Quinolone resistance mutations in topoisomerase IV: relationship to the flqA locus and genetic evidence that topoisomerase IV is the primary target and DNA gyrase is the secondary target of fluoroquinolones in Staphylococcus aureus.

Mutations in the flqA (formerly ofx/cfx) resistance locus of Staphylococcus aureus were previously shown to be common after first-step selections for resistance to ciprofloxacin and ofloxacin and to map on the S. aureus chromosome distinctly from gyrA, gyrB, and norA.grlA and grlB, the genes for the topoisomerase IV of S. aureus, were identified from a genomic lambda library on a common KpnI fragment, and grlB hybridized specifically with the chromosomal SmaI A fragment, which contains the flqA locus. Amplification of grlA sequences (codons 1 to 251) by PCRs from nine independent single-step flqA mutants, one multistep mutant, and the parent strain identified mutations encoding a change from Ser to Phe at position 80 in four mutants, a novel change from Ala to either Glu or Pro at position 116 in three mutants, and no change in three mutants. In the multistep mutant, another resistance locus, flqC, was mapped by transformation to the chromosomal SmaI G fragment by linkage to omega(ch::Tn551)1051 (58%) and nov (97.9%), which encodes resistance to novobiocin. This fragment contains the gyrA gene, and flqC mutants had a mutation in gyrA encoding a change from Ser to Leu at position 84, a change previously found in resistant clinical isolates. In genetic outcrosses, the flqC (gyrA) mutation expressed resistance only in flqA mutants, including those with both types of grla mutations. The silent mutant allele of gyrA was present in a flqA background and expressed resistance only upon introduction of a grlA mutation. At fourfold the MIC of ciprofloxacin, the bactericidal activity of ciprofloxacin was reduced in a grlA mutant and was abolished in gyrA grlA double mutants. These findings provide direct genetic evidence that topoisomerase IV is the primary target of current fluoroquinolones in S. aureus and that this effect may result from the greater sensitivity of topoisomerase IV relative to that of DNA gyrase to these agents. Furthermore, resistance from an altered DNA gyrase requires resistant topoisomerase IV for its expression.

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Mutations in Topoisomerase IV and DNA Gyrase of Staphylococcus aureus: Novel Pleiotropic Effects on Quinolone and Coumarin Activity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.1.121 ◽

1998 ◽

Vol 42 (1) ◽

pp. 121-128 ◽

Cited By ~ 64

Author(s):

Bénédicte Fournier ◽

David C. Hooper

Keyword(s):

Staphylococcus Aureus ◽

Dna Gyrase ◽

Single Step ◽

Pleiotropic Effects ◽

Fluoroquinolone Resistance ◽

Wild Type ◽

Chromosomal Dna ◽

Topoisomerase Iv ◽

Gene Encoding ◽

Double Mutation

ABSTRACT Previous studies have shown that topoisomerase IV and DNA gyrase interact with quinolones and coumarins in different ways. The MICs of coumarins (novobiocin and coumermycin) for MT5, a Staphylococcus aureus nov mutant, are higher than those for wild-type strains. Sequencing the gyrB gene encoding one subunit of the DNA gyrase revealed the presence of a double mutation likely to be responsible for this resistance: at codon 102 (Ile to Ser) and at codon 144 (Arg to Ile). For single-step flqA mutant MT5224c9, previously selected on ciprofloxacin, the fluoroquinolone MIC was higher and the coumarin MIC was lower than those for its parent, MT5. Sequencing the grlB andgrlA genes of topoisomerase IV of MT5224c9 showed a single Asn-470-to-Asp mutation in GrlB. Genetic outcrosses by transformation with chromosomal DNA and introduction of plasmids carrying either the wild-type or the mutated grlB gene indicated that this mutation causes both increased MICs of fluoroquinolones and decreased MICs of coumarins and that the mutant grlBallele is codominant for both phenotypes with multicopy alleles. Integration of these plasmids into the chromosome confirmed the codominance of fluoroquinolone resistance, butgrlB + appeared dominant over grlB(Asp-470) for coumarin resistance. Finally, the gyrA(Leu-84) mutation previously described as silent for fluoroquinolone resistance increased the MIC of nalidixic acid, a nonfluorinated quinolone. Combining the grlA (Phe-80) and grlB(Asp-470) mutations with this gyrA mutation also had differing effects. The findings indicate that alterations in topoisomerases may have pleiotropic effects on different classes of inhibitors as well as on inhibitors within the same class. A full understanding of drug action and resistance at the molecular level must take into account both inhibitor structure-activity relationships and the effects of different classes of topoisomerase mutants.

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Alterations in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV in quinolone-resistant clinical isolates of Klebsiella pneumoniae.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.3.699 ◽

1997 ◽

Vol 41 (3) ◽

pp. 699-701 ◽

Cited By ~ 74

Author(s):

T Deguchi ◽

A Fukuoka ◽

M Yasuda ◽

M Nakano ◽

S Ozeki ◽

...

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Dna Gyrase ◽

Quinolone Resistance ◽

Clinical Isolates ◽

Fluoroquinolone Resistance ◽

Primary Target ◽

Gyra Gene ◽

Topoisomerase Iv ◽

Complementary Role

We determined a partial sequence of the Klebsiella pneumoniae parC gene, including the region analogous to the quinolone resistance-determining region of the Escherichia coli gyrA gene, and examined 26 clinical strains of K. pneumoniae for an association of alterations in GyrA and ParC with susceptibilities to quinolones. The study suggests that in K. pneumoniae DNA gyrase is a primary target of quinolones and that ParC alterations play a complementary role in the development of higher-level fluoroquinolone resistance.

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The Anti-Methicillin-Resistant Staphylococcus aureus Quinolone WCK 771 Has Potent Activity against Sequentially Selected Mutants, Has a Narrow Mutant Selection Window against Quinolone-Resistant Staphylococcus aureus, and Preferentially Targets DNA Gyrase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00641-06 ◽

2006 ◽

Vol 50 (11) ◽

pp. 3568-3579 ◽

Cited By ~ 21

Author(s):

Sachin S. Bhagwat ◽

Lakshmi A. Mundkur ◽

Shrikant V. Gupte ◽

Mahesh V. Patel ◽

Habil F. Khorakiwala

Keyword(s):

Staphylococcus Aureus ◽

Dna Gyrase ◽

Primary Target ◽

Topoisomerase Iv ◽

Mutant Selection ◽

Low Concentrations ◽

Selection Window ◽

High Level ◽

The Impact

ABSTRACT WCK 771 is a broad-spectrum fluoroquinolone with enhanced activity against quinolone-resistant staphylococci. To understand the impact of the target-level interactions of WCK 771 on its antistaphylococcal pharmacodynamic properties, we determined the MICs for genetically defined mutants and studied the mutant prevention concentrations (MPCs), the frequency of mutation, and the cidality against the wild type and double mutants. There was a twofold increase in the MICs of WCK 771 for single gyrA mutants, indicating that DNA gyrase is its primary target. All first- and second-step mutants selected by WCK 771 revealed gyrA and grlA mutations, respectively. The MICs of WCK 771 and clinafloxacin were found to be superior to those of other quinolones against strains with double and triple mutations. WCK 771 was also cidal for high-density double mutants at low concentrations. WCK 771 and clinafloxacin showed narrow mutant selection windows compared to those of the other quinolones. Against a panel of 50 high-level quinolone-resistant clinical isolates of staphylococci (ciprofloxacin MIC ≥ 16 μg/ml), the WCK 771 MPCs were ≤2 μg/ml for 68% of the strains and ≤4 μg/ml for 28% of the strains. Our results demonstrate that gyrA is the primary target of WCK 771 and that it has pharmacodynamic properties remarkably different from those of quinolones with dual targets (garenoxacin and moxifloxacin) and topoisomerase IV-specific quinolones (trovafloxacin). WCK 771 displayed an activity profile comparable to that of clinafloxacin, a dual-acting quinolone with a high affinity to DNA gyrase. Overall, the findings signify the key role of DNA gyrase in determining the optimal antistaphylococcal features of quinolones.

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Differential behaviors of Staphylococcus aureus and Escherichia coli type II DNA topoisomerases.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.12.2714 ◽

1996 ◽

Vol 40 (12) ◽

pp. 2714-2720 ◽

Cited By ~ 106

Author(s):

F Blanche ◽

B Cameron ◽

F X Bernard ◽

L Maton ◽

B Manse ◽

...

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Strong Support ◽

Dna Gyrase ◽

Dna Supercoiling ◽

Type Ii ◽

Topoisomerase Iv ◽

E Coli ◽

Dna Relaxation

Staphylococcus aureus gyrA and gyrB genes encoding DNA gyrase subunits were cloned and coexpressed in Escherichia coli under the control of the T7 promoter-T7 RNA polymerase system, leading to soluble gyrase which was purified to homogeneity. Purified gyrase was catalytically indistinguishable from the gyrase purified from S. aureus and did not contain detectable amounts of topoisomerases from the E. coli host. Topoisomerase IV subunits GrlA and GrlB from S. aureus were also expressed in E. coli and were separately purified to apparent homogeneity. Topoisomerase IV, which was reconstituted by mixing equimolar amounts of GrlA and GrlB, had both ATP-dependent decatenation and DNA relaxation activities in vitro. This enzyme was more sensitive than gyrase to inhibition by typical fluoroquinolone antimicrobial agents such as ciprofloxacin or sparfloxacin, adding strong support to genetic studies which indicate that topoisomerase IV is the primary target of fluoroquinolones in S. aureus. The results obtained with ofloxacin suggest that this fluoroquinolone could also primarily target gyrase. No cleavable complex could be detected with S. aureus gyrase upon incubation with ciprofloxacin or sparfloxacin at concentrations which fully inhibit DNA supercoiling. This suggests that these drugs do not stabilize the open DNA-gyrase complex, at least under standard in vitro incubation conditions, but are more likely to interfere primarily with the DNA breakage step, contrary to what has been reported with E. coli gyrase. Both S. aureus gyrase-catalyzed DNA supercoiling and S. aureus topoisomerase IV-catalyzed decatenation were dramatically stimulated by potassium glutamate or aspartate (500- and 50-fold by 700 and 350 mM glutamate, respectively), whereas topoisomerase IV-dependent DNA relaxation was inhibited 3-fold by 350 mM glutamate. The relevance of the effect of dicarboxylic amino acids on the activities of type II topoisomerases is discussed with regard to the intracellular osmolite composition of S. aureus.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.12.3344-3350.2000 ◽

2000 ◽

Vol 44 (12) ◽

pp. 3344-3350 ◽

Cited By ~ 43

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.2.488-492.2005 ◽

2005 ◽

Vol 49 (2) ◽

pp. 488-492 ◽

Cited By ~ 27

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.

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Topoisomerase Mutations in Trovafloxacin-ResistantStaphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.8.2122 ◽

1998 ◽

Vol 42 (8) ◽

pp. 2122-2124 ◽

Cited By ~ 18

Author(s):

Joseph E. Fitzgibbon ◽

Joseph F. John ◽

Jennifer L. Delucia ◽

Donald T. Dubin

Keyword(s):

Staphylococcus Aureus ◽

Resistance Mutations ◽

Topoisomerase Iv ◽

Dna Topoisomerase ◽

Methicillin Resistant ◽

A Subunit ◽

Resistant Strains

ABSTRACT A total of 201 Staphylococcus aureus isolates were surveyed for susceptibility to ciprofloxacin and trovafloxacin. Of 66 methicillin-resistant isolates, 89% were ciprofloxacin resistant and 6% were also trovafloxacin resistant. Trovafloxacin-resistant strains had unusual patterns of quinoline resistance mutations in DNA topoisomerase genes, including two mutations in the A subunit (encoded by grlA) of topoisomerase IV.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.1.197 ◽

1998 ◽

Vol 42 (1) ◽

pp. 197-198 ◽

Cited By ~ 9

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.

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In Vitro Evaluation of CBR-2092, a Novel Rifamycin-Quinolone Hybrid Antibiotic: Studies of the Mode of Action in Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01649-07 ◽

2008 ◽

Vol 52 (7) ◽

pp. 2313-2323 ◽

Cited By ~ 40

Author(s):

Gregory T. Robertson ◽

Eric J. Bonventre ◽

Timothy B. Doyle ◽

Qun Du ◽

Leonard Duncan ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Rna Polymerase ◽

Mode Of Action ◽

Bacterial Infections ◽

Dna Gyrase ◽

Topoisomerase Iv ◽

Dna Topoisomerase ◽

Resistant Variant ◽

Proven Efficacy

ABSTRACT Rifamycins have proven efficacy in the treatment of persistent bacterial infections. However, the frequency with which bacteria develop resistance to rifamycin agents restricts their clinical use to antibiotic combination regimens. In a program directed toward the synthesis of rifamycins with a lower propensity to elicit resistance development, a series of compounds were prepared that covalently combine rifamycin and quinolone pharmacophores to form stable hybrid antibacterial agents. We describe mode-of-action studies with Staphylococcus aureus of CBR-2092, a novel hybrid that combines the rifamycin SV and 4H-4-oxo-quinolizine pharmacophores. In biochemical studies, CBR-2092 exhibited rifampin-like potency as an inhibitor of RNA polymerase, was an equipotent (balanced) inhibitor of DNA gyrase and DNA topoisomerase IV, and retained activity against a prevalent quinolone-resistant variant. Macromolecular biosynthesis studies confirmed that CBR-2092 has rifampin-like effects on RNA synthesis in rifampin-susceptible strains and quinolone-like effects on DNA synthesis in rifampin-resistant strains. Studies of mutant strains that exhibited reduced susceptibility to CBR-2092 further substantiated RNA polymerase as the primary cellular target of CBR-2092, with DNA gyrase and DNA topoisomerase IV being secondary and tertiary targets, respectively, in strains exhibiting preexisting rifampin resistance. In contrast to quinolone comparator agents, no strains with altered susceptibility to CBR-2092 were found to exhibit changes consistent with altered efflux properties. The combined data indicate that CBR-2092 may have potential utility in monotherapy for the treatment of persistent S. aureus infections.

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