DESPITE A SHARP DECLINE in the incidence of gonococcal infections in developed countries during the past 20 years, gonorrhea remains one of the most common sexually transmitted diseases (STDs) in developing countries and a global health problem. 1–4 The problem is compounded by the development of antimicrobial resistance in Neisseria gonorrhoeae, resulting from both wide dissemination of resistant clones and the emergence of strains with novel resistance mechanisms. 5 Southeast Asia is most likely the origin of several strains of antimicrobial-resistant N gonorrhoeae. Penicillinase-producing strains were first isolated in 1976 in Southeast Asia, and gonococcal isolates highly resistant to spectinomycin and to tetracycline emerged there in the 1980s. 6–8 Fluoroquinolone-resistant gonococci appeared in several Asian countries during the early 1990s and reached high levels in some areas such as Hong Kong, the Philippines, Japan, and Singapore. 7,9–13
Commercial sex workers (CSWs) are the most important reservoir of STDs, including gonorrhea. 14 Periodic monitoring of the antimicrobial susceptibility patterns of gonococci isolated from CSWs provides essential clues regarding drug resistance and treatment options. In this study, we examined the antimicrobial susceptibility patterns of gonococcal isolates obtained from female CSWs in Bandung and Jakarta, Indonesia.
Materials and Methods
A total of 267 N gonorrhoeae strains isolated between January and December 1996, as part of an STD screening program, from cohorts of CSWs in Bandung (n = 85) and Jakarta (n = 182) were studied.
Culture of N gonorrhoeae
Endocervical swabs were inoculated on modified Thayer-Martin agar (Becton Dickinson, Cockeysville, MD), and gonococci were presumptively identified by colony morphology, Gram staining, and oxidase activity. Overnight subcultures on nonselective chocolate agar medium were suspended in skimmed milk, frozen at −70 °C, and shipped on dry ice to the University Hospital in Antwerp (Belgium) for further testing. Definite identification was based on sugar acidification.
MICs were determined by the agar dilution method with use of gonococcal agar base (Difco, Detroit, MI) supplemented with 1% IsoVitaleX (Becton Dickinson). 15 The concentrations of the antibiotics (twofold dilutions) were as follows: penicillin (Sigma, St. Louis, MO), 0.015 to 32 μg/ml; tetracycline (Sigma), 0.25 to 64 μg/ml; spectinomycin (Sigma), 4 to 64 μg/ml; ciprofloxacin (Bayer, Brussels, Belgium), 0.001 to 0.06 μg/ml; cefotaxime (Hoechst, Frankfurt am Main, Germany), 0.001 to 0.06 μg/ml; thiamphenicol (Sigma), 0.125 to 8 μg/ml; kanamycin (Sigma), 4 to 64 μg/ml; azithromycin (Pfizer, Groton, CT), 0.004 to 1 μg/ml; and TMP-SMZ 1:19 (Federa, Brussels, Belgium), 0.25 to 16 μg/ml. TMP-SMZ was tested on Diagnostic Sensitivity Test agar (DST; Oxoid, Basingstoke, Hampshire, England), supplemented with 5% lysed (freezing-thawing) horse blood and 1% Kellogg's supplement. 16 Plates were inoculated as recommended by the NCCLS and incubated for 24 hours at 36 °C in a 5% CO2 incubator. The American Type Culture Collection (ATCC) GC strain 49226 was included for quality control in each test. The breakpoint criteria used for assessing sensitivity for penicillin, spectinomycin, tetracycline, ciprofloxacin, and cefotaxime were based on NCCLS recommendations. 15 The breakpoints for kanamycin and TMP-SMZ were as recommended by the WHO. 16 For thiamphenicol, those of Bogaerts et al were used. 17 The breakpoint for azithromycin was 1 μg/ml (Table 1).
β-lactamase production was detected with BR66A sticks (Oxoid). Isolates producing β-lactamase and having a tetracycline MIC <16 μg/mL were classified as PPNG. Tetracycline resistance was considered to be plasmid-mediated in TRNG β-lactamase-negative isolates with a tetracycline MIC of ≥16 μg/ml. β-lactamase-producing isolates with a tetracycline MIC of ≥16 μg/ml were classified as PPNG-TRNG.
Detection of the Tet M Determinant
Detection of the Tet M determinant was adapted from the method of Xia et al. 18 Five μl of a dense bacterial suspension in water was added to 45 μl of a PCR mixture composed of PCR buffer (50 mmol/l KCl, 20 mmol/l Tris-HCl [pH, 8.3], 3 mmol/l MgCl2, and 0.01% gelatin), 0.2 mmol/l of each deoxynucleotide triphosphate, 100 ng of each primer, and 1 U Super Taq HC (HT Biotechnology Ltd., Cambridge, England) and amplified in a thermal cycler (GeneAmp PCR System 9,600; Perkin Elmer, Foster City, CA). PCR products were visualized on a 1.5% agarose gel. Three control strains (strain ppng 949: Tet M-negative; strain 24064: “Dutch” type Tet M; and strain TR137: “American” type Tet M) were included.
Data were analyzed with Epi Info, version 6 (Centers for Disease Control and Prevention, Atlanta, GA). A P value of 0.05 was used for statistical significance.
A total of 267 isolates of N gonorrhoeae from female CSWs in Indonesia were characterized by their antimicrobial susceptibility profile. The results for each antibiotic tested are shown in Table 2. The resistance phenotype is given in Table 3.
Among the 85 isolates from Bandung, 51 (60.0%) were resistant to penicillin (MIC, ≥2.0 μg/ml), of which 46 were PPNG in combination with TRNG, and 33 (38.8%) isolates showed intermediate resistance (MIC, 0.125–1.0 μg/ml). One strain (1.2%) was still susceptible to penicillin (MIC, ≤0.06 μg/ml). Of the penicillin-resistant strains, 5 (9.8%) were TRNG and 46 (90.2%) were PPNG-TRNG. Of the strains with intermediate resistance to penicillin, 28 (84.8%) were TRNG and 5 (15.2%) were PPNG-TRNG. All isolates were resistant to tetracycline (MIC, ≥2.0 μg/ml), including 34 (40.0%) TRNG. In total, 34 strains (40.0%) were TRNG and 51 (60.0%) were PPNG-TRNG. Thirteen isolates (15.3%) showed chromosomal resistance (MIC ≥2.0 μg/ml) and 25 (29.4%) showed intermediate resistance to thiamphenicol (MIC, 1.0 μg/ml). The remaining 47 strains (55.3%) were still susceptible to thiamphenicol (MIC, ≤0.5 μg/ml). Two strains (2.4%) showed intermediate resistance to TMP-SMZ (MIC, 1/19–2/38 μg/ml).
Of the 182 isolates from Jakarta, 129 (70.9%) were resistant to penicillin (MIC, ≥2.0 μg/ml), of which 108 were PPNG in combination with TRNG, and 49 (26.9%) showed intermediate resistance (MIC, 0.125–1.0 μg/ml). The remaining four strains (2.2%) were susceptible to penicillin (MIC, ≤0.06 μg/ml). Among the penicillin-resistant isolates, 19 (14.7%) were TRNG and 108 (83.7%) were PPNG-TRNG. Two of the penicillin-resistant strains (1.6%) showed chromosomally mediated penicillin resistance. Among the strains with intermediate resistance to penicillin, 45 (91.8%) were TRNG, 2 (4.1%) were PPNG-TRNG, and 2 (4.1%) were neither TRNG nor PPNG-TRNG. Of the 179 isolates (98.4%) resistant to tetracycline (MIC, ≥2 μg/ml), 65 (35.7%) were TRNG; in one isolate (0.6%) resistance to tetracycline was chromosomally mediated, and 113 isolates (62.1%) were PPNG-TRNG. The remaining 3 strains (1.6%) from Jakarta showed intermediate resistance to tetracycline (MIC, 0.5–1.0 μg/ml). Chromosomal resistance to thiamphenicol (MIC, ≥2.0 μg/ml) was detected in 67 isolates (36.8%), whereas 45 (24.7%) showed intermediate resistance (MIC, 1.0 μg/ml). The remaining 70 strains (38.5%) were susceptible to thiamphenicol (MIC, ≤0.5 μg/ml). Resistance to thiamphenicol was significantly higher in Jakarta than in Bandung (P < 0.05). Twenty strains (11.0%) showed intermediate resistance to TMP-SMZ (MIC, 1/19–2/38 μg/ml).
In both in Bandung and Jakarta, isolates were susceptible to spectinomycin (MIC, ≤32 μg/ml), ciprofloxacin (MIC, ≤0.06 μg/ml), cefotaxime (MIC, ≤0.5 μg/ml), kanamycin (MIC, ≤32 μg/ml), and azithromycin (MIC, ≤1 μg/ml).
All isolates, except four from Jakarta, harbored the “Dutch” type (700-bp) Tet M determinant. The 4 isolates from Jakarta (1 showing chromosomal resistance and 3 showing intermediate resistance to tetracycline) were Tet M-negative by PCR.
Resistance of N gonorrhoeae to penicillin and tetracycline is well known and has been increasing over the years. The high rates of plasmid-mediated penicillin and tetracycline resistance in Bandung and Jakarta can be related to inappropriate use of these antibiotics, favored by uncontrolled sale and self-medication by CSWs who take penicillin or tetracycline for STD prevention and treatment or for other illnesses.
In Indonesia, a steady increase in the prevalence of PPNG has been noted since the early 1980s. In 1982, a study conducted in an STD clinic in Jakarta showed an 8% prevalence of PPNG, 19 and among CSWs in Surabaya, the rate of PPNG was 13.5%. 20 A study performed in 1991 in Surabaya found a PPNG prevalence of 17.8% in low-paid CSWs and 44.4% in highly paid CSWs, 21 and in 1993 PPNG rates in Surabaya were as high as 85%. 8 A study in Bandung in 1994 revealed a PPNG rate of 54%, 22 which is similar to our findings (60.0%). In Jakarta we found a PPNG prevalence of 62.1%. It therefore seems that PPNG are less frequent in Bandung and Jakarta than in Surabaya. A rather surprising finding was five PPNG strains from Bandung and two from Jakarta showing intermediate resistance to penicillin (MIC, 0.125–1 μg/ml). The MICs and penicillinase production of the strains were confirmed by repeated testing.
High-level plasmid-mediated tetracycline-resistant N gonorrhoeae (TRNG) was first observed in the United States in 1985 and has since spread worldwide. 23,24 We found a similar TRNG prevalence in Bandung (100%) and in Jakarta (97.8%). These findings are comparable to those of Joesoef et al, 8 who found a prevalence of 98% in Surabaya, and those of Djajakusumah et al, 22 with a TRNG prevalence of 94% in Bandung.
TRNG has been associated with the presence of a plasmid approximately 25.2 MDa in size that carries a Tet M tetracycline resistance determinant. Two different plasmid types, “American” and “Dutch,” have been described, on the basis of the restriction endonuclease digestion pattern. 25 All isolates, except four, harbored the 25.2-MDa plasmid producing a single electrophoretic band of 700 bp after PCR. Xia et al 18 found that the smaller, 700-bp PCR product corresponds to the “Dutch” restriction plasmid type, and the larger, 1600-bp PCR product to the “American” restriction plasmid type. All of our TRNG strains carried the “Dutch” plasmid type, as was the case in the study performed by Djajakusumah et al 22 in Bandung. This type has been predominant among isolates from the Netherlands and Asia, while the larger, “American” type tetracycline resistance plasmid is prevalent in the United Kingdom and east and central Africa. 26
Thiamphenicol (a 3.5-g single oral dose) was frequently used until the mid-1990s; it was still included in the recommended treatments for gonorrhea in 1995. However, the relatively high rates of less susceptible and resistant isolates in this study and other studies in Bandung 22,27 and Jakarta and in Surabaya 8 reduce its efficacy for treatment of N gonorrhoeae.
Other primary drugs for the treatment of gonorrhea in Indonesia are spectinomycin, ofloxacin, and ciprofloxacin. In our study and in a separate study in Bandung 22 no resistance to spectinomycin was detected, in contrast to a prevalence of 18% resistance observed in Surabaya. 8 This underlines the importance of regional surveillance in large countries. Fluoroquinolones have been introduced in Indonesia for the treatment of gonorrhea quite recently and are now the national recommended treatment. The recommended dose of ciprofloxacin was 250 mg until 1995, when it was changed to a 500-mg single oral dose. The quinolones have been used for gonorrhea treatment much later in Indonesia than in most other countries in the region. No resistance was detected in our study and two older studies. 8,22 However, the use of fluoroquinolones elsewhere in Southeast Asia and the Pacific resulted in a rapid stepwise development of resistance due to different mutations in the gonococcus, and fluoroquinolone-resistant strains are now firmly established in these areas. These strains account for 10% of isolates in Hong Kong and Japan and for as many as 49% in the Philippines. 28,29
Because of the high rate of resistance to penicillin and tetracycline, both these drugs are no longer useful for the treatment of N gonorrhoeae infection in Bandung and Jakarta. Fluoroquinolones, spectinomycin, and cephalosporins are still reasonable alternatives, but the last two in particular are considerably more costly and less readily available. As no in vitro resistance was shown to kanamycin, a treatment schedule of 2 g in a single injection remains a potential option for first-line gonorrhea treatment. Kanamycin is widely available in Indonesia; a 2-g single intramuscular injection was recommended in 1995 (Working Party of Ministry of Health—STD Program, Senior Dermatovenerologists, and WHO advisor). These recommendations are endorsed by the Association of Dermatovenerology and are widely distributed. As kanamycin is cheap and widely available in Indonesia, we propose that kanamycin be tested in clinical trials to allow for its eventual recommendation for first-line gonorrhea treatment. Continued surveillance of antimicrobial resistance patterns should be part of gonorrhea control in Indonesia. There are plans to include Indonesia in the inter-regional gonococcal antimicrobial susceptibility monitoring system.
During the preparation of this article, a report by Lesmana et al 30 was published on the antibiotic susceptibilities of 122 N gonorrhoeae isolates from Jakarta, which yielded comparable results concerning tetracycline, thiamphenicol, kanamycin, and penicillin. These authors also tested erythromycin and norfloxacin, while we tested azithromycin and TMP-SMZ.
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