total number of QRDR mutations. However, the effect of a specific mutation varied significantly depending on the agent tested. These data suggest that the heterogeneity of mutations will likely increase as pneumococci are exposed to novel fluoroquinolone structures, complicating the prediction of cross-resistance within this class of antimicrobial agents.

Prevalence and Molecular Analysis of Macrolide and Fluoroquinolone Resistance among Isolates of Streptococcus pneumoniae Collected during the 2000-2001 PROTEKT US Study

“…A novel Q90H change was found in ParC of one isolate, and a novel S80P change was found in another; both changes appeared to play a role in fluoroquinolone resistance. Some isolates (15 of 81; 18.5%) possessed three QRDR A minority of GyrB changes were implicated in resistance, as has been found previously (5,21,38). Surprisingly, 13 instances of ParE alterations (D435N and E474K in the main and one novel A325V change) occurred, which is greater than the number found in previous studies (3,5,21,38).…”

Genetic Relatedness of Levofloxacin-Nonsusceptible Streptococcus pneumoniae Isolates from North America

“…These percentages are reflective of the average susceptibility rates for penicillin and erythromycin in the United States and Canada (9,10,23). Weigel et al reported a similar finding (41). In contrast, Corso et al found that 65% of 20 fluoroquinoloneresistant isolates had reduced susceptibility to penicillin (6).…”

Relative Fitness of Fluoroquinolone-resistantStreptococcus pneumoniae

“…For this study, naturally occurring fluoroquinolone resistance mutations were placed in the serotype 19F strain EF3030 by using established techniques (17). Briefly, 1,325-bp fragments of gyrA and 778 bp of parC were amplified by polymerase chain reaction from 2 previously characterized (18) clinical isolates of QRSP (CT01147 and UAB169; gemifloxacin MIC = 1 µg/mL) by using the primers shown in the Table. Phenotypic expression was carried out for 2 to 24 h. First-step trans-formants were generated by the introduction of a parC or gyrA fragment, and these were selected on 0.06 µg/mL gemifloxacin (SmithKline Beecham Pharmaceuticals, Collegeville, PA, USA).…”