Susceptibility of Strains of Ureaplasma urealyticum and

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUly 1981, p /81/ $02.00/0 Vol. 20, No. 1 Standardized Method for Determining Antimicrobial Susceptibility of Strains of Ureaplasma urealyticum and Their Response to Tetracycline, Erythromycin, and Rosaramicin JANET A. ROBERTSON,* JOHN E. COPPOLA, AND OWEN R. HEISLER Department ofmedical Bacteriology, University ofalberta, Edmonton, Alberta, Canada T6G 2H7 Received 6 January 1981/Accepted 24 April 1981 We describe a method for determining the miniimal inhibitory concentrations (MICs) of antibiotics for Ureaplasma urealyticum which is compatible with current standards of susceptibility testing. A presumptive MIC is available after 24 h of incubation, and the definitive MIC is available at 48 h. The MICs for 9 serotype strains and 27 clinical isolates ranged from O0.5 to 256 pg of tetracycline per ml, cl to 64,ug of erythromycin per ml, and to 4.0,ug of rosaramicin per ml. Of an additional 555 isolates screened for their response to tetracycline, 2% required MICs of greater than 64 pg/ml, which we believe is near the concentration at which in vivo resistance to this agent is expressed. After prolonged exposure to rosaramicin, the resistance of two of three serotype strains of U. urealyticum was increased 8- and 16-fold, but the MICs still did not exceed 1.0 Ag/ml. Strains of Ureaplasma urealyticum can be pathogenic in the human genital tract (29). All mycoplasmas, including U. urealyticum, were considered susceptible to the tetracycline antibiotics (3, 7, 22, 28) until 1974, when Ford and Smith (8) reported the isolation of a strain of ureaplasma from a patient with nongonococcal urethritis which was refractory to tetracycline therapy. Since then, additional strains of this species have been identified as resistant to the tetracyclines (2, 6, 15, 18, 26, 27). Most workers consider ureaplasmas susceptible to erythromycin (6, 11, 22, 28), but reports of strains demonstrating in vitro resistance can be found in the recent literature (2, 26, 27). Because of these findings a new macrolide, rosaramicin, was included in this study. Low concentrations of rosararicin have shown high in vitro activity against a wide range of organisms, including two other genital tract pathogens, Neisseria gonorrhoeae (1, 20) and Chlamydia trachomatis (12, 25). Various methodologies for testing the antimicrobial susceptibility of U. urealyticum have been complicated by the special requirements of the organisms for serum-supplemented medium and a low ph (6.0 ± 0.5) for growth (23). The present study was undertaken to fill the need for a method for an in vitro test which was compatible with current testing standards, was appropriate for use in the clinical laboratory, and also 53 would provide results to the clinician without unreasonable delay. This paper describes such a method and its application to both serotype strains and clinical isolates. (A preliminary account of this work was presented at the 77th Annual Meeting of the American Society for Microbiology, Las Vegas, Nev., 1978). MATERIALS AND METHODS Organisms. U. urealyticum strain T-960-CX8 (serotype 8) was supplied by M. C. Shepard, Camp Lejeune, N.C. All other serotype strains (see Table 1) were supplied by D. K. Ford, Department of Medicine, University of British Columbia, Canada; these are the progenitors of the serotype strains in the American Type Culture Collection. The Boston T strain was obtained from R. B. Kundsin, Peter Bent Brigham Hospital, Boston, Mass. All other clinical isolates were obtained from urethral swabs from patients with urethritis examined between 1977 and 1980 by the Mycoplasma Laboratory, Department of Medical Bacteriology, University of Alberta. U. urealyticum was identified by its colonial morphology on agar and a positive urease spot test (21). Escherichia coli was obtained from the American Type Culture Collection, Rockville, Md. Media and solutions. (i) Media. Bromothymol blue (B) broth and genital mycoplasma agar were used to cultivate U. urealyticum (16). Both media contain 10% (vol/vol) normal horse serum, a requirement of the organism for growth. Nystatin (E. R. Squibb & Sons, New York, N.Y.) and lincomycin hydrochloride

2 54 ROBERTSON, COPPOLA, AND HEISLER (The Upjohn Co. of Canada, Don Mills, Ontario) were added at concentrations of 50 U and 20,ug per ml, respectively, to media used for primary isolation. B broth contained 14 mg of Ca2' and 9 mg of Mg2e per liter. E. coli was cultivated in Mueller-Hinton broth with Ca2' and Mg2+ adjusted to 75 and 20 mg/liter, respectively. Cation concentrations were determined by atomic absorption spectrometry. (ii) Antibiotic solutions. The instability of tetracycline hydrochloride at high concentrations (256,ug/ ml or greater) was indicated by its darkening, especially during incubation, and led us to use tetracycline base instead. Crystalline tetracycline (Sigma Chemical Co., St. Louis, Mo.) was dissolved in ethanol and then diluted 1:10 in broth. This and the aqueous solutions of erythromycin gluceptate (Eli Lilly and Co. [Canada] Ltd., Toronto, Canada) and rosaramicin (Schering Corp., Pointe Claire, Canada) were stored at -20 C and, as required, thawed and further diluted in broth. After the addition of an antibiotic solution the ph of a medium was checked and, if necessary, readjusted to 6.0 with HCI or NaOH. Determination of MICs of antibiotics. (i) Inoculum preparation. Before minimal inhibitory concentrations (MICs) were determined, a series of 1:10 dilutions of each stock culture of U. urealyticum or specimen was made in B broth and incubated overnight. The highest dilution which was chartreuse in color was used for the inoculum. The chartreuse color is intermediate in the change from yellow to green which occurs when ureaplasmas degrade the urea in B broth; it represents a ph of about 6.8, characteristic of a culture in the late logarithmic phase of growth (16). Populations of U. urealyticum were the mean of duplicate color change unit (CCU50) determinations (16). A CCU is the minimum inoculum required to cause growth as indicated by a change in the medium indicator. The CCU50 is the reciprocal of a dilution which has a 50% probability of containing one viable unit. E. coli inocula were logarithmic cultures in Mueller-Hinton broth which were adjusted to an optical density at 600 nm of 0.05 (or about 108 colony-forming units per ml on agar) and then diluted 1:100. (ii) Tube dilution method. For U. urealyticum, doubling dilutions of antibiotic were made in 2-mi volumes of B broth in screw-capped test tubes. Each tube, including the antigen control, received 1 drop from a Pasteur pipette (about 0.05 ml) of the chartreuse-colored culture described above. Controls for broth, organism, and antibiotic were included. For testing tetracycline an additional control, organism in broth with 10% ethanol (the solvent for this antimicrobial agent), was added. Both tests and controls were carried out in duplicate. The tubes were incubated and examined at intervals over several days for color changes. The MIC endpoint was taken as the lowest dilution which had turned green after 48 h of incubation. In instances where the endpoint was debatable, the hydrogen ion concentration was determined; a broth ph of less than 7.0 was considered indicative of inhibition. For determination of the minimal bactericidal concentration, 0.01-ml volumes were removed from appropriate tubes at 24 and 48 h and plated onto genital mycoplasma agar or placed in 1 ml of B broth. These cultures were examined for growth ANTIMICROB. AGENTS CHEMOTHER. after 2 days of incubation. The minimal bactericidal concentration was the lowest dilution in which neither color change in broth nor colonies on agar could be detected after 4 days of incubation (i.e., such cultures contained fewer than 100 survivors per ml). A similar protocol was followed for establishing the MICs for E. coli. The antibiotics were tested in three media: in Mueller-Hinton broth at the usual ph of 7.4 and also at ph 6.0, and in B broth (ph 6.0). An inoculum of 0.1 ml was used. The tubes were examined at intervals for turbidity, and the endpoints were taken at 24 and 48 h. (iii) Abbreviated tube test. For surveying the response of clinical isolates to tetracycline, tubes containing a 2-ml volume of B broth with 0, 2, 16, and 64 ug of the antibiotic per ml were prepared and stored at -20 C. When required, a set of tubes was thawed and received the same size of inoculum used for the full tube dilution method. The tubes were incubated and examined daily for color changes. (iv) Micromethod. The micromethod was a miniaturization of the tube method and yielded the same results for a given strain. Stock solutions of antibiotic were diluted in B broth to contain either 4,096 ug of tetracycline, 1,024 ug of erythromycin, or 32 ug of rosaramicin per ml. A ml volume of antibioticcontaining broth was put into the second and third wells of each row of a microtiter plate (Cooke Engineering Co., Alexandria, Va.). The same volume of plain B broth was put into the 3rd to 12th wells. Using microtiter loops calibrated to hold ml, doubling dilutions of the antibiotic were made from the third to last wells in each row. The first well in each row received ml of B broth, and the others received 0.15 ml to make up that volume. Then all wells were inoculated with ml of the U. urealyticum inoculum culture (described above) which had been diluted 1:10 in broth. The first well served as the Ureaplasma control. Controls for B broth, B broth with antibiotic, and, for testing tetracycline, Ureaplasma in B broth with 1.25% ethanol, were included for each run, as were strains of U. urealyticum requiring a known MIC of each antibiotic (serotypes 1 and 9). Both tests and controls were performed in duplicate. The plates were sealed, vented, incubated, and examined daily for color change. Development of resistance to rosaramicin. Each of three strains of U. urealyticum was tested as follows: 0.5 ml of a chartreuse (logarithmic-phase) culture was introduced into a series of 19.5-ml volumes of B broth in which the rosaramicin concentration was increased by 25% in each successive tube. The initial concentration of rosaramicin was 0.002,g/ml. The series was incubated until no further growth occurred. The organism rescued from the highest concentration of rosaramicin was passed in this level of antibiotic, and the resulting chartreuse culture was used as inoculum for the next series. This procedure was continued until three consecutive passages showed no further increase in the level of antibiotic resistance. Deionized, glass-distilled water was used in all instances. Incubation was carried out at 36 C. Cultures in broth (including all MIC determinations) were incubated in air, and cultures on agar were incubated in 5% CO2 in nitrogen.

3 VOL. 20, 1981 RESULTS Before testing the susceptibility of strains of U. urealyticum, the effects of the components and ph of B broth on MIC determinations were examined by the classic tube dilution method using E. coli ATCC and four E. coli isolates. The responses of the reference strain, ATCC 25922, were representative of the five strains tested (Fig. 1). The MIC of tetracycline after 24 h in Mueller-Hinton broth was consistent with the 1.2 to 1.4,ug/ml obtained with this strain under standard conditions (30); no MICs of erythromycin and rosaramicin were available for this strain. Substitution of B broth (ph 7.4) for Mueller-Hinton broth (ph 7.4) had an apparent effect after 48 h of incubation only on erythromycin, for which the MIC decreased from 128 to 64 Ag/ml for all test strains. Whereas the reduction in the ph of B broth from 7.4 to 6.0 also caused a twofold decrease in the MIC of tetracycline, it quadrupled the MIC of the two macrolide antibiotics. The tube dilution method was also used to establish test conditions for U. urealyticum. The inoculum population was about as large as could be used for rapid results without overwhelming the antibiotic. For instance, strain T-960 required an erythromycin MIC of 4 pug/ml whether the stated inoculum or one tenth that number of cells was used. However, with 10-fold the stated inoculum, growth occurred in 128,ug of the antibiotic per ml, the highest concentration tested. Because the growth of U. urealyticum in broth medium does not cause visually detectable turbidity, a ph indicator of metabolic activity was u FIG. 1. Effect of medium and ph on the MICs of three antibiotics against E. coli ATCC after 24 and 48 h of incubation. Bars represent the MICs at 48 h, the endpoint for U. urealyticum tests. A line through a bar at a lesser concentration of antibiotic indicates a lower reading at 24 h, an acceptable time period for E. coli tests. U. UREALYTICUM SUSCEPTIBILITY TESTING employed. We knew from previous work that chartreuse-colored cultures of stock strains in B broth contain about 107 CCU50/ml (16, 17). Results of CCU50 determinations carried out on the initial chartreuse-colored cultures of four consecutive clinical isolates in B broth ranged between 1.2 x 107 and 2.5 x 107 CCU50/ml, providing evidence that the color of the culture could be relied upon for estimating the inocula of new isolates as well as laboratory-adapted strains. With an inoculum of at least 5.0 x 105 CCU50 of organisms per ml of test broth, the culture control always changed color after 24 h of incubation, and the final MIC could be predicted. The effect of day 2 of incubation on the MICs for U. urealyticum was similar to our findings with E. coli (Fig. 1). The endpoint for tetracycline often changed by one and sometimes by two doublings; the endpoint for erythromycin occasionally was doubled, whereas rosaramicin, with rare exceptions, was unchanged. For all three antibiotics endpoint changes after 48 h of incubation were both unusual and insignificant. Over the initial 48 h of incubation, the ph of the Ureaplasma control changed from 6 to 7.5 ± 0.2. For determination of the miniimum bactericidal concentration, B broth was more sensitive than genital mycoplasma agar for the detection of growth. The three antimicrobial agents had a bactericidal effect at concentrations above the MIC. The miniimal bactericidal concentrations exceeded the MICs by the following degrees: for tetracycline, from one doubling (tube) for strains with high MICs to five tubes for those with low MICs at 24 h, decreasing from three tubes to one tube by 48 h; for erythromycin, from at least four tubes at 24 h to between two and four tubes at 48 h. Rosaramicin was also bactericidal at concentrations above the MIC. The microtiter modification of the tube test was used to determine the MICs of tetracycline, erythromycin, and rosaramicin against 9 serotype strains of U. urealyticum (Table 1) and against 29 consecutive clinical isolates (Fig. 2). The range of MICs of tetracycline for the serotype strains was similar to that the clinical isolates; for the macrolides, the lab-adapted serotypes had a much narrower range than that exhibited by the wild-type strains. At the median MIC, at which 50% of clinical isolates were susceptible to the three antibiotics (see Fig. 2), and on a weight basis, rosaramicin was 16 times as effective as tetracycline and 32 times as effective as erythromycin. Because some of the ureaplasmas required high MICs of tetracycline, the antibiotic commonly used for their treatment, we extended the study and examined a large number of isolates 55

4 56 ROBERTSON, COPPOLA, AND HEISLER TABLE 1. MICs of three antimicrobial agents against serotype strains of U. urealyticum MIC (pg/mi) Serotype Strain Tetra- Erythro- Rosarcycline mycin amicin Pi < Co T-960 (CX8) Vancouvera a Strain Vancouver has been deposited in the American Type Culture Collection as acquisition no ~80,n60l 40 / Rosaramicin O / / otetracycline E20 *Erythromycin : MICs of Antibiotics (pg/mi) FIG. 2. Response of29 clinical isolates of U. ureal- erythromycin, and rosarami- yticum to tetracycline, cin. to determine the incidence of such strains. A total of 555 strains were tested immediately after isolation by the abbreviated tube test. Of these 555, 279 (-50%) were susceptible to c2 jig of tetracycline per ml; 194 (-35%) were susceptible to 2 to 16 ug/ml; 70 (-13%) were susceptible to 16 to 64,ug/ml, and 12 (-2%) were susceptible only to >64,ug/ml. The titers were confirmed by the micromethod. This pattern of response was consistent over the 3-year course of the study. Of the 12 strains resistant to tetracycline at 64,ug/ml, 8 were tested against erythromycin and rosaramicin. For erythromycin, all required an MIC above the median of 2.0 ug/ml (Fig. 2); for rosaramicin, five of the eight strains grew in 0.062,ug/ml, but only one of these grew at 1.0,ug/ml, the other concentration tested. The effectiveness of rosaramicin at such low concentrations led us to investigate the development of rosaramicin resistance in U. urealyticum (Table 2). Inocula of at least 5 x 106 CCU5o/ml of three stock strains that never had been exposed to rosaramicin were transferred in ANTIMICROB. AGENTS CHEMOTHER. increasing concentrations of the antibiotic until a stable endpoint was reached (Table 2). Repeated cultivation in the presence of rosaramicin had no effect on the MIC for strain Pi, but increased the MICs for strains 7 and Vancouver 8- and 16-fold, respectively. Subsequent multiple transfers of these rosaramicin-cultivated strains in rosaramicin-free medium caused no reduction in the MICs. Throughout this study good agreement was obtained in duplicate assays; repeated testing provided endpoints which never differed from the stated titers by more than one doubling dilution. DISCUSSION Applicability to the routine of a clinical laboratory was a prime consideration in designing the test procedures. Because it is cheaper, has a longer shelf life, and is a more sensitive indicator of growth (4, 16), broth was chosen over agar medium. If serial dilutions of a specimen containing U. urealyticum are made in B broth with nystatin and lincomycin, a pure culture of the species is obtained within 12 to 72 h of incubation (16). From the color of the broth a logarithmic-phase culture can be identified; this serves as inoculum for the test. We used the full tube method to establish test conditions. We preferred the efficiency of the abbreviated tube test for the day-to-day screening of clinical isolates and the microtiter method for determining definitive MICs for large numbers of strains. All three modifications provided a presumptive titer at 24 h and an MIC at 48 h. The 48-h reading was more reproducible. Furthermore, cultures containing more than one serotype of U. urealyticum are common on primary culture (10, 14), and the longer incubation period ensured that a resistant strain, present even in very small numbers, would be identified. Initially, we had reservations about testing antimicrobial susceptibility in undefined medium and at the low ph levels required for TABLE 2. Effect of exposure to rosaramicin on rosaramicin MICs against U. urealyticum Rosaramicin MIC (ug/ml) Strain Stock Culture A cultivated Culture B passed in rowith rosara- saramicin- (A) micin (B) free brothb (25)C Pi (26) Vancouver (32) 1.0 a Never exposed to rosaramicin. b Six passages. 'Parentheses indicate number of passages in rosaramicm.

5 VOL. 20, 1981 U. UREALYTICUM SUSCEPTIBILITY TESTING 57 growth of U. urealyticum. The trials with E. coli (Fig. 1) identified the effects of these conditions. The serum had no apparent binding effect on any of the three agents, but the expected decrease in macrolide activity at ph 6.0 was clearly evident. However, this acidity does reflect the level in the genital tract where antimicrobial activity is expressed. In 1974, Ford and Smith (8) reported tetracycline and erythromycin MICs against five serotype strains of U. urealyticum and an isolate that was subsequently named Vancouver (17). Although our MICs for these strains (Table 1) were generally much higher, presumably because of different methodology, the elevated MIC of tetracycline for strain Vancouver was apparent in both laboratories. The type strain, T-960, has been tested in several laboratories (8, 27; Table 1), but because it has demonstrated antigenic heterogeneity (13; Robertson and Stemke, unpublished data) it does not provide a reliable basis for comparison. Spaepen et al. (27) found that the Boston T strain requires MICs of 0.5 to 20 jig of tetracycline per ml and 2 to 40,ug of erythromycin per ml (depending on the time of reading), whereas we found MICs of 2 and 16,ug/ml, respectively, for these antimicrobial agents. Other reported MICs are for wildtype strains. Although the absolute values of MICs may vary, general patterns emerge: the median MIC of tetracycline is less than that for erythromycin (2, 10, 27), and rosaramicin is decidedly more active than erythromycin (11, 24). We found the same results in our study (Fig. 2). Whereas differences in the absolute values for MICs can be resolved by standardizing methodology, the interpretation of results may be a separate issue. The definition of resistance has been an in vitro MIC higher than the level of antibiotic that can be achieved in serum. If one assumes that the usual therapeutic schedules should provide serum levels of about 2,ug of tetracycline and 1.0 to 1.5,ug of erythromycin per ml (9), then about 25 and 70%, respectively, of the isolates we tested (Fig. 2) would be considered resistant to the two antibiotics. Actually, an even wider range of serum levels may be encountered (19). Tetracycline is the antibiotic most commonly prescribed for ureaplasma infections. Ureaplasmas have been reisolated after a course of tetracycline therapy in none of 17 cases (10), in at least 3 of 58 cases (15), in 14 of 141 cases (6), and in 21 of 50 cases (27). Whereas these data could indicate a geographic variation in resistant strains, the problem of differentiating persistent infection from reinfection must be considered. Root et al. have attempted to do this in their recent study (18). We have isolated three strains of U. urealyticum which apparently persisted through tetracycline therapy; they, like strain Vancouver (8), require MICs in excess of 64,ug/ml. Only 2% of the many isolates we have tested have MICs in this range. Erythromycin is not widely used to treat urethritis, and we have not found reports of strains showing in vitro resistance. Strain Vancouver, which is resistant to tetracycline therapy, responds to erythromycin (8). Recently, we isolated a strain of U. urealyticum that required an MIC of 64,tg/ml and persisted during long-term erythromycin therapy. In the present study, only one isolate required an MIC of 64,g/ml (Fig. 2), suggesting that resistance to erythromycin is uncommon. Ureaplasma resistance to rosaramicin has not been documented. We exposed ureaplasmas to rosaramicin for months, but were unable to induce or select for levels of resistance which would likely be of clinical importance. Disagreement obviously exists between the low levels of clinical resistance that have been identified and the outcome of therapy predicted from MIC and serum levels. However, ureaplasmas normally are not found in the blood or interstitial spaces, but in the urethra. They are not exposed to serum, but to urine. Urine levels of greater than 200 jig of tetracycline per ml (5) and from 7 to 46,ig of erythromycin per ml (9) are usual. On the basis of MICs and urine levels, very few isolates would be judged resistant. The relationship between the levels of antibiotic in urethral secretions and the MICs must now be determined. Because U. urealyticum is an etiological agent of nongonococcal urethritis (29) and the absolute number of cases of nongonococcal urethritis is enormous, the antimicrobial resistance of this organism is under continued investigation in our laboratory. ACKNOWLEDGMENTS We thank F. H. Cookson of the University Health Service and his staff for providing us with clinical material, and J. Dakin of the University of Alberta Hospital for the cation determinations. Errol Prasad, May Tso, Sylvia Dublenko, and Adrian Wills provided skilled technical assistance. This work was supported by grant MA 5414 from the Medical Research Council, Ottawa, Canada, and by Schering Corp., Pointe Claire, Quebec, Canada. LITERATURE CMD 1. Biddle, J. W., and C. Thornsberry Activity of rosanicin, josamycin, erythromycin, and clindamycin against f8-lactamase-negative and 18-lactamase-positive strains of Neisseria gonorrhoeae. Antimicrob. Agents Chemother. 15: Bonissol, C., and F. Daoulas-Lesbourdelles Valeur de l'antibiogramme pour le traitement des infections urogenitales a Ureaplasma. Pathol. Biol. 26: Braun, P., J. 0. Klein, and E. H. Kass Susceptibility of genital mycoplasmas to antimicrobial agents. Appl. Microbiol. 19:62-70.

6 58 ROBERTSON, COPPOLA, AND HEISLER ANTIMICROB. AGENTS CHEMOTHER. 4. Braun, P., J. 0. Klein, Y. L. Lee, and E. H. Kass Methodologic investigations and prevalence of genital mycoplasmas in pregnancy. J. Infect. Dis. 121: Bryant, M. C Antibiotics and their laboratory control, 2nd ed. Butterworths, London. 6. Evans, R. T., and D. Taylor-Robinson The incidence of tetracycline-resistant strains of Ureaplasma urealyticum. J. Antimicrob. Chemother. 4: Ford, D. K Culture of human genital "T-strain" pleuropneumonia-like organisms. J. Bacteriol. 84: Ford, D. K., and J. R. Smith Non-specific urethritis associated with a tetracycline-resistant T-mycoplasma. Br. J. Vener. Dis. 50: Garrod, L P., M. P. Lambert, and F. O'Grady Antibiotics and chemotherapy, 4th ed. Churchill Livingstone, London. 10. Graber, C. D., P. Creticos, J. Valianti, and H. 0. Williamson T mycoplasma in human reproductive failure. Obstet. Gynecol. 54: Hill, A. C., and G. Sutton Rosamicin, a macrolide with in vitro activity against Ureaplasma urealyticum. J. Antibiot. 32: Kuo, C.-C., S.-P. Wong, and J. T. Grayston Antimicrobial activity of several antibiotics and a sulfonamide against Chiamydia trachomatis organisms in cell culture. Antimicrob. Agents Chemother. 12: LAn, J.-S., and E. H. Kass Serotypic heterogeneity in isolates of human genital T-mycoplasmas. Infect. Immun. 7: Piot, P Comparison of growth inhibition and immunofluorescence tests in serotyping clinical isolates of Ureaplasma urealyticum. Br. J. Vener. Dis. 63: Prentice, M. J., D. Taylor-Robinson, and G. W. Csonka Non-specific urethritis. A placebo controlled trial of minocycine in conjunction with laboratory investigations. Br. J. Vener. Dis. 52: Robertson, J. A Bromothymol blue broth: improved medium for detection of Ureaplasma urealyticum (T-strain mycoplasma). J. Clin. Microbiol. 7: Robertson, J. A., and G. W. Stemke Modified metabolic inhibition test for serotyping strains of Ureaplasma urealyticum (T-strain mycoplasma). J. Clin. Microbiol. 9: Root, T. E., L. D. Edwards, and P. J. Spengler Nongonococcal urethritis: a survey of clinical and laboratory features. Sex. Trans. Dis. 7: Sabath, L. D Peak serum concentrations frequently obtained with some antimicrobics, p In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and J. P. Truant (ed.), Manual of clinical microbiology, 3rd ed. American Society for Microbiology, Washington, D.C. 20. Sanders, C. C., and W. E. Sanders In vitro activity of rosamicin against Neisseria and Haemophilus, including penicillinase-producing strains. Antimicrob. Agents Chemother. 12: Shepard, M. C., and D. R. Howard Identification of "T" mycoplasmas in primary culture by means of a direct test for urease. Ann. N. Y. Acad. Sci. 174: Shepard, M. C., C. D. Lunceford, and R. L. Baker T-strain mycoplasma. Selective inhibition by erythromycin in vitro. Br. J. Vener. Dis. 42: Shepard, M. C., C. D. Lunceford, D. K. Ford, R. H. Purcell, D. Taylor-Robinson, S. Razin, and F. T. Black Ureaplasma urealyticum gen. nov., sp. nov.: proposed nomenclature for the human T (Tstrain) mycoplasmas. Int. J. Syst. Bacteriol. 24: Smith, T. F In vitro susceptibility of Ureaplasma urealyticum to rosaramicin. Antimicrob. Agents Chemother. 16: Smith, T. F., and H. E. Washton In vitro susceptibility of 30 strains of Chlamydia trachomatis to rosamicin. Antimicrob. Agents Chemother. 14: Spaepen, M. S., and R. B. Kundsin Simple, direct broth-disk method for antibiotic susceptibility testing of Ureaplasma urealyticum. Antimicrob. Agents Chemother. 11: Spaepen, M. S., R. B. Kundsin, and H. W. Horne Tetracycline-resistant T-mycoplasmas (Ureaplasma urealyticum) from patients with a history of reproductive failure. Antimicrob. Agents Chemother. 9: Taylor-Robinson, D Mycoplasmas of various hosts and their antibiotic sensitivities. Postgrad. Med. J. Suppl. 43: Taylor-Robinson, D Pathogenicity of ureaplasmas for animals and man. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 245: Washington, J. A., and A. L. Barry Dilution test procedures, p In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C.