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Gonorrhoea is caused by Neisseria gonorrhoeae which is very infectious sexually transmitted bacteria. Higher rates of infection have been reported due to the increase of antimicrobial resistance to penicillin, tetracyclines so these drugs are not recommended for treatment of gonorrhea (1-4). The cases of treatment failure with fluoroquinolones (ciprofloxacin, ofloxacin) have also been reported. So, floroquinolones are not used as drugs of choice for treatment of gonorrhea (5-7). Currently, extended-spectrum cephalosporins (ceftriaxone, cefixime) are used as primary therapies to treat uncomplicated gonorrhoea.
The Centres for Disease Control and Prevention (CDC) currently recommend treatment of gonorrhoea with combination of two drugs a cephalosporin (cefatriaxone) and a second antibiotic (azithromycin). Cefixime can also be used in combination with azithromycin. However, treatment failures with cefixime have been reported in Japan and Europe due to which cefixime was removed from the treatment guidelines in Japan (8, 9). Despite this, various treatment failures with cephalosporins have been reported in United States. At this time of rising antibiotic resistance, it is important to monitor and track the susceptibility ofN. gonorrhoeaestrain. The Gonococcal Isolate Surveillance Project (GISP) is essential to check the susceptibility of N. gonorrhoeae. GISP is a cooperative group of twenty-six sexually transmitted disease clinics across the United States that collects gonorrhea specimens and sends them to regional offices for resistance diagnosis(10).
There are various methods that determine minimum inhibitory concentration (MIC) values for antibiotics to monitor emerging resistance. As agar dilution is considered as gold standard for susceptibility testing, it is labor intensive and difficult to implement in diagnostic laboratories routinely. The other method used to determine the susceptibility of N. gonorrhoeae is the disk diffusion method. Although this method is feasible, easy to use and less costly but there are certain limitations such as variability in results. So, another method that can be adapted to measure the antimicrobial susceptibility of N. gonorrhoeae is Etest.
The Etest is an in vitro method developed to determine the MIC of antimicrobial agent on agar medium. The Etest consists of thin reagent strip that carries a continuous concentration gradient of stabilized and dried drug (11, 12). It uses the principle of disk diffusion method to perform testing. Etest strips for the antimicrobial agents to be tested are placed on the inoculated agar surface. The Etest approach is well suited for variety of drugs and microorganisms. The Etest method is easy to execute than agar dilution method (13).
In this study, the MIC values from agar dilution test and Etest of three antibiotics namely ceftriaxone, cefixime and cefpodoxime were compared.
Materials and methods
N. gonorrhoeae strains
One hundred and five strains of N. gonorrhoeae from United States, GISP strains, reference strains and ten laboratory derived strains nonsusceptible to cefpodoxime were evaluated for antimicrobial susceptible testing.
The parent strain SPN284 or GC3502 was exposed to increased concentrations of cefpodoxime.
Antimicrobial susceptibility testing
The agar dilution test and Etest were performed in accordance to the Clinical and laboratory standard institute and manufacturer’s instructions respectively. For both Etest and agar dilution testing, suspensions of N. gonorrhoeae were prepared from an overnight chocolate II agar culture into Mueller–Hinton broth (Difco, MI) and adjusted to a turbidity of a 0.5 McFarland standard. For the Etest, the surface of an agar plate was swab inoculated with organism and Etest strips were placed on the inoculated surface in a radial fashion. For the Agar dilution method, Mueller–Hinton agar plates were prepared using GC agar base medium supplemented with 1% IsoVitaleX. The dilution of the suspension was inoculated onto the agar surface within 15 min of preparation. The plates were incubated at 35°C in 5% CO2 for 20 to 24 h. The MICs of antimicrobials were recorded as the lowest concentration of antimicrobial agent that completely inhibited N. gonorrhoeae growth on the agar plates (agar dilution) and strips (Etest).
Statistical data analysis
Simple correlation, linear regression, Wilcoxon rank sums and analysis of variance methods were used for the comparison of MICs of agar dilution and Etest. The strains with MIC value of 96 or 128µg/ml were defined as outliers. The outliers were not illustrative so the tests were reported without outliers. To increase the efficiency of results the in-between values from the Etest were used instead of agar dilution test.
For the clinical isolates the agar dilution test MICs ranged between 0.001 and 0.125 µg/ml, 0.016 and 0.125 µg/ml, and 0.008 and 2 µg/ml and the Etest MICs ranged between 0.002 and 0.25 µg/ml, 0.004 and 0.5 µg/ml, and 0.016 and 1.5 µg/ml for ceftriaxone, cefixime and cefpodoxime respectively.
For the laboratory-generated mutants the agar dilution test MICs ranged between 0.5 and 8 µg/ml, 2 and 16 µg/ml, and 1 and 8 µg/ml and the Etest MICs ranged between 1 and 8 µg/ml, 0.5 and 6 µg/ml, and 3 and 8 µg/ml for ceftriaxone, cefixime and cefpodoxime respectively. A good linear relationship was seen for all the three antibiotics while comparing the MIC values of agar dilution and Etest method.
From the results it is clear that Etest is faster, requires less labor and fewer supplies as compared to the reference agar dilution method. So, it is a method of choice for the health laboratories to test multiple isolates as it has an advantage of providing MICs values using a single culture plate.
Liu et al. measured and compared the MICs of three antibiotics namely ceftriaxone, cefixime and cefpodoxime against N. gonorrhoeae with the agar dilution test and the Etest. Their results showed that the both tests are interchangeable but the Etest has an ability to read an additional one half higher or lower dilution. So, the precision of test can be enhanced using additional reading points. The data presented by them suggests that Etest can be used as an alternative to agar dilution method.
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