a Department of Clinical Microbiology, Albert Szent-Györgyi Medical University, Szeged, Hungary b Central Medical Laboratory, Kiel, Germany
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Abstract |
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Introduction |
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The Etest (AB Biodisk, Solna, Sweden) is an agar-based MIC method which uses a plastic strip with a preformed exponential concentration gradient of an antibiotic. Different studies have validated application of the Etest for susceptibility testing of slow-growing organisms such as anaerobes, Campylobacter jejuniand mycoplasmas. 7 ,8 ,9 The aim of the present study was to evaluate the applicability of the Etest for the determination of MIC of clinically relevant antibiotics for reference isolates and clinical isolates of M. hominisand U. urealyticum,in comparison with the broth microdilution method.
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Materials and methods |
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Consecutive clinical isolates of M. hominis (50) and U. urealyticum (50) were obtained from urogenital specimens in Szeged, Hungary and Kiel, Germany. Reference isolates of M. hominis (PG-21) and U. urealyticum (T-960) were kindly provided by P. A. Mardh (Uppsala, Sweden). The reference isolates and the clinical isolates were stored at -70°C before use in the study.
Broth microdilution method
The broth microdilution method was based on serial dilutions of antibiotics in U-9 broth (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) in a 96-well microtitre plate and inoculated with known concentrations of organisms which were then incubated and observed for growth for 2- 5 days. Erythromycin (Abbott, USA), azithromycin (Pliva-Chinoin, Hungary), doxycycline (Chinoin, Hungary), ofloxacin (Hoechst, Hungary) and ciprofloxacin (Bayer, Hungary) powders were used to prepare fresh stock solutions with a starting concentration of 512 mg/L in 0.1 M phosphate buffer (pH 6.8) for each assay. The antibiotic concentrations tested ranged from 256 mg/L to 0.125 mg/L for each drug. Frozen cultures of M. hominis and U. urealyticumcontrols and clinical isolates were suspended in fresh U-9 medium, incubated for 24 h at 37°C in 5% CO 2, and 200 µL aliquots of these cultures, containing approximately 10 4 cfu/mL, were used to inoculate the wells of the microtitre plates. The MIC was based on the lowest concentration of antibiotic causing a colour change which remained stable after 48 h incubation.
MIC determination by Etest
A7 agar (Sanofi Diagnostics Pasteur), an updated version of A7 differential agar, 10 was used for both M. hominis and U. urealyticum as this medium supports the growth of both species. A 100 µL volume of the same inoculum suspension as used in the broth microdilution method was streaked on to the surface of 90 mm agar plates. Etest strips of erythromycin, azithromycin, doxycycline, ofloxacin and ciprofloxacin were placed on separate plates which were incubated at 37°C in an atmosphere enriched with 5% CO 2. MIC values were read where the lawn of the microcolonies intersected the strip edge after incubation for 2- 4 days, depending on the rate of growth of M. hominis or U. urealyticum.A colony microscope with 100 x magnification was used to visualize the end-point.
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Results and discussion |
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Antibiotic-resistant strains of mycoplasma are becoming increasingly common, and this makes the antibiotic susceptibility testing of mycoplasmas ever more important not only in research laboratories, but also in everyday routine clinical laboratories. The antibiotic susceptibility testing of mycoplasma and ureaplasma isolates requires different methods from those used for rapidly growing bacteria. The broth microdilution method is currently the most widely used procedure. Although this method gives excellent reproducibility in multiple runs carried out on different days, agar dilution methods can be more useful than the broth method. The broth method uses a pH change to mark the end point of the test, making the reading of results subjective. Agar dilution methods provide the potential for visualization of mixtures of susceptible and resistant mycoplasmas in the same population, or for the detection of resistant mutants.
Commercially available systems for testing the susceptibility of clinically important mycoplasmas are based on breakpoint determination. Only one or two concentrations of each antibiotic are used in kits, making interpretation more difficult than with full range MIC tests. The Etest is an established method for MIC determinations on bacteria with different growth rates. Our study has revealed that the Etest performs as well as the broth microdilution procedure for determination of the MICs of antibiotics for both mycoplasmas and ureaplasmas. The technical simplicity, the convenience and the broad dilution range of the Etest make it a viable alternative for determinations of the MICs of antibiotics for M. hominis and U. urealyticum. However, all these methods are suitable only for determination of mycoplasmistatic and not mycoplasmicidal activities of the different antibiotics.
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Acknowledgments |
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Notes |
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References |
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Received 29 April 1998; returned 2 July 1998; revised 31 July 1998; accepted 4 January 1999