scholarly journals ParC and GyrA May Be Interchangeable Initial Targets of Some Fluoroquinolones in Streptococcus pneumoniae

1999 ◽  
Vol 43 (2) ◽  
pp. 302-306 ◽  
Author(s):  
Emmanuelle Varon ◽  
Claire Janoir ◽  
Marie-Dominique Kitzis ◽  
Laurent Gutmann
Keyword(s):  
Streptococcus Pneumoniae ◽  
Susceptible Strain ◽  
Quinolone Resistance ◽  
Topoisomerase Iv ◽  
Double Mutation ◽  
Active Efflux ◽  
Single Target ◽  
Resistant Mutants ◽  
The Individual

ABSTRACT To evaluate the role of known topoisomerase IV and gyrase mutations in the fluoroquinolone (FQ) resistance of Streptococcus pneumoniae, we transformed susceptible strain R6 with PCR-generated fragments encompassing the quinolone resistance-determining regions (QRDRs) of parC orgyrA from different recently characterized FQ-resistant mutants. Considering the MICs of FQs and the GyrA and/or ParC mutations of the individual transformants, we found three levels of resistance. The first level was obtained when a single target, ParC or GyrA, depending on the FQ, was modified. An additional mutation(s) in a second target, GyrA or ParC, led to the second level. The highest increases in resistance levels were seen for Bay y3118 and moxifloxacin with the transformant harboring a double mutation in both ParC and GyrA. When a single modified target was considered, only the ParC mutation(s) led to an increase in the MICs of pefloxacin and trovafloxacin. In contrast, the GyrA or ParC mutation(s) could lead to increases in the MICs of ciprofloxacin, sparfloxacin, grepafloxacin, Bay y3118, and moxifloxacin. These results suggest that the preferential target of trovafloxacin and pefloxacin is ParC, whereas either ParC or GyrA may both be initial targets for the remaining FQs tested. The contribution of the ParC and GyrA mutations to efflux-mediated FQ resistance was also examined. Active efflux was responsible for two- to fourfold increases in the MICs of ciprofloxacin for the transformants, regardless of the initial FQ resistance levels of the recipients.

scholarly journals Grepafloxacin, a Dimethyl Derivative of Ciprofloxacin, Acts Preferentially through Gyrase in Streptococcus pneumoniae: Role of the C-5 Group in Target Specificity

2002 ◽  
Vol 46 (2) ◽  
pp. 582-585 ◽  
Author(s):  
Julia E. Morris ◽  
Xiao-Su Pan ◽  
L. Mark Fisher
Keyword(s):  
Streptococcus Pneumoniae ◽  
Quinolone Resistance ◽  
Target Specificity ◽  
Topoisomerase Iv

ABSTRACT Grepafloxacin, a 5-methyl-7-piperazinyl-3"-methyl analogue of ciprofloxacin, was used to obtain stepwise-selected mutants of Streptococcus pneumoniae 7785. Analysis of the quinolone resistance-determining regions of the gyrA, gyrB, parC, and parE genes in these mutants revealed that gyrA mutations preceded those in parC. Given that ciprofloxacin (5-H,7-piperazinyl) and AM-1121 (5-H,7-piperazinyl-3"-methyl) both act through topoisomerase IV, we conclude that the 5-methyl group of grepafloxacin favors gyrase in S. pneumoniae.

scholarly journals Mechanisms and Frequency of Resistance to Premafloxacin in Staphylococcus aureus: Novel Mutations Suggest Novel Drug-Target Interactions

2000 ◽  
Vol 44 (12) ◽  
pp. 3344-3350 ◽  
Author(s):  
Dilek Ince ◽  
David C. Hooper
Keyword(s):  
Staphylococcus Aureus ◽  
Fold Increase ◽  
Single Step ◽  
The Novel ◽  
Novel Mutations ◽  
Topoisomerase Iv ◽  
Double Mutation ◽  
Fourfold Increase ◽  
Novel Drug

ABSTRACT Premafloxacin is a novel 8-methoxy fluoroquinolone with enhanced activity against Staphylococcus aureus. We found premafloxacin to be 32-fold more active than ciprofloxacin against wild-type S. aureus. Single mutations in either subunit of topoisomerase IV caused a four- to eightfold increase in the MICs of both quinolones. A double mutation (gyrA and eithergrlA or grlB) caused a 32-fold increase in the MIC of premafloxacin, while the MIC of ciprofloxacin increased 128-fold. Premafloxacin appeared to be a poor substrate for NorA, with NorA overexpression causing an increase of twofold or less in the MIC of premafloxacin in comparison to a fourfold increase in the MIC of ciprofloxacin. The frequency of selection of resistant mutants was 6.4 × 10−10 to 4.0 × 10−7 at twofold the MIC of premafloxacin, 2 to 4 log10 less than that with ciprofloxacin. Single-step mutants could not be selected at higher concentrations of premafloxacin. In five single-step mutants, only one previously described uncommon mutation (Ala116Glu), and four novel mutations (Arg43Cys, Asp69Tyr, Ala176Thr, and Pro157Leu), three of which were outside the quinolone resistance-determining region (QRDR) were found. Genetic linkage studies, in which incross ofgrlA + and outcross of mutations were performed, showed a high correlation between the mutations and the resistance phenotypes, and allelic exchange experiments confirmed the role of the novel mutations in grlA in resistance. Our results suggest that although topoisomerase IV is the primary target of premafloxacin, premafloxacin appears to interact with topoisomerase IV in a manner different from that of other quinolones and that the range of the QRDR of grlA should be expanded.

scholarly journals Engineering the Specificity of Antibacterial Fluoroquinolones: Benzenesulfonamide Modifications at C-7 of Ciprofloxacin Change Its Primary Target in Streptococcus pneumoniae from Topoisomerase IV to Gyrase

2000 ◽  
Vol 44 (2) ◽  
pp. 320-325 ◽  
Author(s):  
Fabiana L. Alovero ◽  
Xiao-Su Pan ◽  
Julia E. Morris ◽  
Ruben H. Manzo ◽  
L. Mark Fisher
Keyword(s):  
Streptococcus Pneumoniae ◽  
Resistance Mutation ◽  
Dna Supercoiling ◽  
Quinolone Resistance ◽  
Primary Target ◽  
Topoisomerase Iv ◽  
Enhanced Activity

ABSTRACT We have examined the antipneumococcal mechanisms of a series of novel fluoroquinolones that are identical to ciprofloxacin except for the addition of a benzenesulfonylamido group to the C-7 piperazinyl ring. A number of these derivatives displayed enhanced activity againstStreptococcus pneumoniae strain 7785, including compound NSFQ-105, bearing a 4-(4-aminophenylsulfonyl)-1-piperazinyl group at C-7, which exhibited an MIC of 0.06 to 0.125 μg/ml compared with a ciprofloxacin MIC of 1 μg/ml. Several complementary approaches established that unlike the case for ciprofloxacin (which targets topoisomerase IV), the increased potency of NSFQ-105 was associated with a target preference for gyrase: (i) parC mutants of strain 7785 that were resistant to ciprofloxacin remained susceptible to NSFQ-105, whereas by contrast, mutants bearing a quinolone resistance mutation in gyrA were four- to eightfold more resistant to NSFQ-105 (MIC of 0.5 μg/ml) but susceptible to ciprofloxacin; (ii) NSFQ-105 selected first-step gyrAmutants (MICs of 0.5 μg/ml) encoding Ser-81-to-Phe or -Tyr mutations, whereas ciprofloxacin selects parC mutants; and (iii) NSFQ-105 was at least eightfold more effective than ciprofloxacin at inhibiting DNA supercoiling by S. pneumoniae gyrase in vitro but was fourfold less active against topoisomerase IV. These data show unequivocally that the C-7 substituent determines not only the potency but also the target preference of fluoroquinolones. The importance of the C-7 substituent in drug-enzyme contacts demonstrated here supports one key postulate of the Shen model of quinolone action.

scholarly journals Genetic Characterization of Fluoroquinolone-Resistant Streptococcus pneumoniae Strains Isolated during Ciprofloxacin Therapy from a Patient with Bronchiectasis

2003 ◽  
Vol 47 (4) ◽  
pp. 1419-1422 ◽  
Author(s):  
Adela G. de la Campa ◽  
María-José Ferrandiz ◽  
Fe Tubau ◽  
Román Pallarés ◽  
Federico Manresa ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Genetic Characterization ◽  
Susceptible Strain ◽  
Low Level ◽  
Resistant Strains ◽  
Resistant Mutants ◽  
High Level ◽  
Level Resistance

ABSTRACT Five Spain9V-3 Streptococcus pneumoniae strains were isolated from a patient with bronchiectasis who had received long-term ciprofloxacin therapy. One ciprofloxacin-susceptible strain was isolated before treatment, and four ciprofloxacin-resistant strains were isolated during treatment. The resistant strains were derived from the susceptible strain either by a parC mutation (low-level resistance) or by parC and gyrA mutations (high-level resistance). This study shows that ciprofloxacin therapy in a patient colonized by susceptible S. pneumoniae may select fluoroquinolone-resistant mutants.

scholarly journals Role of Active Efflux in Association with Target Gene Mutations in Fluoroquinolone Resistance in Clinical Isolates of Vibrio cholerae

2002 ◽  
Vol 46 (8) ◽  
pp. 2676-2678 ◽  
Author(s):  
Somesh Baranwal ◽  
Keya Dey ◽  
T. Ramamurthy ◽  
G. Balakrish Nair ◽  
Manikuntala Kundu
Keyword(s):  
Vibrio Cholerae ◽  
Target Gene ◽  
Target Genes ◽  
Gene Mutations ◽  
Proton Motive Force ◽  
Quinolone Resistance ◽  
Clinical Isolates ◽  
Fluoroquinolone Resistance ◽  
Active Efflux

ABSTRACT Quinolones are among the drugs of choice in the management of cholera caused by Vibrio cholerae. In this study, we demonstrate that, in addition to mutations detected in the target genes gyrA and parC, proton motive force-dependent efflux is involved in quinolone resistance in clinical isolates of V. cholerae.

scholarly journals DNA Gyrase and Topoisomerase IV Are Dual Targets of Clinafloxacin Action in Streptococcus pneumoniae

1998 ◽  
Vol 42 (11) ◽  
pp. 2810-2816 ◽  
Author(s):  
Xiao-Su Pan ◽  
L. Mark Fisher
Keyword(s):  
Streptococcus Pneumoniae ◽  
Hot Spots ◽  
Bacterial Resistance ◽  
Low Frequency ◽  
Quinolone Resistance ◽  
Resistance Mutations ◽  
Topoisomerase Iv ◽  
Resistance Pathway ◽  
Fourth Step

ABSTRACT We examined the response of Streptococcus pneumoniae7785 to clinafloxacin, a novel C-8-substituted fluoroquinolone which is being developed as an antipneumococcal agent. Clinafloxacin was highly active against S. pneumoniae 7785 (MIC, 0.125 μg/ml), and neither gyrA nor parC quinolone resistance mutations alone had much effect on this activity. A combination of both mutations was needed to register resistance, suggesting that both gyrase and topoisomerase IV are clinafloxacin targets in vivo. The sparfloxacin and ciprofloxacin MICs for the parC-gyrAmutants were 16 to 32 and 32 to 64 μg/ml, respectively, but the clinafloxacin MIC was 1 μg/ml, i.e., within clinafloxacin levels achievable in human serum. S. pneumoniae 7785 mutants could be selected stepwise with clinafloxacin at a low frequency, yielding first-, second-, third-, and fourth-step mutants for which clinafloxacin MICs were 0.25, 1, 6, and 32 to 64 μg/ml, respectively. Thus, high-level resistance to clinafloxacin required four steps. Characterization of the quinolone resistance-determining regions of thegyrA, parC, gyrB, andparE genes by PCR, HinfI restriction fragment length polymorphism, and DNA sequence analysis revealed an invariant resistance pathway involving sequential mutations in gyrAor gyrB, in parC, in gyrA, and finally in parC or parE. No evidence was found for other resistance mechanisms. The gyrA mutations in first- and third-step mutants altered GyrA hot spots Ser-83 to Phe or Tyr (Escherichia coli coordinates) and Glu-87 to Gln or Lys; second- and fourth-step parC mutations changed equivalent hot spots Ser-79 to Phe or Tyr and Asp-83 to Ala.gyrB and parE changes produced novel alterations of GyrB Glu-474 to Lys and of Pro-454 to Ser in the ParE PLRGK motif. Difficulty in selecting first-step gyrase mutants (isolated with 0.125 [but not 0.25] μg of clinafloxacin per ml at a frequency of 5.0 × 10−10 to 8.5 × 10−10) accompanied by the small (twofold) MIC increase suggested only a modest drug preference for gyrase. Given the susceptibility of defined gyrA or parC mutants, the results suggested that clinafloxacin displays comparable if unequal targeting of gyrase and topoisomerase IV. Dual targeting and the intrinsic potency of clinafloxacin against S. pneumoniaeand its first- and second-step mutants are desirable features in limiting the emergence of bacterial resistance.

scholarly journals In Vitro Activities of Novel Nonfluorinated Quinolones PGE 9262932 and PGE 9509924 against Clinical Isolates of Staphylococcus aureus and Streptococcus pneumoniae with Defined Mutations in DNA Gyrase and Topoisomerase IV

2002 ◽  
Vol 46 (6) ◽  
pp. 1651-1657 ◽  
Author(s):  
Mark E. Jones ◽  
Ian A. Critchley ◽  
James A. Karlowsky ◽  
Renée S. Blosser-Middleton ◽  
Franz-Josef Schmitz ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Streptococcus Pneumoniae ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Clinical Isolates ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Selection Of

ABSTRACT Two 8-methoxy nonfluorinated quinolones (NFQs), PGE 9262932 and PGE 9509924, were tested against contemporary clinical isolates of Staphylococcus aureus (n = 122) and Streptococcus pneumoniae (n = 69) with genetically defined quinolone resistance-determining regions (QRDRs). For S. aureus isolates with wild-type (WT) sequences at the QRDRs, the NFQs demonstrated activities 4- to 32-fold more potent (MICs at which 90% of isolates are inhibited [MIC90s], 0.03 μg/ml) than those of moxifloxacin (MIC90, 0.12 μg/ml), gatifloxacin (MIC90, 0.25 μg/ml), levofloxacin (MIC90, 0.25 μg/ml), and ciprofloxacin (MIC90, 1 μg/ml). Against S. pneumoniae isolates with WT sequences at gyrA and parC, the NFQs PGE 9262932 (MIC90, 0.03 μg/ml) and PGE 9509924 (MIC90, 0.12 μg/ml) were 8- to 64-fold and 2- to 16-fold more potent, respectively, than moxifloxacin (MIC90, 0.25 μg/ml), gatifloxacin (MIC90, 0.5 μg/ml), levofloxacin (MIC90, 2 μg/ml), and ciprofloxacin (MIC90, 2 μg/ml). The MICs of all agents were elevated for S. aureus isolates with alterations in GyrA (Glu88Lys or Ser84Leu) and GrlA (Ser80Phe) and S. pneumoniae isolates with alterations in GyrA (Ser81Phe or Ser81Tyr) and ParC (Ser79Phe or Lys137Asn). Fluoroquinolone MICs for S. aureus strains with double alterations in GyrA combined with double alterations in GrlA were ≥32 μg/ml, whereas the MICs of the NFQs for strains with these double alterations were 4 to 8 μg/ml. The PGE 9262932 and PGE 9509924 MICs for the S. pneumoniae isolates did not exceed 0.5 and 1 μg/ml, respectively, even for isolates with GyrA (Ser81Phe) and ParC (Ser79Phe) alterations, for which levofloxacin MICs were >16 μg/ml. No difference in the frequency of selection of mutations (<10−8 at four times the MIC) in wild-type or first-step mutant isolates of S. aureus or S. pneumoniae was detected for the two NFQs. On the basis of their in vitro activities, these NFQ agents show potential for the treatment of infections caused by isolates resistant to currently available fluoroquinolones.

scholarly journals ATP-Bound Conformation of Topoisomerase IV: a Possible Target for Quinolones in Streptococcus pneumoniae

2003 ◽  
Vol 185 (20) ◽  
pp. 6137-6146 ◽  
Author(s):  
Farid Sifaoui ◽  
Valérie Lamour ◽  
Emmanuelle Varon ◽  
Dino Moras ◽  
Laurent Gutmann
Keyword(s):  
Streptococcus Pneumoniae ◽  
Binding Site ◽  
Active Site ◽  
Random Mutagenesis ◽  
Bacterial Pathogen ◽  
Quinolone Resistance ◽  
Atp Binding ◽  
Topoisomerase Iv ◽  
Atp Binding Site ◽  
Dimeric Interface

ABSTRACT Topoisomerase IV, a C2E2 tetramer, is involved in the topological changes of DNA during replication. This enzyme is the target of antibacterial compounds, such as the coumarins, which target the ATP binding site in the ParE subunit, and the quinolones, which bind, outside the active site, to the quinolone resistance-determining region (QRDR). After site-directed and random mutagenesis, we found some mutations in the ATP binding site of ParE near the dimeric interface and outside the QRDR that conferred quinolone resistance to Streptococcus pneumoniae, a bacterial pathogen. Modeling of the N-terminal, 43-kDa ParE domain of S. pneumoniae revealed that the most frequent mutations affected conserved residues, among them His43 and His103, which are involved in the hydrogen bond network supporting ATP hydrolysis, and Met31, at the dimeric interface. All mutants showed a particular phenotype of resistance to fluoroquinolones and an increase in susceptibility to novobiocin. All mutations in ParE resulted in resistance only when associated with a mutation in the QRDR of the GyrA subunit. Our models of the closed and open conformations of the active site indicate that quinolones preferentially target topoisomerase IV of S. pneumoniae in its ATP-bound closed conformation.

scholarly journals Novel Ser79Leu and Ser81Ile Substitutions in the Quinolone Resistance-Determining Regions of ParC Topoisomerase IV and GyrA DNA Gyrase Subunits from Recent Fluoroquinolone-Resistant Streptococcus pneumoniae Clinical Isolates

2005 ◽  
Vol 49 (6) ◽  
pp. 2479-2486 ◽  
Author(s):  
Nataliya Korzheva ◽  
Todd A. Davies ◽  
Raul Goldschmidt
Keyword(s):  
Streptococcus Pneumoniae ◽  
Amino Acid ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Clinical Isolates ◽  
Fluoroquinolone Resistance ◽  
Amino Acid Substitutions ◽  
Topoisomerase Iv ◽  
Isogenic Strains ◽  
New Mutations

ABSTRACT Resistance of Streptococcus pneumoniae to fluoroquinolones is caused predominantly by amino acid substitutions at positions Ser79 of ParC and Ser81 of GyrA to either Phe or Tyr encoded in the quinolone resistance-determining regions of the parC topoisomerase IV and gyrA DNA gyrase genes. Analysis of highly resistant clinical isolates identified novel second-step substitutions, Ser79Leu (ParC) and Ser81Ile (GyrA). To determine contributions of these new mutations to fluoroquinolone resistance either alone or in combination with other Ser79/81 alleles, the substitutions Ser79Leu/Phe/Tyr in ParC and Ser81Ile/Phe/Tyr in GyrA were introduced into the R6 background, resulting in 15 isogenic strains. Their level of fluoroquinolone resistance was determined by susceptibility testing for ciprofloxacin, levofloxacin, moxifloxacin, gatifloxacin, gemifloxacin, garenoxacin, and norfloxacin. Leu79 and Ile81 alone as well as 79/81Phe/Tyr substitutions did not contribute significantly to resistance, with fluoroquinolone MICs increasing two- to fourfold compared to wild type for all agents tested. Fluoroquinolone MICs for double transformants ParC Ser79Phe/Tyr/Leu-GyrA Ser81Phe/Tyr were uniformly increased by 8- to 64-fold regardless of pairs of amino acid substitutions. However, combinations including Ile81 conferred two- to fourfold-higher levels of resistance than did combinations including any other Ser81 GyrA substitution, thus demonstrating the differential effects of diverse amino acid substitutions at particular hotspots on fluoroquinolone MICs.

scholarly journals In Vitro Assessment of the Further Potential for Development of Fluoroquinolone Resistance in Neisseria meningitidis

2005 ◽  
Vol 49 (5) ◽  
pp. 1753-1760 ◽  
Author(s):  
Tiffany R. Shultz ◽  
Peter A. White ◽  
John W. Tapsall
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Neisseria Meningitidis ◽  
Quinolone Resistance ◽  
Clinical Isolates ◽  
Fluoroquinolone Resistance ◽  
In Vitro Assessment ◽  
Resistant Mutants ◽  
The Individual

ABSTRACT We examined the potential for the development of fluoroquinolone resistance in Neisseria meningitidis by cultivating two clinical isolates of meningococci in the presence of concentrations of ciprofloxacin at and about the predetermined MIC. The quinolone resistance determining regions (QRDRs) of gyrA and parC of 50 stable quinolone-resistant mutants derived in vitro were sequenced and compared with QRDR alterations reported in clinical isolates of quinolone-resistant meningococci and gonococci. MICs to ciprofloxacin and trovafloxacin were determined and sequence changes were correlated with quinolone MICs. Ciprofloxacin and trovafloxacin MICs of the in vitro-derived quinolone-resistant mutants ranged up to 16 mg/liter. Single GyrA alterations were the first change detected and were accompanied by raised MICs, followed by double GyrA changes and still higher MICs. MICs increased further as single ParC substitutions appeared and these were always in the presence of a single or double GyrA change. GyrA changes occurred at positions 91 and 95 with substitutions of Asp-95→Asn and Thr-91→Ala and Ile. Changes in the parC QRDR occurred at positions 85, 86, and 91 with four substitutions, Gly-85→Asp, Asp-86→Asn, Glu-91→Gly, and Glu-91→Lys, detected. The nature of the individual QRDR substitution appeared to influence the level of quinolone resistance expressed, and this varied with the quinolone agent examined. Close similarities occurred between the sequence and nature of QRDR changes in clinical and in vitro-generated quinolone-resistant mutants and with those previously reported for clinical and in vitro-generated quinolone-resistant gonococci. This suggests that quinolone resistance in meningococci may arise in the same manner and reach similar levels in vivo to those seen in quinolone-resistant Neisseria gonorrhoeae.

Sign in / Sign up

Export Citation Format

Share Document