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

2002 ◽  
Vol 46 (2) ◽  
pp. 590-593 ◽  
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.

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

1998 ◽  
Vol 42 (9) ◽  
pp. 2304-2311 ◽  
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.

scholarly journals In Vitro Characterization of the Antibacterial Spectrum of Novel Bacterial Type II Topoisomerase Inhibitors of the Aminobenzimidazole Class

2006 ◽  
Vol 50 (4) ◽  
pp. 1228-1237 ◽  
Author(s):  
Nagraj Mani ◽  
Christian H. Gross ◽  
Jonathan D. Parsons ◽  
Brian Hanzelka ◽  
Ute Müh ◽  
...  
Keyword(s):  
Bacterial Resistance ◽  
Wide Spectrum ◽  
Dna Gyrase ◽  
Antibacterial Activities ◽  
Mechanisms Of Action ◽  
Fluoroquinolone Resistance ◽  
Topoisomerase Iv ◽  
Low Frequencies

ABSTRACT Antibiotics with novel mechanisms of action are becoming increasingly important in the battle against bacterial resistance to all currently used classes of antibiotics. Bacterial DNA gyrase and topoisomerase IV (topoIV) are the familiar targets of fluoroquinolone and coumarin antibiotics. Here we present the characterization of two members of a new class of synthetic bacterial topoII ATPase inhibitors: VRT-125853 and VRT-752586. These aminobenzimidazole compounds were potent inhibitors of both DNA gyrase and topoIV and had excellent antibacterial activities against a wide spectrum of problematic pathogens responsible for both nosocomial and community-acquired infections, including staphylococci, streptococci, enterococci, and mycobacteria. Consistent with the novelty of their structures and mechanisms of action, antibacterial potency was unaffected by commonly encountered resistance phenotypes, including fluoroquinolone resistance. In time-kill assays, VRT-125853 and VRT-752586 were bactericidal against Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, and Haemophilus influenzae, causing 3-log reductions in viable cells within 24 h. Finally, similar to the fluoroquinolones, relatively low frequencies of spontaneous resistance to VRT-125853 and VRT-752586 were found, a property consistent with their in vitro dual-targeting activities.

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

2002 ◽  
Vol 46 (6) ◽  
pp. 1800-1804 ◽  
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.

scholarly journals Role of Mutations in DNA Gyrase and Topoisomerase IV in Fluoroquinolones-Resistance of Mycoplasma gallisepticum Obtained in vitro and in vivo

2012 ◽  
Vol 11 (13) ◽  
pp. 2327-2332
Author(s):  
Hong-Xia Jiang ◽  
Jian Li ◽  
Dian-Hong Lu ◽  
Zhi-Jie Liu ◽  
Xiao-Hua Zhang ◽  
...  
Keyword(s):  
Dna Gyrase ◽  
Mycoplasma Gallisepticum ◽  
Topoisomerase Iv

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

2003 ◽  
Vol 47 (8) ◽  
pp. 2487-2491 ◽  
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.

scholarly journals Mutations in the gyrA, parC, and parE Genes Associated with Fluoroquinolone Resistance in Clinical Isolates of Mycoplasma hominis

1999 ◽  
Vol 43 (4) ◽  
pp. 954-956 ◽  
Author(s):  
Cécile M. Bebear ◽  
Joel Renaudin ◽  
Alain Charron ◽  
Hélène Renaudin ◽  
Bertille de Barbeyrac ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mycoplasma Hominis ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Clinical Isolates ◽  
Fluoroquinolone Resistance ◽  
Amino Acid Substitutions ◽  
Topoisomerase Iv

ABSTRACT Five clinical isolates of Mycoplasma hominis from three different patients were examined for resistance to fluoroquinolones; some of these isolates were probably identical. All five isolates harbored amino acid substitutions in the quinolone resistance-determining regions of both DNA gyrase (GyrA) and topoisomerase IV (ParC or ParE). Furthermore, the novobiocin MIC for three isolates showed a significant increase. This is the first characterization of fluoroquinolone-resistant clinical mycoplasma isolates from humans.

scholarly journals Sequencing of Gyrase and Topoisomerase IV Quinolone-Resistance-Determining Regions of Chlamydia trachomatis and Characterization of Quinolone-Resistant Mutants Obtained In Vitro

1998 ◽  
Vol 42 (10) ◽  
pp. 2474-2481 ◽  
Author(s):  
Sophie Dessus-Babus ◽  
Cécile M. Bébéar ◽  
Alain Charron ◽  
Christiane Bébéar ◽  
Bertille de Barbeyrac
Keyword(s):  
Chlamydia Trachomatis ◽  
Reference Strain ◽  
Fold Increase ◽  
Quinolone Resistance ◽  
Topoisomerase Iv ◽  
Mutant Strains ◽  
Resistant Strains ◽  
Resistant Mutants ◽  
High Level

ABSTRACT The L2 reference strain of Chlamydia trachomatis was exposed to subinhibitory concentrations of ofloxacin (0.5 μg/ml) and sparfloxacin (0.015 μg/ml) to select fluoroquinolone-resistant mutants. In this study, two resistant strains were isolated after four rounds of selection. The C. trachomatis mutants presented with high-level resistance to various fluoroquinolones, particularly to sparfloxacin, for which a 1,000-fold increase in the MICs for the mutant strains compared to the MIC for the susceptible strain was found. The MICs of unrelated antibiotics (doxycycline and erythromycin) for the mutant strains were identical to those for the reference strain. The gyrase (gyrA, gyrB) and topoisomerase IV (parC, parE) genes of the susceptible and resistant strains of C. trachomatis were partially sequenced. A point mutation was found in the gyrAquinolone-resistance-determining region (QRDR) of both resistant strains, leading to a Ser83→Ile substitution (Escherichia coli numbering) in the corresponding protein. ThegyrB, parC, and parE QRDRs of the resistant strains were identical to those of the reference strain. These results suggest that in C. trachomatis, DNA gyrase is the primary target of ofloxacin and sparfloxacin.

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