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Antimicrobial Agents and Chemotherapy, December 2000, p. 3344-3350, Vol. 44, No. 12
Infectious Disease Division and Medical
Services, Massachusetts General Hospital, Harvard Medical School,
Boston, Massachusetts 02114-2696
Received 21 April 2000/Returned for modification 27 June
2000/Accepted 11 September 2000
Premafloxacin is a novel 8-methoxy fluoroquinolone with enhanced
activity against Staphylococcus aureus. We found
premafloxacin to be 32-fold more active than ciprofloxacin against
wild-type S. aureus. Single mutations in either subunit of
topoisomerase IV caused a four- to eightfold increase in the MICs of
both quinolones. A double mutation (gyrA and either
grlA or grlB) caused a 32-fold increase in the
MIC of premafloxacin, while the MIC of ciprofloxacin increased
128-fold. Premafloxacin appeared to be a poor substrate for NorA, with
NorA overexpression causing an increase of twofold or less in the MIC
of premafloxacin in comparison to a fourfold increase in the MIC of
ciprofloxacin. The frequency of selection of resistant mutants was
6.4 × 10 Infections due to highly resistant
Staphylococcus aureus are widespread, and studies to develop
novel antibiotics, including fluoroquinolones, against these organisms
are continuing. Premafloxacin is a novel fluoroquinolone initially
developed for veterinary use (29, 32). Differing from
ciprofloxacin, premafloxacin has a pyrrolidinyl substitute at position
7, which has been reported to increase activity against gram-positive
bacteria (4), and an 8-methoxy group, which has been
associated with increased bactericidal activity in some bacteria
(19, 33, 34). Its activity against gram-negative bacteria
has been reported to be equivalent to that of other quinolones, but its
activity against staphylococci has been shown to be 16- to 32-fold more
than that of ciprofloxacin (32). The interaction of
premafloxacin with its target enzymes and the mechanisms of resistance
of this potent fluoroquinolone, however, have not yet been determined.
Fluoroquinolones exert their bactericidal activity by interacting with
two type 2 topoisomerases, DNA gyrase (subunits encoded by the
gyrA and gyrB genes) and topoisomerase IV
(subunits encoded by the grlA and grlB genes for
S. aureus, and termed parC and parE
for other organisms). These drugs act as topoisomerase poisons, trapping the enzyme reaction intermediate at a step in which the enzyme
is bound to DNA and can be converted to a form in which both strands of
DNA are cleaved (5, 7, 12). Resistance to quinolones arises
from mutations in the primary enzyme target, with mutations in the
secondary target enzyme only contributing to resistance in the presence
of mutations in the primary target (13). Mutations reported
to date in genetically defined resistant mutants for all quinolones
cluster in a domain termed the quinolone resistance-determining region
(QRDR), which is thought to be the quinolone-binding domain. For
S. aureus, the primary target of most quinolones is
topoisomerase IV. In Streptococcus pneumoniae, however, the
primary target of sparfloxacin (22) and gatifloxacin (11) has been shown to be DNA gyrase, and clinafloxacin has been shown to target both enzymes similarly (23). A
quinolone with dual targets and low frequency of resistant mutant
selection in vitro is predicted to carry a lower risk of selection of
resistance in clinical settings.
In this study we evaluated the mechanism of action of and resistance to
premafloxacin. The effect of mutations in topoisomerase IV and DNA
gyrase in genetically defined mutants of S. aureus was
studied, and mutants selected with premafloxacin were characterized. Although topoisomerase IV was found to be the primary drug target, surprisingly, four novel grlA mutations, three of which were
outside the QRDR were identified, suggesting a novel mechanism of
interaction of premafloxacin with the DNA-enzyme complex.
Bacterial strains, plasmids, and growth conditions.
The
bacterial strains and plasmids used are shown in Table
1. Escherichia coli strains
were grown in Luria-Bertani medium, and S. aureus strains
were grown in brain heart infusion medium. All strains were grown at
37°C except the S. aureus strains carrying the
thermosensitive plasmids derived from pCL52.1, which were grown at
30°C. Spectinomycin was used at a concentration of 50 µg/ml, and
tetracycline was used at a concentration of 5 µg/ml except during
integration of the plasmid into the chromosome, when it was used at a
concentration of 3 µg/ml.
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Mechanisms and Frequency of Resistance to
Premafloxacin in Staphylococcus aureus: Novel Mutations
Suggest Novel Drug-Target Interactions
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
10 to 4.0 × 107 at
twofold the MIC of premafloxacin, 2 to 4 log10 less than
that with ciprofloxacin. Single-step mutants could not be selected at
higher concentrations of premafloxacin. In five single-step mutants,
only one previously described uncommon mutation (Ala116Glu), and four
novel mutations (Arg43Cys, Asp69Tyr, Ala176Thr, and Pro157Leu), three
of which were outside the quinolone resistance-determining region
(QRDR) were found. Genetic linkage studies, in which incross of
grlA+ and outcross of mutations were performed,
showed a high correlation between the mutations and the resistance
phenotypes, and allelic exchange experiments confirmed the role of the
novel mutations in grlA in resistance. Our results suggest
that although topoisomerase IV is the primary target of premafloxacin,
premafloxacin appears to interact with topoisomerase IV in a
manner different from that of other quinolones and that the range of
the QRDR of grlA should be expanded.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Strains and plasmids used in this study
Drug susceptibility determinations. Premafloxacin was kindly provided by Pharmacia & Upjohn Pharmaceuticals, and ciprofloxacin was provided by Bayer Corporation. Nalidixic acid, novobiocin, and ethidium bromide were purchased from Sigma Chemical Co. (St. Louis, Mo.). MICs were determined on Trypticase soy agar containing serial twofold dilutions of antibiotics. Incubations were done at 37°C, and growth was scored at 24 and 48 h. MICs of nalidixic acid were used to screen for gyrA mutations, MICs of novobiocin were used to screen for certain grlB mutations (10), and MICs of ethidium bromide were used to screen for NorA overexpression (14).
Frequency of selection of mutants. Mutants were selected by plating S. aureus ISP794 on brain heart infusion agar containing ciprofloxacin or premafloxacin at two-, four-, and eightfold above the MIC of each drug. Selection plates were incubated at 37°C. Mutation frequencies were calculated as the ratio of the number of resistant colonies at 48 h to the number of cells inoculated.
Limit levels of resistance with serial passage. S. aureus ISP794 was serially passaged on brain heart infusion agar containing twofold-increasing concentrations of premafloxacin to define the highest level of resistance achievable.
Sequence analysis. Chromosomal DNA from various single-step and multiple-step mutants of S. aureus ISP794 was used as a template. PCR for the QRDRs of grlA, grlB, gyrA, gyrB, and the promoter region of norA was performed using Vent DNA polymerase (New England Biolabs) (annealing temperature of 53°C for grlA and grlB and gyrA and gyrB and 55°C for the norA promoter) using primers and PCR conditions previously reported (10). DNA sequencing of the PCR products was performed with the ABI fluorescent system and Taq dye terminators (Qiagen). For strains in which no mutation was found in these regions, the entire grlA, grlB, gyrA, and gyrB genes were amplified by PCR and sequenced by the same methods.
DNA transformation.
For transformation in S. aureus, high-molecular-weight DNA was prepared by the method of
Stahl and Pattee (25), and cells were made competent for
transformation as described by Lindberg et al. (17), except
that the original inoculum was grown in Trypticase soy broth at 35°C
overnight in a shaking water bath. Bacteriophage 
55 was maintained
on strain ISP225 and was used to render the S. aureus
strains competent for transformation.
Correlations of resistance phenotype and genotype in genetic crosses. To correlate the resistance phenotype with specific mutations, 10 resistant and 10 susceptible transformants from crosses in which DNA from ISP2133 was used to transform mutants P4, P6, P10, and P21 were selected for PCR amplification and sequencing of the relevant regions of grlA or grlB. PCR primers used for amplification of grlA fragments encompassing the mutations in P4 and P21 were 5'-AGTTGAAGATGAAGTGCGTT-3' (5' nucleotide at position 2196 in the sequence published by Yamagishi et al. [31]) and 5'-ACCATTGGTTCGAGTGTCGT-3' (5' nucleotide at position 2839). For amplification of fragments containing mutations from strains P6 and P10, the primers were 5'-GCTGAAGAGTTATTACGTGA-3' (5' nucleotide at position 2757) and 5'-TGGACGACCATCACTAATAGCGA-3' (5' nucleotide at position 3389). The grlA (Ala176Thr) mutation in mutant P10 is marked by loss of the BstUI site, and the grlA (Asp69Tyr) mutation in mutant P21 is marked by loss of the HphI site (New England Biolabs) within the PCR fragments. For studies with these two mutants, restriction patterns were examined on agarose gels. For the other mutants, DNA sequencing of PCR products was performed to determine the presence and absence of mutations in resistant and susceptible transformants.
Cloning and allelic exchange.
Internal fragments (1,312 bp)
of grlA and grlB from mutants P4, P6, P10, and
P21 were amplified by PCR with the upstream primer containing an
engineered EcoRI site (5'-GAAGAATTCTGGGAAACGACG-3' [position 2140]) and the downstream primer containing an
engineered BamHI site (5'-GCAGGATCCTCAATTTGGTGATT-3'
[position 3451]), using an annealing temperature of 58°C and
an extension time of 1 min 25 s. PCR products were ligated into
the EcoRI and BamHI sites of pGEM3-zf(+), and the
recombinant plasmids were electroporated into E. coli
DH5
. The insert for each mutant was sequenced from the plasmid to
verify that no new mutation was introduced during PCR amplification.
After restriction and purification of the inserts from the pGEM3-zf(+)
by gel extraction, they were ligated to the EcoRI and
BamHI site of pCL52.1, a thermosensitive shuttle plasmid, and introduced into E. coli DH5 again by electroporation.
The resultant plasmids were first electroporated into S. aureus RN4220 and then to ISP794, as described previously
(2). One colony from the resultant strains was incubated at
42°C for 24 h in the presence of 3 µg of tetracycline per ml
of brain heart infusion broth to allow for integration of plasmid into
the chromosome via homologous recombination and then plated on brain
heart infusion agar containing the same concentration of tetracycline
and incubated at 42°C for 36 to 48 h to isolate single colonies
with the integrated plasmid (16). A single colony was then
incubated in brain heart infusion medium without antibiotic selection
for 48 h at 30°C to allow excision of the integrated plasmid
from the chromosome. Appropriate dilutions of the 48-h culture were
plated on medium without antibiotics to isolate single colonies, and
these colonies were screened for tetracycline susceptibility and
quinolone resistance. MICs of ciprofloxacin and premafloxacin for
tetracycline-susceptible, quinolone-resistant colonies were determined,
and the presence of the expected mutation was confirmed by sequencing
of PCR products for the appropriate region amplified from chromosomal DNA.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Quinolone activities against genetically defined mutants.
Table 2 shows the activities of
premafloxacin and ciprofloxacin against previously selected,
genetically defined mutants of S. aureus. Premafloxacin was
32-fold more active than ciprofloxacin against wild-type S. aureus ISP794 and a restriction-deficient mutant, RN4220. MICs of
both premafloxacin and ciprofloxacin increased four- to eightfold in
various grlA and grlB mutants of these two strains with altered subunits of topoisomerase IV. A single mutation in
gyrA of DNA gyrase had no effect on the MIC of ciprofloxacin and about a twofold increase in the MIC of premafloxacin that was
reproducible when an arithmetic series of drug concentrations was
tested. In double mutants with gyrA and either
grlA or grlB mutations, the MIC of premafloxacin
(4.0 µg/ml) increased 32-fold, while the MIC of ciprofloxacin
increased 128-fold. Overexpression of the NorA efflux pump in an
flqB mutant caused an increase of twofold or less in the MIC
of premafloxacin and a fourfold increase in the MIC of ciprofloxacin.
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Frequency of selection of mutants.
Table
3 shows the frequencies of selection of
single-step resistant mutants of wild-type strain ISP794 with
premafloxacin and ciprofloxacin. At twofold the MIC, the selection
frequency was 2 to 4 log10 lower with premafloxacin, and at
fourfold the MIC, mutants could not be selected with premafloxacin. In
contrast, mutants were readily selected at four- and eightfold the MIC
of ciprofloxacin.
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Limit resistance. By stepwise selection on increasing concentrations of ciprofloxacin, the limit level of resistance was 32 µg/ml, 256-fold higher than the MIC of ciprofloxacin for the wild-type strain. In contrast, the limit level of resistance for premafloxacin was 1.0 µg/ml, 128-fold higher than the MIC of premafloxacin for the parent wild-type strain.
Characterization of mutants selected with premafloxacin.
In
single-step mutants, MICs of ciprofloxacin and premafloxacin increased
four- to eightfold. MICs of novobiocin, nalidixic acid, and ethidium
bromide did not change or showed a twofold change. In the
serial-passage mutants, MICs of novobiocin decreased fourfold and MICs
of nalidixic acid and ethidium bromide did not change or decreased
twofold. Only one of the single-step mutants had a previously reported,
albeit uncommon, mutation (Ala116Glu). In four other single-step
mutants, novel mutations, three of which were outside the QRDR, were
found (Table 4). These mutations included
Arg43Cys, Asp69Tyr, Pro157Leu, and Ala176Thr. In two serial passage
mutants, each from a different experiment, commonly occurring mutations
in grlA (Ser80Phe) and gyrA (Ser84Leu) were found, and in a third one, one of the novel mutations in
grlA (Ala176Thr) was found together with two other
mutations, a common gyrA mutation (Ser84Leu) and a novel
mutation in the upstream region of the norA gene (insertion
of five nucleotides), which may account for the increase in the MIC of
ethidium bromide in this mutant.
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Genetic linkage of quinolone resistance loci.
To define the
relationship of these novel mutations to the quinolone resistance
phenotype of the mutants, we performed genetic crosses using
transformation with high-molecular-weight DNA. Tn917 transposon insertions,
(chr::Tn917lac)2 and
(chr::Tn917lac)1, encoding
erythromycin resistance (Ermr) have been previously shown
to be linked to grlA and grlB (21). For an incross experiment, high-molecular-weight chromosomal DNA from
strain ISP2133 (chr::Tn917lac)2
grlA+ grlB+ was used to transform
premafloxacin-selected mutants, selecting for the erythromycin
resistance of Tn917. Linkages similar to those previously
reported for established resistance mutations in grlA
(26) were found and were also shown to be similar to the
linkage to grlA (Ser80Phe) in parallel experiments (Table 5). To demonstrate that these mutant loci
were themselves sufficient to cause resistance, we performed outcross
experiments in which ISP794 (grlA+
grlB+) was transformed with high-molecular-weight
chromosomal DNA prepared from a resistant transformant obtained in the
incross experiments for each mutant. Table
6 shows the linkage results for each
premafloxacin-resistant mutant. In the outcross experiments, as in the
incross experiments, the linkages of the resistance loci to
(chr::Tn917lac)2 and
(chr::Tn917lac)1 were similar to
those reported for known grlBA resistance mutations (21).
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Correlation of the presence of specific mutations in outcross
transformants.
To correlate specific mutations with the resistance
phenotype in the transformants obtained in genetic crosses, we
determined the presence or absence of mutation-specific restriction
fragment length polymorphism patterns or the DNA sequences of the
relevant regions of grlA of selected transformants. The
mutation encoding Ala176Thr (TACACG) in P10 eliminates the
BstUI recognition site, CG
CG, and the mutation encoding
Asp69Tyr (GGTTAT) in P21 eliminates the HphI
recognition site, GGTGA(N)8 (mutations are shown in
boldface type, and vertical arrows show restriction enzyme cutting
site). PCR products of nucleotides 2757 to 3389 of grlA of
10 resistant outcross transformants of ISP794 transformed with
high-molecular-weight DNA from P10, and the P10 mutant all yielded only
one band of 633 bp following BstUI digestion, whereas 10 susceptible transformants all yielded two bands of 488 and 145 bp.
Similarly, PCR products of nucleotides 2196 to 2839 of grlA
of 10 resistant outcross transformants of ISP794 transformed with
high-molecular-weight DNA from P21 and the P21 mutant all yielded only
three bands of 416, 176, and 52 bp, in contrast to the four bands (257, 176, 159, and 52 bp) found with the 10 susceptible transformants.
Direct DNA sequencing of the PCR products of grlA from P4
and P6 revealed the respective presence and absence of the mutations in
10 resistant and 10 susceptible transformants from each outcross experiment.
Confirmation of the role of novel mutations in resistance by allelic exchange. Following integration of donor DNA containing the mutant sequences of grlBA into the chromosome of ISP794 using a thermosensitive shuttle plasmid in cells grown at nonpermissive temperature in the presence of tetracycline, cells were grown at permissive temperature in the absence of tetracycline to allow for plasmid excision. Tetracycline-susceptible cells (lacking the plasmid) were then screened for ciprofloxacin resistance. Tetracycline-susceptible, ciprofloxacin-resistant colonies in which allelic exchange is predicted were then tested for the presence of the expected mutation and for the MICs of ciprofloxacin and premafloxacin in relation to the original mutants. The MICs of ciprofloxacin (1.0 µg/ml) and premafloxacin (0.032 µg/ml) for the original and allele exchange mutants containing the GrlA Arg43Cys (P4 and DIP4), Ala176Thr (P10 and DIP10), and Asp69Tyr (P21 and DIP21) mutations were the same. Comparison of the original mutant P6, which had double mutations [GrlA (Pro157Leu), GyrA (Met538Ile)], with the allele exchange mutant DIP6, which had only the GrlA (Pro157Leu) mutation, revealed the same MIC of ciprofloxacin (1.0 µg/ml) and a slightly lower MIC of premafloxacin (twofold lower) for DIP6 (0.032 versus 0.064 µg/ml). Allelic exchange mutants DIP10, DIP21, DIP4, and DIP6 all contained the expected grlA mutations as shown by direct DNA sequencing of the PCR products of chromosomal DNA.
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DISCUSSION |
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Introduction
Materials and Methods
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Discussion
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We found that premafloxacin was highly potent against S. aureus, being 32-fold more active than ciprofloxacin against both wild-type and grlA mutant strains of S. aureus. A four- to eightfold increase in the MIC of premafloxacin was seen in defined grlA and grlB mutants, but a gyrA mutation had a slight but reproducible effect (approximately twofold) on the activity of premafloxacin in the absence of a grlA mutation. This phenomenon has been reported before with quinolone-resistant mutants of S. pneumoniae (27). These data, together with the identification of grlA mutations in premafloxacin-selected, first-step mutants indicate that topoisomerase IV is the primary target of premafloxacin in S. aureus. In the presence of a mutation in the primary target enzyme, the intrinsic sensitivity of the secondary enzyme may determine the susceptibility of the cell to the quinolone. The increase in the MIC of premafloxacin for strains with grlA gyrA dual mutations in comparison to a strain with a grlA mutation alone was 8-fold, in contrast to a 32-fold increase in the MIC of ciprofloxacin. These findings further suggested that the intrinsic activity of premafloxacin against gyrase was greater than that of ciprofloxacin. This higher intrinsic activity might contribute to the lower frequency of selection of resistant mutants. For other quinolones that, like premafloxacin, have a C-8 methoxy group, the frequency of selection of resistant mutants is also low (6). Thus, the presence of the 8-methoxy group in premafloxacin may contribute to its low frequency of mutant selection.
In addition, the overexpression of the NorA multidrug resistance efflux pump in one mutant (20) had little or no effect on the activity of premafloxacin. Thus, premafloxacin appears to be a poor substrate for NorA and possibly for related pumps, thereby limiting the range of the types of mutations that can contribute to resistance to premafloxacin. This presumption is also supported by our findings that no premafloxacin-selected mutants exhibited a substantial increase in the MIC of ethidium bromide, a common substrate of multidrug resistance pumps.
Clustering of mutations near the 5' ends of grlA and gyrA in S. aureus has led to the QRDRs being defined between residues 68 and 107 in GrlA and between residues 64 and 103 in GyrA (8). In genetic studies, mutations in three codons have been shown to cause quinolone resistance, Ser80Phe or -Tyr, Glu84Lys or -Leu, and Ala116Glu or -Pro (8, 21, 31). Mutations at codon 80 and 84 are more common than codon 116 mutations. Although a range of other mutations has been found in resistant clinical isolates, genetic studies to link the mutations to the resistance phenotype were often not done (13).
Strikingly, we did not select any of the common codon 80 and 84 mutations in grlA in our single-step mutants, and only one mutant had a previously described, but uncommon, Ala116Glu mutation. We have shown by genetic mapping that these novel mutations are indeed highly correlated with the resistance phenotype and by allelic exchange that all four mutations were definitively responsible for the resistance phenotype.
The X-ray crystallographic structure of the breakage-reunion domain of
gyrase in E. coli localizes the QRDR to a positively charged
surface that is predicted to bind the negatively charged phosphate
backbone of DNA and that bridges the two GyrA subunits (3).
The commonly occurring mutations map on -helix 4 of GyrA, and one
mutation (Ser83Trp) has been shown to cause reduced quinolone binding
to the gyrase-DNA complex (30).
The novel resistance mutation in codon 69 in mutant P21 maps to the
3 helix, the first helix in the helix-turn-helix motif, which
dominates the head dimer by packing side by side with the 3 helix of
the other subunit (3). Although no other mutation in this
domain has been reported in genetically defined mutants, the same
mutation (Asp69Tyr) has been reported in a resistant clinical isolate
of Mycoplasma hominis (13), and we have shown here by allelic exchange experiments that this mutation is responsible for resistance.
Mutant P6 carried a particularly unusual double mutation, Pro157Leu in
GrlA and Met538Ile in GyrA. The mutation in GrlA is localized to the
region between the 
5 strand and the 9 helix, which is not highly
conserved and seems to lie on a plane below that of the active site
Tyr. The role of the Met538Ile mutation in GyrA in the resistance
phenotype of this strain was initially uncertain. We were able to
outcross the quinolone resistance phenotype with the grlA
mutation from P6. Also allelic exchange experiments showed that
substitution of only the Pro157Leu mutation caused resistance. Thus,
the gyrA mutation does not itself appear to contribute to
resistance, nor does it appear to serve as a compensatory mutation
necessary for the presence of the grlA mutation.
Another novel mutation at position 176, aligns with the -strand 7 of
the grlA subunit. The crystal structure of the
breakage-reunion domain of DNA gyrase in E. coli has
revealed that isoleucine 174 (-strand 6 in E. coli and
Saccharomyces cerevisiae topoisomerase) and glycine 177 (-strand 7 in yeast topoisomerase II) are also highly conserved and
protein-DNA contacts are also made with the -strand 6 and 7 hairpin.
Although alanine 176 is not a conserved amino acid in E. coli and yeast topoisomerase, by genetic crosses and allelic
exchange we have shown that it is both necessary and sufficient for
quinolone resistance. Interestingly we have seen a similar mutation at
codon 176, Ala176Gly, in a mutant selected with gatifloxacin, a
quinolone which also has an 8-methoxy substitutent (D. Ince and D. C. Hooper, unpublished data).
We also report for the first time a novel resistance mutation at codon
43, which encodes a highly conserved arginine in E. coli
gyrase and yeast topoisomerase II, mapping to the 2 helix in the
helix-turn-helix motif of the catabolite activator protein-like domain
(3). In yeast topoisomerase II, Arg704, the counterpart of
Arg43 of S. aureus GrlA has been modified to alanine by
site-directed mutagenesis and shown to decrease substantially the
relaxation and cleavage activity of the enzyme (18). In
addition, in E. coli, Arg46, the counterpart of Arg43, has
been suggested to serve a role in anchoring the 3' end of the cleaved
DNA together with Arg47, which corresponds to Arg44 in S. aureus (3). Thus, the Arg43Cys mutation might be
predicted to have an effect on enzymatic function.
The molecular mechanisms of quinolone resistance for these novel mutations remain to be determined. These mutants did, however, exhibit low levels of coumarin hypersusceptibility (two- to fourfold for mutant P10 but twofold or less for other mutants), a phenotype reported with GrlB (Asn470Asp) and GrlA (Ala116Glu) mutants that had suggested reduced catalytic efficiency as contributing to resistance (9, 10, 28). In the case of topoisomerase IV reconstituted with the purified GrlB (Asp470) subunit, quinolone resistance and coumarin hypersusceptiblity as well as reduced catalytic efficiency have been demonstrated (X. Zhang and D. C. Hooper, unpublished observations). Thus, it is possible that these novel mutations in the grlA structural gene affect the catalytic activity and structure of topoisomerase IV such that the mutant enzymes form unstable or reduced levels of drug-enzyme-DNA complexes. This concept is supported by previous work in which a mutant yeast topoisomerase II that causes resistance to a quinolone derivative in vivo exhibited reduced stability and cellular levels despite the purified enzyme's having hypersensitivity to the same quinolone derivatives in vitro (24).
Although common mutations in grlA and gyrA (together with grlA) had an effect on the activity of premafloxacin, none of these mutations was selected directly by single-step plating on premafloxacin-containing agar. In contrast, premafloxacin-selected mutants were commonly found outside the conventional QRDR region of GrlA. These findings suggest that premafloxacin interacts with topoisomerase IV-DNA complexes in a manner different from that of many other quinolones, possibly generating unique contact points between the drug and the enzyme-DNA complex. Interestingly, two of three mutants serially passaged on premafloxacin contained common grlA and gyrA mutations, and one contained the novel grlA (Ala176Thr) mutation in addition to a common gyrA mutation. The reasons for the differences in mutational patterns seen with the single-step-selected and the serially passaged mutants is not clear but might be explained by more stringent requirements for fitness in order for mutants to persist under conditions of serial versus single platings on drug-containing agar. Thus, if, as we have suggested above, the novel mutants may have a fitness cost due to catalytic inefficiency of topoisomerase IV or otherwise relative to more commonly identified mutants, then the common mutants will be enriched in serial-passage experiments, which may mimic drug exposures in clinical settings. It is also noteworthy that serially passaged mutants are likely to accumulate multiple mutations (as illustrated by mutant PS3), some in as yet undefined genetic loci, contributing to resistance above that attributable to the identified mutations and some of which might compensate for fitness defects, as could be the case for the grlA (Ala176Thr) mutant identified after serial passage.
In summary, premafloxacin is a highly potent fluoroquinolone that is especially active against even multiply mutant strains of S. aureus. The primary target of premafloxacin in S. aureus is also topoisomerase IV, as is the case with many other quinolones. In five single-step mutants selected with premafloxacin, however, four had novel mutations, three of which were outside the QRDR region of GrlA. Thus, as further development of novel quinolones continues, studies of resistance will need to expand the range of the QRDR region of grlA and possibly gyrA as well. Identification of novel mutations may also further our understanding of drug target interaction and broaden the range of the molecular mechanisms of quinolone resistance due to target alteration.
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ACKNOWLEDGMENTS |
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We thank C. Y. Lee for providing plasmid pCL52.1.
This work was supported in part by a grant from Pharmacia & Upjohn Pharmaceuticals and a grant from the U.S. Public Health Service, National Institutes of Health (RO1 AI23988 to D.C.H.).
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FOOTNOTES |
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* Corresponding author. Mailing address: Infectious Disease Division, Massachusetts General Hospital, 55 Fruit St., Boston, MA 02114-2696. Phone: (617) 726-3812. Fax: (617) 726-7416. E-mail: dhooper{at}partners.org.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Antimicrobial Agents and Chemotherapy, December 2000, p. 3344-3350, Vol. 44, No. 12
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