Antimicrobial Susceptibility of Neisseria gonorrhoeae Isolated in Bangladesh (1997 to 1999): Rapid Shift to Fluoroquinolone Resistance

Periodic monitoring of antimicrobial susceptibility of Neisseriagonorrhoeae is essential for early detection of emergence ofdrug resistance. A total of 343 gonococcal strains isolatedfrom high-risk and general populations in Bangladesh from 1997to 1999 were studied. The MICs of penicillin, tetracycline,ciprofloxacin, ceftriaxone, and spectinomycin for the isolateswere determined by the agar dilution method. Of the isolatesfrom 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 isolatesfrom 1998 and 1999, 1.2 and 3.6%, respectively, both were penicillinaseproducing and displayed plasmid-mediated tetracycline resistance.

INTRODUCTION

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Introduction

Despite a sharp decline in the incidence of gonococcal infectionin developed countries during the last decade, gonorrhea remainsone 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 Neisseriagonorrhoeae, resulting from both wide dissemination of resistantclones 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 ofhighly effective and often single-dose therapy administeredat the time of diagnosis. In response to the emergence of penicillinase-producingN. gonorrhoeae (PPNG), N. gonorrhoeae with plasmid-mediatedtetracycline resistance (TRNG), and N. gonorrhoeae with chromosomallymediated resistance to penicillin and/or tetracycline (CMRNG^PT),the Centers for Disease Control and Prevention, Atlanta, Ga.,has advocated the use of expanded-generation cephalosporinsor fluoroquinolones as the first line of therapy for uncomplicatedgonorrhea (16).

An increase in the prevalence of ciprofloxacin-resistant N.gonorrhoeae with failure to respond to single-dose therapy with500 mg of ciprofloxacin or 400 mg of ofloxacin has been reportedfrom many countries, including Bangladesh (3, 8, 14, 15). Femalesex workers (FSWs) have been considered to be a high-risk populationfor STIs. Due to high-risk behavior they are often infectedby their clients and subsequently may transmit the infectionsto 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 profileof N. gonorrhoeae strains prevalent in high-risk groups suchas FSWs provides essential clues regarding the emergence ofdrug resistance and treatment options. We present here the datafrom our antimicrobial susceptibility surveillance of N. gonorrhoeaeisolates obtained during the period from 1997 to 1999. The resultsenabled us to demonstrate the sudden rise in fluoroquinoloneresistance and the appearance of both PPNG and TRNG isolatesin Bangladesh.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains. N. gonorrhoeae strains isolated from different parts of Bangladeshduring the period from 1997 to 1999 were studied. The identityof 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 with20% glycerol until further study.

ß-Lactamase test. All penicillin-resistant isolates were tested for ß-lactamaseproduction by a paper acidometric method as described earlier(17).

MICs. MICs of penicillin, tetracycline, spectinomycin, ceftriaxone,and ciprofloxacin were determined by an agar dilution methodas described earlier (10, 11). Five N. gonorrhoeae referencestrains, WHO A to WHO E, for which the MICs are known were includedfor quality control in each test. Each test was repeated thrice.The antimicrobial susceptibility was judged according to breakpointcriteria defined by the NCCLS (11). Twofold serial dilutionsof the following antibiotics were used: penicillin (0.03 to64 µg/ml; Sigma, St. Louis, Mo.), tetracycline (0.03 to64 µg/ml; Sigma), ciprofloxacin (0.004 to 4 µg/ml;Bayer, Hampshire, United Kingdom), spectinomycin (2.0 to 128µg/ml; Upjohn, Puurs, Belgium), and ceftriaxone (0.004to 0.5 µg/ml; Sigma).

Phenotypic characterization. The criteria used for phenotypic characterization of N. gonorrhoeaebased on plasmid- and chromosomally mediated resistance to penicillinand tetracycline were as described earlier (13).

RESULTS

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Results

A total of 347 N. gonorrhoeae strains isolated from 1997 to1999 were studied. Of them 137 were from 1997, 73 were from1998, and 137 were from 1999. Among the 1997 isolates, 94 werefrom street-based FSWs in Dhaka, the capital of Bangladesh,and 36 isolates were from brothel-based FSWs from Tangail (located150 km northwest of Dhaka) and from females attending primaryhealth care delivery clinic in Dhaka. All 1998 isolates werefrom street-based FSWs in Dhaka. Among the 1999 isolates, 110were from street-based FSWs in Dhaka, and 27 were from brothel-basedFSWs from Jessore (located 275 km southwest of Dhaka).

Isolates' antimicrobial susceptibilities to penicillin, tetracycline,ciprofloxacin, ceftriaxone, and spectinomycin, determined bythe agar dilution method using the criteria established by theNCCLS, are shown in Fig. 1a to e. The prevalence of PPNG andTRNG among isolates from 1997, 1998, and 1999 was 16 and 6.5%,10 and 13.5%, and 10 and 21.5%, respectively (Fig. 1f), and1.2 and 3.6% of the isolates from 1998 and 1999, respectively,were both PPNG and TRNG.

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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 tociprofloxacin. During 1998 and 1999, 41 and 49% of the isolates,respectively, were resistant to ciprofloxacin (Fig. 1c). Duringthese two years the proportion of isolates with reduced susceptibilitydeclined 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 to1999 were further calculated. The MIC₅₀ was 0.015 µg/mlin 1997 versus 0.5 µg/ml in 1998 and 1999; the MIC₉₀ was1 µg/ml in 1997 versus 8 µg/ml in 1998 and 1999.

Based on plasmid- and chromosomally mediated resistance to penicillinand tetracycline, the isolates were phenotypically categorizedinto seven different groups (Table 1). Patterns of ciprofloxacinsusceptibilities of isolates from different phenotypic categorieswere further analyzed (Table 2). Nine (75%), 24 (85.7%), and39 (57.5%) ciprofloxacin-resistant isolates from 1997, 1998,and 1999 were CMRNG^PT. Among the ciprofloxacin-resistant isolatesfrom 1998 and 1999, 3.6 and 4.5% were PPNG, respectively.

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TABLE 1. Phenotypic categories of N. gonorrhoeae isolates from 1997 to 1999 based on plasmid- and chromosomally mediated resistance to penicillin and tetracycline

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TABLE 2. Ciprofloxacin susceptibility patterns of different phenotypic categories of N. gonorrhoeae isolated from 1997 to 1999

DISCUSSION

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Discussion

The control of gonococcal infection is important given the highincidence of acute infections, complications, and sequelae andthe role of gonococci in facilitating human immunodeficiencyvirus acquisition and transmission (9). The knowledge of antimicrobialsusceptibility of N. gonorrhoeae is a prerequisite for propertreatment and control of the disease. In Bangladesh there isno established antimicrobial susceptibility surveillance forN. gonorrhoeae. In the absence of laboratory data and an establishedmonitoring system, selection of appropriate antibiotics forempirical treatment of gonorrhea is difficult.

Ciprofloxacin and ofloxacin are currently recommended by theWorld Health Organization and the Centers for Disease Controland Prevention for the treatment of uncomplicated gonorrheain areas where multidrug-resistant strains are common, suchas Southeast Asia and Central Africa. Ciprofloxacin has beenused extensively in Bangladesh, since it is relatively cheapand effective and only a single oral dose is required. As aconsequence of the large-scale use of this group of antimicrobialsin areas where over-the-counter availability of drugs withoutprescription is common, a substantial increase of resistantstrains may occur. Self-medication among sex workers in thePhilippines has been shown to play a major role in the developmentof antimicrobial resistance, and a similar practice is alsocommon in Bangladesh (7). The appearance of a high prevalenceof ciprofloxacin-resistant isolates in our study suggests thatFSWs in Bangladesh are exposed to ciprofloxacin and that resistancehas developed under selective antimicrobial pressure.

There has been a remarkable increase in antimicrobial resistanceamong N. gonorrhoeae strains in many developing countries inrecent years (4). The frequency of fluoroquinolone-resistantstrains has also been increased dramatically in recent years.It is interesting that in our present study, 9% of the gonococcalisolates were resistant in 1997, 41% were resistant in 1998,and 49% were resistant in 1999, a significant rise in resistancein a very short period of time.

In our present study 9 (75%), 24 (85.7%), and 39 (57.5%) ciprofloxacin-resistantisolates from 1997, 1998, and 1999 were CMRNG^PT. A similar patternhas also been reported earlier from the United States and Thailand(4, 8). The frequent appearance of elevated ciprofloxacin MICsin CMRNG^PT strains remains unexplained, because the mechanismof ciprofloxacin resistance is different from those of penicillinand tetracycline resistance.

Although a decrease in the prevalence of PPNG and resistanceto penicillin was observed, an increase in the prevalence ofTRNG and PPNG-TRNG isolates appeared in 1998 and 1999.

High-level resistance to penicillin, tetracycline, and ciprofloxacinin the present study has rendered these antimicrobials unacceptablefor empirical treatment of gonorrhea. The isolates tested demonstratedhigh rates of susceptibility to broad-spectrum cephalosporinand spectinomycin, with susceptibility rates of 95 and 100%,respectively.

The present study demonstrates the need for a surveillance systemfor continuous monitoring of the antimicrobial susceptibilityin N. gonorrhoeae for determination of optimal treatment regimens.

ACKNOWLEDGMENTS

This research was funded by the ICDDR, B: Center for Healthand Population Research, which is supported by countries andagencies that share its concern for the health problems of developingcountries. Current donors providing unrestricted support includethe aid agencies of the governments of Australia, Bangladesh,Belgium, Canada, Saudi Arabia, Sweden, Switzerland, the UnitedKingdom, and the United States; international organizationsinclude the United Nations Children's Fund (UNICEF) and theUSAID (grant 207491). ICDDR, B acknowledges with gratitude thecommitment of USAID to the center’s research effort.

FOOTNOTES

* Corresponding author. Mailing address: Laboratory Sciences Division, ICDDR, B, GPO Box-128, Dhaka-1000, Bangladesh. Phone: 880-2-8811751-60. Fax: 880-2-8812529. E-mail: motiur{at}icddrb.org.

REFERENCES

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