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Antimicrobial Agents and Chemotherapy, November 2000, p. 3112-3117, Vol. 44, No. 11
Molecular Genetics Group, Department of
Biochemistry, St. George's Hospital Medical School, University of
London, London SW17 0RE,1 and SmithKline
Beecham Pharmaceuticals, Harlow, Essex CM19
5AW,2 United Kingdom
Received 15 May 2000/Returned for modification 31 July
2000/Accepted 25 August 2000
We investigated the roles of DNA gyrase and topoisomerase IV in
determining the susceptibility of Streptococcus pneumoniae to gemifloxacin, a novel fluoroquinolone which is under development as
an antipneumococcal drug. Gemifloxacin displayed potent activity against S. pneumoniae 7785 (MIC, 0.06 µg/ml) compared
with ciprofloxacin (MIC, 1 to 2 µg/ml). Complementary genetic and
biochemical approaches revealed the following. (i) The gemifloxacin
MICs for isogenic 7785 mutants bearing either parC or
gyrA quinolone resistance mutations were marginally higher
than wild type at 0.12 to 0.25 µg/ml, whereas the presence of both
mutations increased the MIC to 0.5 to 1 µg/ml. These data suggest
that both gyrase and topoisomerase IV contribute significantly as
gemifloxacin targets in vivo. (ii) Gemifloxacin selected first-step
gyrA mutants of S. pneumoniae 7785 (gemifloxacin MICs, 0.25 µg/ml) encoding Ser-81 to Phe or Tyr, or
Glu-85 to Lys mutations. These mutants were cross resistant to
sparfloxacin (which targets gyrase) but not to ciprofloxacin (which
targets topoisomerase IV). Second-step mutants (gemifloxacin MICs, 1 µg/ml) exhibited an alteration in parC resulting in
changes of ParC hot spot Ser-79 to Phe or Tyr. Thus, gyrase appears to be the preferential in vivo target. (iii) Gemifloxacin was at least 10- to 20-fold more effective than ciprofloxacin in stabilizing a cleavable
complex (the cytotoxic lesion) with either S. pneumoniae gyrase or topoisomerase IV enzyme in vitro. These data suggest that
gemifloxacin is an enhanced affinity fluoroquinolone that acts against
gyrase and topoisomerase IV in S. pneumoniae, with gyrase
the preferred in vivo target. The marked potency of gemifloxacin against wild type and quinolone-resistant mutants may accrue from greater stabilization of cleavable complexes with the target enzymes.
Gemifloxacin (SB-265805) is a new
fluoroquinolone which displays impressive activity against
Streptococcus pneumoniae (5, 24), the principal
cause of community-acquired pneumonia and a major player in meningitis,
otitis, sinusitis, and exacerbations of chronic bronchitis
(3). The drug is effective in vitro, not only against
penicillin-susceptible isolates of S. pneumoniae but also
against penicillin-resistant strains, which are now commonly encountered in the clinic (5, 9, 24). A tentative breakpoint for S. pneumoniae of 0.5 µg/ml has been proposed
(35). Recent work has shown that gemifloxacin also retains
activity against multidrug-resistant S. pneumoniae,
including strains resistant to ciprofloxacin (16), a
fluoroquinolone widely used in treating gram-negative infections but
which has borderline activity against pneumococci (31).
Although the incidence of quinolone-resistant pneumococci is presently
low (7), such strains could become important with increased
quinolone usage. The mechanism underlying the susceptibility of
ciprofloxacin-resistant strains to gemifloxacin is not known.
Fluoroquinolones act by inhibiting DNA gyrase and topoisomerase IV, two
enzymes that operate by a double-strand DNA break mechanism (17,
20) and that collaborate in ensuring DNA unwinding and strand
separation during DNA replication (1, 8, 10, 12, 17, 34,
36). Gyrase, an A2B2 tetramer encoded by
the gyrA and gyrB genes, catalyzes negative DNA
supercoiling during the initiation and elongation phases of DNA
replication (13). Topoisomerase IV is made up of two C and
two E subunits specified by the parC and parE
genes and is responsible for the segregation of daughter chromosomes at
cell division (19). Both gyrase and topoisomerase IV form a
ternary complex with quinolones and DNA (termed the "cleavable
complex"), which is converted into a lethal double-strand DNA break
by collision with replication forks or other mechanisms (8).
Resistance to quinolones occurs through stepwise acquisition of
chromosomal mutations in defined segments of the gyrase and
topoisomerase IV genes, termed the quinolone resistance-determining
regions (QRDRs) (23). Within the QRDRs, nucleotide hot spots
are mutated in quinolone-resistant strains leading to particular
changes at the protein level (6, 23), e.g., for S. pneumoniae, S81F or S81Y in GyrA; S79F or S79Y in ParC; E474K in
GyrB; and D435V in ParE (11, 15, 16, 18, 22, 25-28, 30,
33). Many of these mutations have been shown to confer quinolone
resistance when mutant QRDRs are used to transform susceptible S. pneumoniae strains (18, 22).
By establishing the order of topoisomerase QRDR mutations in
consecutive stepwise-selected drug-resistant mutants of S. pneumoniae, we were able to show that quinolones can be grouped
into three archetypal mechanistic classes (27, 28). The
first group, initially identified with ciprofloxacin and now including
levofloxacin, norfloxacin, pefloxacin, and trovafloxacin, selects QRDR
mutations in topoisomerase IV before those in gyrase, suggesting that
the drugs act preferentially through topoisomerase IV in vivo (11, 15, 18, 22, 25, 26, 30, 33). By contrast, a second group of drugs
comprising sparfloxacin, gatifloxacin, and NSFQ-105 [a ciprofloxacin
homologue bearing a 4-(4-aminophenylsulfonyl)-1-piperazinyl group at
C-7] select gyrase mutations before those in topoisomerase IV,
indicating that this class of quinolones acts through gyrase (2,
11, 27). Finally, clinafloxacin acts through both gyrase and
topoisomerase IV (28). Thus, although clinafloxacin selects gyrA or gyrB QRDR mutations in the first step,
these single-step mutants occur at low frequency and display only a
small (<twofold) increase in MIC over wild type, suggesting that both
gyrase and topoisomerase IV contribute substantially to drug action
(28). It appears that the structure of the quinolone defines
its mode of action in S. pneumoniae (2, 27).
In recent work, we and a colleague reported that gemifloxacin retained
activity against S. pneumoniae clinical isolates that were
highly resistant to ciprofloxacin and carried mutations in parC, gyrB, and parE genes
(16). Given the absence of gyrA mutations in
these strains, we speculated that gemifloxacin may act preferentially through gyrase in S. pneumoniae (16). To test
this idea, we have examined the response of an isogenic panel of
S. pneumoniae strains bearing defined quinolone resistance
mutations in topoisomerase genes. In complementary experiments, we have
also determined the order of acquisition of QRDR resistance mutations
in the topoisomerase genes of S. pneumoniae mutants selected
by stepwise challenge with gemifloxacin. Finally, we have investigated
the interaction of recombinant S. pneumoniae gyrase and
topoisomerase IV enzymes with gemifloxacin in vitro.
Bacterial strains.
S. pneumoniae 7785 is a
quinolone-susceptible clinical isolate (26) from which
drug-resistant mutant strains were derived by stepwise challenge.
Mutant strains developed by selection with ciprofloxacin (1C1, 2C6,
2C7, and 3C4) or sparfloxacin (1S1, 1S4, 2S1, and 2S4) have been
described previously (27).
Drug susceptibilities.
Gemifloxacin mesylate was supplied by
SmithKline Beecham Pharmaceuticals, Harlow, Essex, United Kingdom.
Gemifloxacin was dissolved in water at the appropriate concentration
immediately before use. Ciprofloxacin and sparfloxacin were kindly made
available by Bayer U.K., Newbury, United Kingdom, and Dainippon
Pharmaceutical Co., Suita, Japan, respectively. MICs were determined by
twofold agar dilution on brain heart infusion (BHI) plates containing 10% horse blood. Bacteria (104 to 105 CFU)
were spotted onto plates containing the appropriate concentration of
drug. Plates were incubated in air at 37°C for 18 to 24 h and were examined for growth.
Stepwise selection of gemifloxacin-resistant mutants.
Approximately 109 to 1010 CFU of S. pneumoniae strain 7785 (or its drug-resistant mutants) were plated
onto BHI plates containing 10% horse blood and various concentrations
of gemifloxacin. Plates were incubated aerobically for 24 to 48 h.
Colonies were restreaked on plates containing the same drug
concentration. Frequencies of mutant selection were calculated from the
ratio of colonies obtained on drug plates to the number obtained on
drug-free plates. All procedures were as described previously (25,
27).
PCR amplification of QRDRs and RFLP analysis.
Genomic DNA
was isolated from bacterial strains and used as a template in PCR
amplification of the QRDRs of the parC, parE, gyrA, and gyrB genes as previously described
(25). PCR primers used to amplify QRDRs for HinfI
analysis were VGA3 and VGA4 (for gyrA) and M0363 and M4721
(for parC). Primers used for PCR and for sequence analysis
were VGA4 and VGA9 (gyrA), M5884 and M4721 (parC), XS01 and M0361 (parE), and M4025 and
M4026 (gyrB). Primer sequences and PCR conditions have been
reported previously (16). Restriction fragment length
polymorphism (RFLP) analysis was performed as previously described
(27).
Asymmetric PCR and DNA sequencing.
Single-stranded DNA was
generated from QRDR PCR products by asymmetric PCR using the reported
primers and reaction conditions (16). The single-stranded
DNA was sequenced directly by the chain termination method using
Sequenase version 2.0 and appropriate primers (32).
DNA cleavage by S. pneumoniae topoisomerases.
Conditions for the purification and assay of recombinant S. pneumoniae gyrase and topoisomerase IV have been described
previously (29). DNA cleavage assays were carried out as
described earlier (29). The extent of DNA cleavage was
determined from photographic negatives using a Molecular Dynamics
personal densitometer SI and ImageQuant software.
Activity of gemifloxacin against isogenic S. pneumoniae
strains bearing defined quinolone-resistance mutations.
To gain an
understanding of which quinolone resistance mutations affect
susceptibility to gemifloxacin, we made use of quinolone-susceptible clinical isolate 7785 and its characterized mutants obtained by stepwise challenge with ciprofloxacin (strains 1C1, 2C6, 2C7, and 3C4)
or sparfloxacin (1S1, 1S4, 2S1, and 2S4) (Table
1). The susceptibilities of the parent
and mutant strains were determined for ciprofloxacin, sparfloxacin, and
gemifloxacin by twofold agar dilution; the MIC values determined here
for ciprofloxacin and sparfloxacin were identical to, or within one
dilution of, those reported previously (27). The parent
strain 7785 required MICs for ciprofloxacin and sparfloxacin of 1 to 2 and 0.5 µg/ml, respectively (Table 1). However, gemifloxacin was much
more potent, requiring a MIC of only 0.06 µg/ml. This value is in
agreement with previous results obtained for other clinical strains and
underlines the much greater activity of gemifloxacin against S. pneumoniae (16).
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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
and
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Fluoroquinolone susceptibilities and QRDR statuses of
isogenic mutants of S. pneumoniae strain 7785 selected with
ciprofloxacin, sparfloxacin, or gemifloxacin
Gemifloxacin-resistant S. pneumoniae mutants from stepwise challenge. As susceptibility studies on defined mutants proved uninformative about the primary intracellular target of gemifloxacin, we decided to generate and analyze S. pneumoniae strains selected by stepwise drug challenge. Identification of QRDR changes in first-step mutants indicates the preferred in vivo target. The choice of initial drug concentration was guided by the findings in Table 1.
Approximately 109 to 1010 CFU of S. pneumoniae strain 7785 was plated on BHI agar containing 10% horse blood and gemifloxacin at either 0.06 µg/ml (the MIC) or 0.09 µg/ml (1.5 × MIC). After 48 h of incubation in air at 37°C, mutant colonies appeared at each drug concentration. Colonies were restreaked on fresh plates containing the same (selecting) concentration of gemifloxacin. The frequency of first-step mutants selected at either drug concentration was approximately 10
9. Six of the first-step gemifloxacin mutants
1GM4,
1GM5, 1GM7, and 1GM9 (selected at 0.06 µg of drug/ml) and 1GM10 and
1GM11 (selected at 0.09 µg of drug/ml)were chosen for further
characterization (Fig. 1). For the
second-step selection, strain 1GM5 (gemifloxacin MIC, 0.25 µg/ml) was
challenged with drug at 0.25 µg/ml (1 × MIC) and 0.5 µg/ml
(2 × MIC), producing mutants 2GM1-16 and 2GM17-20, respectively
(Fig. 1). The mutant frequencies in both second-step selections were
approximately 109. Mutants 2GM1, 2GM2, 2GM17, and 2GM18
were characterized further.
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First-step mutants carry gyrA QRDR changes; second-step mutants have an additional parC QRDR mutation. All first-step mutants required MICs for gemifloxacin of 0.12 to 0.25 µg/ml, which represents a two- to fourfold increase over that of the wild-type strain 7785 (Table 1). Interestingly, with the exception of strain 1GM4, the other five first-step mutants showed only a small (<twofold) increase in MIC for ciprofloxacin compared with 7785, whereas their sparfloxacin MICs were 4- to 16-fold higher than that of strain 7785. By analogy with the data for ciprofloxacin- and sparfloxacin-selected mutants in Table 1, this result suggested that first-step mutants could have acquired changes in gyrA. Accordingly, the gyrA, parC, gyrB, and parE QRDRs were amplified from each strain by PCR prior to DNA sequence analysis (28). Strain 1GM4 required a ciprofloxacin MIC of 2 to 4 µg/ml and, like 1C1 (ciprofloxacin MIC, 3 µg/ml), did not have any QRDR mutations. To determine whether resistance in these mutants was due to an efflux mechanism, e.g., involving PmrA, their ciprofloxacin MICs and that of parental strain 7785 were determined in the presence of reserpine (7.5 µg/ml), a known efflux pump inhibitor (4, 14). Inclusion of reserpine reduced all the MICs to 0.5 to 1 µg/ml, which is consistent with the operation of an efflux mechanism in both 1C1 and 1GM4. The other five first-step mutants that were examined had acquired single-point changes in the gyrA QRDR specifying S81F, S81Y, or E85K, all of which are known quinolone resistance mutations. None of the first-step mutants had mutations in the parC, gyrB, or parE QRDRs (Table 1).
Second-step mutants 2GM1 to 2GM20 exhibited MICs of ciprofloxacin, sparfloxacin, and gemifloxacin of 64, 32, and 1 µg/ml, respectively. Based on results in Table 1, this phenotype is consistent with the presence of mutations in both gyrA and parC. RFLP analysis by HinfI digestion (28) was performed to detect mutations affecting codon 79 in parC. A 366-bp parC QRDR product was amplified by PCR from all 20 second-step gemifloxacin mutants. In each case, digestion with HinfI produced a 183-bp doublet, indicating mutational loss of a HinfI site encompassing codon 79 (data not shown). DNA sequence analysis of the four topoisomerase QRDRs of selected second-step mutants 2GM1, 2GM2, 2GM17, and 2GM18 (Table 1) confirmed the presence in all the strains of the gyrA (S81F) mutation derived from parent 1GM5. In addition, and in agreement with HinfI RFLP analysis, the strains had acquired a parC mutation encoding S79F or S79Y changes at the protein level. Thus, gemifloxacin selects gyrA and then parC mutations in S. pneumoniae.Gemifloxacin is more potent than ciprofloxacin in stabilizing
cleavable complexes of S. pneumoniae gyrase and
topoisomerase IV.
In principle, the increased antipneumococcal
activity of gemifloxacin could ensue from enhanced stabilization of
type II topoisomerase complexes on DNA. To test this possibility, we
compared the abilities of gemifloxacin and ciprofloxacin to stabilize
complexes of recombinant S. pneumoniae gyrase or
topoisomerase IV with DNA. Supercoiled plasmid pBR322 was incubated
with enzyme in the absence or presence of drug, and DNA cleavage was
effected by the addition of detergent. Samples were digested with
proteinase K, and DNA was analyzed by electrophoresis in 1% agarose
gels. The results are presented for gyrase and for topoisomerase IV in
Fig. 2A and B, respectively. For gyrase,
inclusion of either ciprofloxacin or gemifloxacin resulted in a
dose-dependent increase in the production of linear DNA generated by
disruption of the cleavable complex. There was no drug-dependent
increase in nicked DNA. Quantitative analysis of the bands allowed the
determination of CC25 values (the concentration of drug
that results in conversion of 25% of the substrate DNA to the linear
form under these experimental conditions). The CC25 values
for ciprofloxacin and gemifloxacin were 80 and 5 µM, respectively (Fig. 2A).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Gemifloxacin is a novel antibacterial fluoroquinolone with potent activity against S. pneumoniae. We examined its mechanism of action both in vivo, by using defined S. pneumoniae mutants and stepwise mutant selection, and in vitro, by using purified recombinant S. pneumoniae gyrase and topoisomerase IV proteins. Gemifloxacin selected gyrA QRDR mutants in the first step followed by parC changes in the second step, suggesting that gyrase is the preferred drug target in vivo (Table 1). However, given that S. pneumoniae mutants with defined mutations in parC showed a low-level increment in MIC similar to that of the first-step gyrA mutants (Table 1), it appears that the drug acts substantially through both gyrase and topoisomerase IV, i.e., shows some of the properties expected of a dual-targeting fluoroquinolone. Interestingly, topoisomerase IV was more effective than gyrase in inducing DNA cleavage mediated by gemifloxacin in vitro (Fig. 2). This is the first genetic and enzymatic analysis of gemifloxacin action in S. pneumoniae and has important implications.
One striking feature of the data presented here is the markedly greater activity of gemifloxacin compared with ciprofloxacin and sparfloxacin against wild-type S. pneumoniae and its quinolone-resistant mutants (Table 1). Thus, wild-type strain 7785 was at least 10- to 20-fold more susceptible to gemifloxacin than to the other two agents. The MIC of 0.06 µg/ml is similar to that reported (0.03 to 0.12 µg/ml) for a range of clinical isolates (16). Moreover, the gemifloxacin MICs for single gyrA or parC mutants were elevated only some two- to fourfold, to 0.12 to 0.25 µg/ml (Table 1). Even strains harboring mutations in both parC and gyrA (3C4, 2S1, and 2S4) showed MICs of gemifloxacin of 0.5 to 1 µg/ml (Table 1), some 32- to 64-fold lower than those of ciprofloxacin and sparfloxacin (Table 1).
The factors underlying the enhanced activity of gemifloxacin against S. pneumoniae are not fully understood. However, the DNA cleavage data suggest that the drug exhibits greater affinity for both gyrase and topoisomerase IV than ciprofloxacin in vitro. At least 10- to 20-fold higher levels of ciprofloxacin over gemifloxacin were required to produce comparable levels of DNA cleavage by either enzyme in vitro (Fig. 2). These results are consistent with the idea that enhanced cleavable complex formation is responsible for greater potency in vivo. Interestingly, similar to previous studies with other quinolones (21, 29), we have shown that gemifloxacin stimulates DNA cleavage by topoisomerase IV much more efficiently than by gyrase. Although this observation contrasts with the genetic work showing that both enzymes are targeted in vivo with a preference for gyrase, we assume that inside the bacterium, drug-enzyme affinity is only one aspect determining the killing pathway (29). Moreover, it is conceivable that the relative proficiencies of cleavable complex formation measured in vitro do not properly reflect those that hold under intracellular conditions.
Gemifloxacin is a new addition to those quinolones that select gyrA QRDR changes in first-step challenge of S. pneumoniae. These agents include sparfloxacin, gatifloxacin, and NSFQ-105 (2, 11, 27). Identification of gemifloxacin with these agents may aid structure-activity analysis of how quinolone structure influences in vivo targeting. In fact, the response of the panel of defined gyrA and/or parC mutants to gemifloxacin and the results of enzyme studies (Table 1 and Fig. 2) both show close similarity to data previously obtained for the dual-targeting agent clinafloxacin (28, 29). Thus, similar to gemifloxacin, neither gyrA nor parC mutations alone had much effect on clinafloxacin susceptibility: both mutations were needed to give significant resistance. Moreover, gemifloxacin and clinafloxacin were comparably active against parC-gyrA double mutants, with MICs of 0.5 to 1 µg/ml (28). In enzyme studies, the drugs show very similar CC25 values against gyrase and topoisomerase IV. Such highly potent agents may be especially valuable in attacking mutants selected by previous clinical use of other quinolones, e.g., ciprofloxacin or norfloxacin.
As an example of this approach, we and a colleague recently described two multidrug-resistant S. pneumoniae clinical isolates that were highly resistant to ciprofloxacin (MICs, 64 µg/ml) but remained susceptible to gemifloxacin (MICs, 0.12 µg/ml) (16). Both strains expressed S79F ParC, E474K GyrB, and D435V ParE proteins. Ciprofloxacin preferentially targets topoisomerase IV in S. pneumoniae, and the high-level resistance of these strains presumably arises from the S79F change in ParC, a known quinolone resistance mutation, and D435V, which affects a hot spot for quinolone resistance in the conserved EGDSA motif of ParE. The E474K mutation in GyrB was reported previously in a first-step S. pneumoniae mutant selected with clinafloxacin (28). The mutation lies outside the classical GyrB QRDR and had at most a twofold effect on susceptibility to ciprofloxacin. We hypothesized that the markedly superior activity of gemifloxacin over ciprofloxacin against these strains could be explained if gemifloxacin acts preferentially through gyrase in S. pneumoniae (16). The data presented here support this hypothesis (Table 1).
In summary, gemifloxacin displays some attractive advantages over current quinolones, such as ciprofloxacin, in terms of both its antipneumococcal mechanism and its greatly enhanced potency. These features suggest that the drug could have a promising role in the treatment of S. pneumoniae infections.
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ACKNOWLEDGMENTS |
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We thank Ken Coleman for helpful comments and Xiao-su Pan for purified enzymes.
This work was supported by a project grant from SmithKline Beecham.
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FOOTNOTES |
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* Corresponding author. Mailing address: Molecular Genetics Group, Department of Biochemistry, St. George's Hospital Medical School, University of London, Cranmer Terrace, London SW17 0RE, United Kingdom. Phone: 44 208 725 5782. Fax: 44 208 725 2992. E-mail: lfisher{at}sghms.ac.uk.
Present address: Bayer plc, Stoke Poges, Slough SL2 4LY, United Kingdom.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Antimicrobial Agents and Chemotherapy, November 2000, p. 3112-3117, Vol. 44, No. 11
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