Abstract
The activities of garenoxacin, gatifloxacin, and gemifloxacin were compared with those of four fluoroquinolones against human mycoplasmas and ureaplasmas, including fluoroquinolone-resistant genetically characterized strains. Garenoxacin exhibited the highest activity, followed by gemifloxacin, moxifloxacin, and gatifloxacin. The minimal bactericidal activities of these three compounds were lower than those of the four fluoroquinolones.
Garenoxacin, a des-fluoro(6)-quinolone, and gemifloxacin and gatifloxacin, two fluoroquinolones, are new compounds of the widespread class of fluoroquinolones with potent antibacterial activity against gram-negative, gram-positive, and anaerobic bacteria (6-8, 11). The different fluoroquinolones show various levels of activity against mycoplasmas. In this study, the activities of garenoxacin, gemifloxacin, and gatifloxacin were compared with those of four fluoroquinolones, moxifloxacin, levofloxacin, ciprofloxacin, and ofloxacin and with those of two unrelated antimicrobial agents, doxycycline and erythromycin, against different human Mycoplasma and Ureaplasma species. Furthermore, the activities of these three new compounds were evaluated in a comparison with the activities of other reference fluoroquinolones against several in vivo mutants of Ureaplasma spp. and Mycoplasma hominis and against several in vitro mutants of M. hominis (all mutants genetically characterized). Minimal bactericidal concentrations (MBCs) of fluoroquinolones for five strains of the three major pathogenic species, Mycoplasma pneumoniae, M. hominis, and Ureaplasma isolates, were also determined.
A total of 31 strains of M. pneumoniae (30 clinical respiratory isolates and 1 reference strain FH), 6 strains of Mycoplasma genitalium (5 clinical isolates and 1 reference strain G37), and 40 strains of M. hominis were studied. The 40 M. hominis strains consisted of 20 doxycycline-susceptible clinical strains, 11 doxycycline-resistant clinical isolates, 4 fluoroquinolone-resistant clinical isolates that have been genetically characterized (3, 4) (isolates MHa, MHb1, MHc1, and MHe2), 4 fluoroquinolone-resistant mutants selected in vitro and genetically characterized (2) (isolates IS1, IIS1, IIS3A, and IIIS3A1), and 1 reference strain (PG21). A total of 46 strains of Ureaplasma spp. were studied. The 46 Ureaplasma strains included 17 doxycycline-susceptible clinical isolates, 14 doxycycline-resistant clinical strains, 12 fluoroquinolone-resistant clinical isolates that have been genetically characterized (3) (UUa to UUg5), and 3 reference strains (Ureaplasma parvum, Ureaplasma urealyticum, and U. urealyticum Vancouver serovar 9). Nine strains of Mycoplasma fermentans (seven clinical strains and two reference strains [PG18 and K7]) and two strains of Mycoplasma penetrans (one urethral isolate and one reference strain [GTU-54]) were studied.
All antimicrobial powders tested were obtained from their respective manufacturers and dissolved according to their recommendations. Susceptibility testing was performed as previously described (12), by an agar dilution method for mycoplasmal strains and by a broth dilution method for ureaplasmal strains. MBCs of the different quinolones were determined as previously reported (1, 12) for five strains of M. pneumoniae (one reference strain and four clinical isolates), M. hominis (one reference strain and four doxycycline-susceptible clinical isolates) and Ureaplasma spp. (two reference strains and three doxycycline-susceptible clinical isolates). Bactericidal activity was identified as the MBC being no more than 2 dilutions (fourfold) greater than the MIC.
A comparison of the in vitro activities of garenoxacin, gemifloxacin, gatifloxacin, and other antimicrobials against fluoroquinolone-susceptible mycoplasmal and ureaplasmal strains are shown in Table 1. A concentration of 0.25 μg/ml for garenoxacin and gemifloxacin and a concentration of 0.5 μg/ml for gatifloxacin inhibited 90% of all the strains tested. Garenoxacin MIC90s (MICs at which 90% of the isolates tested are inhibited) for the strains were 1- to 4-fold less than gemifloxacin MIC90s, 2- to 8-fold less than gatifloxacin and moxifloxacin MIC90s, 4- to 66-fold less than levofloxacin MIC90s, 8- to 66-fold less than ofloxacin MIC90s, and 16- to 133-fold less than ciprofloxacin MIC90s, making garenoxacin the most potent quinolone tested against the 114 fluoroquinolone-susceptible mycoplasmal and ureaplasmal strains. Following garenoxacin, the activities of gatifloxacin, moxifloxacin, and gemifloxacin were similar, with at most 1 dilution difference against M. pneumoniae, Mycoplasma genitalium, and Ureaplasma spp. For M. hominis, M. fermentans, and M. penetrans strains, gemifloxacin was 4- to 16-fold more potent than gatifloxacin and moxifloxacin. The three quinolones tested and moxifloxacin clearly exhibited lower MICs than levofloxacin, ciprofloxacin, and ofloxacin, with MIC differences ranging from a factor of 2 to 66.
TABLE 1.
Comparison of the in vitro activities of garenoxacin, gemifloxacin, gatifloxacin, and other antimicrobial agents against fluoroquinolone-susceptible mycoplasmas and Ureaplasma spp.
| Organism (no. of strains tested) and antimicrobial | MIC (μg/ml)a
|
MBC range (μg/ml)b (MIC range for the 5 strains tested) | ||
|---|---|---|---|---|
| Range | 50% | 90% | ||
| M. pneumoniae (31) | ||||
| Garenoxacin | 0.03-0.06 | 0.06 | 0.06 | 0.12-0.25 (0.03-0.06) |
| Gemifloxacin | 0.06-0.25 | 0.25 | 0.25 | 0.12-0.25 (0.06-0.12) |
| Gatifloxacin | 0.06-0.25 | 0.12 | 0.25 | 0.12-0.25 (0.03-0.12) |
| Moxifloxacin | 0.06-0.12 | 0.12 | 0.12 | 0.12-0.25 (0.03-0.06) |
| Levofloxacin | 0.5-1 | 0.5 | 1 | 1-2 (0.5) |
| Ciprofloxacin | 1-2 | 1 | 2 | 2-16 (0.5-1) |
| Ofloxacin | 0.5-2 | 1 | 2 | 1-2 (0.25-0.5) |
| Doxycycline | 0.06-0.25 | 0.12 | 0.25 | —c |
| Erythromycin | ≤0.015 | ≤0.015 | ≤0.015 | — |
| M. genitalium (6) | ||||
| Garenoxacin | 0.06-0.12 | — | — | — |
| Gemifloxacin | 0.12 | — | — | — |
| Gatifloxacin | 0.12 | — | — | — |
| Moxifloxacin | 0.06 | — | — | — |
| Levofloxacin | 0.5 | — | — | — |
| Ciprofloxacin | 1-2 | — | — | — |
| Ofloxacin | 0.5-1 | — | — | — |
| Doxycycline | 0.06 | — | — | — |
| Erythromycin | ≤0.015 | — | — | — |
| M. hominis (32) | ||||
| Garenoxacin | ≤0.015-0.03 | ≤0.015 | ≤0.015 | 0.12-0.5 (≤0.015) |
| Gemifloxacin | ≤0.015-0.06 | 0.03 | 0.03 | 0.03-0.12 (≤0.015-0.03) |
| Gatifloxacin | 0.06-0.25 | 0.12 | 0.12 | 0.5-4 (0.06-0.12) |
| Moxifloxacin | 0.06-0.12 | 0.06 | 0.12 | 0.2-2 (0.03-0.06) |
| Levofloxacin | 0.25-2 | 0.25 | 0.5 | 4-16 (0.12-0.5) |
| Ciprofloxacin | 0.5-2 | 1 | 2 | 8-32 (0.5-1) |
| Ofloxacin | 0.25-1 | 0.5 | 1 | 4-16 (0.12-0.5) |
| Doxycycline | 0.06-16 | — | — | — |
| Erythromycin | >64 | >64 | >64 | — |
| Ureaplasma spp. (34) | ||||
| Garenoxacin | 0.06-0.25 | 0.12 | 0.25 | 0.5-8 (0.25-0.5) |
| Gemifloxacin | 0.03-0.25 | 0.12 | 0.25 | 0.25-1 (0.03-0.25) |
| Gatifloxacin | 0.25-1 | 0.25 | 0.5 | 1-8 (0.5-1) |
| Moxifloxacin | 0.12-0.5 | 0.12 | 0.25 | 0.5-4 (0.25-0.5) |
| Levofloxacin | 0.5-2 | 0.5 | 1 | 0.5-8 (0.5-1) |
| Ciprofloxacin | 2-4 | 1 | 4 | 4-16 (2-4) |
| Ofloxacin | 0.5-2 | 0.5 | 2 | 1-8 (0.5-1) |
| Doxycycline | 0.03-32 | — | — | — |
| Erythromycin | 2-16 | 4 | 16 | — |
| M. fermentans (9) | ||||
| Garenoxacin | ≤0.015 | — | — | — |
| Gemifloxacin | ≤0.015 | — | — | — |
| Gatifloxacin | 0.12-0.25 | — | — | — |
| Moxifloxacin | 0.06 | — | — | — |
| Levofloxacin | 0.12-1 | — | — | — |
| Ciprofloxacin | 0.25 | — | — | — |
| Ofloxacin | 0.25 | — | — | — |
| Doxycycline | 0.03-0.12 | — | — | — |
| Erythromycin | >64 | — | — | — |
| M. penetrans (2) | ||||
| Garenoxacin | ≤0.015 | — | — | — |
| Gemifloxacin | ≤0.015-0.03 | — | — | — |
| Gatifloxacin | 0.06 | — | — | — |
| Moxifloxacin | 0.06-0.12 | — | — | — |
| Levofloxacin | 0.12-0.25 | — | — | — |
| Ciprofloxacin | 0.25-1 | — | — | — |
| Ofloxacin | 0.12-0.5 | — | — | — |
| Doxycycline | 0.12 | — | — | — |
| Erythromycin | 1-4 | — | — | — |
50% and 90%, MIC50 and MIC90, respectively.
The MBCs of garenoxacin, gemifloxacin, gatifloxacin, moxifloxacin, levofloxacin, ciprofloxacin, and ofloxacin were determined for five strains of each species studied.
—, not determined.
For M. pneumoniae, the garenoxacin MIC90 was fourfold less than the doxycycline MIC90, but gatifloxacin and gemifloxacin MIC90s were equal to that of doxycycline (MIC90, 0.25 μg/ml). For M. genitalium, the activities of the three new quinolones were comparable to that of doxycycline. For M. fermentans and M. penetrans, garenoxacin and gemifloxacin were more potent than doxycycline with MICs 2- to 8-fold lower. The three newer quinolones tested were as active against doxycycline-resistant M. hominis or Ureaplasma isolates as they were against doxycycline-susceptible ones. Erythromycin gave the lowest MICs of the antimicrobials tested against M. pneumoniae and M. genitalium (MIC of ≤0.015 μg/ml).
For the five M. pneumoniae isolates tested, the MBCs of garenoxacin, gemifloxacin, and gatifloxacin ranged from 0.12 to 0.25 μg/ml. These MBCs were less than or equal to four times the MICs, indicating bactericidal activity. It should be noted that the MBCs are under the breakpoints accepted for these three new quinolones against usual bacteria (6, 7, 16). The MBCs for the five M. hominis isolates studied were at least eightfold higher than the corresponding MICs, indicating bacteriostatic activity of the three quinolones against this organism. For Ureaplasma isolates, the MBCs of garenoxacin, gemifloxacin, and gatifloxacin ranged from 0.5 to 8 μg/ml, 0.25 to 1 μg/ml, and 1 to 8 μg/ml, respectively. Garenoxacin and gatifloxacin MBCs were within 2 dilutions of the MICs for four of five Ureaplasma isolates, indicating bactericidal activity, whereas gemifloxacin MBCs were less than four times the MICs for only two of the five strains tested. For the three species, the MBCs of moxifloxacin were similar to those of the studied compounds, while levofloxacin, ciprofloxacin, and ofloxacin exhibited distinctly higher MBCs, except for Ureaplasma isolates against which levofloxacin and ofloxacin exhibited the same range of MBCs as gatifloxacin. Overall, our MIC and MBC data are consistent with previously published results for M. pneumoniae (5-7, 9, 10, 13-15). For M. hominis, others described a bactericidal activity of garenoxacin against one of two isolates (13) and a bactericidal activity of gatifloxacin against two of three isolates studied (14). In contrast to the results of Waites et al. (13, 14), we found some bactericidal activities for garenoxacin, gatifloxacin, and gemifloxacin against Ureaplasma spp.
The activities of garenoxacin, gemifloxacin, and gatifloxacin were compared to those of the four other fluoroquinolones against 12 fluoroquinolone-resistant strains of M. hominis and Ureaplasma spp., which have been genetically characterized (Table 2). Among the fluoroquinolones tested, garenoxacin and gemifloxacin exhibited the highest activity against the quinolone-resistant strains, followed by moxifloxacin and gatifloxacin, with the MICs depending on the number of mutations and on the altered positions in the quinolone resistance-determining regions. Garenoxacin and gemifloxacin had the best activity against both Mycoplasma and Ureaplasma species, with MICs of ≤1 μg/ml for all strains with one mutation and most of the strains with two mutations, while gatifloxacin exhibited activity against some strains of M. hominis with one mutation. The gatifloxacin MICs remained ≤1 μg/ml only for the sole single mutant and for two double mutants of M. hominis. At least, several alterations in DNA gyrase and topoisomerase IV, especially two mutations in the DNA gyrase, seemed to be necessary to have high increases in the garenoxacin and gemifloxacin MICs. Our results suggest that development of clinical resistance to new quinolones in human mycoplasmas and ureaplasmas would probably require two or more mutations in the fluoroquinolone targets.
TABLE 2.
Comparison of the in vitro activities of garenoxacin, gemifloxacin, gatifloxacin, and other fluoroquinolones against M. hominis and Ureaplasma clinical isolates and laboratory mutants resistant to fluoroquinolones
| Strain | MIC ranges (μg/ml) of antimicrobiala:
|
Amino acid change in QRDR ofb:
|
||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| GAR | GEM | GAT | MXF | LVX | SPX | CIP | GyrA | ParC | ParE | |
| Ureaplasma spp. | ||||||||||
| Reference strain | 0.12 | 0.25 | 1 | 0.25 | 0.5 | 0.25 | 1 | None | None | None |
| Clinical isolatesc | ||||||||||
| UUe2 | 0.5 | 0.5 | 2 | 1 | 2 | 2 | 8 | None | S80L | None |
| UUb, -d, -f1, -g1 | 0.25-0.5 | 0.25-0.5 | 2 | 0.5-1 | 1 | 0.5 | 4-16 | D95E | None | None |
| UUf2, -g2 to g5 | 0.5-1 | 1 | 2 | 1 | 4 | 2-4 | 16 | D95E | S80L | None |
| UUc | 1 | 0.25 | 4 | 1 | 2 | 2 | 8 | D95E | E84K | None |
| UUa | 4 | 2 | 8 | 4 | 16 | 16 | 64 | Q83R D95E | None | None |
| M. hominis | ||||||||||
| Reference strain | ≤0.015 | 0.03 | 0.06 | 0.12 | 0.5 | 0.06 | 1 | None | None | None |
| Clinical isolatesd | ||||||||||
| MHa | 0.06 | 0.25 | 1 | 0.5 | 2 | 2 | 16 | E87K | E84G | None |
| MHb1 | 0.12 | 0.25 | 1 | 1 | 2 | 2 | 16 | S83L | S81P | None |
| MHc1 | 0.5 | 0.25 | 4 | 2 | 16 | 2 | 32 | S83L | None | D426N |
| MHec | 0.5 | 2 | 4 | 4 | 32 | 4 | 16 | S83W | S80I | A462V |
| In vitro mutantse | ||||||||||
| IS1 | 0.25 | 0.12 | 0.25 | 0.25 | 0.5 | 0.5 | 2 | S83L | None | None |
| IIS1 | 1 | 2 | 8 | 8 | 16 | 16 | 32 | S83L | E84K | None |
| IIS3A | 2 | 4 | 16 | 16 | 32 | 32 | 64 | S83L S84W | None | None |
| IIIS3A1 | 4 | 8 | 32 | 16 | 64 | 32 | 128 | S83L S84W | E84K | None |
GAR, garenoxacin; GEM, gemifloxacin; GAT, gatifloxacin; MXF, moxifloxacin; LVX, levofloxacin; SPX, sparfloxacin; CIP, ciprofloxacin.
The quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE were sequenced for all clinical isolates. Escherichia coli numbering is shown.
Data from reference 3.
Data from reference 2.
Acknowledgments
This study was supported in part by grants from Bristol Myers Squibb, Glaxo SmithKline, and Grunenthal.
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