GONORRHEA IS A MAJOR sexually transmitted infection (STI) worldwide and is an important public health problem. Although the number of reported cases in Western Europe and the United States has declined in recent years, gonorrhea is the second most reported STI in China. 169,715 cases of gonorrhea were reported to the National Center for STD Control, CDC, China, in 2005.1 Increased antimicrobial resistance of Neisseria gonorrhoeae poses a challenge for successful treatment and continuing surveillance of antimicrobial resistance is essential to monitor both the emergence and spread of resistance. Such monitoring has an integral place in helping to determine the most appropriate choice of antibiotics for both individual and public health management.2 A national surveillance program to monitor antimicrobial resistance of N. gonorrhoeae was initiated in China in 1987, which has joined in WHO Western Pacific Regional (WPR) Resistance Surveillance Programme since 1992. Surveillance data from 1993 to 1998 indicated that, although chromosomally mediated resistance to both penicillin and tetracycline was high (>60%), plasmid-mediated resistance was relatively low (<9%) at that time. The average prevalence of ciprofloxacin-resistant isolates was 34.25% during that period.3 The STD clinic at the National Center for STD Control in Nanjing is one of the centers that participate in the national surveillance network. Between 1999 and 2006, a change occurred in the antimicrobial resistance of N. gonorrhoeae isolates from Nanjing; the proportion of plasmid-mediated penicillinase-producing (PPNG) and high-level tetracycline resistant (TRNG) strains of different types increased significantly and isolates resistant to ciprofloxacin became prevalent and endemic. During this period, the primary therapies for uncomplicated gonorrhea recommended by Ministry of Health4 included ceftriaxone 250 mg or spectinomycin 2 g intramuscularly, or ciprofloxacin 500 mg or ofloxacin 400 mg orally, or ceftoxime 1 g intramuscularly. Clinically, the first 2 therapies were commonly used in Nanjing area.
Materials and Methods
N. gonorrhoeae strains were isolated from patients with uncomplicated gonorrhea attending the STD clinic at the National Center for STD Control in Nanjing. Isolates were collected consecutively from March to June and September to November each year. Around 90% of strains were isolated from male patients with urethritis. These isolates represent 40–60% of all culture confirmed cases. Characteristics of patients represented in the samples have remained relatively constant over the period and almost all are heterosexual men and women, and around 85% came from Nanjing area and 15% from other parts in Jiangsu province and adjacent provinces. Urethral or cervical swab specimens were inoculated onto Thayer-Martin medium and cultured in candle jars at 36°C for 24–48 hours. All isolates were identified by colonial morphology, Gram stain, and oxidase testing. Presumptive identification is regarded as sufficient for diagnosis in many setting, particularly for samples from genital tract.5 Gonococcal isolates that met these criteria were picked and subcultured onto GC Agar Base (Difco, Detroit, MI) containing 10% defibrinated sheep blood; colonies were swabbed, suspended in skimmed milk, and frozen at −70°C until used for antimicrobial testing.
Antimicrobial Susceptibility Testing
β-Lactamase production was determined by paper acidometric testing to screen PPNG. Antimicrobial susceptibility testing was performed by agar plate dilution using GC Agar Base (Difco, Detroit, MI) containing 10% defibrinated sheep blood. Concentrations of antibiotics used were: penicillin (SmithKline Beecham), 0.016–4.0 mg/L; ciprofloxacin (Bayer Leverkussen), 0.016–4.0 mg/L; spectinomycin (Upjohn), 2.0–128 mg/L; ceftriaxone (Roche), 0.002–0.50 mg/L; tetracycline (Sigma), 8–16 mg/L (only to screen for strains with high level [plasmid mediated] resistance to tetracycline[TRNG] according to WHO WPR Resistance Surveillance Programme). After thawing and overnight growth on GC Agar Base (Difco, Detroit, MI) containing 10% defibrinated sheep blood, gonococci were suspended in 0.03 mol/L phosphate buffer (pH 7.2), and an inoculum of 104 cfu per spot was added to agar surfaces with a multipoint inoculator. Plates were incubated for 24 hours in a candle jar at 36°C. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of the antibiotic tested that inhibited the growth of the inoculum completely. Control strains of WHO isolates of N. gonorrhoeae: A (spectinomycin-resistant strain), 97QA3 (PPNG) and 97QA4 (TRNG and ciprofloxacin-resistant strain) (kindly provided by Dr. J. W. Tapsall, Prince of Wales Hospital, Sydney, Australia) were included in each assay. We also participated in external quality insurance program by WHO WPR Resistance Surveillance Programme and obtained satisfactory results. Antimicrobial susceptibilities were interpreted according to the criteria by WHO WPR Resistance Surveillance Programme guidelines.6 Isolates with MICs ≥1.0 mg/L to penicillin, ciprofloxacin, were classified as resistant; isolates with MICs of 0.6–0.50 mg/L to penicillin, ciprofloxacin were classified as having decreased susceptibility. Isolates with MICs ≥0.06 mg/L to ceftriaxone were classified as less susceptible.7 Isolates with MICs ≥128 mg/L to spectinomycin were deemed resistant and isolates with MICs of ≥16 mg/L to tetracycline were classified as exhibiting plasmid-mediated resistance (TRNG). Molecular detection and typing of resistant plasmids was further performed for PPNG (β-lactamase positive and tetracycline MIC <16 mg/L), TRNG (tetracycline MIC ≥16mg/L and β-lactamase negative), and PP/TRNG (tetracycline MIC ≥16mg/L and β-lactamase positive).
Subtyping of tetM Gene in TRNG Isolates
Presence of the tetM determinant was investigated by a single tube PCR method.8 Oligonucleotide primers and amplification conditions used to identify American and Dutch types of the tetM gene were the same as those described.8 To prepare the DNA template, whole bacterial cells were harvested from an overnight growth on GC agar and suspended in distilled water and adjusted to an optical density of 0.15 at 670 nm. Bacteria were lysed by heating (100°C for 10 minutes), solutions centrifuged, and supernatants removed and used as templates. Two positive control strains carrying the Dutch tetM (WHO 97QA3) and the American tetM (WHOG) and 2 negative controls (1 strain with chromosomally mediated resistance to tetracycline, and a tube containing distilled water) were included in each assay.
Subtyping of β-Lactamase-Encoding Plasmids in PPNG Isolates
Presence of β-lactamase-encoding plasmids was detected in PPNG isolates by a PCR-based assay, which differentiates among Asian, African, and the Toronto β-lactamase-encoding plasmids of N. gonorrhoeae. Oligonucleotide primers and amplification conditions used to identify the plasmids were the same as those described.9 Control plasmids, pJD4 (Asian type), pJD5 (African type), and pJD7 (Tronto type), were kindly provided by J.R. Dillon (University of Saskatchewan, Canada).
Antimicrobial Susceptibility Testing
One thousand two hundred eight gonococcal isolates collected between 1999 and 2006 were examined. The rate of PPNG increased yearly, from 8.0% (9 of 112) in 1999 to 57.36% (113 of 197) in 2004 but decreased to 44.44% (88 of 198) in 2006 (Table 1). MICs to penicillin for PPNG were ≥8 mg/L. Prevalence of TRNG rose from 1.8% (2 of 112) in 1999 to 32.82% (65 of 198) in 2006. Among 260 TRNG strains, 177 (68.08%) were also PPNG.
Among non-PPNG, the rate of chromosomally mediated resistance to penicillin (PenR) changed in both directions between 57.84% (2003) and 87.80% (2000) during the 6-year period and the change is statistically significant (P = 0.01, Fisher Exact), but the MIC50 and MIC90 to penicillin was unchanged: 1 and 2 mg/L, respectively. Chromosomally mediated resistance to ciprofloxacin (QRNG) was detected in 83.93% (94 of 112) of strains in 1999, rising to 98.99% (196 of 198) in 2006. In total, 46.69% (564 of 1208) of strains had an MIC to ciprofloxacin of ≥8mg/L. Between 1999 and 2000, none of the gonococcal isolates tested had been resistant to spectinomycin but from 2001 onward, 1 or 2 spectinomycin-resistant N. gonorrhoeae strains were identified each year (0.5–1% of the number of isolates annually). The ceftriaxone MICs ranged from 0.002 to 0.25 mg/L. The percentage of gonococcal isolates with decreased susceptibility to ceftriaxone (MIC ≥0.06 mg/L) rose from 17.86% (20 of 112) in 1999 to 57.89% (110 of 190) in 2003, and then decreased to 31% in 2004 and 2005, and rose to 38.38% (76 of 198) in 2006 (Table 2). Among isolates with decreased susceptibility to ceftriaxone 89.9% (413 of 459) also exhibited resistance to both penicillin and ciprofloxaxin, i.e., multiple resistant strains.
Subtyping of the tetM Gene in TRNG Isolates
Two hundred sixty TRNG were investigated. The tetM gene was identified in all strains with tetracycline MICs ≥16 mg/L. 99.23% (258 of 260) contained the Dutch-type tetM gene, and 2 strains, isolated from 2003 and 2005, contained the American-type tetM gene.
Subtyping of β-Lactamase-Encocding Plasmids in PPNG Isolates
Four hundred nine PPNG strains isolated from 2001 to 2006 were examined, and all contained the Asian-type plasmid. Twenty-three PPNG strains were lost during subculture or storage.
Compared with an earlier report that examined antimicrobial susceptibility of strains of N. gonorrhoeae from 1994 to 1995, the prevalence of chromosomally mediated penicillin-resistant strains of N. gonorrhoeae in Nanjing10 remained high between 1999 and 2006, and plasmid-mediated (high-level) resistance to penicillin and tetracycline also increased significantly. From 1994 to 1995, the rate of PPNG was 0.72%, and no TRNG were identified.10 In the current study, prevalence of PPNG increased yearly from 8.0% in 1999 to 57.36% in 2004, and then decreased to 44.44% in 2006. TRNG was first identified in 1999, in Nanjing, and accounted for 1.8% of strains then, increasing to 32.82% of strains in 2006. Another study conducted in nearby Shanghai also showed an increase in the prevalence of PPNG and TRNG strains from 1988 to 2002.11 High-level tetracycline resistance in N. gonorrhoeae is mediated by the tetM determinant, which is integrated into the gonococcal conjugative plasmid. Although a novel tetM determinant has been identified recently from Uruguay,12 2 other plasmid types, the American type and the Dutch type, generally have been found in TRNG.13 Among TRNG strains isolated from Canada,14 the Dutch-type tetM gene predominanted (79.35%) from 1986 to 1997. In Scotland,15 TRNG strains that had originated from Asia or South America between 1992 and 1998, contained the Dutch-type plasmid, whereas strains imported from Africa contained the American type. In the current study, we demonstrated that 258 of 260 (99.23%) of TRNG strains isolated in Nanjing contained the Dutch-type tetM gene. In addition, 21 of 22 (96%) of TRNG strains isolated from Shanghai, Chongqin, Guangzhou, and Fuzhou in 1999–2000, that were subtyped by PCR, carried the Dutch-type tetM gene (unpublished). These data indicate that the Dutch type TRNG predominates in Chinese TRNG, whereas the American type is sporadic. In our study, TRNG strains were screened by recommended criteria for MIC analysis and confirmed by genotype of tetM determinant. It is noteworthy that TRNG strains with low MICs (2.0–8.0 mg/L) for tetracycline but detected by genotyping have been reported.14 Unfortunately, we did not examine the strains with low MICs for tetracycline by PCR-based genotyping.
PPNG strains were first identified in the United States in 197616; in PPNG, resistance to penicillin is mediated by a plasmid carrying the TEM-1 β-lactamase gene. A total of 6 different β-lactamase-encoding plasmids have been identified in PPNG strains but only the Asian and African types have a worldwide distribution. In 1 study conducted in UK, the majority of PPNG from Asian countries harbored the Asian plasmid, and most PPNG known to have been acquired in the UK contained the African plasmid.17 In the current study, we documented that all PPNG strains isolated in Nanjing contained the Asian type β-lactamase-encoding plasmid.
Fluoroquinolones are broad-spectrum antimicrobial agents and have been widely used for the treatment of gonorrhea. However, since the middle 1990s, fluoroquinolone-resistant N. gonorrhoeae (QRNG) have been reported from many countries, particularly in Southeast Asia and other areas located in the Western Pacific.18,19 In Nanjing, surveillance of in vitro susceptibility of N. gonorrhoeae to ciprofloxacin was initiated in 1994. The rate of ciprofloxacin resistance (QRNG) was 2.89% in 1994, increasing to 17.46% in 1995.20 The prevalence of ciprofloxacin-resistant strains increased dramatically during the period of the current study. By 2006, nearly all strains (98.99%) were resistant to ciprofloxacin, and most of the resistance was at a high level (MIC90 ≥8 mg/L). In a study recently reported by Wang et al.,21 clinical isolates collected from several cities in the Jiangsu province of China, including Nanjing, demonstrated complete resistance to ciprofloxacin and 98.9% resistance to levofloxacin. Some studies suggest a correlation between increasing MIC and clinical failure, although the exact MIC cutoff to predict treatment failure still needs to be defined.22 We also found cases of gonorrhea that had failed treatment with fluoroquinolone; MICs to ciprofloxacin of strains isolated from treatment failure patients ranged from 2 to 8 mg/L.23 Quinolone-resistant N. gonorrhoeae (QRNG) strains have now spread widely in China,18 and fluoroquinolones is not recommended for the treatment of gonorrhea in China now.24
Spectinomycin has been recommended as a possible choice for primary therapy for N. gonorrhoeae infection after plasmid-mediated high-level resistance to penicillin and tetracycline emerges and spreads. Spectinomycin resistance has been rare, but after it had been used widely for 3 years in the mid-1980s, clinical treatment failures caused by spectinomycin-resistant strains began to be appear in US military personnel in the Republic of Korea.25 Some spectinomycin resistant strains were detected in Colombia and Venezuela between 1990 and 1998,26 and small numbers have been reported in Papua New Guinea, Vietnam, Bruner, and Korea in WPR GASP surveys.27 Resistance to spectinomycin in N. gonorrhoeae is chromosomally mediated and results from mutations in 16S rRNA genes.28 Spectinomycin is commonly used to treat gonococcal infection in Nanjing and in China at large. A standard treatment regimen is expected to cure 95% or more of gonococcal infections.29 In the current study, 8 spectinomycin-resistant strains were identified from 2001 to 2006. Two were isolated from patients who failed treatment with spectinomycin30; clinical data from the remaining 6 were not available. Currently, the rate of spectinomycin resistance of N. gonorrhoeae is below 5% (0.5–1%) in Nanjing, indicating that spectinomycin is still effective there and can be used as primary treatment of gonorrhea.
Emergence of diminished susceptibility to third-generation cephalosporins among clinical isolates of N. gonorrhoeae has been observed in some countries since the mid-1990s.31–33 Each year WHO reports a small number of gonococcal isolates with decreased susceptibility to third-generation cephalosporins in the western pacific region that includes China,18 and clinical failures have been reported in Japan in patients treated with these antibiotics.34,35 In the current study, the prevalence of isolates with decreased susceptibility to ceftriaxone increased from 1999 to 2003; thereafter, the rate dropped by nearly 50%. Chinese-manufactured spectinomycin was introduced into Nanjing in 2003, and resulted in decrease use of ceftriaxone for the treatment of gonorrhea, prompting speculation that environmental pressure for the selection of relative ceftriaxone resistance of gonococci may be decreasing. Furthermore, most (89.9%) of the gonococcal isolates with decreased susceptibility to ceftriaxone were multiple resistant strains, exhibiting resistance to both penicillin and ciprofloxaxin. Studies in Japan have documented that the mosaic-like structure of the penicillin-binding protein 2 gene (penA) has been associated with a decrease in susceptibility to penicillin and cephalosporins such as ceftriaxone and cefixime.36,37 However, most recent gonococcal isolates with reduced susceptibility to ceftriaxone isolated in Sydney, Australia, lack the mosaic penA sequence38; therefore, additional factors may contribute to altered ceftriaxone susceptibility.
In conclusion, among N. gonorrhoeae strains isolated in Nanjing, plasmid-mediated resistance, including PPNG and TRNG, increased rapidly between 1999 and 2006. Dutch-type TRNG and Asian-type PPNG were predominant. Chromosomally mediated resistance to both penicillin and ciprofloxacin was also high, but resistance to spectinomycin was sporadic. At this time, ceftriaxone and spectinomycin can be considered effective for the treatment of gonorrhea in Nanjing.
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