Susceptibilities of Genital Mycoplasmas to the Newer Quinolones As Determined by the Agar Dilution Method GEORGE E
Total Page：16
File Type：pdf, Size：1020Kb
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1989, p. 103-107 Vol. 33, No. 1 0066-4804/89/010103-05$02.00/0 Copyright © 1989, American Society for Microbiology 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-fluoroquino- lones 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.