SINGLE-DOSE REGIMENS of fluoroquinolones have been recommended as alternative primary therapy for uncomplicated gonorrhea.1 However, emergence of clinical isolates of Neisseria gonorrhoeae with decreased susceptibilities to fluoroquinolones and treatment failures with these regimens have been reported.2–8 Recently, several mechanisms of quinolone resistance have been identified and characterized in N. gonorrhoeae. Initially, an amino acid change was found in the GyrB subunit of DNA gyrase of a laboratory mutant strain with low-level nalidixic acid resistance.9 Reduced fluoroquinolone uptake and accumulation have been reported in clinical isolates with decreased susceptibilities to fluoroquinolones.10 Among the mechanisms analyzed so far, however, alterations of the GyrA subunit of DNA gyrase have a central role in conferring high-level quinolone resistance on laboratory mutants and clinical isolates of N. gonorrhoeae11,12 In addition, alterations of the ParC subunit of topoisomerase IV seem to play a complementary role in increasing resistance to fluoroquinolones.11,13 Until recently, however, there have been no clinical reports that treatment with fluoroquinolones actually selects strains of N. gonorrhoeae with an increase in fluoroquinolone resistance, nor have the isolates that exhibited a high level of fluoroquinolone resistance, resulting in treatment failure, been analyzed for resistance mechanisms. In this communication, we present a case of fluoroquinolone treatment failure in gonorrhea and report a posttreatment isolate that had enhanced fluoroquinolone resistance and exhibited a small decrease in susceptibilities to cephalosporins. We also describe the results of analyzing the isolates for fluoroquinolone resistance mechanisms.
Patient and Methods
A 28-year-old Japanese man presented at the Department of Urology in Tokyo Kyosai Hospital, Tokyo, Japan with urethral discharge and dysuria that had begun a week previously. He had had sexual contact with a woman employee of a massage parlor in Tokyo. His smear showed gram-negative intracellular diplococci. A strain of N. gonorrhoeae (named TK-106) was isolated from a urethral specimen. He was treated with ofloxacin, 200 mg, three times daily for 5 days. When he returned to the clinic with continuing symptoms, gram-negative intracellular diplococci were found in his urethral smear. He denied having any sexual contact since the previous visit and reported that he had completed the prescribed ofloxacin. A strain of N. gonorrhoeae (named TK-109) was isolated again. He was treated with cefoperazone, 2.0 g intravenously, followed by oral minocycline, 100 mg, twice daily for 7 days. This treatment resulted in a clinical cure and a negative urethral culture.
Auxotyping, serotyping, and DNA amplification fingerprinting were performed to assess whether the set of strains (TK-106 and TK-109) isolated before and after the administration of ofloxacin was isogenic. The strains were tested for their nutritional requirement for arginine, hypoxanthine, uracil, proline, and methionine, as described previously.14 Serovar classification was performed by coagglutination using a panel of 14 monoclonal antibodies against protein I (Boule Diagnostics AB, Huddinge, Sweden). For DNA amplification fingerprinting, the primers OPA-03 and OPA-13 reported by Camarena et al.15 were used. β-Lactamase activities of these clinical isolates were tested with nitrocefin disks. Their susceptibilities to ofloxacin, ciprofloxacin, penicillin G, tetracycline, minocycline, ceftriaxone, and cefoperazone were determined by the agar dilution method, as described previously.12
To analyze alterations in DNA gyrase and topoisomerase IV, the regions corresponding to the quinolone resistance-determining regions of the Escherichia coli gyrA and gyrB genes16,17 and the analogous region of the parC gene11 were amplified from chromosomal DNAs of the isolates by polymerase chain reaction (PCR) and their sequences determined. Purification of chromosomal DNAs, PCR amplification, and sequencing of the PCR products were performed as reported previously.13,18
Ofloxacin uptake by strain TK-106 or TK-109 was determined by fluorometric assay, as reported by Chapman and Georgopapadakou.19 In brief, the bacterial cells were incubated with 10 μg ofloxacin/ml for 5 minutes. After the cells were washed extensively and destroyed to release intracellular ofloxacin, the concentration of released ofloxacin was determined by spectrofluorometry. The amount of ofloxacin per milligram (wet weight) of the bacterial cells was calculated as ofloxacin uptake by the cells. The assays were carried out in triplicate.
Statistical analysis was conducted using a t test to compare ofloxacin uptake by TK-106 and TK-109. This comparison was performed as a two-tailed test, with significance set at P < 0.05.
In this case, the 5-day administration of ofloxacin neither eliminated the symptoms nor eradicated the causative microorganism, N. gonorrhoeae. Treatment failure was observed in this case of gonorrhea.
Both the pretreatment and posttreatment isolates of TK-106 and TK-109 required proline, and belonged to the serovar class of Bopyst. Electrophoresis profiles of DNA fragments amplified from the posttreatment isolate, TK-109, with OPA-03 and OPA-13 primers were identical to those from the pretreatment isolate, TK-106. These isolates were assumed to be isogenic, based on the analysis of their phenotypic and genotypic patterns.
TK-106 and TK-109 were enzymatically negative for β-lactamase. Table 1 summarizes antimicrobial susceptibilities of these isolates. TK-106 showed decreased susceptibilities to ofloxacin (minimum inhibitory concentration [MIC], 1.0 mg/l) and ciprofloxacin (MIC, 0.25 mg/l). This strain, which exhibited a penicillin G MIC of 4 mg/l and a tetracycline MIC of 2 mg/l, was assigned to the category of N. gonorrhoeae with chromosomally mediated resistance to penicillin G and tetracycline. However, it was susceptible to ceftriaxone (MIC, 0.015 mg/l).
The posttreatment isolate of TK-109 exhibited higher MICs for ofloxacin (8.0 mg/l) and ciprofloxacin (1.0 mg/l) but did not have increased resistance to penicillin G and tetracycline. Although this strain was still ceftriaxone susceptible (MIC, 0.03 mg/l), it exhibited a small decrease in susceptibility to cephalosporins compared with the pretreatment isolate, TK-106.
The TK-106 isolate was found to have amino acid changes of Ser-91 → Phe in the GyrA subunit of DNA gyrase and Ser-87 → Ile in the ParC subunit of topoisomerase IV (Table 2). The TK-109 isolate also had identical amino acid alterations in GyrA and ParC. In these isolates, however, no mutations were detected in the region of the gyrB gene that was sequenced for this study.
Ofloxacin uptake by the isolates was determined by fluorometric assay.
Ofloxacin uptake by the posttreatment TK-109 isolate (215.29 ± 13.49 ng/mg wet cell weight) was significantly lower than that by the pretreatment TK-106 isolate (148.40 ± 13.68 ng/mg wet cell weight; P < 0.01).
Gonorrhea treatment failures associated with the development of resistance to fluoroquinolones have been observed and reported. In treatment failures initially occurring with single doses of 250 mg ciprofloxacin, strains with ciprofloxacin MICs ranging from 0.05 to 0.25 mg/l have been isolated.5 In three other treatment failures at 500-mg doses, the isolates exhibited ciprofloxacin MICs ranging from 1.0 to 16.0 mg/l.6,8 However, these isolates have not been examined for mechanisms of clinically significant resistance to ciprofloxacin. TK-106, which was isolated before ofloxacin treatment, exhibited an ofloxacin MIC of 1.0 mg/l. This strain was also clinically resistant to fluoroquinolone treatment. We analyzed this isolate for the presence of mutations in the quinolone resistance-determining regions of the gyrA and gyrB genes and in the analogous region of the parC gene, and found amino acid changes in GyrA and ParC. These alterations were analogous to those that have been observed to give rise to the fluoroquinolone resistance phenotypes in N. gonorrhoeae by DNA transformation analysis in laboratory mutants.11 In this case study, therefore, we confirmed the association of alterations in GyrA and ParC in N. gonorrhoeae with a clinically significant resistance to fluoroquinolones.
In this report, the posttreatment isolate, TK-109, which was assumed to be isogenic to the pretreatment TK-106 isolate, exhibited a four- to eightfold increase in resistance to fluoroquinolones. Although TK-109 displayed amino acid changes in GyrA and ParC identical to those found in TK-106, it exhibited a significantly reduced uptake of ofloxacin compared with the pretreatment isolate. Therefore, the increase in resistance to fluoroquinolones in TK-106 was not attributable to alterations of DNA gyrase or topoisomerase IV but to the reduced uptake of ofloxacin.
This report demonstrates that treatment with multiple doses of ofloxacin could bring about selection of a more fluoroquinolone-resistant strain of N. gonorrhoeae in vivo. A posttreatment isolate from a patient in Spain who was treated with ciprofloxacin, 250 mg, twice daily for 5 days, exhibited a ciprofloxacin MIC of 16 mg/l.7 In that report, a pretreatment isolate was not examined for susceptibilities to fluoroquinolones. It was unknown whether the 5-day treatment with ciprofloxacin was associated with the emergence of the posttreatment strain with high-level resistance to ciprofloxacin. However, one of the strains exhibiting the highest level of resistance to ciprofloxacin was isolated after treatment with multiple doses of ciprofloxacin. There have been no reports that treatment with single doses of fluoroquinolones selects a posttreatment strain with an increased fluoroquinolone resistance. Therefore, the heavy continued use of fluoroquinolones might produce more fluoroquinolone-resistant posttreatment strains in vivo.
Currently, quinolone-resistant strains remain susceptible to cephalosporins, including ceftriaxone, and treatment with cephalosporins is efficacious against gonorrhea caused by quinolone-resistant strains.13 However, fluoroquinolone-resistant mutants of Pseudomonas aeruginosa, Klebsiella pneumoniae, and Serratia marcescens selected in vitro after exposure to fluoroquinolones have developed a multiple antibiotic-resistant phenotype, including a cross-resistance to structurally unrelated cephalosporins.20,21 Reduced fluoroquinolone uptake and altered outer membrane proteins have been discovered in these mutants, and the alteration of bacterial outer membrane permeability has been considered to contribute to the development of cross-resistance.21 In clinical isolates of N. gonorrhoeae resistant to quinolones, reduced uptake of quinolones has already been reported.10 The current study also suggests that drug access to bacterial targets can be prevented in N. gonorrhoeae. In a previous study, Carlyn et al.22 reported that the clinical isolates of N. gonorrhoeae with diminished quinolone susceptibility required MICs of ceftriaxone that were four- to eightfold higher than those for the ciprofloxacin-susceptible isolates. We also demonstrated that quinolone-resistant clinical isolates, particularly those having alterations in GyrA and ParC, significantly reduced susceptibilities to cephalosporins.13 The posttreatment TK-109 isolate in this case exhibited a small decrease in susceptibilities to ceftriaxone and cefoperazone, compared with the pretreatment isolate, TK-106. These findings suggest that N. gonorrhoeae strains having quinolone resistance-associated alterations in DNA gyrase and topoisomerase IV might develop cross-resistance to cephalosporins during in vivo exposure to fluoroquinolones.
In conclusion, alterations in DNA gyrase and topoisomerase IV confer high-level resistance, resulting in treatment failure, in N. gonorrhoeae strains. The emergence and dissemination of such strains threatens the efficacy of fluoroquinolones in the treatment of gonorrhea. Treatment with multiple doses of ofloxacin is likely to bring about selection of more fluoroquinolone-resistant strains of N. gonorrhoeae and to influence susceptibilities to cephalosporins. Therefore, a heavy continued use of fluoroquinolones for the treatment of uncomplicated gonorrhea should be prevented, and antimicrobial susceptibility profiles of gonococcal isolates, particularly posttreatment isolates from patients treated unsuccessfully with fluoroquinolones, should be monitored.
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