ANTIMICROBIAL RESISTANCE of Neisseria gonorrhoeae has been a challenge to the treatment of gonococcal infections for the past 25 years. Penicillinase-producing N gonorrhoeae (PPNG) was first identified in 1976 and has spread worldwide since then. N gonorrhoeae with high-level plasmid-mediated resistance to tetracycline (TRNG) was first reported in the United States in 1985 and became widespread. The prevalence of PPNG and TRNG has been high in many countries in Asia 1 and Africa. 2,3N gonorrhoeae strains with decreased susceptibility to fluoroquinolones have been prevalent in Southeast Asia 4,5 and Western Pacific areas, 1 and they have emerged and increased in European countries such as The Netherlands 6 and England. 7
The prevalence of sexually transmitted diseases (STDs) has been increasing in China since the 1980s. 8 Gonorrhea is the most frequently reported STD in China. Although the proportion of gonorrhea cases among all cases of STD declined from 85.84% during the period of 1977–1988 to 40.72% in 1999, the number of gonorrhea cases reported increased from 157,500 (9.93/100,000 residents) in 1989 to 340,960 (27.54/100,000 residents) in 1999. 9 Data on the antimicrobial susceptibility of N gonorrhoeae are important for its clinical management and STD-control policy-making. In order to monitor the antibiotic susceptibility and to find out the characterization and epidemiology of N gonorrhoeae in China, the antimicrobial susceptibilities of N gonorrhoeae isolates have been surveyed nationally since 1987. This work has been sponsored by the World Health Organization Western Pacific Gonococcal Resistance Collaborative Network since 1992. Findings concerning gonococcal resistance during 1987–1992 were published previously. 10
The appearance of penicillinase-producing N gonorrhoeae and chromosomally mediated penicillin resistance in a high proportion of N gonorrhoeae isolates led to the end of administration of penicillin as the therapy for gonorrhea. The currently recommended primary therapeutic agents for gonorrhea in China are ceftriaxone, cefotaxime, spectinomycin, ofloxacin, and ciprofloxacin. Surveillance of the susceptibility to ciprofloxacin of N gonorrhoeae isolates was initiated in 1995. An increase in the prevalence of ciprofloxacin-resistant N gonorrhoeae has been observed since then. This report summarizes the findings concerning antibiotic resistance of N gonorrhoeae isolates in China from 1993 to 1998.
Materials and Methods
Collection of Strains
N gonorrhoeae isolates were collected from patients with gonorrhea at 10 selected STD clinics at facilities in the northeast, northwest, southern, and eastern regions of China: the National Center for STD and Leprosy Control (in Nanjing), Shanghai Control Center for STD, Fuzhou STD Monitoring Center, Guangxi Institute of Dermatology (in Nanning), Guangdong Provincial Institute of Dermatology (in Guangzhou), Guangzhou Municipal Institute of Dermatology (in Guangzhou), Sichuan Provincial Institute of Dermatology (in Chengdu), the First Hospital in Chongqing City, Dalian Municipal Institute of Dermatology, and Xinjiang Provincial Institute of Endemic Diseases (in Urumqi). Some clinics participated in the collaborative program continuously and others participated intermittently. One hundred to two hundred strains were collected from each site annually for standardized antimicrobial susceptibility testing. Most centers performed the testing locally, but two sent the strains (in dry ice or liquid nitrogen) to the reference laboratory at the National Center for STD and Leprosy Control. This national center provided technical support for other monitoring centers as well. A workshop was held annually to summarize the findings.
The specimens were taken from 3 to 4 cm inside the urethra of male patients and 1 to 1.5 cm inside the cervix of female patients. The specimens were then inoculated into selective media (Thayer-Martin medium or Guiming medium) and cultured in candle jars in an incubator at 36 °C or with 5% CO2 for 24 to 36 hours. All isolates were identified on the basis of gram-stained smears, colony morphology, and a positive oxidase reaction. Sugar fermentation tests and direct fluorescent antibody assays were performed as needed for confirmation. The isolates were subcultured in nonselective media, and several colonies were suspended in skim milk and stored in a freezer at −70 °C.
Antimicrobial Susceptibility Tests
The production of β-lactamase was determined by the acidimetric method. 11 MICs of the following antibiotics were determined by the agar plate dilution method 12: penicillin, tetracycline, spectinomycin, ciprofloxacin, and ceftriaxone. The medium (GC Agar Base; Difco, Detroit, MI) was supplemented with 10% defibrinated sheep blood. (The pure antibiotic powder was kindly supplied by Dr. J. W. Tapsall.) Penicillin and ceftriaxone were dissolved in phosphate-buffered saline (pH, 6.5; 0.1 mol/l). Spectinomycin, tetracycline, and ciprofloxacin were dissolved in distilled water. All were stored at −20 °C and tested in 6 months. The antibiotic solutions were double-diluted to appropriate concentrations and added to the media before testing. The concentrations of antibiotics were as follows: penicillin, 0.015 to 4 μg/ml; tetracycline, 0.25 to 16 μg/ml; ciprofloxacin, 0.015 to 16 μg/ml; spectinomycin, 2 to 128 μg/ml; and ceftriaxone, 0.001 to 0.5 μg/ml.
The gonococcal isolates were passaged for several times in nonselective media. The colonies were scraped and suspensions of 108 cfu/ml were made. The suspension was applied to antibiotic-containing media with a multipoint inoculator or special bacterial loop (104 per point). The media were cultured at 36 °C for 24 hours and the results were read. The MIC was defined as the lowest concentration of an antibiotic that inhibited the growth of gonococci. The World Health Organization criteria for resistance are shown in Table 1. Five World Health Organization reference gonococcal strains, A, B, C, D, and E, were included in each run of tests for quality control. Only when the MICs for reference strains were in the known ranges of concentration were we assured that the concentrations of antibiotics were correct in the media, and the results of each antimicrobial susceptibility test were thus validated (Table 2). After 1997 the MIC breakpoint method was used to determine the plasmid-mediated tetracycline resistance. (TRNG [QA97–3] was kindly provided by Dr. J. W. Tapsall and used as a positive control.)
The rate of resistance of gonococcal isolates to five antibiotics during 1993–1998 in China is shown in Table 3.
A total of 3186 strains were tested for antimicrobial susceptibility from 1993 to 1998. Among the 3186 strains tested for penicillin susceptibility, the MICs for 2125 were higher than 1.0 μg/mL. The rate of resistance to penicillin was 66.70%. PPNG accounted for 8.14% of all tested strains. The rate of resistance to tetracycline was 92.03%, and TRNG accounted for 4.65% of tested strains. Ciprofloxacin-resistant strains accounted for 34.25% of all tested strains. The rates of resistance to spectinomycin and ceftriaxone were relatively low: 0.44% and 0.57%, respectively. Spectinomycin- and ceftriaxone-resistant strains were reported to the National Center for STD and Leprosy Control, but their resistance was not confirmed.
The prevalence of PPNG and TRNG varied between regions. The rate of PPNG was highest in Chengdu City, accounting for 45% of isolates. In the other two northwest cities, Chongqing and Urumqi, the proportion of PPNG was approximately 15%. In the other cities the prevalence of PPNG was approximately 5% or less. Plasmid-mediated high-level tetracycline resistance decreased from 20% in 1993 to 4.65% in 1998. The prevalence of chromosomally mediated resistance to both penicillin and tetracycline was high and remained stable at all surveillance sites. The percentage of ciprofloxacin-resistant N gonorrhoeae increased from 15.48% in 1995 to 55.81% in 1998. Resistance to ciprofloxacin was detected in all regions.
N gonorrhoeae has developed several antibiotic-resistance mechanisms over the past several decades. Increased resistance of N gonorrhoeae strains has limited the options for effective treatment of gonorrhea. Surveillance for antimicrobial resistance is crucial to monitoring the emergence and spread of antibiotic resistance in gonococcal isolates and to provide a rational basis for formulating effective therapies for gonorrhea. There have been a number of reports on antimicrobial susceptibility of N gonorrhoeae. 13–15 However, most of those studies involved isolates collected in a certain year. In contrast, we conducted a 10-year surveillance of the antimicrobial susceptibility of N gonorrhoeae with use of standardized testing methods in 10 representative cities. The data may reflect the trends of resistance in gonococcal isolates and the dynamic changes in resistance rates both locally and nationally.
It seems that penicillin and tetracycline are no longer the drugs of choice for the treatment of gonorrhea, because of the high rates of resistance to each (66.70% and 92.03%, respectively). The proportions of strains with plasmid-mediated resistance—PPNG and TRNG—were 8.14% and 4.05%, respectively. The rates of resistance to spectinomycin and ceftriaxone remained less than 1%. Isolates resistant to spectinomycin and ceftriaxone were recorded sporadically at a few centers. It is fortunate that no clinically significant resistance to spectinomycin and the broad-spectrum cephalosporins has been identified. Recently, Li et al 16 reported the percentages of gonococci isolates resistant to spectinomycin (11.11%) and ceftriaxone (16.48%) in Zhanjiang, in the south of China. These percentages are much higher than our findings, probably because a different area was studied. Decreased susceptibility to ceftriaxone was also noted in gonococci isolated from commercial sex workers in Dhaka, Bangladesh. 17 Chromosomally mediated resistance to penicillin is due to the additive effect of mutations at multiple loci, including penA, mtr, and pen 4B. 18 Several studies have shown a close association between chromosomal resistance to penicillin and resistance to cephalosporins. 18,19 Cephalosporins will probably become less effective in areas where chromosomally mediated resistance to penicillin and tetracycline in N gonorrhoeae isolates is highly prevalent. Since high rates of resistance to both penicillin and tetracycline were documented in our study, susceptibility of N gonorrhoeae isolates to cephalosporin and its clinical efficacy should be closely monitored in China in the future.
In recent decades, resistance to quinolones has been reported from many countries in the Western Pacific Region 1 and Europe, 6,7 as well as the United States. 20 In our study we observed a high, increasing prevalence of resistance to ciprofloxacin. This is in accordance with the observations made in other Asian countries. 4,5 For example, isolates with ciprofloxacin resistance increased significantly from 1993–1994 (6.6%) to 1997–1998 (24.4%) in Japan. 5N gonorrhoeae strains resistant to ciprofloxacin in European countries were mainly imported from Southeast Asia. 6,7 The resistance to ciprofloxacin in China may be due to the indiscriminate use of antimicrobials and self-administered antibiotic treatment. Patients can buy antibiotics in drugstores without a prescription from doctors. Indeed, an association between self-prescribed prophylactic antimicrobial use and gonococcal antimicrobial resistance was observed in the Philippines. 21 Ciprofloxacin and ofloxacin are now the drugs recommended for treatment of gonorrhea in China. The Centers for Disease Control and Prevention (Atlanta, GA) recommends that an antibiotic not be selected as the first-line treatment drug if the rate of resistance to it has reached 5% or more. The high rate of ciprofloxacin-resistant isolates documented in this study and the observation of the clinical failure of quinolones such as ofloxacin and levofloxacin in the treatment of inpatients with gonorrhea (unpublished data) indicate that quinolones may not be suitable for first-line treatment of gonorrhea in China.
In summary, our surveillance data show that a high prevalence of chromosomal resistance to both penicillin and tetracycline has been maintained over many years and that the trend of resistance to ciprofloxacin has significantly increased. The third-generation cephalosporins and spectinomycin are now the options for treatment of gonorrhea in China.
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