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.
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.
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
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.
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