scholarly journals Dual Targeting of Topoisomerase IV and Gyrase To Reduce Mutant Selection: Direct Testing of the Paradigm by Using WCK-1734, a New Fluoroquinolone, and Ciprofloxacin

2005 ◽  
Vol 49 (5) ◽  
pp. 1949-1956 ◽  
Author(s):  
Jacob Strahilevitz ◽  
David C. Hooper
Keyword(s):  
Dna Gyrase ◽  
Single Mutation ◽  
Topoisomerase Iv ◽  
Mutant Selection ◽  
Fourfold Increase ◽  
Development Of Resistance ◽  
Dual Targeting ◽  
Gyrase A ◽  
Resistant Strains ◽  
Selection Of

ABSTRACT Quinolones that act equally against DNA gyrase and topoisomerase IV are a desirable modality to decrease the selection of resistant strains. We first determined by genetic and biochemical studies in Staphylococcus aureus that the primary target enzyme of WCK-1734, a new quinolone, was DNA gyrase. A single mutation in gyrase, but not topoisomerase IV, caused a two- to fourfold increase in the MIC. Studies with purified topoisomerase IV and gyrase from S. aureus also showed that gyrase was more sensitive than topoisomerase IV to WCK-1734 (50% inhibitory concentration, 1.25 and 2.5 to 5.0 μg/ml, respectively; 50% stimulation of cleavage complex formation, 0.62 and 2.5 to 5.0 μg/ml, respectively). To test the effect of balanced activity of quinolones against the two target enzymes, we measured the frequency of selection of mutants with ciprofloxacin (which targets topoisomerase IV) and WCK-1734 alone and in combination. With the combination of ciprofloxacin and WCK-1734, each at its MIC, the ratio of frequency of mutants selected was significantly lower than that with each drug alone at two times their respective MICs. We further characterized resistant strains selected with the combination of ciprofloxacin and WCK-1734 and found evidence to suggest the existence of novel mutational mechanisms for low-level quinolone resistance. By use of a combination of differentially targeting quinolones, this study provides novel data in direct support of the paradigm for dual targeting of quinolone action and reduced development of resistance.

scholarly journals Quinolone-resistant mutants of escherichia coli DNA topoisomerase IV parC gene.

1996 ◽  
Vol 40 (3) ◽  
pp. 710-714 ◽  
Author(s):  
Y Kumagai ◽  
J I Kato ◽  
K Hoshino ◽  
T Akasaka ◽  
K Sato ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Gyrase ◽  
Mutant Gene ◽  
Missense Mutations ◽  
Topoisomerase Iv ◽  
Dna Topoisomerase ◽  
E Coli ◽  
A Subunit ◽  
Resistant Strains ◽  
Mutant Genes

Escherichia coli quinolone-resistant strains with mutations of the parC gene, which codes for a subunit of topoisomerase IV, were isolated from a quinolone-resistant gyrA mutant of DNA gyrase. Quinolone-resistant parC mutants were also identified among the quinolone-resistant clinical strains. The parC mutants became susceptible to quinolones by introduction of a parC+ plasmid. Introduction of the multicopy plasmids carrying the quinolone-resistant parC mutant gene resulted in an increase in MICs of quinolones for the parC+ and quinolone-resistant gyrA strain. Nucleotide sequences of the quinolone-resistant parC mutant genes were determined, and missense mutations at position Gly-78, Ser-80, or Glu-84, corresponding to those in the quinolone-resistance-determining region of DNA gyrase, were identified. These results indicate that topoisomerase IV is a target of quinolones in E. coli and suggest that the susceptibility of E. coli cells to quinolones is determined by sensitivity of the targets, DNA gyrase and topoisomerase IV.

scholarly journals Alterations in DNA Gyrase and Topoisomerase IV in Resistant Mutants of Clostridium perfringens Found after In Vitro Treatment with Fluoroquinolones

2005 ◽  
Vol 49 (2) ◽  
pp. 488-492 ◽  
Author(s):  
Fatemeh Rafii ◽  
Miseon Park ◽  
John S. Novak
Keyword(s):  
Clostridium Perfringens ◽  
Allelic Diversity ◽  
Dna Gyrase ◽  
Serial Passage ◽  
Primary Target ◽  
Topoisomerase Iv ◽  
Resistant Mutants ◽  
In Vitro Treatment ◽  
Selection Of

ABSTRACT To compare mutations in the DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) genes of Clostridium perfringens, which are associated with in vitro exposure to fluoroquinolones, resistant mutants were selected from eight strains by serial passage in the presence of increasing concentrations of norfloxacin, ciprofloxacin, gatifloxacin, or trovafloxacin. The nucleotide sequences of the entire gyrA, gyrB, parC, and parE genes of 42 mutants were determined. DNA gyrase was the primary target for each fluoroquinolone, and topoisomerase IV was the secondary target. Most mutations appeared in the quinolone resistance-determining regions of gyrA (resulting in changes of Asp-87 to Tyr or Gly-81 to Cys) and parC (resulting in changes of Asp-93 or Asp-88 to Tyr or Ser-89 to Ile); only two mutations were found in gyrB, and only two mutations were found in parE. More mutants with multiple gyrA and parC mutations were produced with gatifloxacin than with the other fluoroquinolones tested. Allelic diversity was observed among the resistant mutants, for which the drug MICs increased 2- to 256-fold. Both the structures of the drugs and their concentrations influenced the selection of mutants.

scholarly journals Dual Targeting of DNA Gyrase and Topoisomerase IV: Target Interactions of Garenoxacin (BMS-284756, T-3811ME), a New Desfluoroquinolone

2002 ◽  
Vol 46 (11) ◽  
pp. 3370-3380 ◽  
Author(s):  
Dilek Ince ◽  
Xiamei Zhang ◽  
L. Christine Silver ◽  
David C. Hooper
Keyword(s):  
Inhibitory Concentration ◽  
Efflux Pump ◽  
Fold Increase ◽  
Single Step ◽  
The Novel ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Dual Targeting ◽  
Resistant Mutants ◽  
Selection Of

ABSTRACT We determined the target enzyme interactions of garenoxacin (BMS-284756, T-3811ME), a novel desfluoroquinolone, in Staphylococcus aureus by genetic and biochemical studies. We found garenoxacin to be four- to eightfold more active than ciprofloxacin against wild-type S. aureus. A single topoisomerase IV or gyrase mutation caused only a 2- to 4-fold increase in the MIC of garenoxacin, whereas a combination of mutations in both loci caused a substantial increase (128-fold). Overexpression of the NorA efflux pump had minimal effect on resistance to garenoxacin. With garenoxacin at twice the MIC, selection of resistant mutants (<7.4 × 10−12 to 4.0 × 10−11) was 5 to 6 log units less than that with ciprofloxacin. Mutations inside or outside the quinolone resistance-determining regions (QRDR) of either topoisomerase IV, or gyrase, or both were selected in single-step mutants, suggesting dual targeting of topoisomerase IV and gyrase. Three of the novel mutations were shown by genetic experiments to be responsible for resistance. Studies with purified topoisomerase IV and gyrase from S. aureus also showed that garenoxacin had similar activity against topoisomerase IV and gyrase (50% inhibitory concentration, 1.25 to 2.5 and 1.25 μg/ml, respectively), and although its activity against topoisomerase IV was 2-fold greater than that of ciprofloxacin, its activity against gyrase was 10-fold greater. This study provides the first genetic and biochemical data supporting the dual targeting of topoisomerase IV and gyrase in S. aureus by a quinolone as well as providing genetic proof for the expansion of the QRDRs to include the 5′ terminus of grlB and the 3′ terminus of gyrA.

Topoisomerase Sequences of Coagulase-Negative Staphylococcal Isolates Resistant to Ciprofloxacin or Trovafloxacin

1999 ◽  
Vol 43 (7) ◽  
pp. 1631-1637 ◽  
Author(s):  
Donald T. Dubin ◽  
Joseph E. Fitzgibbon ◽  
Massoumeh D. Nahvi ◽  
Joseph F. John
Keyword(s):  
Dna Gyrase ◽  
Fluoroquinolone Resistance ◽  
Topoisomerase Iv ◽  
Dna Topoisomerase ◽  
Methicillin Resistant ◽  
Intraspecies Variation ◽  
Rrna Sequencing ◽  
Identification Of Species ◽  
Resistant Strains ◽  
Ciprofloxacin Resistance

ABSTRACT Coagulase-negative staphylococcal isolates (n = 188) were screened for susceptibility to oxacillin, ciprofloxacin, and trovafloxacin, a new fluoroquinolone. At an oxacillin concentration of ≥4 μg/ml, 43% were methicillin resistant; of these, 70% were ciprofloxacin resistant (MIC, ≥4 μg/ml). Of the methicillin-resistant, ciprofloxacin-resistant isolates, 46% were susceptible to ≤2 μg of trovafloxacin per ml and 32% were susceptible to ≤1 μg of trovafloxacin per ml. Sixteen isolates, including twelve that expressed fluoroquinolone resistance, were chosen for detailed analysis. Identification of species by rRNA sequencing revealed a preponderance of Staphylococcus haemolyticus andS. hominis among fluoroquinolone-resistant strains. Segments of genes (gyrA and grlA) encoding DNA gyrase and DNA topoisomerase IV were sequenced. Considerable interspecies variation was noted, mainly involving noncoding nucleotide changes. Intraspecies variation consisted of coding changes associated with fluoroquinolone resistance. As for S. aureus, ciprofloxacin resistance (MIC, ≥8 μg/ml) and increased trovafloxacin MICs (0.25 to 2 μg/ml) could be conferred by the combined presence of single mutations in each gyrA and grlA gene. Trovafloxacin MICs of ≥8 μg/ml also occurred, but these required an additional mutation in grlA.

scholarly journals Topoisomerase Mutations in Fluoroquinolone-Resistant and Methicillin-Susceptible and -Resistant Clinical Isolates of Staphylococcus aureus

1998 ◽  
Vol 42 (1) ◽  
pp. 197-198 ◽  
Author(s):  
Glenn W. Kaatz ◽  
Susan M. Seo
Keyword(s):  
Staphylococcus Aureus ◽  
Restriction Fragment Length Polymorphism ◽  
Fragment Length Polymorphism ◽  
Dna Gyrase ◽  
Clinical Isolates ◽  
Polymorphism Analysis ◽  
Topoisomerase Iv ◽  
Genes Encoding ◽  
Resistant Strains ◽  
Restriction Fragment Length

ABSTRACT The incidence of the various mutations in the genes encoding topoisomerase IV and DNA gyrase in fluoroquinolone-resistant clinical isolates of Staphylococcus aureus is not known. Using restriction fragment length polymorphism analysis and DNA sequencing, we found that in fluoroquinolone- and methicillin-resistant strains, mutations in grlA and gyrA are quite likely to be present together. For fluoroquinolone-resistant but methicillin-susceptible strains, mutations in grlA alone are more common.

IN VITRO SELECTION OF FLUOROQUINOLONE-RESISTANT NEISSERIA GONORRHOEAE HARBORING ALTERATIONS IN DNA GYRASE AND TOPOISOMERASE IV

2000 ◽  
pp. 847-851
Author(s):  
MITSURU YASUDA ◽  
HIDEYUKI FUKUDA ◽  
SHIGEAKI YOKOI ◽  
SATOSHI ISHIHARA ◽  
YUKIMICHI KAWADA ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
In Vitro Selection ◽  
Dna Gyrase ◽  
Topoisomerase Iv ◽  
Selection Of

scholarly journals DNA Sequence Analysis of DNA Gyrase and DNA Topoisomerase IV Quinolone Resistance-Determining Regions of Salmonella enterica Serovar Typhi and Serovar Paratyphi A

2002 ◽  
Vol 46 (10) ◽  
pp. 3249-3252 ◽  
Author(s):  
Kenji Hirose ◽  
Ai Hashimoto ◽  
Kazumichi Tamura ◽  
Yoshiaki Kawamura ◽  
Takayuki Ezaki ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Dna Sequence ◽  
Salmonella Enterica ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Fluoroquinolone Resistance ◽  
Single Mutation ◽  
Topoisomerase Iv ◽  
Dna Topoisomerase ◽  
Salmonella Enterica Serovar Typhi

ABSTRACT The mutations that are responsible for fluoroquinolone resistance in the gyrA, gyrB, parC, and parE genes of Salmonella enterica serovar Typhi and serovar Paratyphi A were investigated. The sequences of the quinolone resistance-determining region of the gyrA gene in clinical isolates which showed decreased susceptibilities to fluoroquinolones had a single mutation at either the Ser-83 or the Asp-87 codon, and no mutations were found in the gyrB, parC, and parE genes.

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.

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