Mycoplasma hominis, and Ureaplasma urealyticum to Sparfloxacin and PD

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1991, p /91/ $02.00I0 Copyright 1991, American Society for Microbiology Vol. 35, No. 6 In Vitro Susceptibilities of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum to Sparfloxacin and PD K. B. WAITES, L. B. DUFFY, T. SCHMID, D. CRABB, M. S. PATE, AND G. H. CASSELL* Department of Microbiology, University of Alabama Schools of Medicine and Dentistry, Birmingham, Alabama Received 10 December 1990/Accepted 25 March 1991 The in vitro activities of two investigational quinolones, sparfloxacin (previously designated AT 4140) and PD , were determined for 30 strains each of Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum and compared with those of ciprofloxacin, tetracycline, clindamycin, and erythromycin. Erythromycin was the most active compound against M. pneumoniae (maximum MIC, <0.008,ug/ml). PD (MICs, <0.008 to 0.031,ug/ml), sparfloxacin (MICs, <0.008 to 0.25 pg/ml), clindamycin (MICs, <0.008 to 0.5,g/ml), and tetracycline (MICs, to 0.25 pg/ml) were superior to ciprofloxacin (MICs, 0.5 to 2,ug/ml). Sparfloxacin and PD were active against M. hominis (MICs, <0.008 to pg/ml for each) at concentrations comparable to those of clindamycin (MICs, <0.008 to 0.063,g/ml) and at concentrations lower than those of ciprofloxacin (MICs, to 0.5 pg/ml). As expected, M. hominis was resistant to erythromycin (MICs, 32 to.256,ug/ml). For U. urealyticum, PD (MICs, to 0.5,ug/ml) and sparfloxacin (MICs, to 1,ug/ml) were superior to erythromycin (MICs, 0.25 to 4,g/ml), ciprofloxacin (MICs, 0.5 to 8,ug/ml), and clindamycin (MICs, 0.25 to 64,g/ml. Both new quinolones were equally active against tetracyclinesusceptible as well as -resistant strains of M. hominis and U. urealyticum. The possible influence of medium components and/or ph on MICs was evaluated by testing a Staphylococcus aureus reference strain with each antibiotic in SP-4 broth and 10-B broth and comparing the results with published MICs for this strain. MICs determined in 10-B broth for erythromycin were affected most. This study shows that the activities of sparfloxacin and PD are similar to one another and comparable or superior to those of other drugs used to treat mycoplasmal infections. The MICs of both new quinolones were consistently 2 to several dilutions lower than those of ciprofloxacin for each species. Mycoplasma pneumoniae has long been recognized as a common pulmonary pathogen, especially in younger age groups, causing as much as 20% of pneumonia in the general population and up to 50% in military settings (3). Most respiratory infections due to M. pneumoniae are self-limiting, but in some instances life-threatening disease may occur (3). Although naturally occurring strains of M. pneumoniae resistant to erythromycin have not been described to date, the effectiveness of this drug may be limited in some cases because of dose-related nausea, vomiting, or diarrhea, justifying the need for alternative treatment modalities (9). Knowledge of the antibiotic susceptibilities of Mycoplasma hominis and Ureaplasma urealyticum is of increasing importance because of the recognition of these organisms as opportunistic pathogens in newborn infants (4-6, 42, 45) and other immunosuppressed persons (36) and as primary pathogens in sexually transmitted diseases such as nongonococcal urethritis (5) and pelvic inflammatory disease (5). Resistance to traditional drugs such as tetracycline, which has been widely used in treating mycoplasmal infections and other sexually transmitted diseases, is now recognized in both U. urealyticum and M. hominis to some extent (10, 13, 28, 35). Increasing reports of P-lactamase-producing and tetracycline-resistant strains of Neisseria gonorrhoeae have further intensified the search for new agents to treat sexually transmitted infections (19). Fluoroquinolones represent a new class of broad-spectrum antimicrobial agents active against many gram-positive * Corresponding author and gram-negative organisms. These compounds are bactericidal, with generally good pharmacokinetics and safety profiles. However, some pathogens, including mycoplasmas, are not sufficiently susceptible at low concentrations to many of the quinolones tested to date (8, 12, 21, 24, 26, 46). New compounds such as sparfloxacin (previously designated AT 4140) were developed in an attempt to broaden the quinolone antimicrobial spectrum (24). We have described the in vitro susceptibilities of M. pneumoniae, M. hominis, and U. urealyticum to sparfloxacin and PD The activities of these drugs were compared with those of ciprofloxacin, tetracycline, clindamycin, and erythromycin. (Preliminary results of this study have been presented previously [43].) MATERIALS AND METHODS Organisms. Thirty isolates each of U. urealyticum, M. hominis, and M. pneumoniae were tested. The U. urealyticum isolates included 14 reference serotypes (41) and 16 low-passage clinical isolates from the placenta, cervix, urethra, bone, throat, rectum, trachea, cerebrospinal fluid, or amniotic fluid. Four isolates previously shown in our laboratory to be resistant to tetracycline (MIC, -16 ptg/ml) (37) were specifically included. The M. hominis isolates included 7 reference strains (41) and 23 low-passage clinical isolates from the placenta, cervix, urethra, bone, rectum, trachea, cerebrospinal fluid, amniotic fluid, kidney, vascular tissue (thrombophlebitis), or wounds. Each strain had been positively identified by immunoblotting with a monoclonal antibody specific for M. hominis developed in our laboratory

2 1182 WAITES ET AL. (unpublished data). Five isolates previously shown in our laboratory to be tetracycline resistant (MIC,.16,ug/ml) (37) were included. The sources of the M. pneumoniae isolates tested have been described previously (8). Antimicrobial agents. Reference standard powders for in vitro susceptibility testing of the following antimicrobial agents were obtained from their respective manufacturers: tetracycline hydrochloride (Lederle Laboratories, Wayne, N.J.), erythromycin base (Abbott Laboratories, Abbott Park, Ill.), clindamycin hydrochloride (Upjohn Pharmaceuticals, Kalamazoo, Mich.), ciprofloxacin (Miles Laboratories, West Haven, Conn.), and PD and sparfloxacin (Parke-Davis, Ann Arbor, Mich.). A 10-ml stock solution of each antibiotic (2,048,ug/ml),' allowing for purity factors as needed, was prepared fresh on the day of each assay. Erythromycin powder was dissolved in 1 ml of 95% ethanol, and clindamycin, tetracycline, and ciprofloxacin powders were solubilized directly with sterile' tissue culture quality deionized water. PD was dissolved in 0.5 ml ethanol prior to the addition of sterile water to achieve a final volume of 10 ml, in accordance withthe manufacturer's instructions. Sparfloxacin was solubilized by the addition of 0.5 ml of ethanol and then 2 to 3 drops of NaOH prior to final dilution with water. Inoculum preparation. Aliquots of stock cultures were frozen at -70 C, and serial dilutions were made in 10-B broth (ph 6.0) (32) for ureaplasmas or SP-4 broth (ph 7.5) (40) for mycoplasmas to determine the number of colorchanging units present. A color-changing unit was designated as the reciprocal of the highest dilution at which growth was present, as evidenced by an alkaline shift in the presence of the phenol red ph indicator for U. urealyticum and M. hominis and an acidic shift for M. pneumoniae. Stock cultures were thawed to room temperature on the day of the assay and diluted in the appropriate broth without antibiotics to yield approximately 103 to i04 color-changing units per 0.2 ml. This 'concentration has been previously recommended as the optimal organism concentration for reporting susceptibilities of clinical isolates of mycoplasmas (30). Sufficient volumes of organisms were prepared to inoculate 96-well microtiter plates to test all six antibiotics in duplicate at 16 concentrations as well as controls. Organism suspensions were incubated at 37 C under atmospheric conditions for 2 h prior to inoculation into the test system to allow them to become metabolically active. Broth microdilution assay. The broth microdilution assay is essentially equivalent to a metabolism inhibition test (34). Sterile 10-B broth for' U. urealyticum and SP-4 broth for M. hominis and M. pneumoniae were used to perform the assay. Volumes of ml of broth were inoculated in duplicate into microtiter plates (Costar, Cambridge, Mass.) in all except the first well of each of two adjacent rows for each antibiotic, leaving an empty row between each antibiotic. The stock solution (0.025 ml) containing 2,048 jig of each drug per ml was added to the first two wells, and serial twofold dilutions (0.025 ml) were made with a multichannel inoculator beginning with the second well and proceeding until 16 concentrations of each drug were obtained, discarding the final ml' from the last well. A suspension of organisms (0.175 ml) was added to each drug-containing well; the final range of concentrations of antibiotics being tested was to 256,ug/ml. Microtiter plates were sealed with clear acetate and incubated at 37 C under atmospheric conditions. Broth was observed for evidence of growth (color change) after overnight incubation and daily until the endpoint was ANTIMICROB. AGENTS CHEMOTHER. first evident. The MIC was defined as the lowest concentration of antibiotic by which growth was inhibited when the positive control tube first showed a color change, usually after 16 to 20 h of incubation for U. urealyticum but sometimes longer for M. hominis and M. pneumoniae (4 days or more). All assays were repeated in duplicate on a different day to verify reproducibility. Controls. A positive (growth) control consisting of organisms in broth, a negative (sterility) control consisting of uninoculated broth, and a drug control consisting of broth containing the highest concentration of drug were included for each mycoplasmal strain tested. The color-changing units of each strain per ml were determined for the inoculum actually used in the procedure by making 10-fold dilutions (0.1 ml of organism suspension per 0.9 ml of broth) to This back titration of organisms was inoculated. Mycoplasmal requirements for specialized enriched media, along with lower phs for optimal cultivation and testing of ureaplasmas (31), necessitate that considerable deviation be made from the acceptable procedures used for susceptibility testing of conventional bacteria. As a control for potential interactions between antibiotics, medium components, and ph which could potentially affect observed MICs, American Type Culture Collection bacterial reference strain Staphylococcus aureus ATCC 29213, with published MICs obtained in Mueller-Hinton broth for each of the drugs tested (37), was inoculated into microtiter plates containing 10-B broth, SP-4 broth, and appropriate dilutions of the six antibiotics for MIC determinations. These control procedures were repeated each time an assay was performed. RESULTS Susceptibilities of mycoplasmas to antibiotics. The broth microdilution susceptibility testing method has been shown in our laboratory to be an accurate, reproducible means for determining antibiotic susceptibilities of U. urealyticum (44). In the present study, the assay was reproducible within +1 dilution for all MICs reported for both well-to-well testing and batch-to-batch testing. Table 1 shows the MICs obtained for six antibiotics tested against M. pneumoniae, M. hominis, and U. urealyticum. Erythromycin was the most active antibiotic tested against M. pneumoniae, the MIC for all strains being <0.008,ug/ml. Tetracycline and clindamycin also showed favorable activities against all strains at slightly higher levels, with no MIC for any strain being >0.5,ug/ml. Sparfloxacin and PD were active against M. pneumoniae, with no MIC being >0.25,ug/ml. Each of these new quinolones was relatively more active than was ciprofloxacin (MICs, 0.5 to 2,g/ml). The M. hominis strains tested in this study showed the predictable uniform resistance to erythromycin (MICs, 32 to >256,ug/ml) and susceptibility to clindamycin at low levels (MICs, <0.008 to 0.063,ug/ml). Sparfloxacin and PD showed comparable activities against all strains of M. hominis, the MICs being essentially equivalent to those of clindamycin. The MICs of sparfloxacin and PD were equal to or lower than those of tetracycline for every strain, the maximum MIC being 0.031,ug/ml. No difference in the activities of sparfloxacin and PD against tetracyclineresistant and tetracycline-susceptible M. hominis strains was observed. Ciprofloxacin was slightly less active against M. hominis (MICs, to 0.5,ug/ml) than were the two newer quinolones. The U. urealyticum strains were inhibited by erythromycin at MICs ranging from 0.25 to 4,ug/ml, with MICs for 50%

3 VOL. 35, 1991 SUSCEPTIBILITIES OF MYCOPLASMAS TO QUINOLONES 1183 TABLE 1. In vitro susceptibilities of M. pneumoniae, M. hominis, and U. urealyticum to sparfloxacin, PD , and four other antibiotics Organism (no. of strains) Drug MIC (ILg/ml) Range 50%o 90%o M. pneumoniae (30) Tetracycline Erythromycin <0.008 <0.008 <0.008 Clindamycin < Ciprofloxacin Sparfloxacin < PD < M. hominis (30) Tetracycline Erythromycin Clindamycin < < Ciprofloxacin Sparfloxacin < < PD < U. urealyticum (30) Tetracycline Erythromycin Clindamycin Ciprofloxacin Sparfloxacin PD of strains (MIC50s) and MIC%0s of 1 and 2,ig/ml, respectively. Clindamycin was less active (MICs, 0.25 to 64,g/ml). Sparfloxacin and PD were active against all ureaplasmal strains tested, with no MIC being >1 jig/ml, making these drugs superior to ciprofloxacin (MICs, 0.5 to 8,ug/ml; MIC90, 4 jxg/ml). Ciprofloxacin was less active against ureaplasmas than against either of the other two species tested. PD had MIC50s and MIC90s that were 1 dilution (twofold) lower than those of sparfloxacin for U. urealyticum. This slightly greater activity of PD was observed for M. pneumoniae (3 dilutions at the M'C50 and 2 dilutions at the MIC0), while for M. hominis, the MIC90s of the two drugs were equivalent and the MIC50 of sparfloxacin was actually lower than that of PD Each of the new quinolones was active against tetracycline-resistant as well as tetracycline-susceptible ureaplasmal strains, with no appreciable differences in MICs for the two groups. Ranges of MICs for sparfloxacin and PD for U. urealyticum, as well as for the other two species, were relatively narrow, with MIC50s and MIC90s typically varying by 1 or 2 dilutions or not at all. Effect of ph and mycoplasmal media on MICs obtained for a bacterial reference strain. Table 2 shows the acceptable MIC ranges for each antibiotic when tested against S. aureus ATCC in Mueller-Hinton broth (ph 7.2 to 7.4) (37). The MIC ranges obtained in SP-4 broth (ph 7.5) and 10-B broth (ph 6.0) are shown for comparison. SP-4 broth yielded a narrow range of MICs for the reference strain for all antibiotics tested; these MICs matched the acceptable MICs for this strain in Mueller-Hinton broth. However, some MICs achieved in 10-B broth were at variance with those determined in Mueller-Hinton broth or achieved in SP-4 broth. Erythromycin was affected the most, the upper limit of its MIC range being 5 dilutions higher. Ciprofloxacin and clindamycin MICs also tended to be higher, but to a lesser extent, the maximum MICs being only 2 dilutions higher than the upper limits of the recommended ranges for each. Tetracycline MICs were as much as 2 dilutions lower than the lower limit of the acceptable MIC range. Sparfloxacin TABLE 2. Effect of mycoplasmal media and ph on MICs for S. aureus ATCC MIC range (p.g/ml) with the following broth: Drug Mueller-Hinton S)b (ph 7.2 to 7.4)a SP-4 (ph 7.5)b 10-B (ph 6.) Tetracycline (14) (10) Erythromycin (14) 2-16 (10) Clindamycin (15) (11) Ciprofloxacin (14) (12) Sparfloxacin (14) (8) PD (14) (6) a Range of acceptable MICs according to the National Committee for Clinical Laboratory Standards (37). b Numbers in parentheses are numbers of assays. and PD MICs were not affected when the assay was performed with 10-B broth (ph 6.0). DISCUSSION It can be generalized that the quinolones evaluated to date, as a group, show moderate activity against mycoplasmas (12). However, results and interpretations of the numerous publications dealing with in vitro potencies of quinolones against mycoplasmas have varied considerably (1, 2, 7, 8, 11, 15, 17-21, 25-27, 33, 39, 41, 46). The reasons for such disparities, other than extremely small numbers of strains tested in some studies, are likely related to differences in technique, inoculum size, incubation conditions, ph, media, or endpoints measured. Until universally applied standards are in place for performing susceptibility tests on mycoplasmas, further discrepancies can be expected. Studies comparing the activities of various antibiotics with that of erythromycin against M. pneumoniae have shown that the macrolide consistently has the lowest MICs, often by several dilutions (8, 18, 24, 25, 41, 46). Quinolones may have favorable MICs, and some may perform better than others (14, 18, 19, 24-26). Nakamura et al. (24) found sparfloxacin to be highly active (M'C50, 0.1,ug/ml) against M. pneumoniae, its MICs being lower than those of ciprofloxacin, ofloxacin, enoxacin, norfloxacin, and minocycline. Kenny and Cartwright (18) found WIN (MICs, to 0.25 p.g/ml) and sparfloxacin (MICs, 0.25 to 0.5,ug/ml) to be more potent than tetracycline, ofloxacin, lomefloxacin, and fleroxacin. Sparfloxacin and WIN were the only quinolones tested with MICs of <1,ug/ml. These results are generally in agreement with the findings of the present study. M. hominis may be inhibited by theoretically attainable concentrations of ciprofloxacin according to a number of studies (14, 17, 19, 26, 27, 39, 46). MICs for other quinolones have also been reported (17, 19, 20, 26, 27, 33, 39, 41, 46). Nakamura et al. (24) tested the M. hominis type strain PG 21 with sparfloxacin, finding an MIC of 0.05,ug/ml, in agreement with the results of the present study. U. urealyticum has shown variable but generally poor susceptibility to most quinolones tested to date. No previously determined MICs for either PD or sparfloxacin for this organism are available. MICs for other quinolones have been reported (1, 2, 7, 15, 17, 19, 26, 27, 33, 39, 46). The possibility that the specialized media and conditions necessary for optimal cultivation and susceptibility testing of mycoplasmas may exert some effect on the activity and/or stability of various antimicrobial agents cannot be discounted, as our results indicate. Microbial susceptibility

4 1184 WAITES ET AL. tests are best performed near the physiologic ph in welldefined media for standardization purposes (22, 37, 38). Susceptibility testing of ureaplasmas in broth must be carried out at ph 6 to 6.5 because of the growth requirements of the organism and the indicator reaction necessary for the interpretation of the endpoint (31, 32). Erythromycin MICs may be several dilutions higher at ph 6.0 to 6.5 than at the physiologic ph, such that actual activity in vivo may be greater than that indicated by assays performed at a lower ph (22, 38, 41). The reason for the diminished activity of erythromycin in vitro is thought to be due to its greater degree of ionization at a lower ph and the fact that the un-ionized form may be more directly responsible for antimicrobial activity (38). The influence of ph on ureaplasmal growth and interpretation of erythromycin susceptibility is the root of considerable controversy surrounding the utility of this antibiotic in treating infections caused by this organism. It is most likely that ph, rather than medium components in 10-B broth, is primarily responsible for the observed effects on erythromycin MICs, since the MICs of erythromycin for M. pneumoniae in SP-4 broth (ph 7.5), similar in many respects to 10-B broth, were consistently very low (<0.008,g/ml). Erythromycin MICs for S. aureus ATCC in SP-4 broth were also within the acceptable range established for Mueller-Hinton broth (37). We believe that the MICs obtained in this study for erythromycin against U. urealyticum are meaningful, because of the relatively narrow range observed (0.25 to 4,g/ml) and the apparent scarcity of high-level resistance, which would likely be detected even under the present assay conditions. Prospective studies of patients with ureaplasmal infections treated with erythromycin and for whom both in vitro MICs and in vivo microbiological efficacy are determined will be needed to further clarify this complex relationship. Lincosamide MICs may also be elevated at an acidic ph, while tetracyclines may be more active (38). The activities of neither of the new quinolones were affected to a great extent by testing in 10-B broth (ph 6.0). However, other quinolones have shown variable responses to alterations of the medium ph (12, 41). The addition of serum to Mueller-Hinton broth has been shown to significantly diminish the activities of some quinolones, such as difloxacin and temafloxacin, but not ciprofloxacin (12). For most bacterial strains, serum supplementation yields MICs only one- to twofold higher than those yielded without supplementation (12). Similarities between the MICs for the S. aureus reference strain in SP-4 broth containing 20% serum and those within the acceptable range for unsupplemented Mueller-Hinton broth (37) suggested that neither this medium component nor other medium components were of major importance in the present study for any of the antimicrobial agents tested in SP-4 broth. Fluoroquinolones may prove useful for the treatment of mycoplasmal infections because they are potentially effective against all three pathogenic species, including those strains resistant to other drugs, such as tetracycline. The present study has shown that newer quinolone compounds, such as sparfloxacin and PD , have in vitro activities which are comparable or superior to those of other currently available drugs used for the treatment of mycoplasmal and ureaplasmal infections. They have the additional advantage of achieving growth inhibition at concentrations up to several dilutions lower than those of ciprofloxacin, currently the most widely available quinolone in clinical use. The MICs of sparfloxacin and PD against U. urealyticum show these compounds to be perhaps the most potent quinolones tested against this organism to date. ANTIMICROB. AGENTS CHEMOTHER. A limited number of clinical trials assessing the clinical and microbiological efficacies of quinolones in comparison with established treatments for sexually transmitted infections known to have a mycoplasmal etiology or association have shown promise (7, 23). Sparfloxacin is well absorbed orally, has a relatively long half-life in plasma, and has good tissue distribution (29). Preliminary studies have shown that the toxicity of sparfloxacin is low enough to allow the initiation of clinical trials, which will be required to evaluate its clinical utility against pathogenic mycoplasmas (16). ACKNOWLEDGMENT This work was supported by a grant from Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Mich. REFERENCES 1. Aznar, J., M. C. Caballero, M. C. Lozano, C. de Miguel, J. C. Palomares, and E. J. Perea Activities of new quinoline derivatives against genital pathogens. Antimicrob. Agents Chemother. 27: Cantet, P., H. Renaudin, C. Quentin, and C. Bebear Activite comparee in vitro de sept quinolones sur Ureaplasma urealyticum. Pathol. Biol. 31: Cassell, G. H., W. A. Clyde, and J. K. Davis Mycoplasmal respiratory infections, p In S. Razin and M. F. Barile (ed.), The mycoplasmas. Academic Press, Inc., New York. 4. Cassell, G. H., D. T. Crouse, K. B. Waites, P. T. Rudd, and J. K. Davis Does Ureaplasma urealyticum cause respiratory disease in newborns? Pediatr. Infect. Dis. J. 7: Cassell, G. H., J. K. Davis, K. B. Waites, P. T. Rudd, D. Talkington, and S. Horowitz Pathogenesis and significance of urogenital mycoplasmal infections. Adv. Exp. Med. Biol. 24: Cassell, G. H., K. B. Waites, D. T. Crouse, P. T. Rudd, K. C. Canupp, S. Stagno, and G. Cutter Association of Ureaplasma urealyticum infections of the lower respiratory tract with chronic lung disease and death in very low birthweight infants. Lancet ii: Cassell, G. H., K. B. Waites, P. Pappas, K. Baldus, C. Craft, L. Duffy, D. Crabb, T. Schmid, and M. Pate Program Abstr. 30th Intersci. Conf. Antimicrob. Agents Chemother., abstr Casseli, G. H., K. B. Waites, M. S. Pate, K. C. Canupp, and L. B. Duffy Comparative susceptibility of Mycoplasma pneumoniae to erythromycin, ciprofloxacin, and lomefloxacin. Diagn. Microbiol. Infect. Dis. 12: Chow, A. W Erythromycin, p In A. M. Ristuccia and B. A. Cunha (ed.), Antimicrobial therapy. Raven Press, New York. 10. Cummings, M. C., and W. M. McCormack Increase in resistance of Mycoplasma hominis to tetracyclines. Antimicrob. Agents Chemother. 34: Dobson, R. A., J. R. O'Connor, S. A. Poulin, R. B. Kundsin, T. F. Smith, and P. A. Came In vitro antimicrobial activity of rosoxacin against Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum. Antimicrob. Agents Chemother. 18: Eliopoulos, G. M., and C. T. Eliopoulos Quinolone antimicrobial agents: activity in vitro, p In J. S. Wolfson and D. C. Hooper (ed.), Quinolone antimicrobial agents. American Society for Microbiology, Washington, D.C. 13. Evans, R. T., and D. Taylor-Robinson The incidence of tetracycline-resistant strains of Ureaplasma urealyticum. J. Antimicrob. Chemother. 4: Fallon, R. J., and W. M. Brown Sensitivity of Legionellaceae, meningococci and mycoplasmas to the quinolones ciprofloxacin and enoxacin. J. Antimicrob. Chemother. 15: Freeman, S., J. Wormuth, and J. Cornett In vitro activity of amifloxacin against Chlamydia trachomatis and Ureaplasma urealyticum. Eur. J. Clin. Microbiol. 4: Hashimoto, M., A. Minami, K. Nakata, S. Nakamura, K. Ohni-

5 VOL. 35, 1991 SUSCEPTIBILITIES OF MYCOPLASMAS TO QUINOLONES 1185 shi, and M. Shimizu Program Abstr. 28th Intersci. Conf. Antimicrob. Agents Chemother., abstr Hoban, D., P. DeGagne, and E. Witwicki In vitro activity of lomefloxacin against Chlamydia trachomatis, Neisseria gonorrhoeae, Haemophilus ducreyi, Mycoplasma hominis, and Ureaplasma urealyticum. Diagn. Microbiol. Infect. Dis. 12: 83S-86S. 18. Kenny, G. E., and F. D. Cartwright Susceptibility of Mycoplasma pneumoniae to several new quinolones, tetracycline, and erythromycin. Antimicrob. Agents Chemother. 35: Kenny, G. E., T. M. Hooton, M. C. Roberts, F. D. Cartwright, and J. Hoyt Susceptibilities of genital mycoplasmas to the newer quinolones as determined by the agar dilution method. Antimicrob. Agents Chemother. 33: Krausse, R., and U. Ullmann Comparative in vitro activity of fleroxacin (RO ) against Ureaplasma urealyticum and Mycoplasma hominis. Eur. J. Clin. Microbiol. Infect. Dis. 7: Liebowitz, L., J. Saunders, G. Fehler, R. C. Ballard, and H. J. Koornhof In vitro activity of A (difloxacin), A-56620, and other new quinolone antimicrobial agents against genital pathogens. Antimicrob. Agents Chemother. 30: Lorian, V., and L. D. Sabath Effect of ph on the activity of erythromycin against 500 isolates of gram-negative bacilli. Appl. Microbiol. 20: Mogabgab, W. J., B. Holmes, N. Murray, R. Beville, F. B. Lutz, and K. J. Tack Randomized comparison of ofloxacin and doxycycline for chlamydia and ureaplasma urethritis and cervicitis. Chemotherapy (Basel) 36: Nakamura, S., A. Minami, K. Nakata, N. Kurobe, K. Kouno, Y. Sakaguchi, S. Kashimoto, H. Yoshida, T. Kojima, T. Ohue, K. Foujimoto, M. Nakamura, M. Hashimoto, and M. Shimizu In vitro antibacterial activities of AT-4140, a new broad-spectrum quinolone. Antimicrob. Agents Chemother. 33: Osada, Y., and H. Ogawa Antimycoplasmal activity of ofloxacin (DL 8280). Antimicrob. Agents Chemother. 23: Renaudin, H., C. Quentin, B. de Barbeyrac, and C. Bebear Activite in vitro de nouvelles quinolones sur les mycoplasmes pathogenes pour l'homme. Pathol. Biol. 36: Ridgeway, G. L., G. Mumtaz, F. G. Gabriel, and J. D. Oriel The activity of ciprofloxacin and other 4-quinolones against Chlamydia trachomatis and mycoplasmas in vitro. Eur. J. Clin. Microbiol. 3: Roberts, M. C., and G. E. Kenny TetM tetracylineresistant determinants in Ureaplasma urealyticum. Pediatr. Infect. Dis. J. 5:S338-S Sekine, Y., Y. Matsunaga, H. Miyazaki, T. Yamaguchi, Y. Mizuki, T. Itoh, N. Kurobe, S. Nakamura, M. Hashimoto, and M. Shimizu Program Abstr. 28th Intersci. Conf. Antimicrob. Agents Chemother., abstr Senterfit, L. B Antibiotic susceptibility testing of mycoplasmas. Methods Mycoplasmol. 2: Shepard, M. C Culture media for ureaplasmas. Methods Mycoplasmol. 1: Shepard, M. C., and C. D. Lunceford Serological typing of Ureaplasma urealyticum isolates using an agar growth inhibition method. J. Clin. Microbiol. 8: Simon, C., and U. Lindner In vitro activity of norfloxacin against Mycoplasma hominis and Ureaplasma urealyticum. Eur. J. Clin. Microbiol. 2: Taylor-Robinson, D Metabolism inhibition tests. Methods Mycoplasmol. 1: Taylor-Robinson, D., and P. M. Furr Clinical antibiotic resistance of Ureaplasma urealyticum. Pediatr. Infect. Dis. J. 5:S335-S Taylor-Robinson, D., P. M. Furr, and A. D. B. Webster Ureaplasma urealyticum in the immunocompromised host. Pediatr. Infect. Dis. J. 5:S236-S Thornsberry, C., J. Anhalt, A. L. Barry, J. L. Cotton, E. H. Gerlach, R. N. Jones, R. C. Moellering, and R. A. Norton Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard. NCCLS publication no. M7-A. National Committee for Clinical Laboratory Standards, Villanova, Pa. 38. Thrupp, L. D Susceptibility testing of antibiotics in liquid media, p In V. Lorian (ed.), Antibiotics in laboratory medicine, 2nd ed. The Williams & Wilkins Co., Baltimore. 39. Tiiam, K. H., J. H. T. Wagenvoort, B. Van Klingeren, P. Piot, E. Stolz, and M. F. Michel In vitro activity of the two new fluoroquinolones, A and A 56620, against Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, and Gardnerella vaginalis. Eur. J. Clin. Microbiol. 5: Tully, J. G Laboratory diagnosis of Mycoplasma pneumoniae infections. Isr. J. Med. Sci. 17: Waites, K. B., G. H. Cassell, K. C. Canupp, and P. B. Fernandes In vitro susceptibilities of mycoplasmas and ureaplasmas to new macrolides and aryl-fluoroquinolones. Antimicrob. Agents Chemother. 32: Waites, K. B., D. T. Crouse, J. B. Philips, K. C. Canupp, and G. H. Cassell Ureaplasmal pneumonia and sepsis associated with persistent pulmonary hypertension of the newborn. Pediatrics 83: Waites, K. B., L. B. Duffy, T. Schmid, D. Crabb, M. S. Pate, and G. H. Cassell Program Abstr. 30th Intersci. Conf. Antimicrob. Agents Chemother., abstr Waites, K. B., T. A. Figarola, T. Schmid, D. Crabb, L. B. Duffy, and J. W. Simecka Comparison of broth versus agar dilution for determining antibiotic susceptibilities of Ureaplasma urealyticum. Diagn. Microbiol. Infect. Dis. 14: Waites, K. B., P. T. Rudd, D. T. Crouse, K. C. Canupp, K. G. Nelson, C. Ramsey, and G. H. Casseli Chronic Ureaplasma urealyticum and Mycoplasma hominis infection of central nervous system in preterm infants. Lancet i: Yancey, R. F., and L. K. Klein In vitro activity of trospectomycin sulphate against Myoplasma and Ureaplasma species isolated from humans. J. Antimicrob. Chemother. 21: