Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, November 1998, p. 2810-2816, Vol. 42, No. 11
Molecular Genetics Group, Department of
Biochemistry, St. George's Hospital Medical School, University of
London, London SW17 ORE, United Kingdom
Received 1 May 1998/Returned for modification 21 July 1998/Accepted 14 August 1998
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 µg/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 µg/ml, respectively, but the
clinafloxacin MIC was 1 µg/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 µg/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] µg of clinafloxacin per ml at a
frequency of 5.0 × 10 Streptococcus pneumoniae
is an important human pathogen. It is the main cause of
community-acquired pneumonia and is frequently involved in
exacerbations of chronic bronchitis and in meningitis, acute otitis
media, and sinusitis (3). Treatment of S. pneumoniae infections relies heavily on antimicrobial therapy with
penicillin or other beta-lactams. Over the past two decades, the
emergence and, in some areas, the prevalence of pneumococci with
decreased susceptibility to penicillins have emphasized the need for
new therapeutic agents and have focused attention on the
fluoroquinolones (14, 25, 34). However, ciprofloxacin, the
main quinolone in current clinical use, has modest activity against
gram-positive bacteria such as S. pneumoniae. Consequently,
it has had relatively little impact on the treatment of respiratory
tract infections.
Clinafloxacin (AM-1091, CI-960, and PD127391) is a novel
fluoroquinolone with potent broad-spectrum in vitro activity against gram-positive, gram-negative, and anaerobic pathogens (reviewed in
reference 18). The drug has a structure somewhat
different from that of ciprofloxacin Previous studies showed that quinolones inhibit DNA gyrase and DNA
topoisomerase IV (reviewed in reference 6). Both
enzymes act by a double-strand DNA break mechanism and are essential
for bacterial growth (19, 35). They cooperate in DNA
replication to facilitate DNA unlinking and chromosome segregation
(41). Gyrase, an A2B2 tetramer
encoded by the gyrA and gyrB genes, catalyzes negative DNA supercoiling (10, 35) and is thought to act
ahead of the replication fork, neutralizing positive supercoils arising from DNA unwinding and thereby promoting fork movement (41). Topoisomerase IV, a C2E2 complex specified by
the parC and parE genes, allows the segregation
of daughter chromosomes at cell division (1, 17, 41). Point
mutations in discrete regions of the gyrase and topoisomerase IV
genes Given the potency of clinafloxacin against S. pneumoniae, we
have sought to determine its mechanism of action in this pathogen. We
have studied the activity of clinafloxacin against representative S. pneumoniae mutants with characterized mutations in
topoisomerase genes. We also report a detailed analysis of the
gyrA, parC, gyrB, and parE
QRDRs of S. pneumoniae mutants selected stepwise for resistance to clinafloxacin.
Bacterial strains.
S. pneumoniae 7785, a
quinolone-susceptible clinical isolate, has been described previously
(27). Characterization of mutants of strain 7785 selected in
vitro for resistance to ciprofloxacin (1C1, 2C6, 2C7, and 3C4) or to
sparfloxacin (1S1, 1S4, 2S1, and 2S4) also has been reported (26,
28).
Drug susceptibilities.
Clinafloxacin hydrochloride was from
Parke-Davis, Ann Arbor, Mich. A stock solution was made up in water and
stored at Bacterial growth rates.
Strains 7785 and 4CLN9 were taken
from plates, diluted to 50 to 100 CFU/ml in T broth (Sanofi Diagnostics
Pasteur), and grown aerobically on an orbital shaker at 37°C. At time
zero and at 1-h intervals over a 7-h period, 100-µl samples of the
culture were removed and, after appropriate dilution, spread on brain heart infusion plates containing 10% horse blood. Plates were incubated overnight at 37°C for the determination of viable counts. Experiments were done in duplicate, and doubling times were obtained from semilog plots of CFU versus time.
Stepwise selection of clinafloxacin-resistant S. pneumoniae strains.
Mutants were selected by plating
approximately 1011 CFU of strain 7785 or its drug-resistant
derivatives on brain heart infusion plates containing 10% horse blood
and clinafloxacin. Plates were incubated aerobically at 37°C for 24 to 48 h. Mutant frequencies were determined by comparing the
number of colonies that grew on plates containing drug with the number
of colonies obtained in the absence of drug. All procedures were as
described previously (26, 28).
PCR and RFLP analysis.
Conditions for bacterial growth and
the protocol for the isolation of genomic DNA were as described
previously (27). PCR was used to amplify DNA from the QRDRs
of the gyrase and topoisomerase IV genes of clinafloxacin-selected
S. pneumoniae mutants. PCR conditions have been reported
previously, as have the primers: VGA3 and VGA4 for gyrA,
M0363 and M4721 for parC, H4025 and H4026 for
gyrB, and S6398 and S6399 for parE
(28). Restriction fragment length polymorphism (RFLP)
analysis by HinfI digestion of the PCR products was carried
out as reported previously (27).
AsPCR and DNA sequence analysis.
Asymmetric PCR (AsPCR) was
used to generate single-stranded DNA for direct DNA sequencing by the
chain termination method (32). AsPCR conditions,
purification of AsPCR products, and DNA sequencing were as described
previously (28).
Activity of clinafloxacin against S. pneumoniae gyrA
and parC mutants.
As an initial step in defining the
target specificity of clinafloxacin, we examined the susceptibilities
of S. pneumoniae 7785 and its mutants bearing characterized
quinolone resistance mutations in parC, in gyrA,
and in both parC and gyrA (Table
1). These mutants were obtained
previously by stepwise selection for resistance to ciprofloxacin
(strains 1C1, 2C6, 2C7, and 3C4) or sparfloxacin (strains 1S1, 1S4,
2S1, and 2S4) (28). The mutations in these strains affect
hot spots for quinolone resistance (Ser-79 and Asp-83 in ParC and the
residue in S. pneumoniae GyrA equivalent to Ser-83 in
E. coli GyrA). From Table 1, it can be seen that the
parC mutants were about threefold more resistant to
ciprofloxacin (compared to parent 1C1) and that the gyrA
mutants were eightfold more resistant to sparfloxacin. Mutations in
gyrA did not affect the response to ciprofloxacin, and
parC changes had no effect on susceptibility to sparfloxacin
(Table 1). Strains expressing both parC and gyrA
mutations were extremely resistant to both ciprofloxacin and
sparfloxacin; MICs were 16 to 64 µg/ml, i.e., ~64 to 128-fold
higher than those for the wild type.
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
DNA Gyrase and Topoisomerase IV Are Dual Targets of
Clinafloxacin Action in Streptococcus pneumoniae
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
10 to 8.5 × 1010) 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.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
notably, the presence of a
chlorine C-8 substituentand is much more active than ciprofloxacin
against gram-positive species, including S. pneumoniae.
Clinafloxacin has been identified as the most active fluoroquinolone
against S. pneumoniae when compared with ofloxacin,
levofloxacin, sparfloxacin, grepafloxacin, and trovafloxacin
(22a) and is currently being evaluated as an
antipneumococcal agent. However, its mechanism of action has yet to be
examined in detail.
the quinolone resistance-determining regions (QRDRs)are
responsible for the development of resistance (22, 39, 40).
Ciprofloxacin resistance in Staphylococcus aureus and
S. pneumoniae arises through mutation of the parC
or parE genes before changes in the gyrase genes take place,
suggesting that topoisomerase IV is the primary ciprofloxacin target
and that gyrase is the secondary target in these gram-positive species (7, 15, 21, 24, 26, 27, 33). Interestingly, in Escherichia coli and other gram-negative bacteria, gyrase is
the primary target (5, 6, 9, 13, 22), initially suggesting that there could be fundamental differences in drug responses within
the bacterial kingdom. However, in recent work, we have shown that
whereas ciprofloxacin targets topoisomerase IV in S. pneumoniae, sparfloxacin targets gyrase, indicating that the
molecular structure of the quinolone determines the target preference
(28). We and others have proposed that a quinolone acting
equally through gyrase and topoisomerase IV would be desirable, as the
onset of resistance would require selection for two mutations, which
would be a rare event (24, 26). It is not known which if any
of the new antipneumococcal fluoroquinolones exhibits this property.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
70°C. Ciprofloxacin was provided by Bayer U.K., Newbury,
United Kingdom. Sparfloxacin was obtained from Dainippon Pharmaceutical
Co., Suita, Japan. MICs were determined by the twofold dilution method
with brain heart infusion medium supplemented with 10% horse blood. Approximately 105 CFU of bacteria was spotted on plates,
which were examined after overnight aerobic incubation at 37°C.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
TABLE 1.
Quinolone susceptibilities of S. pneumoniae
mutants
Stepwise selection of clinafloxacin-resistant S. pneumoniae mutants. Studies with characterized gyrA and parC mutants have provided evidence that clinafloxacin targets gyrase and topoisomerase IV with approximate parity in vivo but are insufficiently sensitive to distinguish whether one or the other target is favored. Were clinafloxacin to exert a target preference, then in a series of mutants selected in a stepwise manner, we should expect resistance mutations to appear first and invariably in that target. To examine this question, we generated S. pneumoniae 7785 mutants by stepwise challenge using increasing concentrations of clinafloxacin, an approach similar to that adopted in studies of other quinolones (26, 28) (Fig. 1).
|
1CLN1 to 1CLN5 and 1CLN6 to
1CLN8were chosen for analysis (Fig. 1). Strains 1CLN1 and 1CLN2 were
challenged independently with clinafloxacin at 0.5 µg/ml, yielding
second-step mutants 2CLN1 to 2CLN10 and 2CLN11 to 2CLN18, respectively
(Fig. 1). Third-step mutants 3CLN1 to 3CLN3, 3CLN4 to 3CLN6, and 3CLN7
to 3CLN10 were generated from strain 2CLN10 by exposure to
clinafloxacin at 1, 1.5, and 2 µg/ml, respectively. Finally,
fourth-step mutants were selected from strain 3CLN4 with clinafloxacin
at 6, 8, and 16 µg/ml (Fig. 1). The mutant frequencies were similar
for all steps of selection and were in the range of 5 × 1010 to 1.2 × 109 (Table
2). These frequencies were very low,
despite selection with clinafloxacin concentrations that were only one
to three times the MICs for the parent strains. By comparison, the
frequency of mutants selected with ciprofloxacin at similar multiples
of the ciprofloxacin MIC was higher by one to several orders of
magnitude (12, 26).
|
Clinafloxacin has a target preference for DNA gyrase in S. pneumoniae. Resistant strains were characterized for their drug susceptibilities, and the status of their gyrA, parC, gyrB, and parE QRDRs was determined by DNA sequence analysis of PCR products generated with genomic DNA as a template (see reference 26 for details). A rapid RFLP method was particularly useful in the initial screening of S. pneumoniae gyrA and parC PCR products for resistance mutations affecting codons for Ser-83 and Ser-79 (26).
For each of the first-step mutants 1CLN1 to 1CLN8, there was a twofold increase in the clinafloxacin MIC (Table 3). All eight mutants produced a 366-bp parC PCR product which retained the wild-type HinfI digestion pattern, producing 183-, 127-, and 56-bp fragments; this result indicated the likely absence of a mutation affecting ParC Ser-79. All of the mutants (except for 1CLN2 and 1CLN8) yielded a 382-bp gyrA PCR product which did not undergo cleavage at an internal HinfI site overlapping codon 83, suggesting that this codon carried a mutation. PCR products from 1CLN2 and 1CLN8 yielded the 110- and 272-bp fragments seen for the wild-type gyrA gene. By DNA sequence analysis of AsPCR products from the gyrA, parC, gyrB, and parE QRDRs, it could be shown that selected strains 1CLN1, 1CLN3, 1CLN6, and 1CLN7 carried an acquired mutation in gyrA resulting in a change of Ser-83 to Phe or Tyr at the protein level. Strain 1CLN2 carried a GyrA change of Asp-87 to Lys, and 1CLN8 carried a GyrB change of Glu-474 to Lys (Table 3). None of the strains carried parC mutations. These sequencing results are consistent with the RFLP analysis. As 1CLN1 to 1CLN8 were produced in two independent drug challenges, it would seem that the gyrA and gyrB mutations are consistently selected in the first step.
|
Growth properties of quinolone-resistant mutants. Given that gyrase and topoisomerase IV are essential bacterial enzymes, it was of interest to examine whether the growth of resistant mutants was impaired. We found that in the absence of drug, the growth on plates and morphology of fourth-step mutants 4CLN3 and 4CLN9 were indistinguishable from those of the wild type (data not shown). In a more detailed analysis, the generation times for 7785 and 4CLN9 in liquid cultures, determined from duplicate experiments, were 26.6 ± 2.8 and 28.6 ± 3.2 min, respectively (data not shown). Thus, although we cannot rule out the possibility that fourth-step mutants exhibit other phenotypic changes, e.g., altered pathogenicity, it is clear that the altered topoisomerases in these mutants do not have a marked effect on bacterial growth in the laboratory.
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
We have determined the target specificity of clinafloxacin in
S. pneumoniae by using defined gyrA and
parC strains and through analysis of mutants selected in a
stepwise manner. A key finding was that representative gyrA
or parC mutations did not affect the activity of
clinafloxacin (Table 1). The combined presence of the same mutations
led to a small but significant increase in the clinafloxacin MIC. These
results are consistent with the idea that clinafloxacin targets both
gyrase and topoisomerase IV in S. pneumoniae. In further
support of the dual-target hypothesis, it proved very difficult to
isolate clinafloxacin-resistant mutants by stepwise challenge (Tables 2
and 3). First-step mutants could be obtained at a frequency of 5 × 1010 only by challenge with clinafloxacin at 0.125 µg/ml, the MIC for the parent strain. These mutants exhibited an
increase in resistance of only less than or equal to twofold
(clinafloxacin MIC, 0.25 µg/ml); this finding was associated with the
acquisition of a gyrA or gyrB mutation,
suggesting a modest drug preference for gyrase. The subsequent
selection of second-, third-, and fourth-step mutants, again at low
frequencies and involving small incremental changes in clinafloxacin
MICs, was associated with alternate mutations in the topoisomerase IV
and gyrase genes (Table 3). The drug retained potency against first-
and second-step mutants; indeed, four topoisomerase IV mutations were
necessary to generate high-level resistance. These results indicate
that clinafloxacin is intrinsically highly active against S. pneumoniae and exhibits approximate parity in targeting gyrase and
topoisomerase IV in vivo.
Access to a panel of S. pneumoniae mutants expressing ParC and/or GyrA proteins with characterized quinolone resistance mutations proved extremely useful in assessing the activities of clinafloxacin and its target preferences. The responses of mutant strains to clinafloxacin could be rapidly examined and compared with parallel data obtained for sparfloxacin and ciprofloxacin, agents that target gyrase and topoisomerase IV, respectively (Table 1). Clinafloxacin was the most potent of the three agents tested against wild-type S. pneumoniae, parC, or gyrA mutants, and particularly the parC-gyrA double mutants (Table 1). For the parent strain and single mutants, the clinafloxacin MICs were 0.125 and 0.25 µg/ml, respectively. For the double mutants, the clinafloxacin MIC was increased fourfold to 1 µg/ml. However, the sparfloxacin and ciprofloxacin MICs for the double mutants each climbed steeply to 16 to 64 µg/ml, i.e., beyond levels attainable in serum or tissue (Table 1). Although we did not examine trovafloxacin, recent studies with a similar approach have measured trovafloxacin MICs of 0.5 and 6 µg/ml for representative parC and parC-gyrA S. pneumoniae mutants isolated as first-step and second-step mutants by challenge with ciprofloxacin, respectively (12). The fourfold increase in the trovafloxacin MIC for parC mutants indicates that trovafloxacin targets topoisomerase IV, and from human pharmacokinetic data, it appears that the drug retains clinically relevant activity against the first-step mutants (12). For clinafloxacin, pharmacokinetic studies have shown that for twice-daily oral doses of 200 and 400 mg, respective peak concentrations in human serum reached 2.75 and 5.22 µg/ml, with half-lives for elimination of 5.7 and 7.6 h (18, 31). Although further pharmacokinetic studies may be needed, the data presented in Table 1 indicate that clinafloxacin should retain potent clinical activity against gyrA, parC, and gyrA-parC mutants.
One limitation in using mutant strains to test drug activities is that
small preferences in drug targeting are not apparent. Thus, for both
gyrA and parC mutants of S. pneumoniae
there was a twofold increase in the clinafloxacin MIC (Table 1). These small changes are usually considered to be within experimental error
for the twofold dilution method used for MIC determinations. The
stepwise selection of mutants is a more sensitive approach for
determining target preferences. The development of high-level resistance to clinafloxacin required multiple steps with a defined and
invariant sequence of mutations of single residues in gyrase or
topoisomerase IV (Table 3). That first-step mutants displaying an
increase in the clinafloxacin MIC of less than or equal to twofold in
association with a mutation in either gyrA or
gyrB could be selected is consistent with dual targeting
with a modest drug preference for gyrase. Recent work with
trovafloxacin has shown that this agent selects resistant S. pneumoniae mutants at a frequency of ~108 when
used at the MIC. The MICs and QRDRs of the trovafloxacin-resistant mutants were not examined (12).
We found no evidence for clinafloxacin resistance mechanisms other than topoisomerase gene mutations. In contrast, selection with ciprofloxacin led to first-step mutants, such as 1C1, whose resistance was not attributable to topoisomerase changes (Table 1) (26). Interestingly, 1C1 was completely susceptible to clinafloxacin. We have speculated that 1C1 may be a permeation mutant whose resistance accrues from the upregulation of an efflux pump, akin to the NorA protein of S. aureus, for which ciprofloxacin is a substrate. Basal-level expression of NorA in S. aureus is involved in setting wild-type susceptibility to ciprofloxacin (38), and several ciprofloxacin-resistant S. aureus strains in which NorA is upregulated have been identified (16, 23). S. pneumoniae is known to express one or more efflux pumps (2), and our failure to isolate such mutants could suggest that clinafloxacin is not a substrate, a property that would contribute to its intrinsic activity. These issues require further study.
The mutations selected by clinafloxacin challenge were predominantly
those described previously for other quinolones (Tables 1 and 3)
(22). Thus, the gyrA mutations altered GyrA
Ser-83 (to Phe or Tyr) or Glu-87 (to Gln or Lys) (Table 3). The
parC mutations resulted in ParC Ser-79 (to Phe or Tyr) and
Asp-83 (to Ala) changes. These changes occurred at the expected GyrA
and ParC hot spots. In the crystal structure of an N-terminal 59-kDa fragment of the E. coli GyrA protein, the equivalent
residues lie in an 
helix that is adjacent to the catalytic Tyr-122
residues involved in DNA breakage and reunion (20). This
helix likely functions in DNA recognition and binding. It is not known
how the resistance mutations interfere with quinolone action, although this interference may arise from steric inhibition of drug binding (5, 37).
In contrast to the GyrA and ParC changes, the GyrB and ParE mutations
acquired in first-step strain 1CLN8 and fourth-step strain 4CLN3 are
novel. First, the Glu-474
Lys GyrB mutation does not occur in the
EGDSA and P(I/L)RGK motifs of GyrB, commonly implicated in resistance
and identified as the GyrB QRDR (40). Instead, the mutation
lies C terminal to the PLRGK motif. Previous studies of
quinolone-resistant Salmonella typhimurium identified a
Ser-463Lys alteration in GyrB located at a position similar to that
which is altered in GyrB of strain 1CLN8 (11). Moreover, an
Asn-470Asp mutation lying C terminal to the PLRGK motif has been
described for S. aureus ParE (8). Together, the
data suggest that the GyrB QRDR may occupy a more extensive region of
the protein than was previously defined by studies with E. coli (40). Second, the Pro-454Ser ParE mutation lies
within the PLRGK motif but is a novel change, as the Arg residue of
this sequence usually is mutated, at least in GyrB. Other studies with
S. pneumoniae have shown that first-step mutants resistant
to ciprofloxacin carry ParE mutations of Asp-435 to Asn in the EGDSA
motif (30). The functional role of the GyrB or ParE QRDR
region is unknown. The equivalent region in the crystal structure of a
fragment of Saccharomyces cerevisiae topoisomerase II lies
distant from the DNA binding domain, and it is not clear how mutations
in this region may affect drug susceptibility (4). Mutations
in the conserved PLRGK motif and DNA recognition helix A'4 of yeast topoisomerase II confer resistance to the anticancer agents amsacrine and doxorubicin, respectively (29, 36). Thus, there appear to be close similarities in the mechanisms of action of these topoisomerase-targeting antitumor agents and the antibacterial fluoroquinolones.
Interest in drugs that act on two or more targets stems in part from
the possibility of limiting the emergence of resistance. Were a drug to
act with equal potency through two targets, then simultaneous
acquisition of two mutations would be required for the development of
resistance. Given a typical frequency of 108 for single
mutations, the frequency of double mutations would be
1016, an extremely rare occurrence. It may be difficult
to obtain agents that act exactly equally through two topoisomerase
targets. However, comparable if unequal targeting is also advantageous. In this case, the level of resistance arising from mutation of the
preferred target will be severely limited by the appreciable contribution to susceptibility of the second target. The results of the
stepwise selection (Table 3) indicate that clinafloxacin acts in this
manner. To our knowledge, clinafloxacin is the first reported example
of a dual-targeting quinolone for S. pneumoniae and extends
our previous work indicating that quinolone structure determines target
preference in S. pneumoniae (28). Interestingly, recent studies with defined quinolone-resistant S. aureus
mutants suggest that sparfloxacin and DU6895a also act against both
gyrase and topoisomerase IV in S. aureus (8). In
the absence of data from mutants selected in a stepwise manner, it is
an open question whether these fluoroquinolones act equally or with
approximate parity on their two S. aureus targets. However,
the results suggest that dual-targeting quinolones may not be as rare
as might be imagined. It is notable that sparfloxacin targets gyrase in
S. pneumoniae but acts against both gyrase and topoisomerase
IV in S. aureus. Evidently, targeting depends not only on
drug structure but also on relative structural differences in
topoisomerases. It remains to be seen whether dual targeting for one
bacterial species can be retained for other pathogens.
Finally, the target preferences for clinafloxacin described in this paper indicate the relative importance of cell-killing pathways initiated through drug inhibition of gyrase or topoisomerase IV in S. pneumoniae. The molecular determinants favoring one or the other of these pathways are poorly understood. A C-8 substituent on the fluoroquinolone appears to favor cell killing through gyrase in S. pneumoniae. The gyrase-targeting agents sparfloxacin and clinafloxacin have fluorine and chlorine substituents at C-8, respectively. In contrast, neither ciprofloxacin nor trovafloxacin carries a C-8 substituent, and it is significant that both target topoisomerase IV in S. pneumoniae. We do not know whether these preferences arise from enhanced affinity for the target or enhanced lethality, as recently described for the 8-methoxy derivative of ciprofloxacin against E. coli gyrA mutants (42). Our working hypothesis is that the high potency of clinafloxacin against S. pneumoniae and its gyrA, parC, and gyrA-parC mutants arises from intrinsic tight binding of the drug to the target enzyme-DNA complexes, perhaps aided by poor efflux. Biochemical studies on clinafloxacin in progress in our laboratory should provide further information on the factors underlying its potent antipneumococcal activity.
| |
ACKNOWLEDGMENTS |
|---|
We thank Stephen J. Gracheck, Jing Li, and Michael A. Cohen for helpful comments.
This work was supported by a grant from Parke-Davis Co.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Molecular Genetics Group, Department of Biochemistry, St. George's Hospital Medical School, University of London, Cranmer Terrace, London SW17 ORE, United Kingdom. Phone: 44 181 725 5782. Fax: 44 181 725 2992. E-mail: lfisher{at}sghms.ac.uk.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
| 1. | Adams, D. E., E. M. Shekhtman, E. L. Zechiedrich, M. B. Schmid, and N. R. Cozzarelli. 1992. The role of topoisomerase IV in partitioning DNA replicons and the structure of catenated intermediates in DNA replication. Cell 71:277-288[Medline]. |
| 2. | Baranova, N. N., and A. A. Neyfakh. 1997. Apparent involvement of a multidrug transporter in the fluoroquinolone resistance of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 41:1396-1398[Abstract]. |
| 3. |
Bartlett, J. G., and L. M. Grundy.
1995.
Community-acquired pneumonia.
N. Engl. J. Med.
333:1618-1624 |
| 4. | Berger, J. M., S. J. Gamblin, S. C. Harrison, and J. C. Wang. 1996. Structure and mechanism of DNA topoisomerase IV. Nature (London) 379:225-232[Medline]. |
| 5. |
Cullen, M. E.,
A. W. Wyke,
R. Kuroda, and L. M. Fisher.
1989.
Cloning and characterization of a DNA gyrase A gene from Escherichia coli that confers clinical resistance to 4-quinolones.
Antimicrob. Agents Chemother.
33:886-894 |
| 6. | Drlica, K., and X. Zhao. 1997. DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol. Mol. Biol. Rev. 61:377-392[Abstract]. |
| 7. | Ferrero, L., B. Cameron, B. Manse, D. Lagneux, J. Crouzet, A. Famechon, and F. Blanche. 1994. Cloning and primary structure of Staphylococcus aureus DNA topoisomerase IV: a primary target for quinolones. Mol. Microbiol. 13:641-653[Medline]. |
| 8. |
Fournier, B., and D. C. Hooper.
1998.
Mutations in topoisomerase IV and DNA gyrase of Staphylococcus aureus: novel pleiotropic effects on quinolone and coumarin activity.
Antimicrob. Agents Chemother.
42:121-128 |
| 9. |
Gellert, M.,
K. Mizuuchi,
M. H. O'Dea,
T. Itoh, and J.-I. Tomizawa.
1977.
Nalidixic acid resistance: a second genetic character involved in DNA gyrase activity.
Proc. Natl. Acad. Sci. USA
74:4772-4776 |
| 10. |
Gellert, M.,
K. Mizuuchi,
M. H. O'Dea, and H. A. Nash.
1976.
DNA gyrase: an enzyme that introduces superhelical turns into DNA.
Proc. Natl. Acad. Sci. USA
73:3872-3876 |
| 11. | Gensberg, K., Y. F. Jin, and L. J. V. Piddock. 1995. A novel gyrB mutation in a fluoroquinolone-resistant clinical isolate of Salmonella typhimurium. FEMS Microbiol. Lett. 132:57-60[Medline]. |
| 12. | Gootz, T. D., R. Zaniewski, S. Haskell, B. Schmieder, J. Tankovic, D. Girard, P. Courvalin, and R. J. Polzer. 1996. Activity of the new fluoroquinolone trovafloxacin (CP-99,219) against DNA gyrase and topoisomerase IV mutants of Streptococcus pneumoniae selected in vitro. Antimicrob. Agents Chemother. 40:2691-2697[Abstract]. |
| 13. | Heisig, P. 1996. Genetic evidence for a role of parC mutations in development of high-level fluoroquinolone resistance in Escherichia coli. Antimicrob. Agents Chemother. 40:879-885[Abstract]. |
| 14. |
Hofmann, J.,
M. S. Cetron,
M. M. Farley,
W. S. Baughman,
R. R. Facklam,
J. A. Elliott,
K. A. Deaver, and R. F. Breiman.
1995.
The prevalence of drug-resistant Streptococcus pneumoniae in Atlanta.
N. Engl. J. Med.
333:481-486 |
| 15. | Janoir, C., V. Keller, M.-D. Kitzis, N. J. Moreau, and L. Gutmann. 1996. High-level fluoroquinolone resistance in Streptococcus pneumoniae requires mutations in parC and gyrA. Antimicrob. Agents Chemother. 40:2760-2764[Abstract]. |
| 16. | Kaatz, G. W., and S. M. Seo. 1995. Inducible NorA-mediated multidrug resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 39:2650-2655[Abstract]. |
| 17. | Kato, J., Y. Nishimura, R. Imamura, H. Niki, S. Higara, and H. Suzuki. 1990. New topoisomerase essential for chromosomal segregation in E. coli. Cell 63:393-404[Medline]. |
| 18. | Kucers, A., S. M. Crowe, M. L. Grayson, and J. F. Hoy (ed.). 1997. The use of antibiotics. A clinical review of antibacterial, antifungal and antiviral drugs, 5th ed., p. 1169-1171. Butterworth-Heinemann, Oxford, England. |
| 19. |
Mizuuchi, K.,
L. M. Fisher,
M. H. O'Dea, and M. Gellert.
1980.
DNA gyrase action involves the introduction of transient double strand breaks into DNA.
Proc. Natl. Acad. Sci. USA
77:1847-1851 |
| 20. | Morais-Cabral, J. H., A. P. Jackson, C. V. Smith, N. Shikotra, A. Maxwell, and R. C. Liddington. 1997. Crystal structure of the breakage-reunion domain of DNA gyrase. Nature 388:903-906[Medline]. |
| 21. | Munoz, R., and A. de la Campa. 1996. ParC subunit of DNA topoisomerase IV of Streptococcus pneumoniae is a primary target of quinolones and cooperates with DNA gyrase A subunit in forming resistance phenotype. Antimicrob. Agents Chemother. 40:2252-2257[Abstract]. |
| 22. | Nakamura, S. 1997. Mechanisms of quinolone resistance. J. Infect. Chemother. 3:128-138. |
| 22a. | National Committee for Clinical Laboratory Standards. 1998. Performance standards for antimicrobial testing; eighth informational supplement. NCCLS document M100-S8. National Committee for Clinical Laboratory Standards, Wayne, Pa. |
| 23. |
Ng, E. Y.,
M. Trucksis, and D. C. Hooper.
1994.
Quinolone resistance mediated by norA: physiologic characterization and relationship to flqB, a quinolone resistance locus on the Staphylococcus aureus chromosome.
Antimicrob. Agents Chemother.
38:1345-1355 |
| 24. | Ng, E. Y., M. Trucksis, and D. C. Hooper. 1996. Quinolone resistance mutations in topoisomerase IV: relationship to the flqA locus and genetic evidence that topoisomerase IV is the primary target and DNA gyrase is the secondary target of fluoroquinolones in Staphylococcus aureus. Antimicrob. Agents Chemother. 40:1881-1888[Abstract]. |
| 25. |
Pallares, R.,
J. Linares,
M. Vadillo,
C. Carbellos,
F. Manresa,
P. F. Viladrich,
R. Martin, and F. Gudiol.
1995.
Resistance to penicillin and cephalosporin and mortality from severe pneumococcal pneumonia in Barcelona, Spain.
N. Engl. J. Med.
333:474-480 |
| 26. | Pan, X.-S., J. Ambler, S. Mehtar, and L. M. Fisher. 1996. Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 40:2321-2326[Abstract]. |
| 27. |
Pan, X.-S., and L. M. Fisher.
1996.
Cloning and characterization of the parC and parE genes of Streptococcus pneumoniae encoding DNA topoisomerase IV: role in fluoroquinolone resistance.
J. Bacteriol.
178:4060-4069 |
| 28. | Pan, X.-S., and L. M. Fisher. 1997. Targeting of DNA gyrase in Streptococcus pneumoniae by sparfloxacin: selective targeting of gyrase or topoisomerase IV by quinolones. Antimicrob. Agents Chemother. 41:471-474[Abstract]. |
| 29. |
Patel, S.,
A. U. Sprung,
B. A. Keller,
V. J. Heaton, and L. M. Fisher.
1997.
Identification of yeast DNA topoisomerase II mutants resistant to the antitumor drug doxorubicin: implications for the mechanisms of doxorubicin action and cytotoxicity.
Mol. Pharmacol.
52:658-666 |
| 30. | Perichon, B., J. Tankovic, and P. Courvalin. 1997. Characterization of a mutation in the parE gene that confers fluoroquinolone resistance in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 41:1166-1167[Abstract]. |
| 31. | Randinitis, E. J. 1998. Personal communication. |
| 32. |
Sanger, F.,
S. Nicklen, and A. R. Coulson.
1977.
DNA sequencing with chain-terminating inhibitors.
Proc. Natl. Acad. Sci. USA
74:5463-5467 |
| 33. | Tankovic, J., B. Perichon, J. Duval, and P. Courvalin. 1996. Contribution of mutations in the gyrA and parC genes to fluoroquinolone resistance of mutants of Streptococcus pneumoniae obtained in vivo and in vitro. Antimicrob. Agents Chemother. 40:2502-2510. |
| 34. | Tomasz, A. 1995. The pneumococcus at the gates. N. Engl. J. Med. 333:314-315. |
| 35. | Wang, J. C. 1996. DNA topoisomerases. Annu. Rev. Biochem. 65:635-692[Medline]. |
| 36. |
Wasserman, R. A., and J. C. Wang.
1994.
Analysis of yeast DNA topoisomerase II mutants resistant to the antitumor drug amsacrine.
Cancer Res.
54:1795-1800 |
| 37. |
Willmott, C. J., and A. Maxwell.
1993.
A point mutation in the DNA gyrase A protein greatly reduces binding of fluoroquinolones to the gyrase-DNA complex.
Antimicrob. Agents Chemother.
37:126-127 |
| 38. | Yamada, H., S. Kurose-Hamada, Y. Fukuda, J. Mitsuyama, M. Takahata, S. Minami, Y. Watanabe, and H. Narita. 1997. Quinolone susceptibility of norA-disrupted Staphylococcus aureus. Antimicrob. Agents Chemother. 41:2308-2309[Abstract]. |
| 39. |
Yoshida, H.,
M. Bogaki,
M. Nakamura, and S. Nakamura.
1990.
Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli.
Antimicrob. Agents Chemother.
34:1271-1272 |
| 40. |
Yoshida, H.,
M. Bogaki,
M. Nakamura,
L. M. Yamanaka, and S. Nakamura.
1991.
Quinolone resistance-determining region in the DNA gyrase gyrB gene of Escherichia coli.
Antimicrob. Agents Chemother.
35:1647-1650 |
| 41. |
Zechiedrich, E. L., and N. R. Cozzarelli.
1995.
Roles of topoisomerase IV and DNA gyrase in DNA unlinking during replication in Escherichia coli.
Genes Dev.
9:2859-2869 |
| 42. |
Zhao, X.,
C. Xu,
J. Domagala, and K. Drlica.
1997.
DNA topoisomerase targets of the fluoroquinolones: a strategy for avoiding bacterial resistance.
Proc. Natl. Acad. Sci. USA
94:13991-13996 |
Antimicrobial Agents and Chemotherapy, November 1998, p. 2810-2816, Vol. 42, No. 11
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Pan, X.-S., Gould, K. A., Fisher, L. M.
(2009). Probing the Differential Interactions of Quinazolinedione PD 0305970 and Quinolones with Gyrase and Topoisomerase IV. Antimicrob. Agents Chemother.
53: 3822-3831
[Abstract] [Full Text] -
Cambau, E., Matrat, S., Pan, X.-S., Roth Dit Bettoni, R., Corbel, C., Aubry, A., Lascols, C., Driot, J.-Y., Fisher, L. M.
(2009). Target specificity of the new fluoroquinolone besifloxacin in Streptococcus pneumoniae, Staphylococcus aureus and Escherichia coli. J Antimicrob Chemother
63: 443-450
[Abstract] [Full Text] -
German, N., Malik, M., Rosen, J. D., Drlica, K., Kerns, R. J.
(2008). Use of Gyrase Resistance Mutants To Guide Selection of 8-Methoxy-Quinazoline-2,4-Diones. Antimicrob. Agents Chemother.
52: 3915-3921
[Abstract] [Full Text] -
Pan, X. S., Dias, M., Palumbo, M., Fisher, L. M.
(2008). Clerocidin selectively modifies the gyrase-DNA gate to induce irreversible and reversible DNA damage. Nucleic Acids Res
36: 5516-5529
[Abstract] [Full Text] -
Richter, S. N., Giaretta, G., Comuzzi, V., Leo, E., Mitchenall, L. A., Fisher, L. M., Maxwell, A., Palumbo, M.
(2007). Hot-spot consensus of fluoroquinolone-mediated DNA cleavage by Gram-negative and Gram-positive type II DNA topoisomerases. Nucleic Acids Res
35: 6075-6085
[Abstract] [Full Text] -
Escudero, J. A., San Millan, A., Catalan, A., de la Campa, A. G., Rivero, E., Lopez, G., Dominguez, L., Moreno, M. A., Gonzalez-Zorn, B.
(2007). First Characterization of Fluoroquinolone Resistance in Streptococcus suis. Antimicrob. Agents Chemother.
51: 777-782
[Abstract] [Full Text] -
Grossman, T. H., Bartels, D. J., Mullin, S., Gross, C. H., Parsons, J. D., Liao, Y., Grillot, A.-L., Stamos, D., Olson, E. R., Charifson, P. S., Mani, N.
(2007). Dual Targeting of GyrB and ParE by a Novel Aminobenzimidazole Class of Antibacterial Compounds. Antimicrob. Agents Chemother.
51: 657-666
[Abstract] [Full Text] -
Oyamada, Y., Ito, H., Inoue, M., Yamagishi, J.-i.
(2006). Topoisomerase mutations and efflux are associated with fluoroquinolone resistance in Enterococcus faecalis.. J Med Microbiol
55: 1395-1401
[Abstract] [Full Text] -
Oyamada, Y., Ito, H., Fujimoto, K., Asada, R., Niga, T., Okamoto, R., Inoue, M., Yamagishi, J.-i.
(2006). Combination of known and unknown mechanisms confers high-level resistance to fluoroquinolones in Enterococcus faecium. J Med Microbiol
55: 729-736
[Abstract] [Full Text] -
Richter, S. N., Leo, E., Giaretta, G., Gatto, B., Fisher, L. M., Palumbo, M.
(2006). Clerocidin interacts with the cleavage complex of Streptococcus pneumoniae topoisomerase IV to induce selective irreversible DNA damage. Nucleic Acids Res
34: 1982-1991
[Abstract] [Full Text] -
Strahilevitz, J., Robicsek, A., Hooper, D. C.
(2006). Role of the Extended {alpha}4 Domain of Staphylococcus aureus Gyrase A Protein in Determining Low Sensitivity to Quinolones. Antimicrob. Agents Chemother.
50: 600-606
[Abstract] [Full Text] -
Strahilevitz, J., Truong-Bolduc, Q. C., Hooper, D. C.
(2005). DX-619, a Novel Des-Fluoro(6) Quinolone Manifesting Low Frequency of Selection of Resistant Staphylococcus aureus Mutants: Quinolone Resistance beyond Modification of Type II Topoisomerases. Antimicrob. Agents Chemother.
49: 5051-5057
[Abstract] [Full Text] -
Galbraith, K. M., Ng, A. C., Eggers, B. J., Kuchel, C. R., Eggers, C. H., Samuels, D. S.
(2005). parC Mutations in Fluoroquinolone-Resistant Borrelia burgdorferi. Antimicrob. Agents Chemother.
49: 4354-4357
[Abstract] [Full Text] -
Lindgren, P. K., Marcusson, L. L., Sandvang, D., Frimodt-Moller, N., Hughes, D.
(2005). Biological Cost of Single and Multiple Norfloxacin Resistance Mutations in Escherichia coli Implicated in Urinary Tract Infections. Antimicrob. Agents Chemother.
49: 2343-2351
[Abstract] [Full Text] -
Strahilevitz, J., Hooper, D. C.
(2005). Dual Targeting of Topoisomerase IV and Gyrase To Reduce Mutant Selection: Direct Testing of the Paradigm by Using WCK-1734, a New Fluoroquinolone, and Ciprofloxacin. Antimicrob. Agents Chemother.
49: 1949-1956
[Abstract] [Full Text] -
Jimenez, M. R. M., Bellido, J. L. M., Garcia Rodriguez, J. A.
(2005). Risk Factors Associated with Colonization by Pneumococci with Reduced Susceptibility to Fluoroquinolones in Adult Outpatients. J. Clin. Microbiol.
43: 1193-1197
[Abstract] [Full Text] -
Grohs, P., Podglajen, I., Gutmann, L.
(2004). Activities of Different Fluoroquinolones against Bacillus anthracis Mutants Selected In Vitro and Harboring Topoisomerase Mutations. Antimicrob. Agents Chemother.
48: 3024-3027
[Abstract] [Full Text] -
Gould, K. A., Pan, X.-S., Kerns, R. J., Fisher, L. M.
(2004). Ciprofloxacin Dimers Target Gyrase in Streptococcus pneumoniae. Antimicrob. Agents Chemother.
48: 2108-2115
[Abstract] [Full Text] -
Bellon, S., Parsons, J. D., Wei, Y., Hayakawa, K., Swenson, L. L., Charifson, P. S., Lippke, J. A., Aldape, R., Gross, C. H.
(2004). Crystal Structures of Escherichia coli Topoisomerase IV ParE Subunit (24 and 43 Kilodaltons): a Single Residue Dictates Differences in Novobiocin Potency against Topoisomerase IV and DNA Gyrase. Antimicrob. Agents Chemother.
48: 1856-1864
[Abstract] [Full Text] -
Johnson, A. P., Sheppard, C. L., Harnett, S. J., Birtles, A., Harrison, T., Brenwald, N. P., Gill, M. J., Walker, R. A., Livermore, D. M., George, R. C.
(2003). Emergence of a fluoroquinolone-resistant strain of Streptococcus pneumoniae in England. J Antimicrob Chemother
52: 953-960
[Abstract] [Full Text] -
Grohs, P., Houssaye, S., Aubert, A., Gutmann, L., Varon, E.
(2003). In Vitro Activities of Garenoxacin (BMS-284756) against Streptococcus pneumoniae, Viridans Group Streptococci, and Enterococcus faecalis Compared to Those of Six Other Quinolones. Antimicrob. Agents Chemother.
47: 3542-3547
[Abstract] [Full Text] -
Sifaoui, F., Lamour, V., Varon, E., Moras, D., Gutmann, L.
(2003). ATP-Bound Conformation of Topoisomerase IV: a Possible Target for Quinolones in Streptococcus pneumoniae. J. Bacteriol.
185: 6137-6146
[Abstract] [Full Text] -
Komp Lindgren, P., Karlsson, A., Hughes, D.
(2003). Mutation Rate and Evolution of Fluoroquinolone Resistance in Escherichia coli Isolates from Patients with Urinary Tract Infections. Antimicrob. Agents Chemother.
47: 3222-3232
[Abstract] [Full Text] -
Nilius, A. M., Shen, L. L., Hensey-Rudloff, D., Almer, L. S., Beyer, J. M., Balli, D. J., Cai, Y., Flamm, R. K.
(2003). In Vitro Antibacterial Potency and Spectrum of ABT-492, a New Fluoroquinolone. Antimicrob. Agents Chemother.
47: 3260-3269
[Abstract] [Full Text] -
Fisher, L. M., Gould, K. A., Pan, X.-S., Patel, S., Heaton, V. J.
(2003). Analysis of dual active fluoroquinolones in Streptococcus pneumoniae. J Antimicrob Chemother
52: 312-313
[Full Text] -
Smith, H. J., Nichol, K. A., Hoban, D. J., Zhanel, G. G.
(2003). Reply. J Antimicrob Chemother
52: 313-314
[Full Text] -
Price, L. B., Vogler, A., Pearson, T., Busch, J. D., Schupp, J. M., Keim, P.
(2003). In Vitro Selection and Characterization of Bacillus anthracis Mutants with High-Level Resistance to Ciprofloxacin. Antimicrob. Agents Chemother.
47: 2362-2365
[Abstract] [Full Text] -
Drlica, K.
(2003). The mutant selection window and antimicrobial resistance. J Antimicrob Chemother
52: 11-17
[Abstract] [Full Text] -
Bui, M.-H., Stone, G. G., Nilius, A. M., Almer, L., Flamm, R. K.
(2003). PCR-Oligonucleotide Ligation Assay for Detection of Point Mutations Associated with Quinolone Resistance in Streptococcus pneumoniae. Antimicrob. Agents Chemother.
47: 1456-1459
[Abstract] [Full Text] -
Fisher, L. M., Heaton, V. J.
(2003). Dual activity of fluoroquinolones against Streptococcus pneumoniae. J Antimicrob Chemother
51: 463-464
[Full Text] -
Kishii, R., Takei, M., Fukuda, H., Hayashi, K., Hosaka, M.
(2003). Contribution of the 8-Methoxy Group to the Activity of Gatifloxacin against Type II Topoisomerases of Streptococcus pneumoniae. Antimicrob. Agents Chemother.
47: 77-81
[Abstract] [Full Text] -
Ince, D., Zhang, X., Silver, L. C., Hooper, D. C.
(2003). Topoisomerase Targeting with and Resistance to Gemifloxacin in Staphylococcus aureus. Antimicrob. Agents Chemother.
47: 274-282
[Abstract] [Full Text] -
Minnick, M. F., Wilson, Z. R., Smitherman, L. S., Samuels, D. S.
(2003). gyrA Mutations in Ciprofloxacin-Resistant Bartonella bacilliformis Strains Obtained In Vitro. Antimicrob. Agents Chemother.
47: 383-386
[Abstract] [Full Text] -
Ince, D., Zhang, X., Silver, L. C., Hooper, D. C.
(2002). Dual Targeting of DNA Gyrase and Topoisomerase IV: Target Interactions of Garenoxacin (BMS-284756, T-3811ME), a New Desfluoroquinolone. Antimicrob. Agents Chemother.
46: 3370-3380
[Abstract] [Full Text] -
Croisier, D., Chavanet, P., Lequeu, C., Ahanou, A., Nierlich, A., Neuwirth, C., Piroth, L., Duong, M., Buisson, M., Portier, H.
(2002). Efficacy and pharmacodynamics of simulated human-like treatment with levofloxacin on experimental pneumonia induced with penicillin-resistant pneumococci with various susceptibilities to fluoroquinolones. J Antimicrob Chemother
50: 349-360
[Abstract] [Full Text] -
Pan, X.-S., Hamlyn, P. J., Talens-Visconti, R., Alovero, F. L., Manzo, R. H., Fisher, L. M.
(2002). Small-Colony Mutants of Staphylococcus aureus Allow Selection of Gyrase-Mediated Resistance to Dual-Target Fluoroquinolones. Antimicrob. Agents Chemother.
46: 2498-2506
[Abstract] [Full Text] -
Weigel, L. M., Anderson, G. J., Tenover, F. C.
(2002). DNA Gyrase and Topoisomerase IV Mutations Associated with Fluoroquinolone Resistance in Proteus mirabilis. Antimicrob. Agents Chemother.
46: 2582-2587
[Abstract] [Full Text] -
Houssaye, S., Gutmann, L., Varon, E.
(2002). Topoisomerase Mutations Associated with In Vitro Selection of Resistance to Moxifloxacin in Streptococcus pneumoniae. Antimicrob. Agents Chemother.
46: 2712-2715
[Abstract] [Full Text] -
Yague, G., Morris, J. E., Pan, X.-S., Gould, K. A., Fisher, L. M.
(2002). Cleavable-Complex Formation by Wild-Type and Quinolone-Resistant Streptococcus pneumoniae Type II Topoisomerases Mediated by Gemifloxacin and Other Fluoroquinolones. Antimicrob. Agents Chemother.
46: 413-419
[Abstract] [Full Text] -
Morris, J. E., Pan, X.-S., Fisher, L. M.
(2002). Grepafloxacin, a Dimethyl Derivative of Ciprofloxacin, Acts Preferentially through Gyrase in Streptococcus pneumoniae: Role of the C-5 Group in Target Specificity. Antimicrob. Agents Chemother.
46: 582-585
[Abstract] [Full Text] -
Reinhardt, A. K., Bebear, C. M., Kobisch, M., Kempf, I., Gautier-Bouchardon, A. V.
(2002). Characterization of Mutations in DNA Gyrase and Topoisomerase IV Involved in Quinolone Resistance of Mycoplasma gallisepticum Mutants Obtained In Vitro. Antimicrob. Agents Chemother.
46: 590-593
[Abstract] [Full Text] -
Weigel, L. M., Anderson, G. J., Facklam, R. R., Tenover, F. C.
(2001). Genetic Analyses of Mutations Contributing to Fluoroquinolone Resistance in Clinical Isolates of Streptococcus pneumoniae. Antimicrob. Agents Chemother.
45: 3517-3523
[Abstract] [Full Text] -
Takei, M., Fukuda, H., Kishii, R., Hosaka, M.
(2001). Target Preference of 15 Quinolones against Staphylococcus aureus, Based on Antibacterial Activities and Target Inhibition. Antimicrob. Agents Chemother.
45: 3544-3547
[Abstract] [Full Text] -
Fass, R. J., Barnishan, J.
(2001). Comparison of antimicrobial in vitro activities against Streptococcus pneumoniae independent of MIC susceptibility breakpoints using MIC frequency distribution curves, scattergrams and linear regression analyses. J Antimicrob Chemother
48: 609-615
[Abstract] [Full Text] -
Pan, X.-S., Yague, G., Fisher, L. M.
(2001). Quinolone Resistance Mutations in Streptococcus pneumoniae GyrA and ParC Proteins: Mechanistic Insights into Quinolone Action from Enzymatic Analysis, Intracellular Levels, and Phenotypes of Wild-Type and Mutant Proteins. Antimicrob. Agents Chemother.
45: 3140-3147
[Abstract] [Full Text] -
Fluit, A. C., Visser, M. R., Schmitz, F.-J.
(2001). Molecular Detection of Antimicrobial Resistance. Clin. Microbiol. Rev.
14: 836-871
[Abstract] [Full Text] -
Ince, D., Hooper, D. C.
(2001). Mechanisms and Frequency of Resistance to Gatifloxacin in Comparison to AM-1121 and Ciprofloxacin in Staphylococcus aureus. Antimicrob. Agents Chemother.
45: 2755-2764
[Abstract] [Full Text] -
Hartman-Neumann, S., DenBleyker, K., Pelosi, L. A., Lawrence, L. E., Barrett, J. F., Dougherty, T. J.
(2001). Selection and Genetic Characterization of Streptococcus pneumoniae Mutants Resistant to the Des-F(6) Quinolone BMS-284756. Antimicrob. Agents Chemother.
45: 2865-2870
[Abstract] [Full Text] -
Nagai, K., Davies, T. A., Dewasse, B. E., Jacobs, M. R., Appelbaum, P. C.
(2001). Single- and multi-step resistance selection study of gemifloxacin compared with trovafloxacin, ciprofloxacin, gatifloxacin and moxifloxacin in Streptococcus pneumoniae. J Antimicrob Chemother
48: 365-374
[Abstract] [Full Text] -
Roychoudhury, S., Twinem, T. L., Makin, K. M., McIntosh, E. J., Ledoussal, B., Catrenich, C. E.
(2001). Activity of non-fluorinated quinolones (NFQs) against quinolone-resistant Escherichia coli and Streptococcus pneumoniae. J Antimicrob Chemother
48: 29-36
[Abstract] [Full Text] -
Millichap, J. J., Pestova, E., Siddiqui, F., Noskin, G. A., Peterson, L. R.
(2001). Fluoroquinolone Resistance Is a Poor Surrogate Marker for Type II Topoisomerase Mutations in Clinical Isolates of Streptococcus pneumoniae. J. Clin. Microbiol.
39: 2719-2721
[Abstract] [Full Text] -
Fukuda, H., Kishii, R., Takei, M., Hosaka, M.
(2001). Contributions of the 8-Methoxy Group of Gatifloxacin to Resistance Selectivity, Target Preference, and Antibacterial Activity against Streptococcus pneumoniae. Antimicrob. Agents Chemother.
45: 1649-1653
[Abstract] [Full Text] -
Roychoudhury, S., Catrenich, C. E., McIntosh, E. J., McKeever, H. D., Makin, K. M., Koenigs, P. M., Ledoussal, B.
(2001). Quinolone Resistance in Staphylococci: Activities of New Nonfluorinated Quinolones against Molecular Targets in Whole Cells and Clinical Isolates. Antimicrob. Agents Chemother.
45: 1115-1120
[Abstract] [Full Text] -
Boos, M., Mayer, S., Fischer, A., Köhrer, K., Scheuring, S., Heisig, P., Verhoef, J., Fluit, A. C., Schmitz, F.-J.
(2001). In Vitro Development of Resistance to Six Quinolones in Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus. Antimicrob. Agents Chemother.
45: 938-942
[Abstract] [Full Text] -
Blondeau, J. M., Zhao, X., Hansen, G., Drlica, K.
(2001). Mutant Prevention Concentrations of Fluoroquinolones for Clinical Isolates of Streptococcus pneumoniae. Antimicrob. Agents Chemother.
45: 433-438
[Abstract] [Full Text] -
Earnshaw, D. L., Pope, A. J.
(2001). FlashPlate Scintillation Proximity Assays for Characterization and Screening of DNA Polymerase, Primase, and Helicase Activities. J Biomol Screen
6: 39-46
[Abstract] -
Ince, D., Hooper, D. C.
(2000). Mechanisms and Frequency of Resistance to Premafloxacin in Staphylococcus aureus: Novel Mutations Suggest Novel Drug-Target Interactions. Antimicrob. Agents Chemother.
44: 3344-3350
[Abstract] [Full Text] -
Bast, D. J., Low, D. E., Duncan, C. L., Kilburn, L., Mandell, L. A., Davidson, R. J., de Azavedo, J. C. S.
(2000). Fluoroquinolone Resistance in Clinical Isolates of Streptococcus pneumoniae: Contributions of Type II Topoisomerase Mutations and Efflux to Levels of Resistance. Antimicrob. Agents Chemother.
44: 3049-3054
[Abstract] [Full Text] -
Heaton, V. J., Ambler, J. E., Fisher, L. M.
(2000). 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. Antimicrob. Agents Chemother.
44: 3112-3117
[Abstract] [Full Text] -
Yan, S. S., Fox, M. L., Holland, S. M., Stock, F., Gill, V. J., Fedorko, D. P.
(2000). Resistance to Multiple Fluoroquinolones in a Clinical Isolate of Streptococcus pyogenes: Identification of gyrA and parC and Specification of Point Mutations Associated with Resistance. Antimicrob. Agents Chemother.
44: 3196-3198
[Abstract] [Full Text] -
Jones, M. E., Staples, A. M., Critchley, I., Thornsberry, C., Heinze, P., Engler, H. D., Sahm, D. F.
(2000). Benchmarking the In Vitro Activities of Moxifloxacin and Comparator Agents against Recent Respiratory Isolates from 377 Medical Centers throughout the United States. Antimicrob. Agents Chemother.
44: 2645-2652
[Abstract] [Full Text] -
Bébéar, C. M., Grau, O., Charron, A., Renaudin, H., Gruson, D., Bébéar, C.
(2000). Cloning and Nucleotide Sequence of the DNA Gyrase (gyrA) Gene from Mycoplasma hominis and Characterization of Quinolone-Resistant Mutants Selected In Vitro with Trovafloxacin. Antimicrob. Agents Chemother.
44: 2719-2727
[Abstract] [Full Text] -
Nagai, K., Davies, T. A., Pankuch, G. A., Dewasse, B. E., Jacobs, M. R., Appelbaum, P. C.
(2000). In Vitro Selection of Resistance to Clinafloxacin, Ciprofloxacin, and Trovafloxacin in Streptococcus pneumoniae. Antimicrob. Agents Chemother.
44: 2740-2746
[Abstract] [Full Text] -
Sahm, D. F., Peterson, D. E., Critchley, I. A., Thornsberry, C.
(2000). Analysis of Ciprofloxacin Activity against Streptococcus pneumoniae after 10 Years of Use in the United States. Antimicrob. Agents Chemother.
44: 2521-2524
[Abstract] [Full Text] -
Fournier, B., Zhao, X., Lu, T., Drlica, K., Hooper, D. C.
(2000). Selective Targeting of Topoisomerase IV and DNA Gyrase in Staphylococcus aureus: Different Patterns of Quinolone- Induced Inhibition of DNA Synthesis. Antimicrob. Agents Chemother.
44: 2160-2165
[Abstract] [Full Text] -
Guerin, F., Varon, E., Hoï, A. B., Gutmann, L., Podglajen, I.
(2000). Fluoroquinolone Resistance Associated with Target Mutations and Active Efflux in Oropharyngeal Colonizing Isolates of Viridans Group Streptococci. Antimicrob. Agents Chemother.
44: 2197-2200
[Abstract] [Full Text] -
Thomson, K. S.
(2000). Minimizing quinolone resistance: are the new agents more or less likely to cause resistance?. J Antimicrob Chemother
45: 719-723
[Full Text] -
Kaneko, A., Sasaki, J., Shimadzu, M., Kanayama, A., Saika, T., Kobayashi, I.
(2000). Comparison of gyrA and parC mutations and resistance levels among fluoroquinolone-resistant isolates and laboratory-derived mutants of oral streptococci. J Antimicrob Chemother
45: 771-775
[Abstract] [Full Text] -
Alovero, F. L., Pan, X.-S., Morris, J. E., Manzo, R. H., Fisher, L. M.
(2000). Engineering the Specificity of Antibacterial Fluoroquinolones: Benzenesulfonamide Modifications at C-7 of Ciprofloxacin Change Its Primary Target in Streptococcus pneumoniae from Topoisomerase IV to Gyrase. Antimicrob. Agents Chemother.
44: 320-325
[Abstract] [Full Text] -
Jones, M. E., Sahm, D. F., Martin, N., Scheuring, S., Heisig, P., Thornsberry, C., Köhrer, K., Schmitz, F.-J.
(2000). Prevalence of gyrA, gyrB, parC, and parE Mutations in Clinical Isolates of Streptococcus pneumoniae with Decreased Susceptibilities to Different Fluoroquinolones and Originating from Worldwide Surveillance Studies during the 1997-1998 Respiratory Season. Antimicrob. Agents Chemother.
44: 462-466
[Abstract] [Full Text] -
Heaton, V. J., Goldsmith, C. E., Ambler, J. E., Fisher, L. M.
(1999). Activity of Gemifloxacin against Penicillin- and Ciprofloxacin-Resistant Streptococcus pneumoniae Displaying Topoisomerase- and Efflux-Mediated Resistance Mechanisms. Antimicrob. Agents Chemother.
43: 2998-3000
[Abstract] [Full Text] -
Morrissey, I., George, J.
(1999). Activities of Fluoroquinolones against Streptococcus pneumoniae Type II Topoisomerases Purified as Recombinant Proteins. Antimicrob. Agents Chemother.
43: 2579-2585
[Abstract] [Full Text] -
Earnshaw, D. L., Moore, K. J., Greenwood, C. J., Djaballah, H., Jurewicz, A. J., Murray, K. J., Pope, A. J.
(1999). Time-Resolved Fluorescence Energy Transfer DNA Helicase Assays for High Throughput Screening. J Biomol Screen
4: 239-248
[Abstract] -
Kenny, G. E., Young, P. A., Cartwright, F. D., Sjöström, K. E., Huang, W. M.
(1999). Ofloxacin Selects Gyrase Mutations in First-Step Mycoplasma hominis Mutants, whereas Sparfloxacin Selects Topoisomerase IV Mutations. Antimicrob. Agents Chemother.
43: 2493-2496
[Abstract] [Full Text] -
Brisse, S., Fluit, A. C., Wagner, U., Heisig, P., Milatovic, D., Verhoef, J., Scheuring, S., Köhrer, K., Schmitz, F.-J.
(1999). Association of Alterations in ParC and GyrA Proteins with Resistance of Clinical Isolates of Enterococcus faecium to Nine Different Fluoroquinolones. Antimicrob. Agents Chemother.
43: 2513-2516
[Abstract] [Full Text] -
Pan, X.-S., Fisher, L. M.
(1999). Streptococcus pneumoniae DNA Gyrase and Topoisomerase IV: Overexpression, Purification, and Differential Inhibition by Fluoroquinolones. Antimicrob. Agents Chemother.
43: 1129-1136
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»