In Vitro Susceptibility of Ureaplasma urealyticum and Mycoplasma hominis Isolates in Argentina

Objective: Our goal was to determine the in vitro susceptibility of Ureaplasma urealyticum and Mycoplasma hominis isolates to several antibiotics in Argentina. Methods: Ninety-four strains of U. urealyticum and 18 strains of M. hominis isolated from cervical and urethral specimens were studied. Broth microdilution and agar dilution tests for minocycline, tetracycline, erythromycin, ciprofloxacin, and ofloxacin were performed. Results: Both methods proved to be reliable and reproducible for U. urealyticum and M. hominis, with no major differences in results. The U. urealyticurn strains were inhibited by erythromycin at MICs ranging from ≤0.5 to >8 μ/ml. Ofloxacin showed the highest activity against this latter organism. No differences between tetracycline and minocycline MICs were observed with U. urealyticum. Two M. hominis strains displaying high MICs both to tetracycline and to minocycline were detected. Conclusions: The emerging resistance of mycoplasmas to certain antibiotics emphasizes the need to undertake further surveillance studies on the clinical isolates of such organisms.

he antimicrobial susceptibility of the 2 most common genital mycoplasmas, Ureaplasma urealyticum and Mycoplasma hominis, is of interest because they have been recognized as pathogens in infections of the newborn, 1,e arthritis, and particularly sexually transmitted diseases (STDs) such as urethritis 3'4 and pelvic inflammatory disease. 3,4 Since resistance to traditional drugs, such as tetracycline, used for treating these infections is widely known for both U. urealyticum and M. hominis, s-9 alternative antimicrobial agents should be evaluated.
The purpose of the present study was to determine the susceptibility of clinical isolates of U. ure-alyticum and M. hominis to 5 antibiotics and compare the results obtained from agar dilution and broth microdilution tests.

MATERIALS AND METHODS
Ninety-five isolates of U. urealjticum and 19 of el/. aominis strains were tested. The U. urealyticum isolates included reference strain (Mycotrin system) and 94 low-passage clinical isolates from the cervix, urethra, sperm, or first-catch urine. The el//. aominis isolates included 1 M. aominis reference strain (PG 21) obtained from the U.S. National Institutes of Health and 18 low-passage clinical isolates from the cervix, urethra, or sperm. All clinical isolates were U. urealyticum was identified by urease production in U9 broth 1 and typical colonies in Ureaplasma differential agar medium (A7). 1'11 M. hominis was identified by arginine hydrolysis, typical colonies in Hayflick modified-agar medium, 1 and growth inhibition by M. hominis antiserum (provided by David Rose). z

Antimicrobial Agents
The reference standard powers for in vitro susceptibility testing of minocycline (Lederle Laboratories), tetracycline hydrochloride (Pfizer Laboratories, Groton, CT), erythromycin base (Leptit Laboratories), ciprofloxacin (Syntial Laboratories), and ofloxacin (Cilag Laboratories) were obtained from the manufacturers. All 5 were used for U. urealyticure, but erythromycin was excluded for M. hominis because of its natural resistance.

Inoculum
Aliquots of stock culture were frozen at -70C and 6 10-fold serial dilutions in U9 broth (pH 6.0) for ureaplasmas or arginine broth (pH 7.0) 3 for mycoplasmas were prepared in order to determine the number of color-changing units (ccu) present (0.1 ml of organism suspension/0.9 ml of broth). A ccu was designated as the reciprocal of the highest dilution at which growth was present, as evidenced by an alkaline shift in the phenol red pH indicator. The stock cultures were thawed to room temperature on the day of the assay and diluted in the appropriate broth without antibiotics to reach the 104-105 ccu/ ml range. 4 The inoculum size was checked at the time of the assay, and the organism suspensions were "activated" by 2-h incubation prior to inoculating the test media.
Broth Microdilution Test U9 broth (pH 6.0) and arginine broth (pH 7.0) were used for testing U. urealyticum and M. hominis, respectively. [4][5][6][7][8][9][10][11][12][13][14][15][16] Broth volumes of 100 lxl were inoculated into each well of 96-well microtiter plates. A 100-1,1 antibiotic stock solution was added to the first well and serial 2-fold dilutions to the remaining wells in each row were inoculated with 100 I,1 of either organism inoculum until the final antibiotic concentration reached a range of 16-0.5 pg/ml. Separately, the wells without drugs containing serial 10-fold organism dilutions were used as a positive growth control, while the wells lacking organisms and drug were used as a sterility control. Microtiter plates were sealed and incubated for 48 h in an ambient atmosphere at 35C. The plates were observed for evidence of growth (color change). The MIC was defined as the lowest antibiotic concentration at which the color remained unaltered (no growth), as compared with the control growth color change. All assays were performed twice on different days.
Agar Dilution Test Ureaplasma differential agar medium (A7) and Hayflick modified-agar medium (Hm) were used for testing U. urealyticum and M. hominis, respectively. 17 One milliliter of 2-fold serial antibiotic dilution was added to 9 ml of agar medium, to reach a final range of 16-0.5 Ixg/ml. For U. urealyticum and M. hominis, a 10 ccu/ml was placed into each well of a Steers replicator and inoculated on Hm and A7 agar, reaching 104 organism/spot. The plates without antibiotics were used as a positive growth control. All plates were incubated in an ambient atmosphere with 5% COz for 48 h at 35C and were viewed with a microscope at 25 and 100 magnifications. The MIC was defined as the lowest antimicrobial concentration in which no growth (no colony) was observed compared with the growth control plate.

Susceptibili W Test Control
Staphylococcus aureus ATCC 25923 was used as a control for potential interaction of antibiotics, medium components, and pH influence. This strain was tested by broth microdilution and agar dilution tests. The MICs of S. aureus ATCC 25923 were simultaneously performed in Muller-Hinton agar and Muller-Hinton broth by the microdilution method. This control was performed twice on 2 different days.

RESULTS
The broth microdilution and agar dilution tests proved to be reliable and reproducible for U. urealyticum and M. hominis, with no major differences between the methods.

DISCUSSION
The wide spectrum of mycoplasma susceptibility tests so far described has been restricted to differ-ences in technique, inoculum size, pH media, incubation conditions, and endpoint criteria. 5'16'19 The medium used for U. urealyticum and M. hominis is complex, containing 10-20% serum, which makes the MIC values more difficult to compare with those from other bacteria that grow in simple medium. It has been speculated that the complex media and conditions necessary for optimal culture and susceptibility testing of mycoplasmas may exert some effect on the activity or stability of various antimicrobial agents. Robertson et al. 15 failed to find any apparent binding effect when testing the susceptibility of U. urealyticum strains against tetracycline, erythromycin, and rosaramicin, but the expected decrease in macrolide activity at pH 6.0 was observed.
Since mycoplasmas exhibit no turbidity as evidence of growth and both M. hominis and U. urealyticure die soon after reaching peak growth, the preparation of suitable inocula is more difficult than with common bacteria. Using inocula higher than recommended, the MICs frequently were raised over 2-fold. 14 Kenny et al. 17 contended that the agar dilution method was more useful, as it was able to detect mixtures of susceptible and resistant mycoplasmas as well as spontaneous resistant mutants. Furthermore, the agar dilution test affords the advantage of testing many strains at one time. The prevalence of U. urealyticum strains displaying high MICs to erythromycin found in this study was lower than that described by other authors. 15 The 6-fluoroquinolones offer a useful alternative for the treatment of infections caused by gonococci and other genital pathogens that are resistant to penicillin and tetracycline. These quinolones are remarkable for being able to inhibit both 3/1. hominis and U. urealyticum at MIC levels attainable in tissue. In our study, ofloxacin proved more active than ciprofloxacin against U. urealyticum, as reported by Kenny et al. 7 Likewise, we failed to detect any M. hominis strains that were resistant to either ciprofloxacin or ofloxacin, which was in agreement with other authors. 9,7'9 Cummings and McCormack reported that M. hominis isolates in the northeastern United States have become more resistant to tetracycline and minocycline during the past decade, but data available in Argentina are insufficient to anticipate a similar trend.
The low levels of clinical resistance obviously conflict with the outcome of therapy predicted from MIC or serum levels. Since few U. urealyticum and M. hominis infections are septiccmias, the achievable serum drug level may not reflect its concentration at the actual site of infection. Therefore, serum levels exceeding the MIC may be required for a bacteriologic cure.
In conclusion, the emerging resistance of mycoplasmas to certain antibiotics emphasizes the need to undertake further surveillance studies on the clinical isolates of such organisms. Furthermore, in addition to in vitro susceptibility and pharmacokinetic studies, clinical trials including suitable antibi-otics to be used in mycoplasmal or unreaplasmal infections are essential before extrapolating in vitro findings to human treatment.