Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:5.99.1.3 (topoisomerase)
9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The L2 reference strain of Chlamydia trachomatis was exposed to subinhibitory concentrations of ofloxacin (0.5 microg/ml) and sparfloxacin (0.015 microg/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 gyrA quinolone-resistance-determining region (QRDR) of both resistant strains, leading to a Ser83-->Ile substitution (Escherichia coli numbering) in the corresponding protein. The gyrB, 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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PMID:Sequencing of gyrase and topoisomerase IV quinolone-resistance-determining regions of Chlamydia trachomatis and characterization of quinolone-resistant mutants obtained In vitro. 975 44

We examined the response of Streptococcus pneumoniae 7785 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 microg/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-gyrA mutants were 16 to 32 and 32 to 64 microg/ml, respectively, but the clinafloxacin MIC was 1 microg/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 microg/ml, respectively. Thus, high-level resistance to clinafloxacin required four steps. Characterization of the quinolone resistance-determining regions of the gyrA, parC, gyrB, and parE genes by PCR, HinfI restriction fragment length polymorphism, and DNA sequence analysis revealed an invariant resistance pathway involving sequential mutations in gyrA or 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] microg of clinafloxacin per ml at a frequency of 5.0 x 10(-10) to 8.5 x 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. pneumoniae and its first- and second-step mutants are desirable features in limiting the emergence of bacterial resistance.
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PMID:DNA gyrase and topoisomerase IV are dual targets of clinafloxacin action in Streptococcus pneumoniae. 979 8

Ciprofloxacin, 500 mg, was introduced as the first-line therapy for gonorrhea at St. Mary's Hospital, London, in 1989, when a surveillance program was initiated to detect the emergence of resistance. Isolates of Neisseria gonorrhoeae from consecutive patients attending the Jefferiss Wing, Genitourinary Medicine Clinic at St. Mary's Hospital, between 1989 and 1997 have been tested for susceptibility to ciprofloxacin by using an agar dilution breakpoint technique. Isolates considered potentially resistant (MIC, >0.12 microg/ml) were further characterized by determination of the MICs of ciprofloxacin, nalidixic acid, and penicillin, auxotyped and serotyped, and screened for mutations in the DNA gyrase gene, gyrA, and the topoisomerase IV gene, parC. A total of 4,875 isolates were tested. While the majority of isolates were highly susceptible (MIC, </=0.008 microg of ciprofloxacin/ml), there was a drift toward reduced susceptibility in N. gonorrhoeae isolated between 1993 and 1996 (P < 0.001). In 1997 this drift was reduced but remained above pre-1993 levels. Isolates from 18 patients were classed as potentially resistant (MIC, >0.12 microg/ml); all of these belonged to serogroup B, and NR/IB-1 was the most common auxotype/serovar class. The infections in 14 of the 18 patients were known to be acquired abroad, and 5 were known to result in therapeutic failure. The surveillance program has established that ciprofloxacin is still a highly effective antibiotic against N. gonorrhoeae in this population. However, it has identified a drift in susceptibility which may have resulted from increased usage of ciprofloxacin. High-level resistance has now emerged, although treatment failure is still uncommon.
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PMID:Drift in susceptibility of Neisseria gonorrhoeae to ciprofloxacin and emergence of therapeutic failure. 979 26

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.
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PMID:Mutations in the gyrA, parC, and parE genes associated with fluoroquinolone resistance in clinical isolates of Mycoplasma hominis. 1010 8

It has been reported that subinhibitory concentrations (sub-MICs) of some fluoroquinolones are still capable of affecting the topological characteristics of DNA (inhibition DNA-gyrase) and that this leads to a reduction in some of the factors responsible for bacterial virulence (by means of the disruption of protein synthesis and alterations in phenotype expression), even though the microorganisms themselves are not killed. The present study investigated the ability of sub-MICs of rufloxacin, an orally absorbed monofluorinated quinolone with a long half-life (28 to 30 h), to interfere with the bacterial virulence parameters of adhesiveness, hemagglutination, hydrophobicity, motility, and filamentation, as well as their interactions with host neutrophilic defenses such as phagocytosis, killing, and oxidative bursts. It was observed that Escherichia coli adhesiveness was significantly reduced at rufloxacin concentrations of 1/32 MIC, hemagglutination and hydrophobicity were significantly reduced at concentrations of, respectively, 1/4 MIC and 1/8 MIC, and motility was significantly reduced at concentrations of 1/16 MIC; filamentation was still present at concentrations of 1/4 MIC. Phagocytosis was not affected, but killing significantly increased from 1/2 MIC to 1/8 MIC; oxidative bursts measured by means of chemiluminescence were not affected. The fact that sub-MICs are still effective in interfering with the parameters of bacterial virulence is useful information that needs to be correlated with pharmacokinetic data in order to extend our knowledge of the most effective concentrations that can be used to optimize treatment schedules, for example, single administrations, particularly in noncomplicated lower urinary tract infections.
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PMID:Pharmacodynamic effects of subinhibitory concentrations of rufloxacin on bacterial virulence factors. 1022 8

Coagulase-negative staphylococcal isolates (n = 188) were screened for susceptibility to oxacillin, ciprofloxacin, and trovafloxacin, a new fluoroquinolone. At an oxacillin concentration of >/=4 microg/ml, 43% were methicillin resistant; of these, 70% were ciprofloxacin resistant (MIC, >/=4 microg/ml). Of the methicillin-resistant, ciprofloxacin-resistant isolates, 46% were susceptible to </=2 microg of trovafloxacin per ml and 32% were susceptible to </=1 microg 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 and S. 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 microg/ml) and increased trovafloxacin MICs (0.25 to 2 microg/ml) could be conferred by the combined presence of single mutations in each gyrA and grlA gene. Trovafloxacin MICs of >/=8 microg/ml also occurred, but these required an additional mutation in grlA.
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PMID:Topoisomerase sequences of coagulase-negative staphylococcal isolates resistant to ciprofloxacin or trovafloxacin. 1039 Feb 14

Frequencies of mutation to resistance with trovafloxacin and four other quinolones were determined with quinolone-susceptible Staphylococcus aureus RN4220 by a direct plating method. First-step mutants were selected less frequently with trovafloxacin (1.1 x 10(-10) at 2 to 4x the MIC) than with levofloxacin or ciprofloxacin (3.0 x 10(-7) to 3.0 x 10(-8) at 2 to 4x the MIC). Mutants with a change in GrlA (Ser80-->Phe or Tyr) were most commonly selected with trovafloxacin, ciprofloxacin, levofloxacin, or pefloxacin. First-step mutants were difficult to select with sparfloxacin; however, second-step mutants with mutations in gyrA were easily selected when a preexisting mutation in grlA was present. Against 29 S. aureus clinical isolates with known mutations in gyrA and/or grlA, trovafloxacin was the most active quinolone tested (MIC at which 50% of isolates are inhibited [MIC(50)] and MIC(90), 1 and 4 microg/ml, respectively); in comparison, MIC(50)s and MIC(90)s were 32 and 128, 16 and 32, 8 and 32, and 128 and 256 microg/ml for ciprofloxacin, sparfloxacin, levofloxacin, and pefloxacin, respectively. Strains with a mutation in grlA only were generally susceptible to all of the quinolones tested. For mutants with changes in both grlA and gyrA MICs were higher and were generally above the susceptibility breakpoint for ciprofloxacin, sparfloxacin, levofloxacin, and pefloxacin. Addition of reserpine (20 microg/ml) lowered the MICs only of ciprofloxacin fourfold or more for 18 of 29 clinical strains. Topoisomerase IV and DNA gyrase genes were cloned from S. aureus RN4220 and from two mutants with changes in GrlA (Ser80-->Phe and Glu84-->Lys). The enzymes were overexpressed in Escherichia coli GI724, purified, and used in DNA catalytic and cleavage assays that measured the relative potency of each quinolone. Trovafloxacin was at least five times more potent than ciprofloxacin, sparfloxacin, levofloxacin, or pefloxacin in stimulating topoisomerase IV-mediated DNA cleavage. While all of the quinolones were less potent in cleavage assays with the altered topoisomerase IV, trovafloxacin retained its greater potency relative to those of the other quinolones tested. The greater intrinsic potency of trovafloxacin against the lethal topoisomerase IV target in S. aureus contributes to its improved potency against clinical strains of S. aureus that are resistant to other quinolones.
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PMID:Activities of trovafloxacin compared with those of other fluoroquinolones against purified topoisomerases and gyrA and grlA mutants of Staphylococcus aureus. 1042 1

The occurrence of mutations in the genes coding for gyrase (gyrA and gyrB) and topoisomerase IV (parE and parC) of Salmonella typhimurium experimental mutants selected in vitro and in vivo and of 138 nalidixic acid-resistant Salmonella field isolates was investigated. The sequencing of the quinolone resistance-determining region of these genes in highly fluoroquinolone-resistant mutants (MICs of 4 to 16 microg/ml) revealed the presence of gyrA mutations at codons corresponding to Gly-81 or Ser-83, some of which were associated with a mutation at Asp-87. No mutations were found in the gyrB, parC, and parE genes. An assay combining allele-specific PCR and restriction fragment length polymorphism was developed to rapidly screen mutations at codons 81, 83, and 87 of gyrA. The MICs of ciprofloxacin for the field isolates reached only 2 microg/ml, versus 16 microg/ml for some in vitro-selected mutants. The field isolates, like the mutants selected in vivo, had only a single gyrA mutation at codon 83 or 87. Single gyrA mutations were also found in highly resistant in vitro-selected mutants (MIC of ciprofloxacin, 8 microg/ml), which indicates that mechanisms other than the unique modification of the intracellular targets could participate in fluoroquinolone resistance in Salmonella spp. A comparison of experimental mutants selected in vitro, field strains, and mutants selected in vivo suggests that highly fluoroquinolone-resistant strains are counterselected in field conditions in the absence of selective pressure.
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PMID:Comparative studies of mutations in animal isolates and experimental in vitro- and in vivo-selected mutants of Salmonella spp. suggest a counterselection of highly fluoroquinolone-resistant strains in the field. 1047 53

Streptococcus pneumoniae topoisomerase IV and DNA gyrase have been purified from a fluoroquinolone-susceptible Streptococcus pneumoniae strain, from first-step mutants showing low-level resistance to ciprofloxacin, sparfloxacin, levofloxacin, and ofloxacin, and from two clinical isolates showing intermediate- and high-level fluoroquinolone resistance by a gene cloning method that produces recombinant proteins from Escherichia coli. The concentrations of ciprofloxacin, sparfloxacin, levofloxacin, or ofloxacin required to inhibit wild-type topoisomerase IV were 8 to 16 times lower than those required to inhibit wild-type DNA gyrase. Furthermore, low-level resistance to these fluoroquinolones was entirely due to the reduced inhibitory activity of fluoroquinolones against topoisomerase IV. For all the laboratory strains, the 50% inhibitory concentration for topoisomerase IV directly correlated with the MIC. We therefore propose that with S. pneumoniae, ciprofloxacin, sparfloxacin, levofloxacin, and ofloxacin target topoisomerase IV in preference to DNA gyrase. Sitafloxacin, on the other hand, was found to be equipotent against either enzyme. This characteristic is unique for a fluoroquinolone. A reduction in the sensitivities of both topoisomerase IV and DNA gyrase are required, however, to achieve intermediate- or high-level fluoroquinolone resistance in S. pneumoniae.
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PMID:Activities of fluoroquinolones against Streptococcus pneumoniae type II topoisomerases purified as recombinant proteins. 1054 32

Nine penicillin-resistant Streptococcus pneumoniae clinical isolates from Northern Ireland, resistant to ciprofloxacin (MICs, 2 to 64 microg/ml) through topoisomerase- and/or reserpine-sensitive efflux mechanisms, were highly susceptible to gemifloxacin (MICs, 0.03 to 0. 12 microg/ml). Two strains (requiring a ciprofloxacin MIC of 64 microg/ml) carried known quinolone resistance mutations in parC, parE, and gyrB, resulting in S79F, D435V, and E474K changes, respectively. Thus, gemifloxacin is active against clinical strains exhibiting altered topoisomerase and efflux phenotypes.
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PMID:Activity of gemifloxacin against penicillin- and ciprofloxacin-resistant Streptococcus pneumoniae displaying topoisomerase- and efflux-mediated resistance mechanisms. 1058 96


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