Susceptibilities of Genital Mycoplasmas to the Newer Quinolones As Determined by the Agar Dilution Method GEORGE E

Susceptibilities of Genital Mycoplasmas to the Newer Quinolones as Determined by the Agar Dilution Method GEORGE E. KENNY,'* THOMAS M. HOOTON,2 MARILYN C. ROBERTS,' FRANK D. CARTWRIGHT,' AND JEANNE HOYT' Department ofPathobiology SC-38, School ofPublic Health and Community Medicine,' and Department of Medicine, School of Medicine,2 University of Washington, Seattle, Washington 98195 Received 15 August 1988/Accepted 28 October 1988

The increasing resistance of genital mycoplasmas to tetracycline poses a problem because tetracycline is one of the few antimicrobial agents active against Mycoplasma hominis, Ureaplasma urealyticum, chlamydiae, gonococci, and other agents of genitourinary-tract disease. Since the quinolones are a promising group of antimicrobial agents, the susceptibilities ofM. hominis and U. urealyticum to the newer 6-fluoroquinolones were determined by the agar dilution method. Ciprofloxacin, difloxacin, and ofloxacin had good activity against M. hominis, with the MIC for 50% of isolates tested (MlC50) being 1 ,ug/ml. Fleroxacin, lomefloxacin, pefloxacin, and rosoxacin had MIC50s of 2 ,ug/ml. Enoxacin, norfloxacin, and amifloxacin had MIC50s of 8 to 16 ,ug/ml, and cinoxacin and nalidixic acid were inactive (M1C50, .256 ,ig/ml). Overall, the activities of 6-fluoroquinolones for ureaplasmas were similar to those for M. hominis, with MICs being the same or twofold greater. The most active 6-fluoroquinolones against ureaplasmas were difloxacin, ofloxacin, and pefloxacin, with MICsos of 1 to 2 ,Ig/ml. Ciprofloxacin was unusual in that the MIC50 for M. hominis was 1 ,ug/ml, whereas the MIC50 for ureaplasmas was 8 ,Ig/ml. Since the MIC50s for the most active quinolones approximate achievable concentrations in blood and urine, quinolones have promise in treating mycoplasmal infections.

The antibiotic susceptibility of the two most common MATERIALS AND METHODS genital mycoplasmas, Mycoplasma hominis and Ureaplas- Mycoplasmas. Two reference strains of M. hominis were ma urealyticum, is of increasing interest because of the obtained from the American Type Culture Collection recognition that these species are opportunistic pathogens (ATCC), 14027 and 23114T (the type strain). The reference when they extend beyond the genital tract, which they strains of U. urealyticum used included serovars 1 through 7, frequently colonize (34). Both species have been considered which were obtained from the American Type Culture to be generally susceptible to tetracyclines, but an increasing Collection as ATCC strains 27813, 27814, 27815, 27816, number of strains are being recovered which show high-level 27817, 27818, and 27819, respectively. Serovar 8, the type tetracycline resistance due to the presence of the tetracy- strain of the species (28), was obtained from M. Shepard as cline resistance element TetM (15, 21-23, 33). The situation strain 960 (cloned 8X). Serovar 9 (Vancouver) was obtained is further complicated by the fact that treatment of sexually from J. Robertson. The clinical isolates of each species (50 transmitted diseases has become more of a problem because for M. hominis and 50 for U. urealyticum) were obtained of the increase in gonococcal antimicrobial resistance. This from genitourinary specimens tested since 1971. All strains is due to the presence of plasmid-mediated high-level resis- and isolates were stored at -70°C. tance to tetracycline, plasmid-mediated 1-lactamase resis- Media. The broth medium used for growth of M. hominis tance, or chromosomally mediated antibiotic resistance to was soy peptone fresh yeast dialysate broth (12) supple- both penicillin and tetracycline in gonococci (16, 19). Since mented with 20% "agamma" horse serum, 0.001% phenol tetracycline was active against a broad range of sexually red, and 200 U of penicillin per ml. For U. urealyticum, the transmitted organisms (gonococci, Treponema pallidum, dialysate broth was supplemented with 5 mM urea, 10 mM chlamydiae, mycoplasmas, and ureaplasmas), its replace- MES [2-(N-morpholino)ethanesulfonic acid], 10% agamma ment will be difficult. Among the possible candidates for horse serum, 1 mM Na2SO3 (freshly prepared), 0.001% more general use are the quinolones, which are DNA gyrase phenol red, and 200 U of penicillin per ml. The final pH of inhibitors (39). Intense development has produced a large the broth medium was 7.0 to 7.3 for M. hominis and 6.0 to family of newer compounds, the 6-fluoroquinolones, which 6.3 for U. urealyticum. The agar medium employed for are active against a broad range of bacteria (9, 11, 17, 26, 30, antibiotic susceptibility testing for M. hominis was H agar 39). These agents have excellent pharmacokinetics and a (13). The H-agar base contained (per liter of water) 20 g of good safety profile. soy peptone (HySoy, Sheffield Chemical Co., Norwich, The purpose of our study was to determine the suscepti- N.Y.), 5 g of NaCl, 10 g of agarose, and 2 ml of a 1% phenol bilities of clinical isolates of U. urealyticum and M. hominis red solution. The pH was adjusted to 7.3. The complete to the newer quinolones by the agar dilution method. Several H-agar medium contained 70 ml of agar base, 10 ml of fresh quinolones were found to be active against both organisms at yeast dialysate (13), 20 ml of horse serum, and 200 ,ug of concentrations of 1 to 2 ,ug/ml, which are well within penicillin per ml. The final pH of the agar medium incubated attainable concentrations in serum and tissue. in air at 37°C was 7.3. For testing of susceptibilities of ureaplasmas, U agar (13) was used. U-agar base contained (per liter of water) 20 g of soy peptone, 5 g of NaCl, 4.25 g of MES (acid form), 12 g of agarose, and 2 ml of 1% phenol red. * Corresponding author. The pH was adjusted to 5.8 to 6.0. The complete medium 103 104 KENNY ET AL. ANTIMICROB. AGENTS CHEMOTHER. contained 70 ml of U-agar base, 10 ml of fresh yeast extract TABLE 1. Susceptibilities of M. hominis dialysate, 3 mM urea (0.3 ml of filter-sterilized 1 M urea per strains to quinolones 100 ml of agar medium), 200 U of penicillin per ml, and 20% MIC (p.g/ml) No. of strains whole horse serum. The final pH of U agar was 6.0 to 6.3 Quinolonea teted when incubated at 37°C in an atmosphere of 2.5% Co2 in air. 50%o 90o Range este Preparation of mycoplasmal inocula. Because mycoplas- Ciprofloxacin 1.0 1.0 0.5-1.0 34 mas do not show turbidity as evidence of growth in broth, Difloxacin 1.0 1.0 0.25-1.0 44 and because both M. hominis and U. urealyticum rapidly Ofloxacin 1.0 2.0 0.5-4.0 51 suicide after reaching peak growth, preparation of suitable Fleroxacin 2.0 2.0 0.5-4.0 43 inocula was much more difficult than with classical bacteria. Lomefloxacin 2.0 2.0 1.0-4.0 30 For M. hominis, stock frozen cultures (known to be viable at Pefloxacin 2.0 4.0 1.0-4.0 40 high plate counts) were thawed at room temperature, diluted Rosoxacin 2.0 4.0 0.5-4.0 43 Enoxacin 8.0 8.0 1.0-16.0 42 1:100 in fresh medium, and incubated for 24 h at 37°C. This Norfloxacin 8.0 8.0 4.0-8.0 42 tube was subcultured at 1:100 dilution into a new broth tube. Amifloxacin 16.0 32.0 1.0-32.0 52 The two tubes were incubated for an additional 24 h. If the Cinoxacin -128 .128 39 initial tube inoculated showed a haze or a slight alkaline pH Nalidixic acid -256 -256 36 shift after 24 h, the subculture was used for inocula for susceptibility testing. If no reaction was observed, samples a Listed in the order of descending activity (order is alphabetical for from both tubes were mixed and used as inocula. For compounds with the same activity). ureaplasmas, the frozen stock culture was inoculated into the ureaplasmal broth at 1:100 dilution. Eighteen hours later, 4, and 5, the number of colonies was counted. For U. a sample from this culture was serially diluted 1:100 and urealyticum, serial 10-fold dilutions were prepared and the 1:10,000 into two new tubes of medium. These three tubes 10-1, 10-2, and 10-3 dilutions were plated onto U agar. were incubated for 24 h, and the highest dilution which had Ureaplasmal colonies were stained on days 3 and 4 by just showed a color change from yellow to pink was used for spraying the plates with the calcium chloride-urea stain (13). the inoculum for the susceptibility test. For both Mycoplasma isolates and ureaplasmas, plates were Quinolones. The following quinolones (17, 39) were used: viewed with a stereoscopic dissecting microscope (cy- amifloxacin mesylate and rosoxacin (WIN 49375 and WIN cloptic; American Optical Corp., Buffalo, N.Y.) at a magni- 35213; Sterling-Winthrop Research Institute, Rensselaer, fication of x40. The MIC was the concentration of antimi- N.Y.), cinoxacin, (Lilly Research Laboratories, Indianapo- crobial agent (in micrograms per milliliter) which completely lis, Ind.), ciprofloxacin (Miles Laboratories, Inc., West prevented colony formation on spots known to contain 30 to Haven, Conn.), difloxacin (71-326-AL; Abbott Laboratories, 300 CFU as judged by the controls. The MIC50 was the Abbott Park, Ill.), enoxacin (Wamer-Lambert, Ann Arbor, concentration of antimicrobial agent which prevented colony Mich.), fleroxacin (AM 833; Hoffmann-LaRoche, Inc., Nut- formation of 50% of the strains tested, and the MIC90 was ley, N.J.), lomefloxacin (NY 198; G. D. Searle, Mt. Pros- the concentration which prevented growth of 90% of the pect, Ill.), nalidixic acid (Calbiochem-Behring, San Diego, strains tested. Control prototypic strains were included in Calif.), norfloxacin (MK-0366 [AM715]; Merck Sharp & each run along with the clinical isolates. Dohme, Rahway, N.J.), ofloxacin (Ortho Pharmaceutical Co., Raritan, N.J.), and pefloxacin (RP 41982; Rhone-Pou- RESULTS lenc Pharmaceuticals Inc., Princeton, N.J.). On the day of use, each antimicrobial agent was dissolved in a small Effect of inoculum size on susceptibility determinations for amount of 1 N NaOH, and the volume was adjusted with mycoplasmas. The sizes of ureaplasmal colonies decreased water to a final concentration as specified for the specific sharply with increasing density of colonies on agar plates. activity of each antimicrobial agent and sterilized by filtra- Above 300 colonies per spot, the colonies were so small that tion through a 0.22-,um-pore-size filter (Millipore Corp., they were invisible at a magnification of x 40 even after being Bedford, Mass.). stained with the calcium chloride-urea stain. Stained plates Agar dilution susceptibility testing. An agar dilution turned red after overnight incubation at room temperature in method (14) adopted from standard microbiological methods areas where ureaplasmas were present even in the cases (4) was used for determination of susceptibilities of both where colonies were not visible because of high inoculum ureaplasmas and M. hominis isolates to quinolones. In order levels. For M. hominis, crowding was less of a problem to limit dehydration over the 5-day incubation period, 25 ml because confluent lawns of organisms could be recognized of complete agar medium was poured into plastic petri dishes microscopically. Below 300 colonies per spot for both or- (100-mm square), an amount which resulted in an agar ganisms, the results were independent of inoculum size thickness of 2.5 mm. Antimicrobial agents were added in provided that colonies could clearly be distinguished on the appropriate amounts of the molten H or U agar to yield a control plates. When higher inocula were used, MICs fre- twofold serial concentration series beginning at 0.25 ,ug/ml. quently were elevated twofold. The lower limit for number Plates were held in the dark at room temperature for 48 h to of colonies per spot was set at 30 to minimize statistical allow evaporation of excess surface moisture so that the variation. Assays which did not meet these criteria were not inocula could readily be imbibed by the agar. This was included in the data, a factor which accounts for the varia- particularly necessary for visualization of ureaplasmal colo- tions in the numbers of strains tested (Tables 1 and 2). nies. For testing of susceptibilities, serial 10-fold dilutions of Susceptibilities of M. hominis strains to quinolones. Cipro- organisms were prepared in dialysate broth supplemented floxacin, difloxacin, and ofloxacin were most active against with 2% horse serum. For M. hominis, samples of the 10-2 strains of M. hominis with MIC50s of 1.0 ,ug/ml (Table 1). and io-3 dilutions were placed into the wells of a Steers Fleroxacin, lomefloxacin, pefloxacin, and rosoxacin showed replicator (32) and plated onto the H-agar surface (0.025 ml intermediate activities, with MIC50s of 2 ,ugIml. Enoxacin per spot). Cultures were incubated in air at 37°C. On days 3, and norfloxacin had MIC50s of 8 ,ug/ml. Amifloxacin was less VOL. 33, 1989 SUSCEPTIBILITIES OF MYCOPLASMAS TO QUINOLONES 105

TABLE 2. Susceptibilities of U. urealyticum other genital pathogens resistant to penicillin and tetracy- strains to quinolones cline (2, 11, 24). They have been demonstrated to be very in the treatment of infections but MIC (GLg/ml) No. of strains effective gonococcal gen- Quinolone'a erally have performed poorly against chlamydial infections. 50% 90% Range tested It is important to know their effects on U. urealyticum and Difloxacin 1 2 0.5-2.0 29 M. hominis because both species appear to be opportunistic Ofloxacin 2 2 0.5-4.0 58 pathogens in the perinatal period, being involved in infec- Pefloxacin 2 4 1.0-4.0 40 tions of the bloodstream, the upper female genital tract, and Fleroxacin 4 4 2.0-8.0 50 the fetus (34, 37). U. urealyticum may be associated with Lomefloxacin 4 4 0.5-8.0 32 nonchlamydial nongonococcal urethritis in men (34). Rosoxacin 4 4 2.0-4.0 51 The susceptibilities of M. hominis and U. urealyticum to Ciprofloxacin 8 16 2.0-64.0 56 the newer quinolones are similar to the susceptibilities of Enoxacin 8 16 4.0-16.0 32 gram-positive organisms, such as streptococci and staphylo- Norfloxacin 8 16 4.0-16.0 31 in that are less than Amifloxacin 16 32 1.0-64.0 52 cocci (39), they susceptible gram- Cinoxacin -128 .128 49 negative enteric organisms by a factor of 5- to 10-fold. For Nalidixic acid 64 256 60 the original quinolone, nalidixic acid, gram-negative enteric bacteria are susceptible to 2 to 8 ,ig/ml; gram-positive a Listed in the order of descending activity (order is alphabetical for organisms and mycoplasmas are resistant to 64 to -256 ,ug/ compounds with the same activity). ml (39). The newer 6-fluoroquinolones show MICs as low as 0.05 ,ug/ml for gram-negative enteric organisms, compared effective, and negligible activity was shown by both cinoxa- with 1 Vig/ml for gram-positive organisms. If the differences cin and nalidixic acid. Overall, the MIC90 was either the between gram-negative and gram-positive organisms in sus- same as the MIC50 (in six cases) or twofold greater (in four ceptibilities to quinolones result from fundamental genetic cases), with the exceptions of cinoxacin and nalidixic acid, differences, then U. urealyticum and M. hominis appear to for which endpoints were not reached. be more related to gram-positive organisms than to gram- Susceptibilities of U. urealyticum strains to quinolones. negative organisms, an idea in accord with the proposal that Difloxacin, ofloxacin, and pefloxacin showed MIC50s against mycoplasmas (in general) descended from gram-positive U. urealyticum of 1 to 2 ,.g/ml (Table 2). Fleroxacin, organisms, as evidenced by comparison of the rRNAs of lomefloxacin, and rosoxacin had MIC50s of 4 ,ug/ml. Cipro- procaryotes (38). floxacin, enoxacin, and norfloxacin had MIC50s of 8 [.g/ml, The order of activity of quinolones against M. hominis and amifloxacin had an MIC50 of 16 ,.g/ml. As with M. (Table 1) closely paralleled the order observed with classical hominis (Table 1), cinoxacin and nalidixic acid had little bacteria (26) in that quinolones which were most active activity against U. urealyticum (Table 2). The MIC9,s were against other bacteria were also most active against M. twofold greater than the MIC,os for 7 out of 11 quinolones, hominis. The results were different with U. urealyticum with the exception of nalidixic acid, for which an endpoint (Table 2); ciprofloxacin was not among the most active was not reached (Table 2). agents. Ciprofloxacin was eightfold less active against U. Comparison of the susceptibilities of M. hominis and U. urealyticum than against M. hominis, whereas for the other urealyticum to quinolones. Overall, the susceptibilities of M. fluoroquinolones, the MIC50s for U. urealyticum were either hominis and U. urealyticum showed close parallels; the the same or twofold greater than those for M. hominis. MIC50s for U. urealyticum strains were the same as those for Recently, Renaudin et al. (18) have shown similar differ- M. hominis for half of the quinolones and twofold higher for ences in the comparative susceptibilities of ureaplasmas and the remainder (with the exceptions of cinoxacin and nalidixic M. hominis to ciprofloxacin. This difference with ciproflo- acid). Ciprofloxacin was also an exception to this generali- xacin might have been a function of pH, since the plates used zation, because its M'C50 for ureaplasmas was eight times for ureaplasmas were at pH 6.3 and those used for M. greater than the M'C50 for M. hominis. Difloxacin showed hominis were at pH 7.3. Smith and Ratcliffe (31) have shown the greatest activity against both species. Five quinolones that the MIC of ciprofloxacin for Escherichia coli increases (fleroxacin, lomefloxacin, ofloxacin, pefloxacin, and rosoxa- as the pH decreases. Although it is not possible to grow cin) with MIC50s of 1 to 2 ,.g/ml for M. hominis had MIC50s ureaplasmas at pH 7.3, it was possible to test M. hominis at of 2 to 4 jig/ml for U. urealyticum. The quinolones next in pH 6.3 on U agar. Ciprofloxacin was only a twofold dilution effectiveness were amifloxacin, enoxacin, and norfloxacin, less active against M. hominis at pH 6.3 than it was at pH which were about twofold less effective. One apparent trend 7.3. In general, the effect of pH on the susceptibilities of was that quinolones which demonstrated the least activity gram-positive organisms to quinolones is much less than the showed the broadest range in MICs (Tables 1 and 2). effect of pH on susceptibilities of gram-negative organisms Relationships of quinolone susceptibility to tetracycline sus- (9). ceptibility. To determine whether resistance to tetracycline The previous studies on the susceptibility of U. urealyti- was related to quinolone susceptibility, six tetracycline- cum to quinolones have used a liquid medium system, and resistant ureaplasmal strains which carried the TetM deter- the endpoint has been judged by a color change in the pH minant (21) were included in the study. The results for these indicator (3, 20, 36). The MIC50s in a number of reports for strains were similar to those obtained with tetracycline- quinolones such as rosoxacin (5, 6, 10), difloxacin (36), susceptible strains. Likewise, 10 tetracycline-resistant M. pefloxacin (5), norfloxacin (3, 5), and amifloxacin (10) corre- hominis strains with the TetM determinant (22) behaved the sponded to the results of this study within a twofold dilution, same as tetracycline-susceptible strains. although they were usually higher than those of this study. For ciprofloxacin, two studies found MIC50s of 1 to 2 jig/ml DISCUSSION (20, 29, 36) and two others found MIC50s of 16 to 32 p.g/ml (3, The newer 6-fluoroquinolones offer an important alterna- 7), in contrast to the M'C50 of 8 found in this study. For tive for treatment of infections caused by gonococci and ofloxacin, MIC50s of 4 to 8 p.g/ml (3, 7) have been reported, 106 KENNY ET AL. ANTIMICROB. AGENTS CHEMOTHER. which are considerably higher than the 1 pug/ml found in this 3. Aznar, J., M. C. Caballero, M. C. Lozano, C. de Miguel, J. C. study. Polomares, and E. J. Perea. 1985. Activities of new quinoline In studies with M. hominis, MIC50s of 0.25 to 2.0 ,ug/ml derivatives against genital pathogens. Antimicrob. Agents Che- were found by broth dilution for ciprofloxacin (7, 20, 36). mother. 27:76-78. 4. Barry, A. L. 1986. Procedure for testing antimicrobial agents in MIC50s of 0.25 to 1 ,ug/ml were found for difloxacin (36). By agar media: theoretical considerations, p. 1-26. In V. Lorian agar dilution, Fallon and Brown obtained an MIC50 of 2.5 ,ug/ (ed.), Antibiotics in laboratory medicine. The Williams & Wil- ml for ciprofloxacin and >10 ,ug/ml for enoxacin (8). These kins Co., Baltimore. MIC50s can be compared with our findings of 1 ,ug/ml for 5. Cantet, P., H. Renaudin, C. Quentin, and C. Bebear. 1983. ciprofoxacin, 1 ,ug/ml for difloxacin, and 8 ,ug/ml for enoxa- Activite comparee in vitro de sept quinolones sur Ureaplasma cin. For ofloxacin, when the broth dilution method was used, urealyticum. Pathol. Biol. 31:501-503. MIC50s of 0.5 (29) and 8 to 16 ,ug/ml (7) have been reported. 6. Dobson, R. A., J. R. O'Connor, S. A. Poulin, ft. B. Kundsin, Antibiotic susceptibility testing of Mycoplasma isolates T. F. Smith, and P. E. Came. 1980. In vitro antimicrobial and ureaplasmas is distinctly different from such testing of activity of rosoxacin against Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum. Antimicrob. Agents conventional bacteria, because the mycoplasmas and urea- Chemother. 18:738-740. plasmas grow slowly and form recognizable colonies only in 7. Escalante, A., J. Aznar, C. DeMiguel, and E. J. Perea. 1985. 2 to 4 days. A diffusion method (disk method) cannot be Activity of nine antimicrobial agents against Mycoplasma ho- recommended, because the antibiotic will equilibrate in the minis and Ureaplasma urealyticum. Eur. J. Sex. Transm. Dis. agar plate before colonies appear (4). The medium for 2:85-87. mycoplasmas and ureaplasmas is complex, containing 10 to 8. Fallon, R. J., and W. M. Brown. 1985. In vitro sensitivity of 20% serum, which makes the MICs for mycoplasmas and legionellas, meningococci and mycoplasmas to ciprofloxacin ureaplasmas difficult to compare with those for bacteria and enoxacin. J. Antimicrob. Chemother. 15:767-789. which can be grown in much simpler media. The U-agar 9. Fernandes, P. B. 1988. Mode of action and in vitro and in vivo activities of fluoroquinolones. J. Clin. Pharmacol. 28:156-168. medium (13) used in the present study did not contain added 10. Freeman, S., J. Wormuth, and J. Cornett. 1985. In vitro activity Ca2" as an intrinsic stain for ureaplasmas (27) because of amifloxacin against Chlamydia trachomatis and Ureaplasma divalent cations can greatly change susceptibilities to some urealyticum. Eur. J. Clin. Microbiol. 4:515-516. antimicrobial agents (35). The agar dilution method as used 11. Hooper, D. C., and J. S. Wolfson. 1985. The fluoroquinolones: in the present study appears to be more useful than the broth pharmacology, clinical uses, and toxicities in humans. Antimi- dilution method in which a pH change is used as an endpoint crob. Agents Chemother. 28:716-721. to the test (25) because the agar dilution method can detect 12. Kenny, G. E. 1967. Heat lability and organic solvent solubility of mixtures of susceptible and resistant mycoplasmas and Mycoplasma antigens. Ann. N.Y. Acad. Sci. 143:676-681. provide for the detection of spontaneous resistant mutants. 13. Kenny, G. E. 1985. Mycoplasmas, p. 407-411. In E. H. Len- nette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), The agar dilution method has the advantage that many Manual of clinical microbiology, 4th ed. American Society for strains can be tested at a single time, but it has the disad- Microbiology, Washington, D.C. vantage that testing of one or two strains takes nearly as 14. Kenny, G. E., F. D. Cartwright, and M. C. Roberts. 1986. Agar much time as testing many strains. dilution method for determination of antibiotic susceptibility of Fluoroquinolones may be useful for the treatment of Ureaplasma urealyticum. Pediatr. Infect. Dis. 5:S338-S340. genital infections caused by mycoplasmas because quino- 15. Magalhaes, M., and A. Veras. 1984. Minocycline resistance lones are one of the few groups of antimicrobial agents which among clinical isolates of Ureaplasma urealyticum. J. Infect. inhibit both M. hominis and ureaplasmas at MICs which Dis. 149:117. appear to be within attainable levels in tissue (11). The in 16. Morse, S. A., S. R. Johnson, J. W. Biddle, and M. C. Roberts. vitro data may be helpful in determining the potential use- 1986. High-level tetracycline resistance in Neisseria gonorrhoeae due to the acquisition of the tetM determinant. Antimi- fulness of the various quinolones. In a recent study (1), crob. Agents Chemother. 30:664-670. treatment with ciprofloxacin was effective for the elimination 17. Nix, D. E., and J. J. Schentag. 1988. The quinolones: an of M. hominis, but not U. urealyticum, from the vaginal flora overview and comparative appraisal of their pharmacokinetics of women. Although this result might have been predicted and pharmacodynamics. J. Clin. Pharmacol. 28:169-178. from our in vitro data, controlled clinical trials will be 18. Renaudin, H., C. Quentin, B. deBarbeyrac, and C. Bebear. 1988. necessary to evaluate the usefulness of quinolones in the Activite in vitro de nouvelles quinolones sur les mycoplasmas treatment of infections by Mycoplasma isolates and urea- pathogenes pour l'homme. Pathol. Biol. 36:496-499. plasmas. 19. Rice, R. J., J. W. Biddle, Y. A. Jean Locies, W. E. deWitt, J. H. Blount, and S. A. Morse. 1986. Chromosomally mediated resistance in Neisseria gonorrhoeae in the United States: results of ACKNOWLEDGMENTS surveillance and reporting, 1983-1984. J. Infect. Dis. 153:340- This study was supported in part by Public Health Service grants 345. AI-06720 and AI-12192 from the National Institute of Allergy and 20. Ridgway, G. L., G. Mumtaz, F. G. Gabriel, and J. D. Oriel. Infectious Diseases; Miles Pharmaceuticals Div., West Haven, 1984. The activity of ciprofloxacin and other 4-quinolones Conn.; Hoffmann-LaRoche, Inc., Nutley, N.J.; G. D. Searle, Mt. against Chlamydia trachomatis and mycoplasmas in vitro. Eur. Prospect, Ill.; Ortho Pharmaceutical Co., Raritan, N.J.; and Rhone- J. Clin. Microbiol. 3:344-346. Poulenc Pharmaceuticals Inc., Princeton, N.J. 21. Roberts, M. C., and G. E. Kenny. 1986. Dissemination of the tetM tetracycline resistance determinant to Ureaplasma urealy- LITERATURE CITED ticum. Antimicrob. Agents Chemother. 29:350-352. 1. Ahmed-Jushuf, I. H., 0. Parya, D. Hobson, B. C. Pratt, C. A. 22. Roberts, M. C., L. A. Koutsky, K. K. Holmes, D. L. LeBlanc, Hart, S. J. How, I. A. Tait, and P. M. S. Rao. 1988. Ciproflo- and G. E. Kenny. 1985. Tetracycline resistant Mycoplasma xacin treatment of chlamydial infections of urogenital tracts of hominis strains contain tetM sequences. Antimicrob. Agents women. Genitourin. Med. 64:14-17. Chemother. 28:141-143. 2. Arcieri, G., R. August, N. Becker, C. Doyle, E. Griffith, G. 23. Robertson, J. A., G. W. Stemke, S. G. Maclellan, and D. E. Gruenwaldt, A. Heyd, and B. O'Brien. 1986. Clinical experience Taylor. 1988. Characterization of tetracycline-resistant strains with ciprofloxacin in the USA. Eur. J. Clin. Microbiol. 5:220- of Ureaplasma urealyticum. J. Antimicrob. Chemother. 21:319- 225. 332. VOL. 33, 19.89 SUSCEPTIBILITIES OF MYCOPLASMAS TO QUINOLONES 107

24. Romanowski, B., H. Wood, J. Draker, and M. C. Tisanco. 1986. ity to antibiotics. Antibiot. Chemother. 9:307-311. Norfloxacin in the therapy of uncomplicated gonorrhea. Anti- 33. Taylor-Robinson, D., and P. M. Furr. 1986. Clinical antibiotic microb. Agents Chemother. 30:514-515. resistance of Ureaplasma urealyticum. Pediatr. Infect. Dis. 25. Senterfit, L. B. 1983. Antibiotic sensitivity testing of mycoplas- 5(Suppl.):S335-S337. mas. Methods Mycoplasmol. 2:397-401. 34. Taylor-Robinson, D., and W. M. McCormack. 1979. Mycoplas- 26. Shannon, K. P., and I. Phillips. 1985. The antimicrobial spec- mas in human genitourinary infections, p. 307-366. In J. F. trum of the quinolones, p. 29-35. In J. G. Collee (ed.), DNA- Tully and R. F. Whitcomb (ed.), The mycoplasmas, vol. 2. gyrase inhibitors: the present and the future. Research and Academic Press, Inc., New York. Clinical Forums, Kent, England. 35. Thrupp, L. D. 1986. Susceptibility testing of antibiotics in liquid 27. Shepard, M. C. 1983. Culture media for urea,plasmas. Methods media, p. 93-150. In V. Lorian (ed.), Antibiotics in laboratory Mycoplasmol. 1:137-146. medicine, 2nd ed. The Williams & Wilkins Co., Baltimore. 28. Shepard, M. C., D. C. Lunceford, D. K. Ford, R. H. Purcell, D. 36. Tijam, K. H., J. H. T. Wagenvoort, B. van Klingeren, P. Piot, E. Taylor-Robinson, S. Razin, and F. T. Black. 1974. Ureaplasma Stolz, and M. F. Michel. 1986. In vitro activity of the two new urealyticum gen. nov., sp. nov.: proposed nomenclature for the 4-quinolones A56619 and A56620 against Neisseria gonor- human T (T-strain) mycoplasmas. Int. J. Syst. Bacteriol. 24: rhoeae, Chlamydia trachomatis, Mycoplasma hominis, Urea- 160-171. plasma urealyticum, and Gardnerella vaginalis. Eur. J. Clin. 29. Simon, S. 1984, In-vitro-Aktivitat von Gyrase-Hemmern gegen Microbiol. 5:498-501. Mycoplasmem. FAC Fort. Antimikr. Antineoplast. Chemother. 37. Waites, K. B., P. T. Rudd, D. T. Crouse, K. C. Canupp, K. G. Band 3-5:629-632. Nelson, C. Ramsey, and G. H. Cassell. 1988. Chronic Ureaplas- 30. Smith, J. T. 1984. Awakening the slumbering potential of the ma urealyticum and Mycoplasma hominis infections of the 4-quinolone antibacterials. Pharm. J. 233:299-305. central nervous system in preterm infants. Lancet i:17-21. 31. Smith, J. T., and N. T. Ratcliffe. 1986. Einfluz von pH-wert and 38. Woese, C. R. 1987. Bacterial evolution. Microbiol. Rev. 51:221- Magnesium auf die antibackterielle Aktivitat von Chinolonpra- 271. paraten. Infection 14(Suppl. I):31-35. 39. Wolfson, J. S., and D. C. Hooper. 1985. The fluoroquinolones: 32. Steers, E., E. L. Foltz, and B. S. Graves. 1959. An inocula structures, mechanisms of action and resistance, and spectra of replicating apparatus for routine testing of bacterial susceptibil- activity in vitro. Antimicrob. Agents Chemother. 28:581-586.