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

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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Inhibitory Activities of Quinolones against DNA Gyrase and Topoisomerase IV of Enterococcus faecalis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.6.1800-1804.2002 ◽

2002 ◽

Vol 46 (6) ◽

pp. 1800-1804 ◽

Cited By ~ 21

Author(s):

Yoshikuni Onodera ◽

Jun Okuda ◽

Mayumi Tanaka ◽

Kenichi Sato

Keyword(s):

Enterococcus Faecalis ◽

Recombinant Dna ◽

Dna Gyrase ◽

Primary Target ◽

Topoisomerase Iv ◽

Bacterial Killing ◽

Drug Concentrations ◽

Inhibitory Activities ◽

Resistant Mutants

ABSTRACT We have cloned the DNA gyrase and topoisomerase IV genes of Enterococcus faecalis to examine the actions of quinolones against E. faecalis genetically and enzymatically. We first generated levofloxacin-resistant mutants of E. faecalis by stepwise selection with increasing drug concentrations and analyzed the quinolone resistance-determining regions of gyrA and parC from the resistant mutants. Isogenic mutants with low-level resistance contained a mutation in gyrA, whereas those with higher levels of resistance had mutations in both gyrA and parC. These results suggested that gyrA is the primary target for levofloxacin in E. faecalis. We then purified the recombinant DNA gyrase and topoisomerase IV enzymes of E. faecalis and measured the in vitro inhibitory activities of quinolones against these enzymes. The 50% inhibitory concentrations (IC50s) of levofloxacin, ciprofloxacin, sparfloxacin, tosufloxacin, and gatifloxacin for DNA gyrase were found to be higher than those for topoisomerase IV. In conflict with the genetic data, these results indicated that topoisomerase IV would be the primary target for quinolones in E. faecalis. Among the quinolones tested, the IC50 of sitafloxacin (DU-6859a), which shows the greatest potency against enterococci, for DNA gyrase was almost equal to that for topoisomerase IV; its IC50s were the lowest among those of all the quinolones tested. These results indicated that other factors can modulate the effect of target affinity to determine the bacterial killing pathway, but the highest inhibitory actions against both enzymes correlated with good antienterococcal activities.

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Alterations in Topoisomerase IV and DNA Gyrase in Quinolone-Resistant Mutants of Mycoplasma hominis Obtained In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.9.2304 ◽

1998 ◽

Vol 42 (9) ◽

pp. 2304-2311 ◽

Cited By ~ 40

Author(s):

Cécile M. Bébéar ◽

Hélène Renaudin ◽

Alain Charron ◽

Joseph M. Bové ◽

Christiane Bébéar ◽

...

Keyword(s):

Mycoplasma Hominis ◽

Dna Gyrase ◽

Quinolone Resistance ◽

Primary Target ◽

Topoisomerase Iv ◽

The Third ◽

Fourth Step ◽

Resistant Mutants

ABSTRACT Mycoplasma hominis mutants were selected stepwise for resistance to ofloxacin and sparfloxacin, and their gyrA,gyrB, parC, and parE quinolone resistance-determining regions were characterized. For ofloxacin, four rounds of selection yielded six first-, six second-, five third-, and two fourth-step mutants. The first-step mutants harbored a single Asp426→Asn substitution in ParE. GyrA changes (Ser83→Leu or Trp) were found only from the third round of selection. With sparfloxacin, three rounds of selection generated 4 first-, 7 second-, and 10 third-step mutants. In contrast to ofloxacin resistance, GyrA mutations (Ser83→Leu or Ser84→Trp) were detected in the first-step mutants prior to ParC changes (Glu84→Lys), which appeared only after the second round of selection. Further analysis of eight multistep-selected mutants of M. hominis that were previously described (2) revealed that they carried mutations in ParE (Asp426→Asn), GyrA (Ser83→Leu) and ParE (Asp426→Asn), GyrA (Ser83→Leu) and ParC (Ser80→Ile), or ParC (Ser80→Ile) alone, depending on the fluoroquinolone used for selection, i.e., ciprofloxacin, norfloxacin, ofloxacin, or pefloxacin, respectively. These data indicate that in M. hominis DNA gyrase is the primary target of sparfloxacin whereas topoisomerase IV is the primary target of pefloxacin, ofloxacin, and ciprofloxacin.

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

The Journal of Urology ◽

10.1097/00005392-200009010-00060 ◽

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

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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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.6.1651-1657.2002 ◽

2002 ◽

Vol 46 (6) ◽

pp. 1651-1657 ◽

Cited By ~ 18

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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Cefotaxime Acts Synergistically with Levofloxacin in Experimental Meningitis Due to Penicillin-Resistant Pneumococci and Prevents Selection of Levofloxacin-Resistant Mutants In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.8.2487-2491.2003 ◽

2003 ◽

Vol 47 (8) ◽

pp. 2487-2491 ◽

Cited By ~ 16

Author(s):

F. Kühn ◽

M. Cottagnoud ◽

F. Acosta ◽

L. Flatz ◽

J. Entenza ◽

...

Keyword(s):

Induced Resistance ◽

Fold Increase ◽

Low Doses ◽

Topoisomerase Iv ◽

Pneumococcal Strain ◽

Genes Encoding ◽

Resistant Strains ◽

Resistant Mutants ◽

Selection Of

ABSTRACT Cefotaxime, given in two doses (each 100 mg/kg of body weight), produced a good bactericidal activity (−0.47 Δlog10 CFU/ml · h) which was comparable to that of levofloxacin (−0.49 Δlog10 CFU/ml · h) against a penicillin-resistant pneumococcal strain WB4 in experimental meningitis. Cefotaxime combined with levofloxacin acted synergistically (−1.04 Δlog10 CFU/ml · h). Synergy between cefotaxime and levofloxacin was also demonstrated in vitro in time killing assays and with the checkerboard method for two penicillin-resistant strains (WB4 and KR4). Using in vitro cycling experiments, the addition of cefotaxime in sub-MIC concentrations (one-eighth of the MIC) drastically reduced levofloxacin-induced resistance in the same two strains (64-fold increase of the MIC of levofloxacin after 12 cycles versus 2-fold increase of the MIC of levofloxacin combined with cefotaxime). Mutations detected in the genes encoding topoisomerase IV (parC and parE) and gyrase (gyrA and gyrB) confirmed the levofloxacin-induced resistance in both strains. Addition of cefotaxime in low doses was able to suppress levofloxacin-induced resistance.

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Characterization of Mutations in DNA Gyrase and Topoisomerase IV Involved in Quinolone Resistance of Mycoplasma gallisepticum Mutants Obtained In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.2.590-593.2002 ◽

2002 ◽

Vol 46 (2) ◽

pp. 590-593 ◽

Cited By ~ 24

Author(s):

A. K. Reinhardt ◽

C. M. Bébéar ◽

M. Kobisch ◽

I. Kempf ◽

A. V. Gautier-Bouchardon

Keyword(s):

Target Genes ◽

Dna Gyrase ◽

Mycoplasma Gallisepticum ◽

Quinolone Resistance ◽

Mycoplasma Species ◽

Topoisomerase Iv ◽

Genes Encoding ◽

Resistant Mutants

ABSTRACT Mycoplasma gallisepticum enrofloxacin-resistant mutants were generated by stepwise selection in increasing concentrations of enrofloxacin. Alterations were found in the quinolone resistance-determining regions of the four target genes encoding DNA gyrase and topoisomerase IV from these mutants. This is the first description of such mutations in an animal mycoplasma species.

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Potent Antipneumococcal Activity of Gemifloxacin Is Associated with Dual Targeting of Gyrase and Topoisomerase IV, an In Vivo Target Preference for Gyrase, and Enhanced Stabilization of Cleavable Complexes In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.11.3112-3117.2000 ◽

2000 ◽

Vol 44 (11) ◽

pp. 3112-3117 ◽

Cited By ~ 55

Author(s):

Victoria J. Heaton ◽

Jane E. Ambler ◽

L. Mark Fisher

Keyword(s):

Hot Spot ◽

Dna Gyrase ◽

Second Step ◽

Wild Type ◽

Resistance Mutations ◽

Topoisomerase Iv ◽

Dual Targeting ◽

Resistant Mutants

ABSTRACT We investigated the roles of DNA gyrase and topoisomerase IV in determining the susceptibility of Streptococcus pneumoniaeto gemifloxacin, a novel fluoroquinolone which is under development as an antipneumococcal drug. Gemifloxacin displayed potent activity against S. pneumoniae 7785 (MIC, 0.06 μg/ml) compared with ciprofloxacin (MIC, 1 to 2 μg/ml). Complementary genetic and biochemical approaches revealed the following. (i) The gemifloxacin MICs for isogenic 7785 mutants bearing either parC orgyrA quinolone resistance mutations were marginally higher than wild type at 0.12 to 0.25 μg/ml, whereas the presence of both mutations increased the MIC to 0.5 to 1 μg/ml. These data suggest that both gyrase and topoisomerase IV contribute significantly as gemifloxacin targets in vivo. (ii) Gemifloxacin selected first-stepgyrA mutants of S. pneumoniae 7785 (gemifloxacin MICs, 0.25 μg/ml) encoding Ser-81 to Phe or Tyr, or Glu-85 to Lys mutations. These mutants were cross resistant to sparfloxacin (which targets gyrase) but not to ciprofloxacin (which targets topoisomerase IV). Second-step mutants (gemifloxacin MICs, 1 μg/ml) exhibited an alteration in parC resulting in changes of ParC hot spot Ser-79 to Phe or Tyr. Thus, gyrase appears to be the preferential in vivo target. (iii) Gemifloxacin was at least 10- to 20-fold more effective than ciprofloxacin in stabilizing a cleavable complex (the cytotoxic lesion) with either S. pneumoniaegyrase or topoisomerase IV enzyme in vitro. These data suggest that gemifloxacin is an enhanced affinity fluoroquinolone that acts against gyrase and topoisomerase IV in S. pneumoniae, with gyrase the preferred in vivo target. The marked potency of gemifloxacin against wild type and quinolone-resistant mutants may accrue from greater stabilization of cleavable complexes with the target enzymes.

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Activity of the new fluoroquinolone trovafloxacin (CP-99,219) against DNA gyrase and topoisomerase IV mutants of Streptococcus pneumoniae selected in vitro.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.12.2691 ◽

1996 ◽

Vol 40 (12) ◽

pp. 2691-2697 ◽

Cited By ~ 85

Author(s):

T D Gootz ◽

R Zaniewski ◽

S Haskell ◽

B Schmieder ◽

J Tankovic ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Parent Strain ◽

Low Frequency ◽

Dna Gyrase ◽

Second Step ◽

Wild Type ◽

Topoisomerase Iv ◽

Resistant Mutants ◽

Lethal Doses

The MICs of trovafloxacin, ciprofloxacin, ofloxacin, and sparfloxacin at which 90% of isolates are inhibited for 55 isolates of pneumococci were 0.125, 1, 4, and 0.5 microgram/ml, respectively. Resistant mutants of two susceptible isolates were selected in a stepwise fashion on agar containing ciprofloxacin at 2 to 10 times the MIC. While no mutants were obtained at the highest concentration tested, mutants were obtained at four times the MIC of ciprofloxacin (4 micrograms/ml) at a frequency of 1.0 x 10(-9). Ciprofloxacin MICs for these first-step mutants ranged from 4 to 8 micrograms/ml, whereas trovafloxacin MICs were 0.25 to 0.5 microgram/ml. Amplification of the quinolone resistance-determining region of the grlA (parC; topoisomerase IV) and gyrA (DNA gyrase) genes of the parents and mutants revealed that changes of the serine at position 80 (Ser80) to Phe or Tyr (Staphylococcus aureus coordinates) in GrlA were associated with resistance to ciprofloxacin. Second-step mutants of these isolates were selected by plating the isolates on medium containing ciprofloxacin at 32 micrograms/ml. Mutants for which ciprofloxacin MICs were 32 to 256 micrograms/ml and trovafloxacin MICs were 4 to 16 micrograms/ml were obtained at a frequency of 1.0 x 10(-9). Second-step mutants also had a change in GyrA corresponding to a substitution in Ser84 to Tyr or Phe or in Glu88 to Lys. Trovafloxacin protected from infection mice whose lungs were inoculated with lethal doses of either the parent strain or the first-step mutant. These results indicate that resistance to fluoroquinolones in S. pneumoniae occurs in vitro at a low frequency, involving sequential mutations in topoisomerase IV and DNA gyrase. Trovafloxacin MICs for wild-type and first-step mutants are within clinically achievable levels in the blood and lungs of humans.

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Contribution of Topoisomerase IV and DNA Gyrase Mutations in Streptococcus pneumoniae to Resistance to Novel Fluoroquinolones

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.8.2000 ◽

1999 ◽

Vol 43 (8) ◽

pp. 2000-2004 ◽

Cited By ~ 65

Author(s):

Ekaterina Pestova ◽

Rebecca Beyer ◽

Nicholas P. Cianciotto ◽

Gary A. Noskin ◽

Lance R. Peterson

Keyword(s):

Streptococcus Pneumoniae ◽

Molecular Basis ◽

Inhibitory Concentration ◽

Dna Gyrase ◽

Structural Genes ◽

Primary Target ◽

Topoisomerase Iv ◽

Dna Topoisomerase ◽

Enhanced Activity

ABSTRACT In this study, we assessed the activity of ciprofloxacin, levofloxacin, sparfloxacin, and trovafloxacin against clinical isolates of Streptococcus pneumoniae that were resistant to the less-recently developed fluoroquinolones by using defined amino acid substitutions in DNA gyrase and topoisomerase IV. The molecular basis for resistance was assessed by using mutants selected with trovafloxacin, ciprofloxacin, and levofloxacin in vitro. This demonstrated that the primary target of trovafloxacin in S. pneumoniae is the ParC subunit of DNA topoisomerase IV, similar to most other fluoroquinolones. However, first-step mutants bearing the Ser79→Phe/Tyr substitution in topoisomerase IV subunit ParC were susceptible to trovafloxacin with a minimum inhibitory concentration of 0.25 μg/ml, and mutations in the structural genes for both topoisomerase IV subunit ParC (parC) and the DNA gyrase subunit (gyrA) were required to achieve levels of resistance above the breakpoint. The data also suggest that enhanced activity of trovafloxacin against pneumococci is due to a combination of factors that may include reduced efflux of this agent and an enhanced activity against both DNA gyrase and topoisomerase IV.

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