Abstract
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.
MATERIALS AND METHODS
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, ≤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.
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→Ile and the Ser-87→Leu mutations (underlined codons), respectively. The mutant plasmids were confirmed by DNA sequencing.
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%.
RESULTS AND DISCUSSION
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.
TABLE 1.
Amino acid changes encoded by mutations in gyrA, gyrB, parC, and parE
| Group | No. of strainsa | Replacement in QRDR
|
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| GyrA at position:
|
GyrB at position:
|
ParC at position:
|
ParE at position:
|
||||||||||
| 83 (Thr) | 87 (Asp) | Other | 468 (464)c (Ser) | 470 (466) (Glu) | Other | 87 (80) (Ser) | 91 (84) (Glu) | Other | 419 (420) (Asp) | 425 (426) (Ala) | Other | ||
| I | ≥4 (22) | —b | — | — | — | — | — | — | — | ||||
| II | 2 | — | — | — | Asp | — | — | — | — | ||||
| (2) | — | — | Phe | — | — | — | — | — | |||||
| (1) | — | — | — | — | — | — | — | — | Ala-473→Val | ||||
| III | ≥14 (5) | Ile | — | — | — | — | — | — | — | ||||
| ≥1 (1) | Ala | — | — | — | — | — | — | — | |||||
| ≥1 (1) | — | Tyr | — | — | — | — | — | — | |||||
| (1) | — | Asn | — | — | — | — | — | — | |||||
| IV | (1) | Ile | — | Phe | — | — | — | — | — | ||||
| 1 | Ile | — | — | — | — | — | Leu-95→Gln | — | — | ||||
| 2 | Ile | — | — | — | — | — | — | — | Glu-459→Val | ||||
| 1 | Ile | — | — | — | — | — | — | — | Glu-459→Lys | ||||
| 1 | — | Asn | — | — | — | — | — | — | Ala-473→Val | ||||
| V | 48 | Ile | — | — | — | Leu | — | — | — | ||||
| VI | 3 | Ile | — | — | Asp | Leu | — | — | — | ||||
| 7 | Ile | — | — | Asp | Trp | — | — | — | |||||
| 7 | Ile | — | — | — | Ala-477→Val | Leu | — | — | — | ||||
| 1 | Ile | — | — | — | Thr-473→Met | Leu | — | — | — | ||||
| 1 | Ile | — | — | — | Gln-469→Val | Leu | — | — | — | ||||
| 1 | Ile | — | — | — | Gln-459→Arg | Leu | — | — | — | ||||
| VII | 4 | Ile | Asn | — | — | — | — | Asn | — | ||||
| 1 | Ile | Asn | — | — | — | — | — | — | |||||
| VIII | 6 | Ile | Asn | — | — | Leu | — | — | — | ||||
| 1 | Ile | Asn | — | — | Trp | — | — | — | |||||
| 1 | Ile | Gly | — | — | Leu | — | — | — | |||||
| 1 | Ile | Tyr | — | — | Trp | — | — | — | |||||
| IX | 3 | Ile | — | Glu-54→Lys | — | — | Leu | — | Asn | Val | |||
| 1 | Ile | Asn | — | — | Leu | Lys | — | — | |||||
| 1 | Ile | Asn | — | — | Leu | — | Ala-88→Pro | — | — | ||||
| 1 | Ile | — | — | Asp | Leu | — | — | — | Glu-459→Val | ||||
| 1 | — | Gly | Ala-67→Ser | Tyr | — | Leu | — | — | — | ||||
Numbers in parentheses are numbers of strains intermediately resistant to levofloxacin (MIC, 4 μg/ml). The total number of strains was ≥116 (with 34 being intermediately resistant to levofloxacin).
—, no amino acid change.
The number in parentheses is the number of the corresponding codon in E. coli.
gyrA mutations.
An amino acid replacement(s) in the QRDR of GyrA (Thr-83→Ile or Ala, or Asp-87→Asn, 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-83→Ile 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-83→Ile in GyrA plus Ser-87→Leu 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-87→Leu) to the single mutation in gyrA (Thr-83→Ile) 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-87→Asn, Gly, or Tyr) (Table 2; group VIII). Thus, the alterations in GyrA (Thr-83→Ile and Asp-87→Asn, Gly, or Tyr) and ParC (Ser87→Leu or Trp) seem to play significant roles in fluoroquinolone resistance in clinical P. aeruginosa isolates.
TABLE 2.
Number of isolates corresponding to the MICs of each fluoroquinolone
| Antibiotic | No. of isolates for which MICs (μg/ml) were:
|
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ≤0.12 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | >128 | |
| I (no substitution) (n = 26) | ||||||||||||
| Levofloxacin | — | 22 | 4 | |||||||||
| Sitafloxacin | —a | 6 | 15 | 4 | 1 | |||||||
| Ciprofloxacin | — | 2 | 17 | 5 | 2 | |||||||
| Sparfloxacin | — | 2 | 14 | 6 | 4 | |||||||
| II (GyrB or ParE) (n = 5) | ||||||||||||
| Levofloxacin | 3 | 1 | 1 | |||||||||
| Sitafloxacin | 2 | 2 | 1 | |||||||||
| Ciprofloxacin | 2 | 2 | 1 | |||||||||
| Sparfloxacin | 1 | 2 | 1 | 1 | ||||||||
| III (GyrA) (n = 24) | ||||||||||||
| Levofloxacin | 8 | 1 | 6 | 2 | 6 | |||||||
| Sitafloxacin | 10 | 1 | 7 | 6 | ||||||||
| Ciprofloxacin | 2 | 6 | 5 | 4 | 1 | 4 | 2 | |||||
| Sparfloxacin | 4 | 4 | 8 | 1 | 1 | 6 | ||||||
| IV (GyrA and other locations) (n = 6) | ||||||||||||
| Levofloxacin | 1 | 1 | 2 | 1 | 1 | |||||||
| Sitafloxacin | 1 | 1 | 3 | 1 | ||||||||
| Ciprofloxacin | 1 | 1 | 4 | |||||||||
| Sparfloxacin | 1 | 3 | 1 | 1 | ||||||||
| V (GyrA [position 83] and ParC [position 87]) (n = 48) | ||||||||||||
| Levofloxacin | 1 | 4 | 17 | 19 | 4 | 3 | ||||||
| Sitafloxacin | 1 | 6 | 24 | 17 | ||||||||
| Ciprofloxacin | 1 | 10 | 30 | 7 | ||||||||
| Sparfloxacin | 6 | 23 | 17 | 2 | ||||||||
| VI (GyrA [position 83], ParC [position 87], and other locations) (n = 20) | ||||||||||||
| Levofloxacin | 1 | 3 | 3 | 13 | ||||||||
| Sitafloxacin | 6 | 13 | 1 | |||||||||
| Ciprofloxacin | 1 | 7 | 3 | 2 | 7 | |||||||
| Sparfloxacin | 1 | 3 | 6 | 10 | ||||||||
| VII (GyrA [positions 83 and 87] and other locations) (n = 5) | ||||||||||||
| Levofloxacin | 2 | 3 | ||||||||||
| Sitafloxacin | 3 | 2 | ||||||||||
| Ciprofloxacin | 4 | 1 | ||||||||||
| Sparfloxacin | 4 | 1 | ||||||||||
| VIII (GyrA [positions 83 and 87] and ParC [position 87]) (n = 9) | ||||||||||||
| Levofloxacin | 1 | 8 | ||||||||||
| Sitafloxacin | 1 | 7 | 1 | |||||||||
| Ciprofloxacin | 1 | 4 | 3 | 1 | ||||||||
| Sparfloxacin | 9 | |||||||||||
| IX (4 locations) (n = 7) | ||||||||||||
| Levofloxacin | 1 | 2 | 4 | |||||||||
| Sitafloxacin | 1 | 2 | 4 | |||||||||
| Ciprofloxacin | 2 | 1 | 1 | 3 | ||||||||
| Sparfloxacin | 2 | 5 | ||||||||||
—, MIC for reference strain (P. aeruginosa PAO1).
On the basis of biochemical, genetic, and epidemiological studies, DNA gyrase is known to be the primary target enzyme for fluoroquinolones and topoisomerase IV is known to be the secondary target in P. aeruginosa (1, 19, 20). Thus, the alteration in ParC occurred after GyrA alteration and is associated with the development of higher-level fluoroquinolone resistance. In the present study, we confirmed that the additional replacement in ParC led to a higher level of fluoroquinolone resistance in P. aeruginosa. Moreover, we demonstrated that secondary replacements in GyrA occur as the third step in strains already having double replacements in GyrA and ParC and lead to multifold increases in these strains' fluoroquinolone resistance levels.
All 109 of the levofloxacin-resistant isolates (for which MICs of levofloxacin were ≥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-83→Ile type in GyrA and the Ser-87→Leu 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.
TABLE 3.
Inhibitory effects of quinolones against type II topoisomerases
| Compound | IC50 (μg/ml) for:
|
|||
|---|---|---|---|---|
| DNA gyrase
|
Topoisomerase IV
|
|||
| Wild type | T83I | Wild type | S87L | |
| Levofloxacin | 0.88 | 10.4 | 4.96 | 49.2 |
| Sitafloxacin | 0.42 | 1.85 | 2.12 | 8.62 |
| Ciprofloxacin | 0.55 | 8.29 | 4.06 | 33.0 |
| Sparfloxacin | 0.75 | 17.5 | 6.14 | 52.3 |
ACKNOWLEDGMENTS
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.
REFERENCES
- 1.Akasaka T, Onodera Y, Tanaka M, Sato K. Cloning, expression, and enzymatic characterization of Pseudomonas aeruginosa topoisomerase IV. Antimicrob Agents Chemother. 1999;43:530–536. doi: 10.1128/aac.43.3.530. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Breines D M, Ouabdesselam S, Ng E Y, Tankovic J, Shah S, Soussy C J, Hooper D C. Quinolone resistance locus nfxD of Escherichia coli is a mutant allele of the parE gene encoding a subunit of topoisomerase IV. Antimicrob Agents Chemother. 1997;41:175–179. doi: 10.1128/aac.41.1.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Cambau E, Perani E, Dib C, Petinon C, Trias J, Jarlier V. Role of mutations in DNA gyrase genes in ciprofloxacin resistance of Pseudomonas aeruginosa susceptible or resistant to imipenem. Antimicrob Agents Chemother. 1995;39:2248–2252. doi: 10.1128/aac.39.10.2248. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Drlica K, Zhao X. DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol Mol Biol Rev. 1997;61:377–392. doi: 10.1128/mmbr.61.3.377-392.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Fukuda H, Hosaka M, Iyobe S, Gotoh N, Nishino T, Hirai K. nfxC-type quinolone resistance in a clinical isolate of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1995;39:790–792. doi: 10.1128/AAC.39.3.790. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Gensberg K, Jin Y F, Piddock L J. A novel gyrB mutation in a fluoroquinolone-resistant clinical isolate of Salmonella typhimurium. FEMS Microbiol Lett. 1995;132:57–60. doi: 10.1111/j.1574-6968.1995.tb07810.x. [DOI] [PubMed] [Google Scholar]
- 7.Hancock R E. Resistance mechanisms in Pseudomonas aeruginosa and other nonfermentative gram-negative bacteria. Clin Infect Dis. 1998;27:S93–S99. doi: 10.1086/514909. [DOI] [PubMed] [Google Scholar]
- 8.Hooper D C. Clinical applications of quinolones. Biochim Biophys Acta. 1998;1400:45–61. doi: 10.1016/s0167-4781(98)00127-4. [DOI] [PubMed] [Google Scholar]
- 9.Jalal S, Ciofu O, Hoiby N, Gotoh N, Wretlind B. Molecular mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrob Agents Chemother. 2000;44:710–712. doi: 10.1128/aac.44.3.710-712.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Jalal S, Wretlind B. Mechanisms of quinolone resistance in clinical strains of Pseudomonas aeruginosa. Microb Drug Resist. 1998;4:257–261. doi: 10.1089/mdr.1998.4.257. [DOI] [PubMed] [Google Scholar]
- 11.Jorgensen J H, Weigel L M, Ferraro M J, Swenson J M, Tenover F C. Activities of newer fluoroquinolones against Streptococcus pneumoniae clinical isolates, including those with mutations in the gyrA, parC, and parE loci. Antimicrob Agents Chemother. 1999;43:329–334. doi: 10.1128/aac.43.2.329. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Kato J, Nishimura Y, Imamura R, Niki H, Hiraga S, Suzuki H. New topoisomerase essential for chromosome segregation in E. coli. Cell. 1990;63:393–404. doi: 10.1016/0092-8674(90)90172-b. [DOI] [PubMed] [Google Scholar]
- 13.Kitamura A, Hoshino K, Kimura Y, Hayakawa I, Sato K. Contribution of the C-8 substituent of DU-6859a, a new potent fluoroquinolone, to its activity against DNA gyrase mutants of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1995;39:1467–1471. doi: 10.1128/aac.39.7.1467. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Kohler T, Michea-Hamzehpour M, Henze U, Gotoh N, Curty L K, Pechere J C. Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa. Mol Microbiol. 1997;23:345–354. doi: 10.1046/j.1365-2958.1997.2281594.x. [DOI] [PubMed] [Google Scholar]
- 15.Kohler T, Michea-Hamzehpour M, Plesiat P, Kahr A L, Pechere J C. Differential selection of multidrug efflux systems by quinolones in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1997;41:2540–2543. doi: 10.1128/aac.41.11.2540. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Kureishi A, Diver J M, Beckthold B, Schollaardt T, Bryan L E. Cloning and nucleotide sequence of Pseudomonas aeruginosa DNA gyrase gyrA gene from strain PAO1 and quinolone-resistant clinical isolates. Antimicrob Agents Chemother. 1994;38:1944–1952. doi: 10.1128/aac.38.9.1944. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Levine C, Hiasa H, Marians K J. DNA gyrase and topoisomerase IV: biochemical activities, physiological roles during chromosome replication, and drug sensitivities. Biochim Biophys Acta. 1998;1400:29–43. doi: 10.1016/s0167-4781(98)00126-2. [DOI] [PubMed] [Google Scholar]
- 18.Mine T, Morita Y, Kataoka A, Mizushima T, Tsuchiya T. Expression in Escherichia coli of a new multidrug efflux pump, MexXY, from Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1999;43:415–417. doi: 10.1128/aac.43.2.415. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Mouneimne H, Robert J, Jarlier V, Cambau E. Type II topoisomerase mutations in ciprofloxacin-resistant strains of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1999;43:62–66. doi: 10.1128/aac.43.1.62. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Nakano M, Deguchi T, Kawamura T, Yasuda M, Kimura M, Okano Y, Kawada Y. Mutations in the gyrA and parC genes in fluoroquinolone-resistant clinical isolates of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1997;41:2289–2291. doi: 10.1128/aac.41.10.2289. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A5. 5th ed. Wayne, Pa: National Committee for Clinical Laboratory Standards; 2000. [Google Scholar]
- 22.Perichon B, Tankovic J, Courvalin P. Characterization of a mutation in the parE gene that confers fluoroquinolone resistance in Streptococcus pneumoniae. Antimicrob Agents Chemother. 1997;41:1166–1167. doi: 10.1128/aac.41.5.1166. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Piddock L J. Mechanisms of fluoroquinolone resistance: an update, 1994–1998. Drugs. 1999;2:11–18. doi: 10.2165/00003495-199958002-00003. [DOI] [PubMed] [Google Scholar]
- 24.Poole K, Gotoh N, Tsujimoto H, Zhao Q, Wada A, Yamasaki T, Neshat S, Yamagishi J, Li X Z, Nishino T. Overexpression of the mexC-mexD-oprJ efflux operon in nfxB-type multidrug-resistant strains of Pseudomonas aeruginosa. Mol Microbiol. 1996;21:713–724. doi: 10.1046/j.1365-2958.1996.281397.x. [DOI] [PubMed] [Google Scholar]
- 25.Poole K, Krebes K, McNally C, Neshat S. Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon. J Bacteriol. 1993;175:7363–7372. doi: 10.1128/jb.175.22.7363-7372.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Takenouchi T, Sakagawa E, Sugawara M. Detection of gyrA mutations among 335 Pseudomonas aeruginosa strains isolated in Japan and their susceptibilities to fluoroquinolones. Antimicrob Agents Chemother. 1999;43:406–409. doi: 10.1128/aac.43.2.406. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Tanaka M, Onodera Y, Uchida Y, Sato K. Quinolone resistance mutations in the GrlB protein of Staphylococcus aureus. Antimicrob Agents Chemother. 1998;42:3044–3046. doi: 10.1128/aac.42.11.3044. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.West K L, Meczes E L, Thorn R, Turnbull R M, Marshall R, Austin C A. Mutagenesis of E477 or K505 in the B′ domain of human topoisomerase II beta increases the requirement for magnesium ions during strand passage. Biochemistry. 2000;39:1223–1233. doi: 10.1021/bi991328b. [DOI] [PubMed] [Google Scholar]
- 29.Yamagishi J, Yoshida H, Yamayoshi M, Nakamura S. Nalidixic acid-resistant mutations of the gyrB gene of Escherichia coli. Mol Gen Genet. 1986;204:367–373. doi: 10.1007/BF00331012. [DOI] [PubMed] [Google Scholar]
- 30.Yamaguchi K, Miyazaki S, Kashitani F, Iwata M, Kanda M, Tsujio Y, Okada J, Tazawa Y, Watanabe N, Uehara N, Igari J, Oguri T, Kaimori M, Kawamura C, Iinuma Y, Nisawataira T, Tashiro H, Ueno K, Ishigo S, Yasujima M, Kawahara S, Itoh C, Yoshida T, Yamanaka K, Toyoshima S, Katoh J, Kudoh M, Matsushima T, Niki Y, Miyashita N, Funato T, Kaku M, Sato N, Saito Y, Ishii K, Kuwabara M, Hongo T, Negayama K, Kamihira S, Miyazaki Y, Takii M, Ishii M, Nakagawa K, Ono J, Takada T, Murakami N, Taira M, Tamaki I, Matsudou Y, Tadano J, Nagasawa Z, Kusano N, Nakasone I. Activities of antimicrobial agents against 5,180 clinical isolates obtained from 26 medical institutions during 1998 in Japan. Jpn J Antibiot. 2000;53:387–408. [PubMed] [Google Scholar]
- 31.Yonezawa M, Takahata M, Matsubara N, Watanabe Y, Narita H. DNA gyrase gyrA mutations in quinolone-resistant clinical isolates of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1995;39:1970–1972. doi: 10.1128/aac.39.9.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Yoshida H, Bogaki M, Nakamura M, Nakamura S. Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli. Antimicrob Agents Chemother. 1990;34:1271–1272. doi: 10.1128/aac.34.6.1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Yoshida H, Bogaki M, Nakamura M, Yamanaka L M, Nakamura S. Quinolone resistance-determining region in the DNA gyrase gyrB gene of Escherichia coli. Antimicrob Agents Chemother. 1991;35:1647–1650. doi: 10.1128/aac.35.8.1647. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Yoshida H, Kojima T, Yamagishi J, Nakamura S. Quinolone resistant mutations of the gyrA gene of Escherichia coli. Mol Gen Genet. 1988;211:1–7. doi: 10.1007/BF00338386. [DOI] [PubMed] [Google Scholar]