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.

Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae.

1996 ◽  
Vol 40 (10) ◽  
pp. 2321-2326 ◽  
Author(s):  
X S Pan ◽  
J Ambler ◽  
S Mehtar ◽  
L M Fisher
Keyword(s):  
Streptococcus Pneumoniae ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Clinical Isolates ◽  
Resistant Isolate ◽  
Topoisomerase Iv ◽  
E Coli ◽  
Low Level ◽  
Drug Concentrations ◽  
Level Resistance

Ciprofloxacin-resistant mutants of Streptococcus pneumoniae 7785 were generated by stepwise selection at increasing drug concentrations. Sequence analysis of PCR products from the strains was used to examine the quinolone resistance-determining regions of the GyrA and GyrB proteins of DNA gyrase and the analogous regions of the ParC and ParE subunits of DNA topoisomerase IV. First-step mutants exhibiting low-level resistance had no detectable changes in their topoisomerase quinolone resistance-determining regions, suggesting altered permeation or another novel resistance mechanism. Nine of 10 second-step mutants exhibited an alteration in ParC at Ser-79 to Tyr or Phe or at Ala-84 to Thr. Third- and fourth-step mutants displaying high-level ciprofloxacin resistance were found to have, in addition to the ParC alteration, a change in GyrA at residues equivalent to Escherichia coli GyrA resistance hot spots Ser-83 and Asp-87 or in GyrB at Asp-435 to Asn, equivalent to E. coli Asp-426, part of a highly conserved EGDSA motif in GyrB. No ParE changes were observed. Complementary analysis of two S. pneumoniae clinical isolates displaying low-level resistance to ciprofloxacin revealed a ParC change at Ser-79 to Phe or Arg-95 to Cys but no changes in GyrA, GyrB, or ParE. A highly resistant isolate, in addition to a ParC mutation, had a GyrA alteration at the residue equivalent to E. coli Asp-87. Thus, in both laboratory strains and clinical isolates, ParC mutations preceded those in GyrA, suggesting that topoisomerase IV is a primary topoisomerase target and gyrase is a secondary target for ciprofloxacin in S. pneumoniae.

scholarly journals Characterization of a mutation in the parE gene that confers fluoroquinolone resistance in Streptococcus pneumoniae.

1997 ◽  
Vol 41 (5) ◽  
pp. 1166-1167 ◽  
Author(s):  
B Perichon ◽  
J Tankovic ◽  
P Courvalin
Keyword(s):  
Streptococcus Pneumoniae ◽  
Fluoroquinolone Resistance ◽  
Primary Target ◽  
Topoisomerase Iv ◽  
Low Level ◽  
Sequential Acquisition ◽  
Level Resistance

We report a mutation in the parE genes of two in vitro mutants of Streptococcus pneumoniae responsible for low-level resistance to fluoroquinolones. Sequential acquisition of mutations in parE and gyrA leads to higher levels of resistance. This confirms that topoisomerase IV is the primary target of fluoroquinolones in S. pneumoniae.

scholarly journals High-level fluoroquinolone resistance in Streptococcus pneumoniae requires mutations in parC and gyrA.

1996 ◽  
Vol 40 (12) ◽  
pp. 2760-2764 ◽  
Author(s):  
C Janoir ◽  
V Zeller ◽  
M D Kitzis ◽  
N J Moreau ◽  
L Gutmann
Keyword(s):  
Streptococcus Pneumoniae ◽  
Quinolone Resistance ◽  
Fluoroquinolone Resistance ◽  
Clinical Samples ◽  
Low Frequencies ◽  
Low Level ◽  
Resistant Strains ◽  
High Level ◽  
Level Resistance

The mechanism of high-level fluoroquinolone resistance was studied in strains of Streptococcus pneumoniae, either selected in vitro or isolated from clinical samples. By using DNA from these high-level-resistant strains, low-level-resistant transformants (MIC of pefloxacin, > or = 32 micrograms/ml; MIC of ciprofloxacin, 4 micrograms/ml; MIC of sparfloxacin, 0.50 micrograms/ml) were obtained at high frequencies (ca.10(-2)), while high-level-resistant transformants (MIC of pefloxacin, > or = 64 micrograms/ml; MIC of ciprofloxacin, 16 to 64 micrograms/ml; MIC of sparfloxacin, > or = 8 micrograms/ml) were obtained only at low frequencies (ca.10(-4)). This suggested that mutations in at least two unlinked genes were necessary to obtain high-level resistance. Low-level resistance was associated with ParC mutations (change from Ser to Tyr at position 79 [Ser79Tyr], Ser79Phe, or Asp83Gly). ParC mutations were associated, in high-level-resistant strains and transformants, with alterations in the quinolone resistance-determining region of GyrA (Ser84Tyr, Ser84Phe, and/or Glu88Lys). Low-level resistance was shown to be necessary for expression of the gyrA mutations. No mutation in the region corresponding to the quinolone resistance-determining region of GyrB and no alteration of drug accumulation were found.

scholarly journals Genetic Characterization of Fluoroquinolone-Resistant Streptococcus pneumoniae Strains Isolated during Ciprofloxacin Therapy from a Patient with Bronchiectasis

2003 ◽  
Vol 47 (4) ◽  
pp. 1419-1422 ◽  
Author(s):  
Adela G. de la Campa ◽  
María-José Ferrandiz ◽  
Fe Tubau ◽  
Román Pallarés ◽  
Federico Manresa ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Genetic Characterization ◽  
Susceptible Strain ◽  
Low Level ◽  
Resistant Strains ◽  
Resistant Mutants ◽  
High Level ◽  
Level Resistance

ABSTRACT Five Spain9V-3 Streptococcus pneumoniae strains were isolated from a patient with bronchiectasis who had received long-term ciprofloxacin therapy. One ciprofloxacin-susceptible strain was isolated before treatment, and four ciprofloxacin-resistant strains were isolated during treatment. The resistant strains were derived from the susceptible strain either by a parC mutation (low-level resistance) or by parC and gyrA mutations (high-level resistance). This study shows that ciprofloxacin therapy in a patient colonized by susceptible S. pneumoniae may select fluoroquinolone-resistant mutants.

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 Low-Level Resistance to Rifampin in Streptococcus pneumoniae

2003 ◽  
Vol 47 (3) ◽  
pp. 863-868 ◽  
Author(s):  
Patricia Stutzmann Meier ◽  
Silvia Utz ◽  
Suzanne Aebi ◽  
Kathrin Mühlemann
Keyword(s):  
Streptococcus Pneumoniae ◽  
Point Mutations ◽  
Low Level ◽  
Development Of Resistance ◽  
Rifampin Resistance ◽  
High Level ◽  
Tissue Compartments ◽  
Level Resistance

ABSTRACT Rifampin is recommended for combination therapy of meningitis due to β-lactam-resistant Streptococcus pneumoniae. High-level rifampin resistance (MIC, ≥4 mg/liter) has been mapped to point mutations in clusters I and III of rpoB of the pneumococcus. The molecular basis of low-level resistance (MICs, ≥0.5 and <4 mg/liter) was analyzed. Spontaneous mutants of clinical pneumococcal isolates were selected on Columbia sheep blood agar plates containing rifampin at 0.5, 4, 10, or 50 mg/liter. Low-level resistance could be assigned to mutations in cluster II (I545N, I545L). Sensitive (MIC, <0.048 mg/liter) wild-type strains acquired low-level resistance at a rate approximately 10 times higher than that at which they acquired high-level resistance (average mutation frequencies, 2.4 × 10−7 for low-level resistance versus 2.9 × 10−8 for high-level resistance [P < 0.0001]). In second-step experiments, the frequencies of mutations from low- to high-level resistance were over 10 times higher than the frequencies of mutations from susceptibility to high-level resistance (average mutation frequencies, 7.2 × 10−7 versus 5.0 × 10−8 [P < 0.001]). Mutants with low-level resistance were stable upon passage. Sequencing of a clinical isolate with low-level resistance (MIC, 0.5 mg/liter) revealed a Q150R mutation upstream of cluster I. The frequencies of mutations to high-level resistance for this strain were even higher than the rates observed for the in vitro mutants. Therefore, a resistance-mediating mutation located outside clusters I, II, and III has been described for the first time in the pneumococcus. In vitro low-level rifampin resistance in S. pneumoniae could be mapped to cluster II of rpoB. Mutants of pneumococcus with low-level resistance may be selected in vivo during therapy in tissue compartments with low antibiotic concentrations and play a role in the development of resistance.

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

1998 ◽  
Vol 42 (1) ◽  
pp. 121-128 ◽  
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.

scholarly journals Novel Ser79Leu and Ser81Ile Substitutions in the Quinolone Resistance-Determining Regions of ParC Topoisomerase IV and GyrA DNA Gyrase Subunits from Recent Fluoroquinolone-Resistant Streptococcus pneumoniae Clinical Isolates

2005 ◽  
Vol 49 (6) ◽  
pp. 2479-2486 ◽  
Author(s):  
Nataliya Korzheva ◽  
Todd A. Davies ◽  
Raul Goldschmidt
Keyword(s):  
Streptococcus Pneumoniae ◽  
Amino Acid ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Clinical Isolates ◽  
Fluoroquinolone Resistance ◽  
Amino Acid Substitutions ◽  
Topoisomerase Iv ◽  
Isogenic Strains ◽  
New Mutations

ABSTRACT Resistance of Streptococcus pneumoniae to fluoroquinolones is caused predominantly by amino acid substitutions at positions Ser79 of ParC and Ser81 of GyrA to either Phe or Tyr encoded in the quinolone resistance-determining regions of the parC topoisomerase IV and gyrA DNA gyrase genes. Analysis of highly resistant clinical isolates identified novel second-step substitutions, Ser79Leu (ParC) and Ser81Ile (GyrA). To determine contributions of these new mutations to fluoroquinolone resistance either alone or in combination with other Ser79/81 alleles, the substitutions Ser79Leu/Phe/Tyr in ParC and Ser81Ile/Phe/Tyr in GyrA were introduced into the R6 background, resulting in 15 isogenic strains. Their level of fluoroquinolone resistance was determined by susceptibility testing for ciprofloxacin, levofloxacin, moxifloxacin, gatifloxacin, gemifloxacin, garenoxacin, and norfloxacin. Leu79 and Ile81 alone as well as 79/81Phe/Tyr substitutions did not contribute significantly to resistance, with fluoroquinolone MICs increasing two- to fourfold compared to wild type for all agents tested. Fluoroquinolone MICs for double transformants ParC Ser79Phe/Tyr/Leu-GyrA Ser81Phe/Tyr were uniformly increased by 8- to 64-fold regardless of pairs of amino acid substitutions. However, combinations including Ile81 conferred two- to fourfold-higher levels of resistance than did combinations including any other Ser81 GyrA substitution, thus demonstrating the differential effects of diverse amino acid substitutions at particular hotspots on fluoroquinolone MICs.

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.

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