scholarly journals Primary Targets of Fluoroquinolones inStreptococcus pneumoniae

1999 ◽  
Vol 43 (2) ◽  
pp. 410-412 ◽  
Author(s):  
Hideyuki Fukuda ◽  
Keiichi Hiramatsu
Keyword(s):  
Streptococcus Pneumoniae ◽  
Dna Gyrase ◽  
The Other ◽  
Wild Type ◽  
Primary Target ◽  
Topoisomerase Iv ◽  
Other Hand

ABSTRACT Mutants of wild-type Streptococcus pneumoniae IID553 with mutations in parC were obtained by selection with trovafloxacin, levofloxacin, norfloxacin, and ciprofloxacin. All of theparC mutants were cross-resistant to the selecting agents but were not resistant to gatifloxacin and sparfloxacin. On the other hand, gyrA mutants were isolated by selection with gatifloxacin and sparfloxacin. The gyrA mutants were cross-resistant to gatifloxacin and sparfloxacin but were not resistant to the other fluoroquinolones tested. These results suggest that in wild-type S. pneumoniae the primary target of trovafloxacin, levofloxacin, norfloxacin, and ciprofloxacin is topoisomerase IV, whereas the primary target of gatifloxacin and sparfloxacin is DNA gyrase.

scholarly journals Contributions of the 8-Methoxy Group of Gatifloxacin to Resistance Selectivity, Target Preference, and Antibacterial Activity against Streptococcus pneumoniae

2001 ◽  
Vol 45 (6) ◽  
pp. 1649-1653 ◽  
Author(s):  
Hideyuki Fukuda ◽  
Ryuta Kishii ◽  
Masaya Takei ◽  
Masaki Hosaka
Keyword(s):  
Antibacterial Activity ◽  
Streptococcus Pneumoniae ◽  
Type Strain ◽  
Methoxy Group ◽  
Wild Type Strain ◽  
Dna Gyrase ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Mutant Strains

ABSTRACT Gatifloxacin (8-methoxy, 7-piperazinyl-3′-methyl) at the MIC selected mutant strains that possessed gyrA mutations at a low frequency (3.7 × 10−9) from wild-type strainStreptococcus pneumoniae IID553. AM-1147 (8-methoxy, 7-piperazinyl-3′-H) at the MIC or higher concentrations selected no mutant strains. On the other hand, the respective 8-H counterparts of these two compounds, AM-1121 (8-H, 7-piperazinyl-3′-methyl) and ciprofloxacin (8-H, 7-piperazinyl-3′-H), at one and two times the MIC selected mutant strains that possessed parC mutations at a high frequency (>2.4 × 10−6). The MIC of AM-1147 increased for the gyrA mutant strains but not for theparC mutant strains compared with that for the wild-type strain. These results suggest that fluoroquinolones that harbor 8-methoxy groups select mutant strains less frequently and prefer DNA gyrase, as distinct from their 8-H counterparts. The in vitro activities of gatifloxacin and AM-1147 are twofold higher against the wild-type strain, eight- and twofold higher against the first-stepparC and gyrA mutant strains, respectively, and two- to eightfold higher against the second-step gyrA andparC double mutant strains than those of their 8-H counterparts. These results indicate that the 8-methoxy group contributes to enhancement of antibacterial activity against target-altered mutant strains as well as the wild-type strain. It is hypothesized that the 8-methoxy group of gatifloxacin increases the level of target inhibition, especially against DNA gyrase, so that it is nearly the same as that for topoisomerase IV inhibition in the bacterial cell, leading to potent antibacterial activity and a low level of resistance selectivity.

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 Contribution of the 8-Methoxy Group to the Activity of Gatifloxacin against Type II Topoisomerases of Streptococcus pneumoniae

2003 ◽  
Vol 47 (1) ◽  
pp. 77-81 ◽  
Author(s):  
Ryuta Kishii ◽  
Masaya Takei ◽  
Hideyuki Fukuda ◽  
Katsuhiko Hayashi ◽  
Masaki Hosaka
Keyword(s):  
Antibacterial Activity ◽  
Streptococcus Pneumoniae ◽  
Methoxy Group ◽  
Dna Gyrase ◽  
Type Ii ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Methoxy Groups ◽  
Dual Inhibition ◽  
Inhibitory Activities

ABSTRACT The inhibitory activities (50% inhibitory concentrations [IC50s]) of gatifloxacin and other quinolones against both DNA gyrase and topoisomerase IV of the wild-type Streptococcus pneumoniae IID553 were determined. The IC50s of 10 compounds ranged from 4.28 to 582 μg/ml against DNA gyrase and from 1.90 to 35.2 μg/ml against topoisomerase IV. The inhibitory activity against DNA gyrase was more varied than that against topoisomerase IV among fluoroquinolones. The IC50s for DNA gyrase of the 8-methoxy quinolones gatifloxacin and AM-1147 were approximately seven times lower than those of their 8-H counterparts AM-1121 and ciprofloxacin, whereas the IC50s for topoisomerase IV were 1.5 times lower. Moreover, the IC50 ratios (IC50 for DNA gyrase/IC50 for topoisomerase IV) of gatifloxacin, AM-1147, and moxifloxacin, which possess 8-methoxy groups, were almost the same. The 8-methoxy quinolones showed higher antibacterial activity and less mutant selectivity against IID553 than their 8-H counterparts. These results suggest that the 8-methoxy group enhances both target inhibition, especially for DNA gyrase, leading to potent antipneumococcal activity and dual inhibition against both DNA gyrase and topoisomerase IV in the bacterial cell.

scholarly journals Activities of Fluoroquinolones againstStreptococcus pneumoniae Type II Topoisomerases Purified as Recombinant Proteins

1999 ◽  
Vol 43 (11) ◽  
pp. 2579-2585 ◽  
Author(s):  
Ian Morrissey ◽  
John George
Keyword(s):  
Streptococcus Pneumoniae ◽  
Recombinant Proteins ◽  
Dna Gyrase ◽  
Fluoroquinolone Resistance ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Low Level ◽  
Cloning Method ◽  
High Level ◽  
Level Resistance

ABSTRACT Streptococcus pneumoniae topoisomerase IV and DNA gyrase have been purified from a fluoroquinolone-susceptibleStreptococcus 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 fromEscherichia 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 withS. 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.

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

1999 ◽  
Vol 43 (8) ◽  
pp. 2000-2004 ◽  
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.

scholarly journals Streptococcus pneumoniae DNA Gyrase and Topoisomerase IV: Overexpression, Purification, and Differential Inhibition by Fluoroquinolones

1999 ◽  
Vol 43 (5) ◽  
pp. 1129-1136 ◽  
Author(s):  
Xiao-Su Pan ◽  
L. Mark Fisher
Keyword(s):  
Streptococcus Pneumoniae ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Topoisomerase Iv ◽  
T7 Promoter ◽  
Bacterial Killing ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Comparable Activity

ABSTRACT Streptococcus pneumoniae gyrA and gyrBgenes specifying the DNA gyrase subunits have been cloned into pET plasmid vectors under the control of an inducible T7 promoter and have been separately expressed in Escherichia coli. Soluble 97-kDa GyrA and 72-kDa GyrB proteins bearing polyhistidine tags at their respective C-terminal and N-terminal ends were purified to apparent homogeneity by one-step nickel chelate column chromatography and were free of host E. coli topoisomerase activity. Equimolar amounts of the gyrase subunits reconstituted ATP-dependent DNA supercoiling with comparable activity to gyrase of E. coli and Staphylococcus aureus. In parallel, S. pneumoniae topoisomerase IV ParC and ParE subunits were similarly expressed in E. coli, purified to near homogeneity as 93- and 73-kDa proteins, and shown to generate efficient ATP-dependent DNA relaxation and DNA decatenation activities. Using the purified enzymes, we examined the inhibitory effects of three paradigm fluoroquinolones—ciprofloxacin, sparfloxacin, and clinafloxacin—which previous genetic studies with S. pneumoniae suggested act preferentially through topoisomerase IV, through gyrase, and through both enzymes, respectively. Surprisingly, all three quinolones were more active in inhibiting purified topoisomerase IV than gyrase, with clinafloxacin showing the greatest inhibitory potency. Moreover, the tested agents were at least 25-fold more effective in stabilizing a cleavable complex (the relevant cytotoxic lesion) with topoisomerase IV than with gyrase, with clinafloxacin some 10- to 32-fold more potent against either enzyme, in line with its superior activity againstS. pneumoniae. The uniform target preference of the three fluoroquinolones for topoisomerase IV in vitro is in apparent contrast to the genetic data. We interpret these results in terms of a model for bacterial killing by quinolones in which cellular factors can modulate the effects of target affinity to determine the cytotoxic pathway.

Activities of Trovafloxacin Compared with Those of Other Fluoroquinolones against Purified Topoisomerases and gyrA and grlA Mutants ofStaphylococcus aureus

1999 ◽  
Vol 43 (8) ◽  
pp. 1845-1855 ◽  
Author(s):  
Thomas D. Gootz ◽  
Richard P. Zaniewski ◽  
Suzanne L. Haskell ◽  
Frank S. Kaczmarek ◽  
Alison E. Maurice
Keyword(s):  
Dna Cleavage ◽  
Dna Gyrase ◽  
The Other ◽  
Second Step ◽  
Ofstaphylococcus Aureus ◽  
Topoisomerase Iv ◽  
Direct Plating ◽  
Clinical Strains ◽  
Plating Method ◽  
Susceptibility Breakpoint

ABSTRACT Frequencies of mutation to resistance with trovafloxacin and four other quinolones were determined with quinolone-susceptibleStaphylococcus aureus RN4220 by a direct plating method. First-step mutants were selected less frequently with trovafloxacin (1.1 × 10−10 at 2 to 4× the MIC) than with levofloxacin or ciprofloxacin (3.0 × 10−7 to 3.0 × 10−8 at 2 to 4× 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 [MIC50] and MIC90, 1 and 4 μg/ml, respectively); in comparison, MIC50s and MIC90s were 32 and 128, 16 and 32, 8 and 32, and 128 and 256 μg/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 μg/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.

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.

Nutritional Requirement for 4-Aminobenzoate Caused by Mutation of Dihydropteroate Synthetase

1976 ◽  
Vol 31 (5-6) ◽  
pp. 285-287 ◽  
Author(s):  
Helmut Rappold ◽  
Adelbert Bacher
Keyword(s):  
Parent Strain ◽  
The Other ◽  
Synthetase Activity ◽  
Nutritional Requirement ◽  
Wild Type ◽  
Aerobacter Aerogenes ◽  
Low Level ◽  
Other Hand ◽  
High Concentrations

Abstract Aerobacter aerogenes mutant 62-1 AC requires high concentrations of 4-aminobenzoate for growth. The mutant accumulates N-glucosyl-4-aminobenzoate and has an intact 4-aminobenzoate synthetase (Bacher, Gilch, Rappold, and Lingens, Z. Naturforsch. 28c, 614 - 617 [1973]). On the other hand the ability of the mutant to synthesize dihydropteroate is markedly reduced. The dihydropteroate synthetase level of mutant 62-1 AC is 1% as compared to the parent strain. Spontaneous revertants of mutant 62-1 AC show wild type levels of dihydropteroate synthetase. We conclude that the requirement for 4-aminobenzoate in mutant 62-1 AC is due to poor utilization of 4-aminobenzoate as a consequence of the low level of dihydropteroate synthetase activity.

H-Ras Is Involved in the Inside-out Signaling Pathway of Interleukin-3–Induced Integrin Activation

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1540-1548 ◽  
Author(s):  
Hirohiko Shibayama ◽  
Naoyuki Anzai ◽  
Stephen E. Braun ◽  
Seiji Fukuda ◽  
Charlie Mantel ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Dominant Negative ◽  
The Other ◽  
Wild Type ◽  
Integrin Activation ◽  
Mapk Kinase ◽  
Interleukin 3 ◽  
Other Hand ◽  
Inside Out ◽  
Constitutively Active

Abstract The proto-oncogene product, p21ras, has been implicated in the cellular mechanism of adhesion, although its precise role has been controversial. Numerous cytokines and growth-factors activate Ras, which is an important component of their growth-promoting signaling pathways. On the other hand, the role of Ras in cytokine-induced adhesion has not been elucidated. We therefore investigated the function of H-Ras in the inside-out signaling pathway of interleukin-3 (IL-3)–induced integrin activation in the murine Baf3 cell line after transfection of cells with either constitutively active, dominant-negative, or wild-type H-Ras cDNAs. Adhesion of Baf3 cells to fibronectin was induced by IL-3 in a dose-dependent manner via very late antigen-4 (VLA-4; 4β1 integrins) and VLA-5 (5β1 integrins) activation. On the other hand, IL-4 did not induce the adhesion of Baf3 cells to fibronectin, although IL-4 did stimulate the cell proliferation of Baf3 cells. Constitutively active H-Ras–transfected Baf3 cells adhered to fibronectin without IL-3 stimulation through VLA-4 and VLA-5, whereas dominant-negative H-Ras–transfected Baf3 cells showed significantly less adhesion induced by IL-3 compared with wild-type and constitutively active H-Ras–transfected Baf3 cells. Anti-β1 integrin antibody (clone; 9EG7), which is known to change integrin conformation and activate integrins, induced the adhesion of dominant-negative H-Ras–transfected Baf3 cells as much as the other types of H-Ras–transfected Baf3 cells. 8-Br-cAMP, Dibutyryl-cAMP, Ras-Raf-1 pathway inhibitors, and PD98059, a MAPK kinase inhibitor, suppressed proliferation and phosphorylation of MAPK detected by Western blotting with anti–phospho-MAPK antibody, but not adhesion of any type of H-Ras–transfected Baf3 cells, whereas U-73122, a phospholipase C (PLC) inhibitor, suppressed adhesion of these cells completely. These data indicate that H-Ras and PLC, but not Raf-1, MAPK kinase, or the MAPK pathway, are involved in the inside-out signaling pathway of IL-3–induced VLA-4 and VLA-5 activation in Baf3 cells.

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