DEVELOPMENT OF RESISTANCE to antimicrobial agents has become a significant problem in the treatment and control of gonorrhea.1 With the emergence of penicillinase-producing Neisseria gonorrhoeae, other drugs like cefotaxime, cefuroxime, and spectinomycin have been used successfully to treat N. gonorrhoeae infection.2 Azithromycin, a new antibiotic belonging to the azalides, a new class of compounds resembling macrolides, is active against N. gonorrhoeae. A single dose of azithromycin has been shown to be effective in the treatment of uncomplicated gonorrhea.3
With the increasing emergence of antimicrobial resistance seen with N. gonorrhoeae, there is a need to simplify and standardize the in vitro antimicrobial susceptibility testing of N. gonorrhoeae to monitor the situation. The agar dilution method has been the standard method recommended for quantitative monitoring of such strains. However, it is usually laborious and impractical for routine use.4 The E-test is a new technique for quantitative antimicrobial susceptibility testing that allows for the direct determination of minimum inhibitory concentration (MIC).5 It consists of a plastic carrier strip with a predefined continuous exponential antibiotic gradient immobilized on one side. It is an attractive alternative to the agar dilution method for gonococcal antimicrobial susceptibility testing.6
This study was undertaken to determine the in vitro susceptibility of N. gonorrhoeae to azithromycin and to compare the E-test and agar dilution methods in the determination of the MIC of azithromycin.
Clinical isolates of N. gonorrhoeae were obtained from patients who attended sexually transmitted disease clinics in seven major hospitals representing five geographic locations throughout Malaysia. Strains were isolated using modified Thayer Martin media and identified by colonial morphology, Gram stain appearance, production of oxidase, and utilization of glucose. Strains were kept in liquid nitrogen until performance of the test. From January, 1994 to June, 1995, 135 viable strains were received and included in this study.
Antimicrobial agents used for susceptibility pattern tests, obtained from local agents, included penicillin, cefuroxime (Glaxo, Research Triangle Park, NC), ceftriaxone (Roche Laboratories, Nutley, NJ), norfloxacin (Astra Pharmaceutical Products, Westborough, MA), tetracycline (National Pharmaceutical Control Laboratory, Kuala Lumpur, Malaysia) kanamycin (Meiji Seika Kaisha, Ltd., Japan), spectinomycin (Upjohn, Kalamazoo, MI), and azithromycin (Pfizer Laboratories, New York, NY). All drug solutions were prepared and stored according to the manufacturers' instructions.
Antimicrobial Susceptibility Testing
The antimicrobial susceptibilities of the gonococci were established by determination of the MICs by an agar dilution method recommended by the World Health Organization/Venereal Disease Technique.7 The MIC of azithromycin was determined by both agar dilution method and E-test. Inocula of 108 bacteria/ml were prepared by the direct suspension of colonies, grown overnight on gonococcal agar, in phosphate-buffered saline (PBS), pH 7.2, to achieve a turbidity equivalent to a 0.5 McFarland standard. For the agar dilution technique, the bacterial suspensions were further diluted 1: 10 in PBS, and then spotted with a Denley multipoint inoculator onto the test plates containing twofold concentration increments of the antimicrobial agents to give final inocula of 104 bacteria/spot.
E-test was performed following the modified Kirby-Bauer method recommended by the National Committee for Clinical Laboratory Standards.8 The bacterial suspension of 108 bacteria/ml was streaked onto the entire surface of the culture plates by cotton swab, and the E-test strip was applied 15 minute later.
Both E-test and the agar dilution method were using GC medium base agar (Difco, Detroit, MI) supplemented with Isovitalex and without hemoglobin. Tests were completed simultaneously; test plates were incubated at 35 °C in a 5% CO2 atmosphere and the MIC results were read after 18 to 24 hours. In the E-test, the intercept of the antibiotic gradient strip and the zone of inhibition is taken as the MIC. The MIC determination by the agar dilution method was completed as described previously.7 Production of β-lactamase was determined using the chromogenic cephalosporin substrate, nitrocephin (Oxoid, Hampshire, England).
The MIC of antibiotics against all the clinical gonococcal isolates obtained by agar dilution method are listed in Table 1.
Seventy-six of the isolates were penicillinase-producing N. gonorrhoeae, and 69 were high-level tetracycline-resistant N. gonorrhoeae (i.e., MICs ≥ 16 μg/ml).
All the isolates were susceptible to azithromycin based on the susceptible MIC breakpoint of no more than 2.0 μg/ml.9 The MICs range from 0.0078 to 0.25 μg/ml by agar dilution method and from 0.016 to 0.50 μg/ml by E-test. The MIC for 50% (MIC50) of the isolates was 0.064 μg/ml by both testing methods, and the MICs for 90% (MIC90) of the isolates was 0.125 μg/ml by agar dilution method and 0.25 μg/ml by E-test. The percentage agreement, within the acceptable ±1 log2 dilution, between MICs obtained by E-test and those obtained by the agar dilution method was 97.8% (Table 2).
A scatter diagram (Fig. 1) shows the linear relationship between the MIC values obtained by the two testing methods. Correlation analysis (Spearman) gives a correlation coefficient (r) value equal to +0.89.
It has been suggested that azithromycin be given strong consideration in the management of the common, uncomplicated sexually transmitted diseases caused by Chlamydia trachomatis, N. gonorrhoeae and Ureaplasma urealyticum3 because of its good activity and favorable dosage regime, although the number of clinical trials10–13 is limited. The findings in this study are in agreement with previous studies12 that showed a good in vitro activity of azithromycin against N. gonorrhoeae. All of the 135 N. gonorrhoeae isolates we tested are susceptible to azithromycin, with MICs ranging from 0.0078 to 0.25 μg/ml (agar dilution method), similar to the observations by Baker et al.5 The MIC50 and MIC90 were one dilution lower compared with the observation by Steingrimsson et al.,3 even though the isolates were a mixture of penicillin-sensitive and penicillin-resistant strains.
The MIC of azithromycin against N. gonorrhoeae as determined by E-test is comparable to the result obtained by the standard agar dilution method. The same was seen when testing other antimicrobials against this organism.7,14 The MIC of azithromycin is not influenced by the presence of the β-lactamase enzyme or the possession of the tetracycline-resistant plasmid.
The single-dose regime and good antigonococcal and antichlamydial activity make azithromycin a suitable choice for treatment. Because the antimicrobial susceptibility pattern of N. gonorrhoeae may change rapidly,1 it is important to monitor the susceptibility pattern to detect the emergence of resistance. In this study, we have shown that the determination of MICs of azithromycin by E-test is as reliable as the agar dilution method.
In conclusion, azithromycin has a very good in vitro antigonococcal activity, and the E-test is a reliable method for determining the MIC of azithromycin against N. gonorrhoeae.
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