Evaluation of the Etest for susceptibility testing of Mycoplasma hominis and Ureaplasma urealyticum

Erika Dósaa, Elisabeth Nagya,*, Wolfgang Falkb, Ildikó Szókea and Uwe Balliesb

a Department of Clinical Microbiology, Albert Szent-Györgyi Medical University, Szeged, Hungary b Central Medical Laboratory, Kiel, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The Etest was used for antibiotic susceptibility testing of Mycoplasma hominisand Ureaplasma urealyticum isolates and the results were compared with those obtained with the broth microdilution method. For 50 clinical isolates of M. hominis the MICs of doxycycline, ofloxacin and ciprofloxacin agreed within ± one dilution and ± two dilutions in 82-98% and 98-100% of cases, respectively. The MICs of erythromycin, azithromycin, doxycycline, ofloxacin and ciprofloxacin were evaluated for 50 clinical isolates of U. urealyticum. The corresponding levels of agreement were 70-98% and 94- 100%, respectively. Reference isolates M. hominis PG-21 and U. urealyticum T-960 were also used. The Etest seems to be an alternative method for determination of MICs of antibiotics with M. hominis and U. urealyticum.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The urogenital mycoplasmas Mycoplasma hominisand Ureaplasma urealyticummay be implicated in a large variety of infections and sexually transmitted conditions, such as non-gonococcal urethritis, pelvic inflammatory disease, endometritis and bacterial vaginosis in coexistence with other pathogens. 1 They may also be involved in infections of premature and low birth weight infants with clinical manifestations of septicaemia, respiratory tract disease or neurological disorders. 2 ,3 As antibiotic resistance among urogenital mycoplasmas can arise similarly to other bacterial species through gene mutation or the acquisition of new genetic material, 4 ,5 the in-vitro testing of the responses of these isolates to both older and currently available drugs is assuming ever greater importance. A standardized method for the antibiotic susceptibility testing of mycoplasmas is not readily available. The disc diffusion method cannot be utilized because M. hominis and U. urealyticum grow very slowly and the antibiotic concentration equilibrates throughout the agar plate before colonies appear. Agar dilution and broth microdilution methods have been recommended, 6 but these methods are mainly used in reference laboratories.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial isolates

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.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The MICs of the different antibiotics with the broth microdilution and the Etest method for the control isolates of M. hominis (PG-21) and U. urealyticum (T-960) were within one dilution when tested on five different days. The Table shows the MIC ranges and MIC 90 of the antibiotics obtained by the two methods and the percentage agreement between the methods. The M. hominis isolates were found to be uniformly resistant to both macrolides (data not shown). Doxycycline, ofloxacin and ciprofloxacin were active against M. hominisisolates, with MIC 90 values of 0.5- 1 mg/L by Etest. Doxycycline inhibited most M. hominis at concentrations of 0.016- 8 mg/L. Only one of the 50 M. hominis isolates, from chronic vulvo-vaginitis, was resistant to doxycycline (MIC 32 mg/L), and two isolates were resistant to ofloxacin (MICs 16 mg/L and 32 mg/L, respectively). All but one of the M. hominisisolates were sensitive to ciprofloxacin.


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Table. MICs of different antibiotoics for M. hominis (50) and U. urealyticum (50) isolates by broth microdilution and Etest methods
 
Ten U. urealyticum isolates were highly resistant to erythromycin with MICs >=8 mg/L. Five of these isolates also exhibited resistance to azithromycin (MIC >=4 mg/L). Doxycycline inhibited all U. urealyticum isolates (MIC 90 0.25 or 0.5 mg/L). The MIC 90 values of ofloxacin and ciprofloxacin measured with the Etest and the broth microdilution method for the U. urealyticum isolates were 2 mg/L and 4 mg/L, respectively. The Figure shows an Etest result for one U. urealyticum isolate.



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Figure Etest MICs of ofloxacin (OF) and erythromycin (EM) for a clinical isolate of Ureaplasma urealyticum.

 
Reading of MIC values by the Etest and the broth microdilution methods was blinded. For U. urealyticum the agreement between methods was ± one dilution or ± two dilutions in 70- 98%and 94- 100% of cases, respectively. For M. hominis, the corresponding levels of agreement were 82- 98% and 98- 100%. The Etest MICs of ofloxacin and ciprofloxacin gave the best agreement with the reference method in our study (100% ± two dilutions and 96- 98% ± one dilution). The worst agreement (95% ± two dilutions and 80% ± one dilution) was observed for erythromycin tested with U. urealyticum.

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.


    Acknowledgments
 
These studies were supported in part by a grant from the Hungarian Ministry of Welfare (ETT No. T-10-555). We thank Mrs E. Mészáros Kasza for technical assistance. Preliminary reports of this work were presented at the 7th Congress of the European Society of Clinical Microbiology and Infectious Diseases and at the 4th International Conference on the Macrolides, Streptogramins and Ketolides.


    Notes
 
* Corresponding author. Tel and Fax: +36-62-310-981; E-mail: nagye{at}mlab.szote.u-szeged.hu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Krausse, D. C. & Taylor-Robinson, D. (1992). Mycoplasmas which infect humans. In Mycoplasmas: Molecular Biology and Pathogenesis (Maniloff, J., Ed.), pp. 417–45. American Society for Microbiology, Washington, DC.

2 . Cassell, G. H., Waites, K. B., Watson, H. L., Crouse, D. T. & Harasawa, R. (1993). Ureaplasma urealyticum intrauterine infection: role in prematurity and disease in newborns.Clinical Microbiology Reviews 6 , 69 –87.[Abstract]

3 . Romero, R., Mazor, M., Oyarzun, E., Sirtori, M., Wu, Y. K. & Hobbins, J. C. (1989). Is genital colonization with Mycoplasma hominis and Ureaplasma urealyticum associated with prematurity/ low birth weight? Obstetrics and Gynecology 73 , 532 –6.[Abstract]

4 . Roberts, M. C. & Hillier, S. L. (1990). Genetic basis of tetracycline resistance in urogenital bacteria. Antimicrobial Agents and Chemotherapy 34 , 261 –4.[ISI][Medline]

5 . Palu, G., Valisena, S., Barile, M. F. & Meloni, G. A. (1989). Mechanisms of macrolide resistance in Ureaplasma urealyticum : a study on collection and clinical strains. European Journal of Epidemiology 5 , 146 –53.[ISI][Medline]

6 . Roberts, M. C. (1992). Antibiotic resistance. In Mycoplasmas: Molecular Biology and Pathogenesis (Maniloff, J., Ed.), pp. 513–23. American Society for Microbiology, Washington, DC.

7 . Citron, D. M., Ostovari, M. I., Karlsson, A. & Goldstein, E. J. (1991). Evaluation of the E test for susceptibility testing of anaerobic bacteria. Journal of Clinical Microbiology 29 , 2197 –203.[ISI][Medline]

8 . Baker, C. N. (1992). The E-test and Campylobacter jejuni. Diagnostic Microbiology and Infectious Disease 15 , 469 –72.[ISI][Medline]

9 . Waites, K. B., Crabb, D. M., Duffy, L. B. & Cassell, G. H. (1997). Evaluation of the Etest for detection of tetracycline resistance in Mycoplasma hominis. Diagnostic Microbiology and Infectious Disease 27 , 117 –22.[ISI][Medline]

10 . Shepard, M. C. & Lunceford, C. D. (1976). Differential agar medium (A7) for identification of Ureaplasma urealyticum (human T mycoplasmas) in primary cultures of clinical material. Journal of Clinical Microbiology 3 , 613 –25.[ISI][Medline]

Received 29 April 1998; returned 2 July 1998; revised 31 July 1998; accepted 4 January 1999





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