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Antimicrobial Agents and Chemotherapy, August 2001, p. 2263-2268, Vol. 45, No. 8
New Product Research Laboratories I, Daiichi
Pharmaceutical Co., Ltd., Edogawa-ku, Tokyo
134-8630,1 and Department of Cell
Membrane Biology, Institute of Scientific and Industrial Research,
Osaka University, Ibaraki-shi, Osaka 567-0047,2
Japan
Received 27 December 2000/Returned for modification 12 March
2001/Accepted 8 May 2001
The major mechanism of resistance to fluoroquinolones for
Pseudomonas aeruginosa is the modification of type II
topoisomerases (DNA gyrase and topoisomerase IV). We examined the
mutations in quinolone-resistance-determining regions (QRDR) of
gyrA, gyrB, parC, and parE genes of recent
clinical isolates. There were 150 isolates with reduced
susceptibilities to levofloxacin and 127 with reduced susceptibilities
to ciprofloxacin among 513 isolates collected during 1998 and 1999 in
Japan. Sequencing results predicted replacement of an amino acid in the
QRDR of DNA gyrase (GyrA or GyrB) for 124 of the 150 strains (82.7%);
among these, 89 isolates possessed mutations in parC or
parE which lead to amino acid changes. Substitutions of
both Ile for Thr-83 in GyrA and Leu for Ser-87 in ParC were the
principal changes, being detected in 48 strains. These replacements
were obviously associated with reduced susceptibilities to
levofloxacin, ciprofloxacin, and sparfloxacin; however, sitafloxacin showed high activity against isolates with these replacements. We
purified GyrA (The-83 to Ile) and ParC (Ser-87 to Leu) by site-directed mutagenesis and compared the inhibitory activities of the
fluoroquinolones. Sitafloxacin showed the most potent inhibitory
activities against both altered topoisomerases among the
fluoroquinolones tested. These results indicated that, compared with
other available quinolones, sitafloxacin maintained higher activity
against recent clinical isolates with multiple mutations in
gyrA and parC, which can be explained by
the high inhibitory activities of sitafloxacin against both mutated enzymes.
Fluoroquinolones are often used in
therapy for various bacterial infections (8). The targets
of quinolones are considered to be the type II topoisomerases (DNA
gyrase and topoisomerase IV), which are essential enzymes responsible
for controlling the topological state of DNA during its replication and
transcription (17). DNA gyrase, a heterotetramer, is
composed of two A and B subunits, which are encoded by the
gyrA and gyrB genes, respectively. Topoisomerase
IV is homologous to DNA gyrase and is also composed of two subunits,
ParC and ParE, which are encoded by the parC and
parE genes, respectively (12).
Pseudomonas aeruginosa is a ubiquitous environmental
organism and is a major opportunistic pathogen causing human
infections. Fluoroquinolones have been widely used for the treatment of
P. aeruginosa infections in hospitals; however, P. aeruginosa is capable of acquiring resistance during antibiotic
therapy (8, 30). In P. aeruginosa, the
mechanisms of resistance to fluoroquinolones are known to be the
modification of DNA gyrase and topoisomerase IV, decreased permeability
of the cell wall, and multidrug efflux systems (4, 7).
Alterations in DNA gyrase or topoisomerase IV caused by mutations in
the so-called quinolone-resistance-determining region (QRDR)
(32) appear to play a major role in fluoroquinolone resistance in clinical isolates of P. aeruginosa (3,
10, 19). While many studies have focused on A subunits (GyrA and ParC) (3, 10, 13, 16, 20, 26, 31), less is known about
subunit B (gyrB and parE) in P. aeruginosa. Recently, Mouneimne et al. (19) reported
that a gyrB mutation in P. aeruginosa clinical strains, leading to the substitution of Phe for Ser-464, may be associated with fluoroquinolone resistance. There are, however, no
reports of a parE mutation in clinical isolates of P. aeruginosa that are resistant to fluoroquinolones. Thus, we
started to analyze mutations in the four genes encoding the target
enzymes using recent clinical isolates.
To characterize the prevalence of mutations in type II topoisomerase
genes and their impact on fluoroquinolone resistance, we analyzed the
QRDRs of the gyrA, gyrB, parC, and parE genes of
150 isolates with reduced susceptibility or resistance to levofloxacin from 513 P. aeruginosa isolates. Furthermore, we purified
mutant enzymes which were most frequently detected in clinical isolates and compared the inhibitory activities of fluoroquinolones against these purified enzymes.
Antibacterial agents and bacterial strains.
All
fluoroquinolones tested in this study were synthesized at New Product
Research Laboratories I, Daiichi Pharmaceutical Co., Ltd., Tokyo,
Japan. A total of 5,180 clinical isolates were obtained from 26 geographically distinct medical institutions in Japan in a study
undertaken by the Levofloxacin Surveillance Group during 1998 and 1999 (30). From these strains, 513 isolates of P. aeruginosa were obtained from individual patients with respiratory tract infections (RTI) or urinary tract infections (UTI) and 75.2% of
all of the P. aeruginosa isolates were susceptible to
ciprofloxacin (MIC, <2 µg/ml). To classify the strains according to
the breakpoint of the National Committee for Clinical Laboratory
Standards, 363 isolates (128 from UTI patients and 235 from RTI
patients) were susceptible to levofloxacin (MIC, Determination of MIC.
MICs were determined by the standard
agar dilution assay according to guidelines of the National Committee
for Clinical Laboratory Standards (21) with Mueller-Hinton
agar (Difco Laboratories, Detroit, Mich.). Drug-containing agar plates
were incubated with one loopful (5 µl) of an inoculum corresponding
to about 104 CFU per spot and were incubated at 35°C for
18 h. The MIC was defined as the lowest drug concentration that
prevented visible growth of bacteria.
PCR amplification and DNA sequence determination and
analysis.
Primers were designed to amplify the fragment including
the putative QRDR. For the QRDR of gyrA (GenBank accession
number L29417), the forward primer was
5'-AGTCCTATCTCGACTACGCGAT-3' (nucleotides [nt] 320 to 341)
and the reverse primer was 5'-AGTCGACGGTTTCCTTTTCCAG-3' (nt
676 to 697). These primers amplifed the fragment of P. aeruginosa gyrA from positions 421 to 630. Two primers,
5'-TGCGGTGGAACAGGAGATGGGCAAGTAC-3' (nt 1053 to 1080) and
5'-CTGGCGGAAGAAGAAGGTCAACAGCAGGGT-3' (nt 1534 to 1563), were
used to amplify the fragment of P. aeruginosa gyrB (GenBank
accession number AB00581) from positions 1213 to 1455. Primers for the
parC gene (GenBank accession number AB003428) were
5'-CGAGCAGGCCTATCTGAACTAT-3' (nt 214 to 235) and
5'-GAAGGACTTGGGATCGTCCGGA-3' (nt 496 to 517) and were used
to amplify the fragment from positions 350 to 481. Primers for the
parE gene (GenBank accession number AB003429) were
5'-CGGCGTTCGTCTCGGGCGTGGTGAAGGA-3' (nt 1223 to 1250) and
5'-TCGAGGGCGTAGTAGATGTCCTTGCCGA-3' (nt 1787 to 1814) and
were used to amplify the fragment from positions 1378 to 1620. The
amplification procedure comprised denaturation at 94°C for 3 min;
this was followed by 35 cycles of denaturation for 30 s at 94°C,
annealing for 30 s at 55°C, and polymerization for 1 min at 68 or 72°C. The reactions were performed in a final volume of 50 µl
with 2.5 U of LA Taq DNA polymerase (Takara Syuzo, Shiga, Japan). PCR-amplified DNA was directly sequenced by the dideoxy chain
termination method employing a Thermo Sequenase II dye terminator cycle
sequencing kit (Amersham Pharmacia Biotech, Piscataway, N.J.) according
to the manufacturer's protocol. The products were automatically
analyzed in a model 373A DNA autosequencer (Perkin-Elmer, Applied
Biosystems Division, Foster City, Calif.). The above-mentioned operations were performed together with Mitsubishi Kagaku Bio-Clinical Laboratories, Inc.
Site-directed mutagenesis.
Mutated gyrA and
parC genes prepared separately by site-directed mutagenesis
with Mutan-Super Express Km (Takara) were also used to construct
expression vectors. Two primers,
5'-GCACGGCGACATCGCGGTCTACGA-3' and
5'-ACGGCGACTTGGCCTGCTAC-3', were used to
introduce the Thr-83 Purification of the enzymes.
The altered GyrA (The-83 to
Ile) and ParC (Ser-87 to Leu) and wild-type GyrB and ParE were
overexpressed and separately purified by a fusion system with
maltose-binding proteins as described previously (1). The
fusion proteins were purified according to the manufacturer's protocol.
Inhibitory activities of quinolones against DNA gyrase and
topoisomerase IV.
Supercoiled pBR322 plasmid DNA purchased from
Boehringer Mannheim GmbH (Mannheim, Germany) was relaxed by
topoisomerase I (TopoGEN, Inc., Columbus, Ohio) before testing for the
supercoiling activity of DNA gyrase. The inhibitory activities of
fluoroquinolones against DNA gyrase and topoisomerase IV were assayed
electrophoretically as described previously (1). For the
supercoiling assay of DNA gyrase, the reaction mixture (20 µl),
containing subunits A and B (1 U each, which brought 50% of the pBR322
plasmid to the supercoiled form), drug solution, 20 mM Tris
hydrochloride (pH 7.5), 20 mM KCl, 4 mM MaCl2, 1 mM
spermidine hydrochloride, 1 mM ATP, 1 mM dithiothreitol, 20 µg of
bovine serum albumin per ml, and 0.2 µg of relaxed pBR322 plasmid
DNA, was incubated at 37°C for 1 h. The DNA in each band was
quantified, and the amount of supercoiled plasmid DNA treated with each
concentration of quinolone was measured to determine the 50%
inhibitory concentration (IC50) against DNA gyrase. The
IC50s against topoisomerase IV were determined as the drug
concentrations that reduced the decatenation activity seen with
drug-free controls by 50%.
The amino acid alterations found in GyrA, GyrB, ParC, and ParE
QRDRs of the 150 isolates that were intermediate and resistant to
levofloxacin are described in Table 1.
The isolates were classified into nine groups. Group I isolates
contained no mutation; group II isolates contained a mutation in
gyrB or parE only; group III isolates contained a
mutation in gyrA alone; group IV isolates contained
mutations in gyrA and gyrB, parC, or
parE; group V isolates contained mutations in
gyrA and parC; group VI isolates contained mutations in gyrA, gyrB, and parC; group VII
isolates contained two mutations in gyrA and one or no
mutation in parE; group VIII isolates contained two
mutations in gyrA and one mutation in parC; and
group IX isolates contained four mutations.
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.8.2263-2268.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Type II Topoisomerase Mutations in Fluoroquinolone-Resistant
Clinical Strains of Pseudomonas aeruginosa Isolated in
1998 and 1999: Role of Target Enzyme in Mechanism of
Fluoroquinolone Resistance
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
2 µg/m), 34 isolates (9 from UTI patients and 25 from RTI patients) were
intermediately resistant (MIC, 4 µg/m), and 116 isolates (82 from UTI
patients and 34 from RTI patients) were resistant (MIC, 
8 µg/m).
From these strains, 150 isolates with reduced susceptibilities to
levofloxacin were used in this study.
Ile and the Ser-87Leu mutations (underlined
codons), respectively. The mutant plasmids were confirmed by DNA sequencing.
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
TABLE 1.
Amino acid changes encoded by mutations in gyrA,
gyrB, parC, and parE
gyrA mutations.
An amino acid replacement(s) in
the QRDR of GyrA (Thr-83Ile or Ala, or Asp-87Asn, Gly, or Tyr)
was predicted for 119 of 150 isolates (79.3%). Substitution of Ile for
Thr-83 in GyrA was the principal replacement (112 of 150 isolates;
74.7%), while other substitutions were rare. This result was in
accordance with previous reports on clinical isolates of P. aeruginosa (10, 19, 20, 26, 31). Twenty isolates
possessed double point mutations in gyrA; among these, we
predicted the replacements of Thr-83 and Asp-87 in 16 isolates. A novel
mutation predicting the alteration of Glu-54 to Lys was found in three
of the isolates with a Thr-83Ile replacement. The change of
negatively charged Glu to positively charged Lys may participate in the
quinolone-gyrase interaction and be responsible for quinolone
resistance. However, the detailed role of this substitution will be
clarified by further study, such as by a site-directed mutagenesis
study. An Ala-67-to-Ser substitution found in only one isolate has
been previously described for quinolone-resistant strains of P. aeruginosa (26) and Escherichia coli
(34). No strains intermediately resistant to levofloxacin (for which MICs were 4 µg/ml) possessed gyrA double point
mutations. Three types of silent mutations were observed at codons 68 (CGT to CGA; two strains), 79 (CCG to CCA; two strains), and 103 (GTA to GTG; two strains).
gyrB mutations. In GyrB, we found changes of Gln-459 to Arg in 1 isolate, Ser-468 to Tyr in 1 isolate and to Phe in 3 isolates, Gln-469 to Val in 1 isolate, Glu-470 to Asp in 13 isolates, Thr-473 to Met in 1 isolate, and Ala-477 to Val in 7 isolates. None of the isolates, however, had mutations at codon Asp-430, which corresponds to Asp-426 in E. coli GyrB, or Lys-451, which corresponds to Lys-447 in E. coli GyrB, where mutations were responsible for quinolone resistance in E. coli (29). Mouneimne et al. (19) recently reported the absence of mutations in codons 430 and 451 and the substitution of Phe for Ser at amino acid position 468 (reported as Ser-464 to Phe) in P. aeruginosa. The same mutation was found in three isolates with reduced susceptibilities to levofloxacin. We found that, for another novel replacement, Ser-468 to Tyr, a corresponding alteration in quinolone-resistant strains of Salmonella enterica serovar Typhimurium, Ser-464 to Tyr has been described (6). Furthermore, silent mutations were found at codons 410 (AAA to AAG, 43 isolates), 421 (CTC to CTT, 2 isolates), 440 (CGC to CGT, 11 isolates), 448 (CTG to TTG, 1 isolate), 458 (GAA to GAG, 42 isolates), 460 (GCG to GCA, 33 isolates), 476 (ACC to ACT, 2 isolates), 483 (GGC to GGT, 2 isolates), and 487 (TCC to TCT, 1 isolate).
parC mutations.
The amino acid sequences in the
QRDR of ParC showed a high frequency of replacement of Ser-87
(equivalent to Ser-80 in E. coli ParC) to Leu (75 isolates)
or Trp (9 isolates), while other replacements were rare. It is notable
that all of the isolates with ParC alterations had a alteration in
GyrA. Thus, we confirmed that alterations in ParC occurred at a second
step in strains already having a single alteration in GyrA in P. aeruginosa. More noteworthy is that strains with two alterations
(Thr-83Ile in GyrA plus Ser-87Leu in ParC) were most frequently
identified in clinical isolates (48 isolates). Strains with this
predominant alteration, in particular, may become clinically and
epidemiologically important. In the parC gene of P. aeruginosa, we identified the first double mutations, which led to
the following amino acid changes: Ser-87 to Leu plus Glu-91 to Lys and
Ser-87 to Leu plus Ala-88 to Pro. Also, a single alteration (Glu-91 to
Lys) has been described for clinical isolates (19, 20) and
a single alteration similar to Glu-91 to Lys has been described to
occur in quinolone-resistant strains of E. coli GyrA: Ala-84
to Pro (32). Two isolates contained a silent mutation at
codon 106 (CTG to TTG), which does not lead to an amino acid substitution.
parE mutations. We identified a novel mutation in the parE gene of P. aeruginosa, which changed Asp-419 to Asn, located in the EGDSA motif, which is highly conserved in type II topoisomerase B subunits (28). An alteration in this motif has been implicated in a fluoroquinolone-resistant E. coli GyrB (33). Also, similar replacements have been described for quinolone-resistant strains of Streptococcus pneumoniae ParE (Asp-435 to Asn [11, 22]) and Staphylococcus aureus GrlB (ParE) (Ala-432 to Asn [27]). In S. aureus, our previous site-directed mutagenesis study (27) showed that the change of Asp to Asn at position 432 in GrlB (ParE) was responsible for a low level of quinolone resistance. Therefore, we assumed that the change of Asp to Asn at position 419 is responsible for quinolone resistance in P. aeruginosa. Other mutations leading to amino acid changes were found at codons 425 (Ala to Val, 3 isolates), 459 (Glu to Val, 3 isolates; Glu to Lys, 1 isolate), and 473 (Ala to Val, 2 isolates). However, these mutations have not been reported for other bacteria. Of special note, the change of Ala-473 to Val, which was found in the strains intermediately resistant to levofloxacin (MIC, 4 µg/ml), was located away from the QRDR. Thus, we speculated that such replacements are clinically rare or not obviously associated with fluoroquinolone resistance. None of the isolates had a replacement at codon 444, where a replacement (Leu-445 to His) was described for E. coli ParE (2), but a silent mutation was found (CTG to TTG, 1 isolate). The other silent mutations in the QRDR were found at codons 397 (CCC to CCT, 1 isolate), 401 (GCC to GCT, 2 isolates), 432 (GAA to GAG, 4 isolates), 445 (AAC to AAT, 2 isolates), 448 (GAA to GAG, 12 isolates), 451 (GGC to GGT, 2 isolates), 455 (CTC to CTT, 6 isolates), 465 (GTG to GTA, 40 isolates), 472 (GGT to GGC, 57 isolates), and 474 (AGT to AGC, 59 isolates). The mutations in gyrB or parE are also rare in other clinical isolates (23). In P. aeruginosa, it is confirmed that mutations in type II topoisomerase subunit B are rare.
Relation to MIC.
The MIC distributions of each fluoroquinolone
tested are shown in Table 2. Overall,
sitafloxacin MICs for the quinolone-resistant P. aeruginosa
isolates were lower than the MICs of the other fluoroquinolones tested.
The distributions of the MICs for the isolates possessing no alteration
were slightly different from those for the isolates possessing a
replacement in GyrB or ParE alone (Table 2; groups I and II).
Therefore, no gyrB or parE single mutation that
conferred significantly reduced susceptibility to any of the
fluoroquinolones was found. In contrast, susceptibility was noticeably
increased when the MICs for isolates with a mutation in gyrA
(Table 2; group III) are considered. The addition of a parC
mutation (Ser-87Leu) to the single mutation in gyrA
(Thr-83Ile) appeared to have significant effects on the MICs of
ciprofloxacin and sparfloxacin, while it had a slight influence on the
activity of sitafloxacin (Table 2; groups III and V). Moreover, the
susceptibilities of these isolates to all fluoroquinolones tested were
reduced even more by the addition of a second gyrA mutation
(Asp-87Asn, Gly, or Tyr) (Table 2; group VIII). Thus, the
alterations in GyrA (Thr-83Ile and Asp-87Asn, Gly, or Tyr) and
ParC (Ser87Leu or Trp) seem to play significant roles in
fluoroquinolone resistance in clinical P. aeruginosa
isolates.
|
16 µg/ml) had substitutions in the QRDRs of type
II topoisomerases. The rate of intermediate resistance to levofloxacin
(MIC, 4 µg/ml) in isolates with no amino acid replacement was high
(22 of 26 isolates; 84.6%). These results emphasized that the main
mechanisms of high-level fluoroquinolone resistance in clinical strains
of P. aeruginosa are replacements in DNA gyrase or
topoisomerase IV.
For many isolates with a significant replacement(s), the MICs of
fluoroquinolones showed a wide range. Multiple efflux pump systems,
namely, MexAB-OprM (25), MexCD-OprJ (24),
MexEF-OprN (14), and MexXY-OprM (18), have
been identified in P. aeruginosa. Overexpression of these
pumps has been described for clinical fluoroquinolone-resistant
isolates (5, 9, 10, 15). It was likely that some of the
isolates had various levels of expression of these pumps or had another
unknown mutation that confers resistance to fluoroquinolones. It was
interesting that a range of MICs of sitafloxacin for isolates with the
same replacement in group III was slightly shorter than those of the
other fluoroquinolones tested. It suggested that the other mutational
mechanisms may have somewhat less of an effect on sitafloxacin than on
the other fluoroquinolones tested. The detailed role of the efflux pump or the other mutation, however, will be clarified by further study of
clinical isolates of P. aeruginosa.
Comparison of inhibitory activities of fluoroquinolones against
type II topoisomerases.
In this study, alterations of the
Thr-83Ile type in GyrA and the Ser-87Leu type in ParC were the
principal alterations in clinical isolates of P. aeruginosa
with decreased susceptibilities to fluoroquinolones. Thus, to identify
the amino acid changes conferring fluoroquinolone resistance, we
compared the inhibitory activities of fluoroquinolones against the
purified wild-type and the altered P. aeruginosa type II
topoisomerases. Site-directed mutagenesis was used to obtain enzymes
containing these replacements. The inhibitory activities of
fluoroquinolones against these enzymes are shown in Table
3. The replacement of Thr-83 by Ile in
GyrA induced 12-, 4-, 15-, and 23-fold increases and the replacement of
Ser-87 by Leu in ParC induced 10-, 4-, 8-, and 9-fold increases in the
IC50s of levofloxacin, sitafloxacin, ciprofloxacin, and sparfloxacin, respectively. These results clearly emphasize the key
role of Thr at position 83 in DNA gyrase and the corresponding site at
position 87 in topoisomerase IV for determining fluoroquinolone resistance. Our previous study (13) showed that
sitafloxacin had high inhibitory activity against various purified
gyrases from fluoroquinolone-resistant clinical isolates of P. aeruginosa collected in 1990. Furthermore, we demonstrated that
sitafloxacin maintained high inhibitory activities against recent
clinical P. aeruginosa isolates and had the highest
inhibitory activities against both wild-type and altered target enzymes
among the fluoroquinolones tested.
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ACKNOWLEDGMENTS |
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We thank I. Kobayashi, A. Kanayama, M. Shimazu, and K. Matsuda of Mitsubishi-kagaku BCL, Inc., for DNA sequencing analysis. We also thank E. Yamazaki, S. Ueha, and K. Yoshihara for technical assistance during this work.
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FOOTNOTES |
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* Corresponding author. Mailing address: New Product Research Laboratories I, Daiichi Pharmaceutical Co., Ltd., 16-13, Kitakasai 1-Chome, Edogawa-ku, Tokyo 134-8630, Japan. Phone: 81-3-3680-0151. Fax: 81-3-5696-4264. E-mail: akasa94k{at}daiichipharm.co.jp.
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Abstract
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Antimicrobial Agents and Chemotherapy, August 2001, p. 2263-2268, Vol. 45, No. 8
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.8.2263-2268.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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