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First Isolate of a Neisseria gonorrhoeae Strain Associated With an Ofloxacin Treatment Failure in Spain

Case Report

OTERO, LUIS PhD, MD,*; ALCALA, BELEN MD,†; VARELA, JOSE ANTONIO MD,‡; MIGUEL, MARIA DOLORES MD,*; VAZQUEZ, JULIO ALBERTO PhD, MD,†; VAZQUEZ, FERNANDO PhD, MD,§‖‖

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FLUOROQUINOLONE REGIMENS, recommended for the treatment of uncomplicated gonorrhea, 1 have been used widely for this purpose. The recent emergence of clinical isolates resistant to fluoroquinolones has become a major problem for the treatment of gonococcal infections in several countries. 2 Genetic characterization of a treatment failure in Spain resulting from a high-level fluoroquinolone-resistant strain of Neisseria gonorrhoeae is reported.

Case Report

The patient, a 32-year-old heterosexual man, had experienced sexual contact with a Brazilian woman prostitute in Gijón (Asturias, Spain) but had no history of foreign travel. On April 20, 2000, he presented at the sexually transmitted disease (STD) clinic with dysuria and purulent discharge of approximately 1 month duration. A Gram-stained smear of the discharge showed gram-negative intracellular diplococci. The man was treated in the STD clinic with ofloxacin, 400 mg orally in a single dose, and tinidazole 1 g. From the urethral discharge, a β-lactamase-negative N gonorrhoeae was cultured.

On April 24, the patient returned to the STD clinic because of persistent discharge. A Gram-stained smear again was positive. He denied any sexual contact since the previous visit. Once again a β-lactamase-negative N gonorrhoeae was cultured from the discharge. The patient was treated with cefixime, 400 mg orally in a single dose, along with clarithromycin, 500 mg orally twice a day for 7 days. The Brazilian woman prostitute could not be located for follow-up assessment, and her travel history was unknown, although she had come recently from Brazil (fewer than 3 months before).

As a first step in the clinical laboratory, both isolates were screened for ciprofloxacin susceptibility using a 5-μg disc on Mueller-Hinton agar. The two isolates showed no zone of inhibition. We performed a Sensititre (Trek Diagnostic System, East Grinstead, UK) and antibiogram (ATB) NH (bioMeríeux, France) analysis. They showed MICs, respectively, of 2 mg/l or more to ciprofloxacin and 4 mg/l or more to pefloxacin. Because of this resistance, the patient was called back to the STD clinic on May 4 and 24. At that time, his symptoms had resolved, and both the Gram stain and culture showed negative results.

Both isolates were sent to the Neisseria reference laboratory for characterization and MIC determination. 3 Briefly, inocula were prepared by growing isolates on supplemented gonococcal agar plates and then suspending the growth in Mueller-Hinton broth until an optical density equivalent to 108 cfu/ml was obtained. Inoculation was performed with an automatic multi-inoculator device that dispensed a final inoculum of 105 cfu/spot. Incubation was performed at 37 °C in a 5% carbon dioxide atmosphere for 18 to 20 hours. Both strains were characterized as belonging to the prototrophic/IBopyst auxotype/serovar class (Table 1), and the MIC to ciprofloxacin was 16 mg/l. Mutations in the gyrA and parC genes of these ciprofloxacin-resistant strains were identified by polymerase chain reaction and direct sequencing of amplified products that included the quinolone resistance–determining regions of these genes.

Table 1
Table 1:
Characteristics of the Two Isolates With High-Level Fluoroquinolone Resistance

The oligonucleotide primers for the polymerase chain reaction amplification were as follows:gyrA gene (forward primer, 5′-CGGCGCGTACTGTACGCGATGCA-3′, reverse primer, and 5′-AATGTCTGCCAGCATTTCATGTGAGA-3′) and parC gene (forward primer, 5′-ATGCGCGATATGGGTTTGAC-3′, and reverse primer, 5′-GGACA- ACAGCAATTCCGCAA-3′). These primers were produced with a DNA synthesizer according to sequences previously reported by Tanaka et al. 4

The gyrA gene sequence was determined from nucleotides 160 to 438, which correspond to amino acids 54 to 146 of the GyrA protein. This includes the quinolone resistance–determining region (amino acids 55 to 110 of the gonococcal GyrA protein). The parC gene sequence also was 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 (amino acids 66 to 119 of the gonococcal ParC protein).

Polymerase chain reaction gene amplification and automatic sequencing 4 showed a serine-to-phenylalanine substitution at position 91 and an aspartic acid-to-glycine substitution at position 95 on the first gene and a serine-to-arginine substitution at position 87 on the second gene. These gyr A mutations have been particularly associated with a high level of resistance to ciprofloxacin. 5

Discussion

Gonococcal resistance to antimicrobial agents is an increasing problem, and a high prevalence of plasmid-mediated high-level or a chromosomally mediated low-level resistance to penicillin or tetracycline has been recognized. The development and use of newer fluoroquinolone antibiotics was opportune inasmuch as they have a low rate of adverse effects, a lower cost than some other regimens, and excellent activity against N gonorrhoeae.6 However, the emergence of strains with decreased susceptibilities to fluoroquinolones has been reported. 7–9 These strains are associated with mutations in the gyr A subunit of DNA gyrase and in the par C subunit of DNA topoisomerase IV. 5,10–12

Strains with reduced susceptibility (0.125–0.5 mg/l) have been widely reported, including cases from Rwanda, Hong Kong, Bangkok, Japan, the United States, the United Kingdom, Canada, Hawaii, the Philippines, and Australia. 6 In Spain, a 6.5% prevalence of intermediate resistance to ciprofloxacin was found in a recent study. 13 Treatment failure for isolates with reduced susceptibility, although rarely reported, has been more common in patients with isolates of 1 mg/l from Australia and the Philippines. 6

This discussion reports the first strain of N gonorrhoeae with a high level of resistance to ciprofloxacin isolated in Spain. Other strains isolated in United Kingdom but acquired in Bilbao (northern Spain) had been described previously. 14 Because the phenotype of the two strains is not the same, we do not believe they are related.

We used Mueller-Hinton agar, Sensititre, and ATB-NH systems for the initial susceptibility studies, but these methods are not recommended and may not be reliable. For this reason, all our strains are sent to the Neisseria reference laboratory for confirmation of the initial results.

Genetic characterization of the gyr A and par C subunits showed mutations particularly associated with a high level of resistance to ciprofloxacin. 15 Continuous surveillance monitoring the level of susceptibility to ciprofloxacin must be maintained to detect and control these types of strains, particularly when a high level of resistance appears from time to time.

References

1. Centers for Disease Control and Prevention. 1998 Guidelines for treatment of sexually transmitted diseases. MMWR Morb Mortal Wkly Rep 1998; 47 (RR-1):59–63.
2. Knapp JS. Neisseria gonorrhoeae resistant to ciprofloxacin and ofloxacin. Sex Transm Dis 1998; 25: 425–426.
3. De La Fuente L, Vázquez JA. Analysis of genetic variability of penicillinase nonproducing Neisseria gonorrhoeae strains with different levels of resistance to penicillin. J Med Microbiol 1992; 37: 96–99.
4. Tanaka M, Nakayama H, Haraoka M, et al. Antimicrobial resistance of Neisseria gonorrhoeae and high prevalence of ciprofloxacin-resistant isolates in Japan, 1993–1998. J Clin Microbiol 2000; 38: 521–525.
5. Corkill JE, Percival A, Lind M. Reduced uptake of ciprofloxacin in a resistant strain of Neisseria gonorrhoeae and transformation of resistance to other strains. J Antimicrob Chemother 1991; 28: 601–604.
6. Ehret JM, Judson FN. Quinolone-resistant Neisseria gonorrhoeae: the beginning of the End?. Sex Transm Dis 1998; 25: 522–526.
7. Knapp JS, Washington JA, Doyle LJ, Neal SW, Parekh MC, Rice RJ. Persistence of Neisseria gonorrhoeae strains with decreased susceptibilities to ciprofloxacin and ofloxacin in Cleveland, Ohio, from 1992 through 1993. Antimicrob Agents Chemother 1994; 38: 2194–2196.
8. Kam KM, Wong PW, Cheung MM, Ho NKY, Lo KK. Quinolone-resistant Neisseria gonorrhoeae in Hong Kong. Sex Transm Dis 1996; 23: 103–108.
9. Tapsall JW, Phillips EA, Shultz TR, Thacker C. Quinolone-resistant Neisseria gonorrhoeae isolated in Sydney, Australia, 1991 to 1995. Sex Transm Dis 1996; 23: 425–428.
10. Belland RJ, Morrison SG, Ison C, Huang WM. Neisseria gonorrhoeae acquires mutations in analogous regions of gyr A and par C in fluoroquinolone-resistant isolates. Mol Microbiol 1994; 14: 371–380.
11. Deguchi T, Yasuda M, Asano M, et al. DNA gyrase mutations in quinolone-resistant clinical isolates of Neisseria gonorrhoeae. Antimicrob Agents Chemother 1995; 39: 561–563.
12. Tanaka M, Fukuda H, Hirai K, Hosaka M, Matsumoto T, Kumazawa J. Reduced uptake and accumulation of norfloxacin in resistant strains of Neisseria gonorrhoeae isolated in Japan. Genitourin Med 1994; 70: 253–255.
13. Berrón S, Vázquez JA, Giménez MJ, de la Fuente L, Aguilar L. In vitro susceptibilities of 400 Spanish isolates of Neisseria gonorrhoeae to Gemifloxacin and 11 other antimicrobial agents. Antimicrob Agents Chemother 2000; 44: 2543–2544.
14. Birley H, McDonald P, Carey P, Fletcher J. High-level ciprofloxacin resistance in Neisseria gonorrhoeae. Genitourin Med 1994; 70: 292–293.
15. Deguchi T, Yasuda M, Nakano M, et al. Quinolone-resistant Neisseria gonorrhoeae: correlation of alterations in the gyr A subunit of DNA gyrase and the par C subunit of topoisomerase IV with antimicrobial susceptibility profiles. Antimicrob Agents Chemother 1996; 40: 1020–1023.
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