Neisseria gonorrhoeae is the causative agent of one of the most important sexually transmitted diseases, gonorrhea. Gonorrhea remains an important clinical and public health problem throughout the world. The incidence of gonorrhea has decreased in Europe1,2 and in the United States3 over the past 2 decades. However, in Korea, the prevalence among university students and adolescents accommodated in youth shelters was 0.4% and 28%, respectively.4,5 Gonococcal urethritis is more common among young persons and persons held in correctional facilities.6 Historically, gonorrhea has responded well to antimicrobial agents, including penicillin G and fluoroquinolones, but the emergence of antimicrobial resistance in N. gonorrhoeae has compromised the treatment and control of the disease.7,8
The proportion of penicillinase-producing N. gonorrhoeae (PPNG) has gradually decreased in some countries.9,10 In certain countries, fluoroquinolones are no longer recommended for empirical treatment of gonorrhea because of rapid rise in resistance.11,12 The World Health Organization (WHO) recommends the use of antigonococcal agents that can cure >95% of patients,6,13 but with the increasing prevalence of resistant isolates, empirical selection of antimicrobial agents has become almost impossible. Trends in antimicrobial resistance vary significantly with time and depending on geographic location.12,14–18 Nevertheless, routine isolation and susceptibility testing of N. gonorrhoeae is rarely performed because of the fastidious nature of the organism, cost of procedure, use of nucleic acid amplification assays, and syndromic diagnosis. Therefore, national and international surveillance data of antimicrobial resistance is required for the empirical treatment and prevention of gonococcal infections,19 but only a few surveillance studies have been conducted in Korea since 1992.20–22
The aims of this study were to determine recent trends in antimicrobial susceptibility of N. gonorrhoeae, particularly to β-lactams and to fluoroquinolones; to analyze recent use of antigonococcal agents in Korea; and to investigate the relationship between fluoroquinolone resistance and amino acid substitutions in the fluoroquinolone resistance-determining regions (QRDR) of gyrA and parC.
MATERIALS AND METHODS
N. gonorrhoeae Isolates
All laboratory tests were performed at a central laboratory using regional isolates or specimens. A total of 977 N. gonorrhoeae isolates were collected from male patients with urethritis (n = 104) and prostitutes (n = 873) between 2000 and 2006 in Korea. The annual numbers of isolates were 190 in 2000, 177 in 2001, 210 in 2002, 214 in 2003, 91 in 2004, 48 in 2005, and 47 in 2006. Among the 104 isolates from male patients with urethritis, 84 were from a tertiary care hospital in Seoul, and the remaining 9 and 11 isolates were from private urology clinics, 1 in Seoul and 5 in other cities, respectively. Among the 873 isolates from prostitutes, 793 were from a public health center in Seoul, and the rest were from 5 public health centers in other cities, including Busan.
Modified Thayer-Martin agar plates (Becton Dickinson, Cockeysville, MD) were used for isolation, and conventional biochemical tests and the Vitek NHI system (bioMerieux, Marcy l'Etoile, France) were used for species identification. The isolates were stored in 20% skim milk (Difco, Detroit, MI) at −70°C until they were used for this study.
Antimicrobial Susceptibility Testing
The antimicrobial susceptibility of all 997 isolates was determined by the disk diffusion methods recommended by the Clinical and Laboratory Standards Institute (CLSI),23 and by the Western Pacific Region Gonococcal Antimicrobial Surveillance Program of the WHO.13 Following the CLSI method, the disks used were penicillin G (10 units), spectinomycin (100 μg), tetracycline (30 μg), and ciprofloxacin (5 μg) (Becton Dickinson). To detect decreased susceptibility to ceftriaxone, disks of 0.5-μg ceftriaxone (susceptible breakpoint of >18 mm) were used according to the Western Pacific Region Gonococcal Antimicrobial Surveillance Program method.13
Minimum inhibitory concentrations (MICs) of antimicrobial agents were determined to detect subtle changes in susceptibility by the CLSI agar dilution method using GC II agar base supplemented with 1% IsoVitaleX (Becton Dickinson) for 145 randomly selected isolates stratified by year with a statistically random method.24 The antimicrobial agents used were penicillin G (Sigma Chemical, Saint Louis, MO), ceftriaxone (Hanmi, Seoul, Korea), spectinomycin (Kuk Je, Seoul, Korea), cefixime (Dong-A, Seoul, Korea), tetracycline (Pfizer Korea, Seoul, Korea), and ciprofloxacin (Bayer Korea, Seoul, Korea). The approximate inocula of 104 CFU were applied with a Steer's replicator (Craft Machine, Chester, PA) to the agar plates followed by incubation in a 5% CO2 atmosphere at 35°C for 24 hours. β-lactamase production was determined using a cefinase disk (Becton Dickinson). N. gonorrhoeae strain ATCC 49,226, WHO A 100A6, WHO B 100A7, WHO G 100I1, WHO J 100I4, WHO K 300J4 and WHO L 00G 1,003 OKKA20 were used as quality control strains.
Sequencing the QRDR of gyrA and parC
To determine amino acid substitution in QRDR, nucleotide sequences of gyrA and parC were analyzed for 25 ciprofloxacin-resistant (MIC, 2–16 μg/mL), 2 intermediate (MIC, 0.1–0.25 μg/mL), and 3 susceptible (MIC, ≤0.008 μg/mL) isolates in 2006. The primers used were identical to those used by Tanaka et al.25 Polymerase chain reaction (PCR) was carried out in a total volume of 20 μL containing 1 μL of heat-extracted template DNA, 10 pmol of each primer, and PreMix (Bioneer, Cheongwon, Korea) containing 1 U of Taq DNA polymerase. The PCR conditions were 35 cycles of 93°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute, using a thermal cycler (Eppendorf, Hamburg, Germany). The PCR products were purified using a DNA extraction kit (Qiagen, Hilden, Germany) and the nucleotide sequences of the PCR-generated amplicons were analyzed at a commercial laboratory (Macrogen, Seoul, Korea).
Antimicrobial Agents Used to Treat Gonococcal Infection
Recent trends in the usage of antimicrobial agents for the treatment of gonorrhea in Korea were analyzed to determine appropriateness in the context of recent trends of resistance. The analysis was based on prescription data from the Korea Health Insurance Review and Assessment Service for the period of 2002 to 2006. The data are highly reliable because antimicrobial agents are not available without prescription.
Disk diffusion testing of 997 isolates showed that none of the isolates was susceptible to penicillin G, and that the proportion of PPNG decreased significantly, from 64% to 21% during the study period (Table 1). The resistance rate to ciprofloxacin increased significantly from 26% to 83%. None of the isolates was resistant to spectinomycin and ceftriaxone, but the majority (93% to 100%) showed resistance to tetracycline, and 1% to 10% of them were high-level tetracycline-resistant N. gonorrhoeae, depending on the year of isolation. The susceptibility patterns for isolates from men and women were similar (data not shown).
Agar dilution penicillin G susceptibility testing for 145 randomly selected isolates showed an MIC range of 0.12 to >128 μg/mL, MIC50 of 2 μg/mL, and MIC90 of 64 μg/mL (Table 2). MIC90s of penicillin G decreased 16-fold from 64 μg/mL in 2001 to 4 μg/mL in 2006. The MICs of both cefixime and ceftriaxone ranged very wide indicating MIC creep (Table 2). Furthermore, the MIC range of cefixime was slightly higher than that of ceftriaxone. All isolates were susceptible to ceftriaxone, but for 1 isolate, the MIC was 0.25 μg/mL, which is the upper limit of the susceptible breakpoint, whereas 1 isolate was nonsusceptible to cefixime, for which the MIC was 0.5 μg/mL. During the study period, the MIC50s of ciprofloxacin increased 64-fold, from 0.25 to 16 μg/mL, which resulted in an increase in resistant isolates from 17% to 88%, but MIC90s increased only 2-fold, from 8 to 16 μg/mL (Table 2). All isolates were susceptible to spectinomycin; in contrast, majority of the isolates were resistant to tetracycline.
Genetic Alterations in the QRDRs
Ciprofloxacin-resistant and -intermediate isolates showed amino acid substitutions of serine at position 91, and aspartic acid at position 95 in Gyrase A (GyrA), and of aspartic acid at position 86 and of serine at position 87 in ParC subunit of topoisomerase IV (ParC). Among the 25 ciprofloxacin-resistant isolates (MIC 2–16 μg/mL), all had amino acid substitutions (Ser-91-Phe) and 23 had substitutions (Asp-95-Gly in GyrA). Twenty-one isolates had Ser-87-Arg in ParC.
The most common substitution was Ser-91-Phe and Asp-95-Gly in GyrA, and Ser-87-Arg in ParC (20/25, 80%). Two isolates had substitutions only in GyrA (Ser-91-Phe and Asp-95-Gly) with ciprofloxacin MIC of 8 μg/mL. These 2 types were observed in our previous study in 2004, but the others were not. One had the substitutions Ser-91-Phe in GyrA, and Ser-87-Arg in ParC. Another isolate with ciprofloxacin MIC of 4 μg/mL had substitutions at Ser-91-Phe and Asp-95-Ala in GyrA, and Ser-87-Asn in ParC. The third isolate with ciprofloxacin MIC of 2 μg/mL had substitutions at Ser-91-Phe and Asp-95-Gly in GyrA, and Asp-86-Asn in ParC. Two isolates intermediate to ciprofloxacin contained only 1 or 2 amino acid substitutions (Ser-91-Tyr or Ser-91-Phe in GyrA, and Asp-86-Asn in ParC). No amino acid substitutions were detected in the ciprofloxacin-susceptible (MIC ≤0.015 μg/mL) control isolates.
Antimicrobial Agents Used for Gonorrhea Treatment
Spectinomycin was most frequently used to treat gonorrhea (Table 3). The proportion of patients with spectinomycin treatment increased from 53.4% in 2002 to 58.1% in 2006. The use of fluoroquinolones decreased somewhat, from 36.8% in 2002 to 25% in 2006. A comparison of the use of ceftriaxone and cefixime in 2002 and 2006 showed increases from 8.4% to 12.9% and from 1.1% to 3.2%, respectively. Penicillin G and other cephalosporins (e.g., cefotaxime, cefpodoxime, and ceftizoxime) were rarely used.
The prevalence of N. gonorrhoeae infection has decreased substantially in developed countries, but it is still prevalent in many developing countries with an estimated prevalence of 2% in young males in Sub-Saharan Africa, 1% in South and Southeast Asia, and 0.6% in South and Central America.26 The incidence of gonorrhea has decreased in Korea,27 but in 2006 the incidence of gonorrhea was still as high as 12.6% in certain patient groups, such as adolescents accommodated in youth shelters.5 According to data from the Korea Health Insurance Review and Assessment Service, among 35,394 patients treated for gonorrhea in 2006, 19,762 were solely gonorrhea patients excluding patients coinfected with other sexually transmitted diseases. The antimicrobial resistance of N. gonorrhoeae had been a serious problem in Korea. PPNG had been as prevalent as 84% until the late 1990s, and ciprofloxacin-resistant isolates has been increasingly detected since early 2000s.21,22
PPNG produce a TEM-1-type β-lactamase, which confers high-level resistance to penicillin G. The prevalence of PPNG increased steadily through the 1990s and remains higher in many Asian countries than in other developed countries.18 In Korea, proportions of PPNG reached 84% in 1999,21 and had rapidly decreased to 21% in 2006. The decline was also reported in Hong Kong, China, and Japan.9,10,12 Although the prevalence of PPNG decreased, all non-PPNG isolates were nonsusceptible to penicillin G, indicating that penicillin G cannot be used for treatment.
Fluoroquinolones were effectively used for many years to treat gonorrhea, but fluoroquinolone-resistant N. gonorrhoeae (QRNG) increased in some regions.15,18,28–30 In 2008, proportions of QRNG in the Western Pacific region were generally high, although the rates were highly variable, that is, from 0% in Papua New Guinea to 99.9% in China.18 A European surveillance study involving 17 countries from 2006 to 2008 showed an overall resistance rate of 51.2% and a range of 29.3 to 83.3% in 2008.16 In the United States in 2009, 9.6% of 5630 isolates were resistant to fluoroquinolones.3 The 2002 guidelines from the Centers for Disease Control and Prevention in the United States recommended that quinolones not be used for the treatment of infections acquired in Asia and the Pacific, including Hawaii.31
In Korea, in 2000, 90.5% isolates were not susceptible to fluoroquinolone.22 Before the Korean guidelines for gonorrhea treatment were made available in 2008, relevant clinicians became aware of the increase in fluoroquinolone nonsusceptible isolates and started to switch from fluoroquinolone to third generation cephalosporins or to spectinomycin according to the 2002 Centers for Disease Control and Prevention guideline,31 resulting in decreased use of fluoroquinolone from 36.8% in 2002 to 25.0% in 2006.
The CLSI disk diffusion method can accurately discriminate ceftriaxone- and cefixime-susceptible gonococci, but cannot differentiate isolates with subtle decrease in susceptibility. Gonorrhea due to isolates with decreased cefixime susceptibility may not respond to this antimicrobial agent. Therefore, we determined the MICs of these antimicrobial agents by the agar dilution method. Based on the agar dilution method, disk diffusion test showed 20 (14%) of 145 isolates with minor error (an isolate with one susceptibility category difference) for penicillin susceptibility, and 30 (20%) isolates with minor, 1 with major (an isolate with false resistance), and 2 with very major (an isolate with false susceptible) error for ciprofloxacin susceptibility. No discrepancy was found in other antibiotic susceptibilities.
All of the ciprofloxacin-resistant isolates had amino acid substitutions at the 91 and 95 positions in GyrA and a single substitution at position at 86 or 87 in ParC. Of the 7 isolates identified in 2006, 5 had new amino acid substitutions that had not been observed in our previous study.22 This could suggest slow but continued evolution of the genes due to selection for fluoroquinolone resistance, but it was more possible that new QRDR substitutions resulted from importation of new strains rather than de novo evolution. The MIC of fluoroquinolones could be affected by mutations outside the sequence covered by the sensor probe.32 Overexpression of norM can also elevate the MIC of ciprofloxacin and norfloxacin, albeit at a low level.33 The rapid increase of ciprofloxacin-nonsusceptible isolates documented in our study (Table 1) could be due to the use of fluoroquinolones (Table 3) without testing for susceptibility, or due to the spread of ciprofloxacin-resistant isolates from asymptomatic female carriers. In the United States, the prevalence of gonorrhea among males and females age 18 to 29 was 2.3%, and 45% of cases were asymptomatic.34 A literature review showed that even patients in family planning clinics had positive rates of 0.1% to 2.8% for N. gonorrhoeae.35 In a previous Korean study, based on comparisons of PFGE types, an increase in fluoroquinolone-resistant isolates was considered to be due to local spread rather than importation.22 It was also reported that fluoroquinolone resistance in London is no longer associated with importation, but with endemic spread among high risk groups.36
Although the proportion of tetracycline-resistant N. gonorrhoeae in Korea was much lower than in other countries in the Western Pacific region,18 almost all isolates were nonsusceptible to tetracycline, indicating that this antimicrobial agent is not useful for the treatment of gonorrhea. Multidrug resistance in N. gonorrhoeae led to a slight increase in the use of third-generation cephalosporins and spectinomycin in Korea. In 2002, only 8.4% and 1.1% of patients were treated with parenteral ceftriaxone and oral cefixime, respectively. In 2006, the rates increased slightly to 12.9% and 3.2%. The MIC range of ceftriaxone was slightly lower than that of cefixime, but MIC creep was apparent: there were ≥32-fold difference between the lowest and highest MICs. The MIC range of ceftriaxone for isolates collected from 1997 to 1999 in our previous study21 was ≤0.008 to 0.06 μg/mL, but it was ≤0.008 to 0.25 μg/mL in the current study. The proportion of isolates inhibited by ≥0.06 μg/mL increased from less than 10% of isolates in 1997–1999 to 42% of the isolates in the current study. In a study in the United Kingdom, the proportion of isolates for which MICs of ceftriaxone and cefixime were 0.125 to 0.25 μg/mL increased from none in 2005 to 0.4% and 2.8%, respectively, in 2008. Emergence of cefixime-nonsusceptible isolate in Korea underscores the need to monitor the trend further.
In conclusion, during the study period of 2000 to 2006 in Korea, the incidence of PPNG decreased significantly, but none of the isolates were susceptible to penicillin G. Inappropriate use of fluoroquinolone was frequent due to a lack of awareness of the rapid increase in resistance to this antimicrobial agent. All isolates were susceptible to spectinomycin. The MICs of ceftriaxone was within the susceptible range for all isolates, but MIC creep was apparent. The MICs of cefixime were slightly higher than those of ceftriaxone, and it was 0.5 μg/mL (nonsusceptible) for 1 isolate.
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