Abstract
Periodic monitoring of antimicrobial susceptibility of Neisseria gonorrhoeae is essential for early detection of emergence of drug resistance. A total of 343 gonococcal strains isolated from high-risk and general populations in Bangladesh from 1997 to 1999 were studied. The MICs of penicillin, tetracycline, ciprofloxacin, ceftriaxone, and spectinomycin for the isolates were determined by the agar dilution method. Of the isolates from 1997, 9% were resistant (MIC ≥ 1.0 μg/ml) to ciprofloxacin, while 41 and 49% of the isolates from 1998 and 1999, respectively, were resistant to ciprofloxacin. Of the N. gonorrhoeae isolates from 1998 and 1999, 1.2 and 3.6%, respectively, both were penicillinase producing and displayed plasmid-mediated tetracycline resistance.
Despite a sharp decline in the incidence of gonococcal infection in developed countries during the last decade, gonorrhea remains one of the most common sexually transmitted infections (STIs) in developing countries and is a global health problem (5). The emergence of resistance to antimicrobial agents in Neisseria gonorrhoeae, resulting from both wide dissemination of resistant clones and the emergence of strains with novel resistance mechanisms, is a major obstacle in the control of gonorrhea (6).
Strategies for control of gonorrhea have relied on the use of highly effective and often single-dose therapy administered at the time of diagnosis. In response to the emergence of penicillinase-producing N. gonorrhoeae (PPNG), N. gonorrhoeae with plasmid-mediated tetracycline resistance (TRNG), and N. gonorrhoeae with chromosomally mediated resistance to penicillin and/or tetracycline (CMRNGPT), the Centers for Disease Control and Prevention, Atlanta, Ga., has advocated the use of expanded-generation cephalosporins or fluoroquinolones as the first line of therapy for uncomplicated gonorrhea (16).
An increase in the prevalence of ciprofloxacin-resistant N. gonorrhoeae with failure to respond to single-dose therapy with 500 mg of ciprofloxacin or 400 mg of ofloxacin has been reported from many countries, including Bangladesh (3, 8, 14, 15). Female sex workers (FSWs) have been considered to be a high-risk population for STIs. Due to high-risk behavior they are often infected by their clients and subsequently may transmit the infections to other clients. In most parts of Asia and Africa, 80 to 90% of the STIs, including gonorrhea, are transmitted through FSWs (1, 6). It has recently been shown that as many as 35 to 42% FSWs in Bangladesh are culture positive for N. gonorrhoeae (2, 12).
Periodic monitoring of the antimicrobial susceptibility profile of N. gonorrhoeae strains prevalent in high-risk groups such as FSWs provides essential clues regarding the emergence of drug resistance and treatment options. We present here the data from our antimicrobial susceptibility surveillance of N. gonorrhoeae isolates obtained during the period from 1997 to 1999. The results enabled us to demonstrate the sudden rise in fluoroquinolone resistance and the appearance of both PPNG and TRNG isolates in Bangladesh.
MATERIALS AND METHODS
Bacterial strains.
N. gonorrhoeae strains isolated from different parts of Bangladesh during the period from 1997 to 1999 were studied. The identity of the organism was confirmed by colony morphology, Gram staining, oxidase and catalase tests, and carbohydrate utilization test. Isolates were kept at −70°C in Trypticase soy broth with 20% glycerol until further study.
β-Lactamase test.
All penicillin-resistant isolates were tested for β-lactamase production by a paper acidometric method as described earlier (17).
MICs.
MICs of penicillin, tetracycline, spectinomycin, ceftriaxone, and ciprofloxacin were determined by an agar dilution method as described earlier (10, 11). Five N. gonorrhoeae reference strains, WHO A to WHO E, for which the MICs are known were included for quality control in each test. Each test was repeated thrice. The antimicrobial susceptibility was judged according to breakpoint criteria defined by the NCCLS (11). Twofold serial dilutions of the following antibiotics were used: penicillin (0.03 to 64 μg/ml; Sigma, St. Louis, Mo.), tetracycline (0.03 to 64 μg/ml; Sigma), ciprofloxacin (0.004 to 4 μg/ml; Bayer, Hampshire, United Kingdom), spectinomycin (2.0 to 128 μg/ml; Upjohn, Puurs, Belgium), and ceftriaxone (0.004 to 0.5 μg/ml; Sigma).
Phenotypic characterization.
The criteria used for phenotypic characterization of N. gonorrhoeae based on plasmid- and chromosomally mediated resistance to penicillin and tetracycline were as described earlier (13).
RESULTS
A total of 347 N. gonorrhoeae strains isolated from 1997 to 1999 were studied. Of them 137 were from 1997, 73 were from 1998, and 137 were from 1999. Among the 1997 isolates, 94 were from street-based FSWs in Dhaka, the capital of Bangladesh, and 36 isolates were from brothel-based FSWs from Tangail (located 150 km northwest of Dhaka) and from females attending primary health care delivery clinic in Dhaka. All 1998 isolates were from street-based FSWs in Dhaka. Among the 1999 isolates, 110 were from street-based FSWs in Dhaka, and 27 were from brothel-based FSWs from Jessore (located 275 km southwest of Dhaka).
Isolates' antimicrobial susceptibilities to penicillin, tetracycline, ciprofloxacin, ceftriaxone, and spectinomycin, determined by the agar dilution method using the criteria established by the NCCLS, are shown in Fig. 1a to e. The prevalence of PPNG and TRNG among isolates from 1997, 1998, and 1999 was 16 and 6.5%, 10 and 13.5%, and 10 and 21.5%, respectively (Fig. 1f), and 1.2 and 3.6% of the isolates from 1998 and 1999, respectively, were both PPNG and TRNG.
FIG. 1.
Antimicrobial susceptibilities and prevalence of drug resistance among isolates. (a to e) Antimicrobial susceptibilities of penicillin (a), tetracycline (b), ciprofloxacin (c), ceftriaxone (d), and spectinomycin (e) for N. gonorrhoeae strains in this study from 1997 to 1999. Susceptible strains (open bars), strains with reduced susceptibility (striped bars), and resistant strains (solid bars) are indicated. The breakpoint criteria used for assessing susceptibility were as recommended previously (10, 11). (f) Distribution of PPNG and TRNG among the isolates in this study from 1997 to 1999. PPNG (open bars), TRNG (striped bars), and PPNG/TRNG (solid bars) are indicated.
Among the isolates from 1997, 9% were resistant (MIC ≥ 1.0 μg/ml), 10% had reduced susceptibility, and 81% were susceptible to ciprofloxacin. During 1998 and 1999, 41 and 49% of the isolates, respectively, were resistant to ciprofloxacin (Fig. 1c). During these two years the proportion of isolates with reduced susceptibility declined to 8 and 4%, respectively.
The MICs at which 50 and 90% of the isolates tested were inhibited (MIC50 and MIC90, respectively) by ciprofloxacin from 1997 to 1999 were further calculated. The MIC50 was 0.015 μg/ml in 1997 versus 0.5 μg/ml in 1998 and 1999; the MIC90 was 1 μg/ml in 1997 versus 8 μg/ml in 1998 and 1999.
Based on plasmid- and chromosomally mediated resistance to penicillin and tetracycline, the isolates were phenotypically categorized into seven different groups (Table 1). Patterns of ciprofloxacin susceptibilities of isolates from different phenotypic categories were further analyzed (Table 2). Nine (75%), 24 (85.7%), and 39 (57.5%) ciprofloxacin-resistant isolates from 1997, 1998, and 1999 were CMRNGPT. Among the ciprofloxacin-resistant isolates from 1998 and 1999, 3.6 and 4.5% were PPNG, respectively.
TABLE 1.
Phenotypic categories of N. gonorrhoeae isolates from 1997 to 1999 based on plasmid- and chromosomally mediated resistance to penicillin and tetracycline
| Category | Criteria | No (%) of isolates meeting criteria in yr
|
||
|---|---|---|---|---|
| 1997 | 1998 | 1999 | ||
| PPNG | β-Lactamase positive; MIC of tetracycline ≤ 16 μg/ml | 22 (16.1) | 6 (8.3) | 11 (8.0) |
| Plasmid-mediated TRNG | β-Lactamase negative; MIC of tetracycline ≥ 16 μg/ml | 10 (7.3) | 10 (14) | 24 (17.4) |
| PPNG-TRNG | β-Lactamase positive; MIC of tetracycline ≥ 16 μg/ml | 0 | 1 (1.2) | 5 (3.6) |
| CMRNGPT | Non-PPNG, non-TRNG; MIC of penicillin ≥ 2 μg/ml; MIC of tetracycline ≥ 2 μg/ml | 33 (24.1) | 24 (32) | 13 (9.4) |
| CMRNGP | Non-PPNG, non-TRNG; MIC of penicillin ≥ 2 μg/ml; MIC of tetracycline ≤ 2 μg/ml | 9 (6.6) | 5 (6.8) | 6 (4.3) |
| CMRNGT | Non-PPNG, non-TRNG; MIC of tetracycline ≥ 2 μg/ml | 34 (24.8) | 19 (26) | 47 (34.1) |
| N. gonorrhoeae susceptible to both penicillin and tetracycline | MIC of penicillin ≤ 2 μg/ml; MIC of tetracycline ≤ 2 μg/ml | 29 (21.2) | 9 (12.3) | 31 (22.6) |
TABLE 2.
Ciprofloxacin susceptibility patterns of different phenotypic categories of N. gonorrhoeae isolated from 1997 to 1999
| Isolation yr | Susceptibility | No (%) of isolates belonging to indicated phenotype(s)
|
||||||
|---|---|---|---|---|---|---|---|---|
| PPNG | TRNG | PPNG-TRNG | CMRNGPT | CMRNGP | CMRNGT | Penicillin and/or tetra- cycline susceptible | ||
| 1997 | Susceptible (n = 111) | 16 (14.4) | 8 (7.2) | 0 (0) | 23 (20.7) | 7 (6.3) | 30 (27.0) | 27 (24) |
| Intermediate (n = 14) | 6 (42.9) | 1 (7.1) | 0 (0) | 6 (42.9) | 0 (0) | 1 (7.1) | 0 (0) | |
| Resistant (n = 12) | 0 (0) | 1 (8.3) | 0 (0) | 4 (33.3) | 2 (16.7) | 3 (25) | 2 (16.7) | |
| 1998 | Susceptible (n = 37) | 3 (8.1) | 7 (18.9) | 0 (0) | 12 (32.4) | 2 (5.4) | 8 (21.6) | 5 (13.5) |
| Intermediate (n = 6) | 2 (33.3) | 2 (33.3) | 0 (0) | 0 (0) | 0 (0) | 1 (16.7) | 1 (16.7) | |
| Resistant (n = 30) | 1 (3.6) | 1 (3.6) | 1 (3.6) | 11 (39.3) | 3 (10.7) | 10 (35.7) | 3 (10) | |
| 1999 | Susceptible (n = 64) | 8 (12.5) | 8 (12.5) | 5 (7.8) | 5 (7.8) | 4 (6.3) | 16 (25) | 18 (28.1) |
| Intermediate (n = 6) | 0 (0) | 2 (33.3) | 0 (0) | 1 (16.7) | 1 (16.7) | 0 (0) | 2 (33.3) | |
| Resistant (n = 67) | 3 (4.5) | 14 (20.9) | 0 (0) | 7 (10.4) | 1 (1.5) | 31 (45.6) | 11 (16.4) | |
DISCUSSION
The control of gonococcal infection is important given the high incidence of acute infections, complications, and sequelae and the role of gonococci in facilitating human immunodeficiency virus acquisition and transmission (9). The knowledge of antimicrobial susceptibility of N. gonorrhoeae is a prerequisite for proper treatment and control of the disease. In Bangladesh there is no established antimicrobial susceptibility surveillance for N. gonorrhoeae. In the absence of laboratory data and an established monitoring system, selection of appropriate antibiotics for empirical treatment of gonorrhea is difficult.
Ciprofloxacin and ofloxacin are currently recommended by the World Health Organization and the Centers for Disease Control and Prevention for the treatment of uncomplicated gonorrhea in areas where multidrug-resistant strains are common, such as Southeast Asia and Central Africa. Ciprofloxacin has been used extensively in Bangladesh, since it is relatively cheap and effective and only a single oral dose is required. As a consequence of the large-scale use of this group of antimicrobials in areas where over-the-counter availability of drugs without prescription is common, a substantial increase of resistant strains may occur. Self-medication among sex workers in the Philippines has been shown to play a major role in the development of antimicrobial resistance, and a similar practice is also common in Bangladesh (7). The appearance of a high prevalence of ciprofloxacin-resistant isolates in our study suggests that FSWs in Bangladesh are exposed to ciprofloxacin and that resistance has developed under selective antimicrobial pressure.
There has been a remarkable increase in antimicrobial resistance among N. gonorrhoeae strains in many developing countries in recent years (4). The frequency of fluoroquinolone-resistant strains has also been increased dramatically in recent years. It is interesting that in our present study, 9% of the gonococcal isolates were resistant in 1997, 41% were resistant in 1998, and 49% were resistant in 1999, a significant rise in resistance in a very short period of time.
In our present study 9 (75%), 24 (85.7%), and 39 (57.5%) ciprofloxacin-resistant isolates from 1997, 1998, and 1999 were CMRNGPT. A similar pattern has also been reported earlier from the United States and Thailand (4, 8). The frequent appearance of elevated ciprofloxacin MICs in CMRNGPT strains remains unexplained, because the mechanism of ciprofloxacin resistance is different from those of penicillin and tetracycline resistance.
Although a decrease in the prevalence of PPNG and resistance to penicillin was observed, an increase in the prevalence of TRNG and PPNG-TRNG isolates appeared in 1998 and 1999.
High-level resistance to penicillin, tetracycline, and ciprofloxacin in the present study has rendered these antimicrobials unacceptable for empirical treatment of gonorrhea. The isolates tested demonstrated high rates of susceptibility to broad-spectrum cephalosporin and spectinomycin, with susceptibility rates of 95 and 100%, respectively.
The present study demonstrates the need for a surveillance system for continuous monitoring of the antimicrobial susceptibility in N. gonorrhoeae for determination of optimal treatment regimens.
Acknowledgments
This research was funded by the ICDDR, B: Center for Health and Population Research, which is supported by countries and agencies that share its concern for the health problems of developing countries. Current donors providing unrestricted support include the aid agencies of the governments of Australia, Bangladesh, Belgium, Canada, Saudi Arabia, Sweden, Switzerland, the United Kingdom, and the United States; international organizations include the United Nations Children's Fund (UNICEF) and the USAID (grant 207491). ICDDR, B acknowledges with gratitude the commitment of USAID to the center’s research effort.
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