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Antibiotic Susceptibility Patterns and Plasmid Profiles of Penicillinase-ProducingNeisseria gonorrhoeaeStrains in Durban, South Africa, 1990–1993

CHENIA, HAFIZAH Y. BSc (HONS); PILLAY, BALAKRISHNA DR. REF NAT; HOOSEN, ANWAR A. MSc MBChB MMEd; PILLAY, DORSAMY PhD

Sexually Transmitted Diseases: January 1997 - Volume 24 - Issue 1 - p 18–22
Original Article
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Background and Objectives: The appearance of strains ofNeisseria gonorrhoeaeresistant, both chromosomally and plasmidmediated, to penicillin and other antibiotics makes this versatile pathogen difficult to treat. There is, therefore, a need for surveillance ofN. gonorrhoeaestrains to determine the efficacy of current therapeutic measures.

Goals: To survey the antibiotic susceptibilities and plasmid profiles of penicillinase-producingN. gonorrhoeaestrains isolated over a 4-year period.

Study Design: Penicillinase-producingN. gonorrhoeaestrains were detected by the chromogenic cephalosporin test. Minimum inhibitory concentrations to penicillin G, tetracycline, ceftriaxone, and ciprofloxacin were determined using the E-test. Plasmid DNA was obtained by the alkaline lysis method and profiles generated.

Results: Penicillinase-producingN. gonorrhoeaestrains increased from 16.4% to 19.0% in the period from 1990 through 1993. Although all strains were resistant to penicillin, strains were susceptible to varying levels of ciprofloxacin, ceftriaxone, and even tetracycline. All penicillinase-producingN. gonorrhoeaestrains possessed the 2.6-megadalton cryptic plasmid, and in addition 87.7% contained the 24.5-megadalton conjugative plasmid. Of the six known gonococcal β-lactamase plasmids, the 4.4-megadalton Asian and 3.2-megadalton African plasmids were predominant. The most prevalent plasmid profile contained the 2.6-megadalton cryptic, 24.5-megadalton conjugative, and 4.4-megadalton Asian plasmids.

Conclusions: To ensure effective treatment of gonorrhea, continued surveillance of the antimicrobial susceptibilities and plasmid profiles of penicillinase-producingN. gonorrhoeaestrains is necessary.

From the Departments of Microbiology, University of Durban-Westville and Medical Microbiology, University of Natal, Durban, South Africa

The authors thank Mrs. M. Pillay, Mr. C. Pillay, and Mrs. A. Kharsany for their technical assistance during the initial stages of this study.

Reprint requests: Dorsamy Pillay, PhD, Department of Microbiology, University of Durban-Westville, Private Bag X54001, Durban 4000, South Africa.

Received for publication November 29, 1995, revised April 15, 1996, and accepted April 22, 1996.

GONORRHEA REPRESENTS one of the most widely disseminated sexually transmitted diseases worldwide. Before the widespread use and abuse of penicillin, Neisseria gonorrhoeae was susceptible to less than 0.03 g/ml penicillin.1,2 By the early 1970s, 1 g/ml penicillin administered in vivo was ineffective against gonococci isolated in the Far East and Africa.3,4 In 1976, plasmid-mediated resistance to penicillin emerged with penicillinase-producing N. gonorrhoeae (PPNG) strains being isolated from patients infected in the Far East and West Africa.3,4 Penicillinase-producing N. gonorrhoeae strains have since become prevalent throughout the world.5 The emergence of PPNG strains drew the penicillin era of therapy for N. gonorrhoeae, which began in 1943, to a close.

Six gonococcal β-lactamase plasmids have been described, including the 3.2-megadalton (MDa) “African,” the 4.4-MDa “Asian,” the 2.9-MDa “Rio,” the 3.05-MDa “Toronto,” the 4.0-MDa “Nimes,” and the 6.0-MDa “New Zealand” plasmids.6 In addition, PPNG strains may possess a 2.6-MDa cryptic plasmid, a 24.5-MDa conjugative plasmid, or a 25.2-MDa TetM-conjugative plasmid or all three.7

Penicillinase-producing N. gonorrhoeae strains first were detected in 1977 in South Africa with isolated reports from Durban8 and Johannesburg.9 Since then, PPNG strains have been reported from various centers in South Africa,10–12 where there is no formal surveillance program to study changes in the levels of susceptibility of gonococci to various antibiotics and efficacy of treatment. Neither are changes in the plasmid content of PPNG strains and their influence on therapeutic measures monitored.

Antimicrobial susceptibility testing is performed to detect strains resistant to standard therapy or to establish patterns of susceptibility that may influence the choice of therapy or to do both.13 Antimicrobial susceptibility testing patterns are used to discriminate among gonococcal strains and, in conjunction with other typing methods, are essential for the description of the diversity of antibiotic-resistant strains in communities. Plasmid analysis and documentation of temporal changes in the distribution and prevalence of strains harboring them can shed light on the genesis and spread of strains from different geographic areas.14

There appears to be a rising incidence of gonorrhea in Durban, with 12,500 cases reported in 1990 and the number of cases increasing to 15,000 in 1992.15 This study was, therefore, carried out to determine the in vitro susceptibility patterns and plasmid profiles of PPNG strains isolated in Durban from 1990 through 1993.

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Materials and Methods

Three hundred twenty-one gonococcal strains were isolated from urethral exudates of adult men presenting with acute urethritis to the Sexually Transmitted Diseases Clinic at King Edward VIII Hospital, Durban, South Africa, from 1990 through 1993. The sampling procedure included strains from consecutive patients in the annual surveillance studies of acute urethritis. These strains were confirmed previously as N. gonorrhoeae by morphologic features of the colonies, a positive oxidase test, characteristic gram-negative staining, and carbohydrate utilization testing. Strains were cultured on New York City agar plates and incubated at 37 °C in 6% carbon dioxide atmosphere. Fifty-eight PPNG strains were identified by the chromogenic cephalosporin, nitrocefin (Oxoid Ltd., Basingstoke, England) test.16 Control strains included five PPNG strains (515P3, SB5, ST2, GR4103, and SB16) obtained from the Medical University of South Africa, Escherichia coli ATCC 10418, and Staphylococcus aureus ATCC 25923.

Antimicrobial susceptibility testing of the PPNG strains only was performed using the in vitro E-test (PDM Epsilometer, AB Biodisk, Solna, Sweden) for penicillin G, ceftriaxone, ciprofloxacin, and tetracycline strips. The medium used was GC agar base (Oxoid, Basingstoke, England), 5% saponin-lysed horse blood, and 1% Iso VitaleX (Becton Dickinson, Cockeysville, MD). Duplicate tests were performed on all PPNG strains as well as on the five N. gonorrhoeae control strains. Results were recorded after incubation at 37 °C for 24 hours in a 6% carbon dioxide atmosphere. Inoculum concentration and quality control parameters for the E-test were according to the manufacturer's recom-mendations. The interpretive criteria used were according to the National Committee for Clinical Laboratory Standards.17

For plasmid isolation, PPNG strains were subcultured after 24 hours from New York City agar plates onto chocolate agar plates, cultures harvested directly using phosphate-buffered saline, and plasmids isolated by the alkaline lysis method.18 Plasmid profiles were obtained by electrophoresis in a 1% agarose gel in 0.5x TBE (0.045-M Tris-borate, 0.001-M ethylenediaminetetra-acetic acid, pH 8.0) at 2 V/cm. The gel was stained in a solution of ethidium bromide (0.5 g/ml) for 15 minutes, destained for 15 minutes in excess tap water, viewed on an ultraviolet transilluminator (312 nm), and photo-graphed. Penicillinase-producing N. gonorrhoeae strains with the following plasmid profiles were included as control specimens: 515P3 (24.5 and 7.8 MDa), SB16 (2.6 and 4.4 MDa), ST2 (2.6 and 3.2 MDa), SP5 (2.6, 24.5, and 4.4 MDa), and GR4103 (2.6, 24.5, and 3.2 MDa).

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Results

The chromogenic cephalosporin test results showed 58 (17.8%) PPNG strains among the 321 isolates examined. A prevalence of 16.4% (9/55), 17.7% (6/34), 18.2% (24/132), and 19.0% (19/100) PPNG strains was found among the local strains for the years 1990 through 1993, respectively.

For the antimicrobial susceptibility testing, drop-shaped inhibition zones or inhibition ellipses were observed on agar plates. Zones were generally clear, with sharp endpoints, which intersected the carrier strip at specific antibiotic concentrations and made reading of minimum inhibitory concentrations (MICs) easy. The MIC results were interpreted according to the recom-mendations of the National Committee for Clinical Laboratory Standards17 and graded accordingly. All PPNG strains showed high MIC values for penicillin G (MIC ≥ 32 g/ml). According to National Committee for Clinical Laboratory Standards-recommended criteria, 96.5% (55/57) of strains were sensitive to ceftriaxone with MICs of ≤ 0.25 g/ml. Only 3.51% (2/57) of strains showed MICs of 0.34 g/ml. Of the 1990, 1991, 1992, and 1993 strains, 55.6%, 83.3%, 100%, and 94.8%, respectively, had MIC values between 0.002 and 0.006 g/ml for ceftriaxone. Overall, 87.7% (50/57) of strains were susceptible to 0.002 g/ml to 0.006 g/ml ceftriaxone. For tetracycline, 82.5% (47/57) of strains were susceptible with MICs ≤ 0.25 g/ml. Overall, 10.53% (6/57) of strains showed low-level chromosomally mediated tetracycline resistance with MICs of 1.25 g/ml to 4.5 g/ml. Low-level tetracycline resistance was seen in 3 of a total of 9 strains from 1990 exhibiting MICs of 1.25 g/ml to 1.75 g/ml and 2 of 19 strains from 1993 exhibiting MICs of 3.0 g/ml to 4.5 g/ml. The susceptibilities for ciprofloxacin were interpreted according to the recom-mendations of the National Committee for Clinical Laboratory Standards, which define gonococcal isolates for which MICs are ≤ 0.06 g/ml as susceptible to ciprofloxacin; no interpretive criteria for intermediate or resistant categories currently are available for ciprofloxacin.19 There were 94.7% (54/57) of the strains sensitive to ciprofloxacin with MIC values ≤ 0.06 g/ml. The MIC values between 0.002 and 0.008 g/ml for ciprofloxacin were found in 66.7%, 83.3%, 91.3%, and 73.7% of strains isolated in 1990, 1991, 1992 and 1993, respectively. There were 71.9% (41/57) of strains susceptible to 0.002 g/ml ciprofloxacin and 1.75% (1/57) of strains showed MICs of 0.094 g/ml. Clinically significant resistance was found in two isolates showing MICs of 0.125 g/ml and 0.19 g/ml.Figure 1.

Fig. 1

Fig. 1

Three types of plasmids were found in this study, that is, (1) the 2.6-MDa cryptic plasmid, (2) the 24.5-MDa conjugative plasmid, and (3) the gonococcal β-lactamase plasmid. Distribution of the PPNG strains according to their plasmid profile and year of isolation is listed in Table 1. Only two of the six known types of β-lactamase plasmids were evident, that is, (1) the 4.4-MDa Asian and (2) the 3.2-MDa African plasmids. The predominant β-lactamase type was the 4.4-MDa Asian plasmid (44, 77.2%) found in combination with the 2.6-MDa cryptic plasmid. Of these strains, 68.4% (39) also possessed the 24.5-MDa conjugative plasmid. Eleven (19.3%) of 57 PPNG strains showed a 3.2-MDa plasmid, 9 (15.8%) of which also carried the 24.5-MDa conjugative plasmid. Overall, 100.0% and 87.7% of the strains possessed the 2.6-MDa cryptic and the 24.5-MDa conjugative plasmids, respectively. Table 2.

TABLE 1

TABLE 1

TABLE 2

TABLE 2

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Discussion

Before 1985, the prevalence of PPNG strains was reported to be between 5% and 13% in South Africa.20 A 1983 study10 in Durban showed a 5% prevalence of PPNG strains, which increased to 29% in a 1985 study20 in Durban. In the present study, the incidence of PPNG strains appeared to increase from 16.4% in 1990 to 19.0% in 1994. The prevalence of PPNG strains in Durban is low compared with reports from other countries worldwide. In Nigeria, PPNG strains account for as many as 85.5% of all strains21; in Ndola, Zambia, a prevalence of 54% PPNG strains was found,22 and PPNG strains are known to constitute 35% to 50% of all gonococcal strains in certain Asian countries.20

Until 1988, penicillin still was being used as first-line therapy for gonococci in Durban and elsewhere in South Africa.20 The findings of that study showed a 29% prevalence of PPNG strains and led to the recommendation that penicillin therapy be replaced by agents active against PPNG and non-PPNG strains, that is, the cephalosporin, rosoxacin, or spectinomycin.20 Current treatment for urethritis in South Africa includes ciprofloxacin, ceftriaxone, spectinomycin, or ofloxacin followed by tetracycline to treat coinfection by Chlamydia trachomatis concurrently.

The E-test is an in vitro method for quantitative antimicrobial susceptibility testing that combines the simplicity of agar diffusion with the ability to produce an MIC result.23 Its usefulness and simplicity have been shown in our laboratories, and the values obtained are more precise than are conventional MICs based on discontinuous twofold serial dilutions. It has been shown that the total quantitative accuracy (±1 log2 dilution step) between the E-test and agar dilution results was 87%.24 Their E-test MICs generally were the same or twofold lower for penicillin and tetracycline, whereas ciprofloxacin had the highest E-test quantitative accuracy within ± log2 dilution of 100%.24.Table 3

TABLE 3

TABLE 3

Penicillinase-producing N. gonorrhoeae strains tested in this study showed penicillin MICs of ≥ 32 g/ml compared with MICs of 1 g/ml to 2 g/ml reported in 1984.10 Plasmid-mediated high-level tetracycline resistance was first observed in N. gonorrhoeae (MIC 16–64 g/ml) in 1985 in the United States.25 Subsequently, these strains of N. gonorrhoeae have been isolated from Canada,26 the Netherlands,27 England,28 France,29 Zaire,30 and Kenya.31 However, in the present study, there were no signs of high-level plasmid-mediated tetracycline resistance. The percentage of chromosomal tetracycline resistance shown by these strains (10.53%) is low compared to the 45% chromosomally resistant tetracycline strains in Kinshasa, Zaire.30

To counter the resistance of gonococci to penicillins and tetracyclines, broad-spectrum cephalosporins and selected quinolones are being used as primary therapies for uncomplicated gonorrhea. There have been reports of decreased susceptibility (MIC 0.06 g/ml) to ceftriaxone in Southeast Asia.32 However, in this study, 96.5% of strains appear to be sensitive to ceftriaxone. Presently, we have not seen strains with decreased susceptibility or resistance to ceftriaxone. Overall, ciprofloxacin was found to be highly active against 94.7% of all the strains (MIC ≤ 0.002–0.06 g/ml). Clinically significant resistance to ciprofloxacin has been suggested for strains with MICs ≥ 0.125 mg/ml to 0.25 mg/ml when patients are treated with 250 mg of ciprofloxacin.33 Three PPNG strains in Hawaii have been shown to possess ciprofloxacin MICs ≥ 2 g/ml.34 Although the origin of these strains appears to be the Republic of the Philippines, isolated reports of strains possessing similar MICs have been received from Thailand and Canada.34 In Durban, 250-mg dose of ciprofloxacin is recommended for gonococcal infection; therefore, the presence of two strains with MICs of 0.125 g/ml and 0.19 g/ml exhibits clinically significant resistance or decreased susceptibility to ciprofloxacin.33,34 In Durban, local patterns of susceptibility to ceftriaxone and tetracycline appear to be different in comparison to the antimicrobial susceptibility patterns of PPNG strains from other countries. However, the development of resistance to current treatment measures in other countries mandates continual monitoring of local isolates to detect the development of resistance before treatment failure becomes widespread. In this retrospective study of 57 PPNG strains, it was found overall that PPNG strains that carried the 2.6-MDa, 4.4-MDa, and 24.5-MDa plasmids outnumbered those that carried only the 2.6-MDa and 4.4-MDa plasmids. Table 1 lists that from 1990 through 1992, there was an increase in the number of strains possessing the above three plasmids, followed by a decrease in 1993. In contrast, from 1990 through 1992, there was a decrease in the number of strains possessing only the 4.4-MDa and 2.6-MDa plasmids, followed by a slight increase in 1993. The finding that all of the PPNG strains possessed the 2.6-MDa cryptic plasmid suggests that this plasmid confers a selective advantage to the gonococcus. The 24.5-MDa conjugative plasmid is responsible for mobilizing itself and the small gonococcal β-lactamase plasmids to other N. gonorrhoeae strains, as well as mobilizing only the gonococcal β-lactamase plasmids to E. coli and other Neisseria species.7 High prevalence of the conjugative plasmid in these PPNG strains may be responsible for the increased prevalence of PPNG strains.Table 4

TABLE 4

TABLE 4

This study shows that the β-lactamase plasmid of Far East origin is more common than the plasmid of West African origin. Durban is an important port city and tourist destination in Africa. The influx of sailors and tourists from Asia, Europe, and the United States could be one of the factors influencing the prevalence of the Asian 4.4-MDa plasmid.

Continued surveillance of the efficacy of currently recommended therapeutic measures is important. Resistance to these agents can be expected to develop, as was the case with penicillin and tetracycline.35 Because N. gonorrhoeae is a versatile pathogen displaying several antibiotic resistance mechanisms, the effectiveness of single-dose treatment should be monitored and surveillance of trends in resistance is necessary to achieve effective control of this pathogen.

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