Abstract
Forty-two men with gonococcal urethritis were treated with an oral dosage of 200 mg of pazufloxacin, a new fluoroquinolone, three times daily for 3 days. Only 28 of the 42 men (66.7%) had negative culture results for Neisseria gonorrhoeae during follow-up. Of the 42 isolates, 41 could be recultured for antibiotic susceptibility testing and DNA sequencing. In 26 of the 41 isolates (63.4%), GyrA mutations with or without ParC mutations were identified. Among the 26 isolates, 23 contained a single GyrA mutation, 1 contained two GyrA mutations, and 2 contained three mutations including double GyrA and single ParC mutations. A single Ser-91-to-Phe mutation, which was detected in 14 of the 26 isolates, was the most common GyrA mutation, followed by an Ala-75 to Ser mutation and an Asp-95 to Asn or Gly mutation in GyrA. All three isolates with two or three mutations contained the Ser-91-to-Phe GyrA mutation. Eleven of the 14 isolates with the single Ser-91-to-Phe mutation within GyrA and all 3 isolates with two or three mutations persisted after pazufloxacin treatment. On the other hand, all 15 wild-type and 9 mutant isolates with a substitution at codon Ala-75 or Asp-95 were eradicated. The mean MIC of pazufloxacin for mutants with the single Ser-91-to-Phe mutation in GyrA was 66-fold higher than that for the wild type. The results obtained in this study suggest that a high prevalence of fluoroquinolone-resistant gonococcal isolates with the Ser-91-to-Phe mutation in GyrA reduced the efficacy of pazufloxacin as treatment for gonococcal urethritis.
The increasing frequency of occurrence of Neisseria gonorrhoeae isolates with plasmid- and chromosome-mediated resistance to penicillin or tetracycline is a serious problem worldwide. Fluoroquinolones such as norfloxacin, ofloxacin, and ciprofloxacin demonstrate excellent in vitro activities against N. gonorrhoeae, including penicillin- and tetracycline-resistant strains, and are highly effective for the oral treatment of gonococcal infections caused not only by strains sensitive to penicillin or tetracycline but also by strains resistant to these antibiotics (4, 6, 20). Therefore, over the past decade, fluoroquinolone regimens have increasingly been used in various countries for the treatment of gonococcal infections. Although the emergence of isolates of N. gonorrhoeae showing reduced susceptibility to fluoroquinolones has recently been reported in several countries including Japan (3, 5, 7, 9–12, 22, 23), high failure rates for fluoroquinolone treatment of gonococcal urethritis have not yet been reported.
Pazufloxacin (T-3761) is a new orally administered fluoroquinolone which has excellent in vitro activity against β-lactamase-producing and -nonproducing clinical isolates of N. gonorrhoeae, with an MIC at which 50% of isolates are inhibited (MIC50) of <0.006 μg/ml and an MIC90 of 0.1 μg/ml (16). A single oral dose of 200 mg of pazufloxacin results in a mean peak level in plasma of 2.0 μg/ml in 1.2 h, with the half-life averaging 1.9 h (17). These data suggest that pazufloxacin might be effective for the treatment of gonococcal urethritis in men. An open clinical trial was undertaken to assess the efficacy and safety of pazufloxacin in men with acute urethritis caused by N. gonorrhoeae. However, a high failure rate was obtained for the treatment of gonococcal urethritis with pazufloxacin. Thus, we examined the prevalence of clinically fluoroquinolone-resistant N. gonorrhoeae isolates with mutations in the GyrA protein with or without mutations in the ParC protein, both of which confer quinolone resistance (2, 24), and investigated the relationship between the clinical efficacy of pazufloxacin and mutations within the GyrA and ParC proteins of these bacteria.
MATERIALS AND METHODS
Study population and design.
Forty-seven men who presented with urethral discharge and whose urethral discharge showed gram-negative intracellular diplococci upon Gram staining and five or more polymorphonuclear leukocytes per high-power field were enrolled in this study. The men ranged in age from 19 to 47 years (mean age, 30.3 years). The diagnosis of gonococcal urethritis was confirmed by recovery of N. gonorrhoeae by culture. All participants provided oral or written informed consent before they were enrolled in the study.
Patients were treated with an oral dosage of 200 mg pazufloxacin three times daily for 3 days and were asked to return for reexamination on days 4 and 7 after the initiation of treatment. At each visit, patients were examined, urethral smears were collected for microscopy and the detection of N. gonorrhoeae, and first-void urine specimens were also obtained for the detection of the Chlamydia trachomatis antigen.
Microbiological examination.
Two urethral specimens for the detection of N. gonorrhoeae and a first-void urine sample for the detection of the C. trachomatis antigen were collected from each patient. The detection of N. gonorrhoeae was performed by recovery by culture and a DNA probe test (Gen Probe). A commercial kit (IDEIA; Dako, Cambrigeshire, United Kingdom) was used for the C. trachomatis antigen assay. Specimens for culture of N. gonorrhoeae were immediately inoculated onto Thayer-Martin agar (Beckton Dickinson). The culture plates were incubated at 35°C for 24 to 48 h in a 5% CO2 atmosphere. N. gonorrhoeae was identified by colony morphology, oxidase reaction, and appearance upon Gram staining and was confirmed by sugar utilization reactions. β-Lactamase production was assayed by the chromogenic cephalosporin technique (15, 18).
N. gonorrhoeae isolates were stored at −70°C until antibiotic susceptibility testing. The MICs for all isolates were determined by an agar dilution technique with a GC agar base containing 1% IsoVitaleX (Beckton Dickinson) and twofold dilutions of the antibiotic (8). The plates were inoculated with 5 μl of 106 CFU of each isolate per ml with a multipoint inoculator. The plates were incubated at 35°C for 24 h in a 5% CO2 atmosphere. The MICs were defined as the lowest antibiotic concentration that inhibited bacterial growth. Three fluoroquinolones were tested: pazufloxacin (Toyama Chemical Company, Tokyo, Japan), norfloxacin (Kyorin, Tokyo, Japan), and ciprofloxacin (Bayer, Osaka, Japan).
Molecular biology-based study.
PCR and direct DNA sequencing were performed to identify mutations in the gyrA and parC genes of the gonococcal strains isolated before and after treatment. Chromosomal DNA was extracted by standard methods (1) and was subjected to PCR. The oligonucleotide primers for the PCR amplification were as follows: for the gyrA gene the forward primer was 5′-CGGCGCGTACTGTACGCGTTGAC-3′ and the reverse primer was 5′-AATGTCTGCCAGCATTTCATGTGAGA-3′, and for the parC gene the forward primer was 5′-ATGCGCGATATGGGTTTGAC and the reverse primer was 5′-GGACAACAGCAATTCCGCAA. These primers were produced with a DNA synthesizer and were based on the sequences previously reported by Belland et al. (2). The gyrA gene sequence was determined from nucleotides 160 to 439, which correspond to amino acids 54 to 147 of the GyrA protein. This includes the quinolone resistance-determining region (amino acids 55 to 110 of the gonococcal GyrA protein) (2). The parC gene sequence was also determined from nucleotides 166 to 420, which correspond to amino acids 56 to 140 of the gonococcal ParC protein. This includes the quinolone resistance-determining region of the ParC protein (amino acids 66 to 119 of the gonococcal ParC protein) (2).
PCR amplification was performed with 25 μl of a reaction mixture which contained 2.5 μl of 10× Taq polymerase buffer (500 mM KCl, 100 mM Tris-HCl [pH 8.3], 15 mM MgCl2, 0.1% gelatin), 0.25 μl of each of the two primers (25 pmol/μl), 0.5 μl of each of the four deoxynucleotide triphosphates (10 mM), 0.2 μl of Taq DNA polymerase (5 U/μl; Takara, Shiga, Japan), 2.5 μl of Triton X-100 (2 mg/ml), and 1.0 μl of template DNA (100 ng/μl). Thirty-five cycles were performed for each reaction. Each cycle consisted of 30 s at 93°C, 1 min at 52°C, and 1 min at 72°C.
The PCR amplification products were directly sequenced by the dideoxy-chain termination method (13) with the Taq DyeDeoxy Terminator Cycle Sequencing Kit and a model 373A autosequencer (ABI).
RESULTS
Clinical results.
Of the 47 men enrolled in the study and treated with pazufloxacin, 5 could not be evaluated due to protocol violation. Among the 42 patients evaluated, only 1 had been treated with another fluoroquinolone before this study. The remaining 41 patients were not treated with any antimicrobial agent before pazufloxacin treatment. Seven were infected with penicillinase-producing N. gonorrhoeae strains. N. gonorrhoeae was not detected again in only 28 of the 42 patients (66.7%) during follow-up. Thus, the response rate to pazufloxacin treatment of gonococcal urethritis was very low. Table 1 presents the relationship between the MICs of pazufloxacin for N. gonorrhoeae isolates and the bacteriological responses. Forty-one isolates could be recultured for antibiotic susceptibility testing. All 23 isolates for which the MICs of pazufloxacin were 0.1 μg/ml or less were eradicated by treatment. Of the 11 isolates for which the MICs of pazufloxacin ranged from 0.2 to 0.78 μg/ml, only 4 were eradicated. All seven isolates for which the pazufloxacin MICs exceeded 1.56 μg/ml were persistent. The MIC of pazufloxacin for an isolate from the patient treated with another fluoroquinolone before this study was 1.56 μg/ml. These results indicate that the gonococcal isolates for which the MICs of pazufloxacin were higher than 0.2 μg/ml are possibly clinically resistant to pazufloxacin treatment. C. trachomatis was also present in 8 of the 42 patients (19.0%). The C. trachomatis antigen was not detected again in five of these eight patients during follow-up. One patient from whom C. trachomatis was eradicated had been given 200 mg of pazufloxacin three times daily for 7 days.
TABLE 1.
Relationship between MIC and bacteriological response to pazufloxacin treatment
| Pazufloxacin MIC (μg/ml) | No. of strains eradicated/no. of strains isolated (%) |
|---|---|
| 0.006 | 2/2 (100) |
| 0.013 | 12/12 (100) |
| 0.025 | 1/1 (100) |
| 0.05 | 3/3 (100) |
| 0.1 | 5/5 (100) |
| 0.2 | 2/3 (66.7) |
| 0.39 | 1/5 (20) |
| 0.78 | 1/3 (33.3) |
| 1.56 | 0/4 (0) |
| 3.13 | 0/2 (0) |
| 6.25 | 0/0 |
| 12.5 | 0/1 (0) |
| NDa | 1/1 (100) |
| Total | 28/42 (66.7) |
ND, not determined.
The treatment was well tolerated. No side effects related to the treatment were observed in these patients.
Prevalences of the gyrA and parC gene mutations.
Table 2 lists the mutations within the gyrA and parC genes of 41 gonococcal strains isolated before treatment. In 26 of the 41 isolates (63.4%), GyrA mutations with or without ParC mutations were identified. Among these 26 isolates, 23 (56.1%) contained a single GyrA mutation, 1 (2.4%) contained two GyrA mutations, and 2 (4.9%) contained three mutations including double GyrA and single ParC mutations. A Ser-91-to-Phe mutation was the most common GyrA mutation, followed by an Ala-75-to-Ser mutation and an Asp-95-to-Asn or -Gly mutation in GyrA. The detected mutations within ParC were a Ser-88-to-Pro mutation and a Glu-91-to-Gly mutation. One type of silent mutation was frequently detected: the DNA sequence was substituted at codon 131, but the ParC protein sequence was unchanged.
TABLE 2.
Mutations in the gyrA and parC genes in N. gonorrhoeae strains isolated before treatment
|
gyrA
|
parC
|
No. (%) of strains | ||||
|---|---|---|---|---|---|---|
| Codon(s) | Nucleotide mutation | Amino acid mutation | Codon | Nucleotide mutation | Amino acid mutation | |
| 75 | GCG→TCG | Ala→Ser | 131 | CTC→CTG | Leu (silent) | 5 (12.2) |
| 91 | TCC→TTC | Ser→Phe | 131 | CTC→CTG | Leu (silent) | 14 (34.1) |
| 95 | GAC→AAC | Asp→Asn | 131 | None or CTC→CTG | None or Leu (silent) | 2 (4.9) |
| 95 | GAC→GGC | Asp→Gly | 131 | None or CTC→CTG | None or Leu (silent) | 2 (4.9) |
| 91 and 95 | TCC→TTC, GAC→GGC | Ser→Phe, Asp→Gly | None | None | 1 (2.4) | |
| 91 and 95 | TCC→TTC, GAC→GGC | Ser→Phe, Asp→Gly | 91 | GAG→GGG | Glu→Gly | 1 (2.4) |
| 91 and 95 | TCC→TTC, GAC→AAC | Ser→Phe, Asp→Asn | 88 | TCC→CCC | Ser→Pro | 1 (2.4) |
| None | None | 131 | None or CTC→CTG | None or Leu (silent) | 15 (36.6) | |
Of the 41 strains, 18 were isolated in Tokyo and the remaining 23 were isolated in Fukuoka city, located in the northern part of Kyushu Island, which is far from Tokyo. There was no significant difference in the incidence of isolates with GyrA mutations between Fukuoka (69.6%) and Tokyo (61.1%). It seems that fluoroquinolone-resistant gonococci carrying GyrA mutations are widespread in Japan. We did not know whether the isolates carrying the Ser-91-to-Phe GyrA mutation or the double mutations were clonal.
We also analyzed the DNA sequences of isolates not eradicated by pazufloxacin treatment. All 14 strains which were isolated after treatment with pazufloxacin had GyrA mutations with or without ParC mutations. However, there were no differences in the GyrA and ParC mutation patterns or in susceptibility to pazufloxacin between pretherapy and posttherapy isolates from the same patient.
Bacteriological responses of mutants to pazufloxacin.
The relationships between mutations in GyrA and ParC and the bacteriological responses to pazufloxacin are presented in Table 3. All 15 wild-type isolates, 5 containing the Ala-75-to-Ser mutation in GyrA and 4 containing the Asp-95-to-Asn or -Gly mutation in GyrA, were eradicated by pazufloxacin treatment. On the other hand, of the 14 isolates containing the single Ser-91-to-Phe mutation, only 3 (21.4%) were eradicated. Moreover, all three isolates with the double mutations in GyrA or the triple mutations involving both GyrA and ParC were persistent. These three mutants contained the Ser-91-to-Phe mutation in GyrA. These results indicate that although the mutants with the Ala-75-to-Ser, Asp-95-to-Asn, or Asp-95-to-Gly substitution in GyrA were still clinically susceptible to pazufloxacin treatment, the mutants containing the Ser-91-to-Phe substitution in GyrA with or without another substitution were clinically resistant to this treatment.
TABLE 3.
Relationship between mutations in GyrA and ParC and bacteriological response
| Mutation
|
Total no. of strains | Bacteriological response (no. [%] of isolates)
|
||
|---|---|---|---|---|
| GyrA | ParC | Eradicated | Persistent | |
| Wild type | Wild type | 15 | 15 (100) | 0 |
| Ala75Ser | Wild type | 5 | 5 (100) | 0 |
| Ser91Phe | Wild type | 14 | 3 (21.4) | 11 (78.6) |
| Asp95Asn | Wild type | 2 | 2 (100) | 0 |
| Asp95Gly | Wild type | 2 | 2 (100) | 0 |
| Ser91Phe and Asp95Gly | Wild type | 1 | 0 | 1 (100) |
| Ser91Phe and Asp95Gly | Glu91Gly | 1 | 0 | 1 (100) |
| Ser91Phe and Asp95Asn | Ser88Pro | 1 | 0 | 1 (100) |
Susceptibilities of the mutants to fluoroquinolones.
Table 4 describes relationships between mutations in GyrA and ParC and the pazufloxacin MICs for N. gonorrhoeae. The mean MICs of pazufloxacin for the mutants with the Ala-75-to-Ser, Ser-91-to-Phe, Asp-95-to-Asn, and Asp-95-to-Gly substitutions in GyrA were 0.08, 0.86, 0.2, and 0.075 μg/ml, respectively, while that for the wild-type isolate was 0.013 μg/ml. This result indicates that gonococcal isolates which acquired the single Ser-91-to-Phe mutation are more resistant to pazufloxacin than the other mutants with a single GyrA substitution. The mutants with double GyrA substitutions or triple substitutions involving GyrA and ParC were extremely resistant to pazufloxacin compared to the level of resistance of the wild type. These mutants were cross-resistant to other fluoroquinolones: norfloxacin and ciprofloxacin.
TABLE 4.
Relationship between mutations in GyrA and ParC and the pazufloxacin MIC for N. gonorrhoeae
| Mutation
|
No. of strains | Mean MIC (μg/ml)
|
|||
|---|---|---|---|---|---|
| GyrA | ParC | Pazufloxacin | Norfloxacin | Ciprofloxacin | |
| Wild type | Wild type | 15 | 0.013 | 0.032 | 0.0069 |
| Ala75Ser | Wild type | 5 | 0.08 (6×)a | 0.14 (4×) | 0.019 (3×) |
| Ser91Phe | Wild type | 14 | 0.86 (66×) | 1.72 (54×) | 0.28 (41×) |
| Asp95Asn | Wild type | 2 | 0.2 (15×) | 0.59 (18×) | 0.075 (11×) |
| Asp95Gly | Wild type | 2 | 0.075 (6×) | 0.3 (9×) | 0.038 (6×) |
| Ser91Phe and Asp95Gly | Wild type | 1 | 3.13 (240×) | 3.13 (98×) | 0.39 (57×) |
| Ser91Phe and Asp95Gly | Glu91Gly | 1 | 3.13 (240×) | 12.5 (390×) | 3.13 (454×) |
| Ser91Phe and Asp95Asn | Ser88Pro | 1 | 12.5 (961×) | 12.5 (390×) | 3.13 (454×) |
The values in parentheses refer to the increase in the MIC compared with the MICs for the wild-type isolates.
DISCUSSION
Until recently, fluoroquinolones have been shown to have excellent antimicrobial activities against N. gonorrhoeae isolates. Thus, fluoroquinolone regimens have increasingly been used in Japan. The emergence of gonococcal isolates showing reduced susceptibility to fluoroquinolones in vitro has recently been reported in Japan (22, 23). However, the widespread existence of gonococcal isolates clinically resistant to treatment with fluoroquinolones has not yet been recognized.
In this study, we evaluated the clinical efficacy of pazufloxacin, a new fluoroquinolone, for the treatment of gonococcal urethritis. This treatment eradicated N. gonorrhoeae isolates from only 66.7% of patients with gonococcal urethritis. This response rate would appear to be very low, because until recently the effectiveness rates of various fluoroquinolones for the treatment of uncomplicated gonorrhea were approximately 100% (4, 6, 14, 19–21). To determine the cause of this high rate of failure of pazufloxacin treatment, we investigated the frequency of occurrence of isolates with mutations in GyrA and ParC proteins which confer fluoroquinolone resistance on these bacteria. Surprisingly, in 26 (63.4%) of the 41 isolates tested, GyrA mutations with or without ParC mutations were identified. The identified mutations in GyrA were Ala-75-to-Ser, Ser-91-to-Phe, Asp-95-to-Asn, and Asp-95-to-Gly substitutions. Of these mutations, the Ser-91-to-Phe mutation was the most common GyrA mutation, and it was detected in 17 (65.4%) of the 26 isolates with GyrA substitutions. Among these 17 isolates, 14 contained the single Ser-91-to-Phe GyrA mutation, 1 contained two GyrA mutations, and 2 contained three mutations including double GyrA and single ParC mutations. The eradication rate for the isolates with the Ser-91-to-Phe mutation was only 17.6% (3 of 17 isolates), while that for wild-type isolates or isolates containing the other GyrA mutations was 100% (24 of 24 isolates). These results indicate that the Ser-91-to-Phe mutation in GyrA is a critical mutation which confers on N. gonorrhoeae clinical resistance to fluoroquinolones. Antibiotic susceptibility tests of these mutants corroborated the clinical significance of the Ser-91-to-Phe mutation in GyrA. The mean MICs of pazufloxacin for the mutants with the Ser-91-to-Phe substitution were increased 66-fold compared to that for the wild type. However, the level of reduction of the susceptibility of the mutants with substitutions at codon Ala-75 or Asp-95 was 6- or 15-fold less, respectively, than that of the wild type.
The ParC mutation was identified in only 2 of the 41 isolates. The double GyrA mutations coexisted in the two isolates with the ParC mutation. No isolates contained the ParC mutation without the simultaneous presence of at least one GyrA mutation. The mutants with both GyrA and ParC mutations were extremely resistant to fluoroquinolones. The ParC mutation may be essential for the bacteria to acquire high-grade fluoroquinolone resistance.
In this study we were not able to compare pazufloxacin therapy with ciprofloxacin therapy, because this study was undertaken as an open clinical trial. Treatment failure with ciprofloxacin has been reported for isolates for which MICs are ≥0.05 or 0.06 μg/ml (7, 9). Therefore, the isolates containing a single Ser-91-to-Phe GyrA mutation, double GyrA mutations, or triple mutations within GyrA and ParC may be refractory to ciprofloxacin treatment, because the ciprofloxacin MICs for these mutants ranged from 0.1 to 3.13 μg/ml.
To our knowledge, this is the first study to examine the relationship between the clinical efficacies of fluoroquinolones against gonorrhea and mutations involving the GyrA and ParC proteins of these bacteria. The high prevalence of fluoroquinolone-resistant N. gonorrhoeae isolates with the GyrA mutation limits the clinical efficacy of pazufloxacin against gonococcal infections in Japan.
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