Levonadifloxacin (WCK 771) was evaluated against 68 type strains and clinical isolates of Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma pneumoniae, and Ureaplasma spp. in comparison with moxifloxacin, levofloxacin, tetracycline, and azithromycin or clindamycin.
KEYWORDS: levonadifloxacin, WCK 771, quinolone, Mycoplasma, Ureaplasma, antimicrobial resistance
ABSTRACT
Levonadifloxacin (WCK 771) was evaluated against 68 type strains and clinical isolates of Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma pneumoniae, and Ureaplasma spp. in comparison with moxifloxacin, levofloxacin, tetracycline, and azithromycin or clindamycin. Levonadifloxacin MICs were ≤0.5 μg/ml for M. genitalium. MIC90s were 1 μg/ml for M. hominis, 0.125 μg/ml for M. pneumoniae, and 2 μg/ml for Ureaplasma spp. Levonadifloxacin merits further study for treating infections caused by these organisms.
TEXT
Levonadifloxacin (WCK 771) and its oral prodrug alalevonadifloxacin (WCK 2349) are being developed by Wockhardt Bio AG for the treatment of acute bacterial skin and soft tissue infections, community-acquired bacterial pneumonias, and other types of infections. In vitro data indicate that levonadifloxacin has a broad spectrum of in vitro activity against Gram-positive and Gram-negative bacteria, including fluoroquinolone-resistant staphylococci (1). In addition to the bacterial species already evaluated, it is important to determine the in vitro activity of levonadifloxacin against other important pathogens of the urogenital and respiratory tracts, including Mycoplasma and Ureaplasma spp.
Mycoplasma pneumoniae is an important cause of tracheobronchitis and community-acquired pneumonia in people of all ages (2). Mycoplasma hominis and Ureaplasma spp. are causes of various urogenital conditions in adults and can be systemic pathogens in infants and other immunosuppressed individuals (3). Mycoplasma genitalium is an important urogenital pathogen that causes urethritis, cervicitis, and pelvic inflammatory disease (4). Treatment options for mycoplasmal and ureaplasmal infections have become more complicated in recent years as a result of the emergence of antimicrobial resistance. Macrolide resistance due to gene mutations in 23S rRNA is now almost universal in M. pneumoniae clinical isolates in China and Japan and has been documented in Europe and North America (5). Tetracycline resistance mediated by tet(M) has been known to occur in M. hominis and Ureaplasma spp. since the 1980s, and resistance to macrolides and fluoroquinolones has been reported in these organisms in recent years (5). Declining efficacy of macrolides and fluoroquinolones in M. genitalium due to mutations in 23S rRNA genes and in the quinolone resistance-determining regions (QRDRs), primarily parC/parE, has been documented over the past few years (5). For these reasons, new antimicrobial agents are needed for treatment of Mycoplasma and Ureaplasma infections.
Currently, fluoroquinolones are the only FDA-approved antimicrobials for use against human mycoplasmas and ureaplasmas that target DNA replication. Fluoroquinolones have the advantage of also being bactericidal against these organisms (5).
We performed an in vitro study to test the activity of the investigational benzoquinolizine fluoroquinolone levonadifloxacin (WCK 771) against a collection of 8 laboratory-adapted M. genitalium strains, 20 clinical isolates of M. pneumoniae, 20 M. hominis isolates, 10 Ureaplasma parvum isolates, and 10 Ureaplasma urealyticum isolates. With the exception of the 8 M. genitalium strains, all clinical isolates were obtained between 2004 and 2017, with more than half obtained since 2015. Levofloxacin, moxifloxacin, tetracycline, and azithromycin were used as comparators. Clindamycin was substituted for azithromycin for M. hominis. Organisms tested included clinical isolates containing tet(M), the only known mechanism for naturally occurring tetracycline resistance in human mycoplasmas and ureaplasmas; isolates with 23S rRNA gene mutations conferring macrolide resistance; and strains with parC/parE mutations conferring fluoroquinolone resistance. A summary of acquired antimicrobial resistance mechanisms previously shown to be present in various isolates of M. hominis, M. pneumoniae, U. parvum, and U. urealyticum is provided in Table 1.
TABLE 1.
Mechanisms of acquired antimicrobial resistance in various isolates of Mycoplasma and Ureaplasma species
| Laboratory accession no., by species | MIC (μg/ml) for: |
Resistance mechanism(s) | |
|---|---|---|---|
| Selected antimicrobial | Levonadifloxacin | ||
| M. pneumoniae | |||
| 54265 | Azithromycin, 16 | 0.125 | A2063G 23S rRNA gene |
| 60898 | Azithromycin, 16 | 0.125 | A2063G 23S rRNA gene |
| 61653 | Azithromycin, 16 | 0.125 | A2063G 23S rRNA gene |
| 70636 | Azithromycin, 16 | 0.125 | A2063G 23S rRNA gene |
| 66200 | Azithromycin, 32 | 0.125 | A2063G 23S rRNA gene |
| 66201 | Azithromycin, 32 | 0.125 | A2063G 23S rRNA gene |
| 66224 | Azithromycin, 32 | 0.125 | A2063G 23S rRNA gene |
| M. hominis | |||
| 66014 | Levofloxacin, 16; moxifloxacin, 8 | 16 | G272T (S91I) parC, G1249A (V417I) gyrA |
| 68674 | Levofloxacin, 8; moxifloxacin, 4 | 1 | G193A (R65K), A2612G (N871S), G2707A (E903K) gyrA; C938T (T313I) gyrB; A431G (K144R) parC; G1276A (D426N) parE; 2 plasmids: aac(6′)-lb and qepA |
| 51962 | Tetracycline, 16 | 0.125 | tet(M) |
| 52128 | Tetracycline, 32 | 0.125 | tet(M) |
| 57405 | Tetracycline, 16 | 1 | tet(M) |
| U. parvum | |||
| 50186 | Tetracycline, 16; levofloxacin, 4; moxifloxacin, 1 | 0.25 | tet(M), C5T (A2V) parE |
| 61605 | Tetracycline, 16 | 0.063 | tet(M) |
| 66378 | Levofloxacin, 8; moxifloxacin, 4 | 2 | C248T (S83I), G943A (V315I), C1657T (T553L) parC |
| 71094 | Levofloxacin, 4; moxifloxacin, 2 | 2 | C248T (S83L) parC |
| U. urealyticum | |||
| 25353 | Tetracycline, 32; levofloxacin, 8; moxifloxacin, 4; azithromycin, 8 | 4 | tet(M), A2066G (OP1), C2713T (OP2) 23S rRNA gene; A208C (T70P) ribosomal protein L4 gene; A2399T (N800I) gyrA; T355C (L119S) gyrB; C248T (S83L) parC; A208C (T70P) ribosomal protein L4 gene |
| 69255 | Azithromycin, 32 | 0.125 | A2066G 23S rRNA gene |
| ATCC 33175 | Tetracycline, 16 | 0.063 | tet(M) |
Antimicrobial agents were obtained in powdered form of known purity and diluted in accordance with their respective manufacturer's instructions. MICs were determined by broth microdilution, and quality control was performed in accordance with Clinical and Laboratory Standards Institute guideline M43-A (6). Microdilution plates were incubated aerobically at 37°C and examined daily for color change in the growth control wells. MICs were recorded as the lowest concentration of antimicrobial inhibiting color change in 10B (for Ureaplasma spp.) or SP4 (for Mycoplasma spp.) broth at the time the growth control well demonstrated a color change. Two strains of each Mycoplasma species and one of each Ureaplasma species were tested to determine the minimum bactericidal concentrations (MBCs) of levonadifloxacin using techniques described previously (7). The MBC was defined as the lowest concentration of antimicrobial at which there was no evidence of broth color change after prolonged incubation. Positive and negative controls for the MBC assay consisted of tetracycline (bacteriostatic) and levofloxacin (bactericidal). When the MBC was ≥3 dilutions greater than the MIC, the drug was considered bacteriostatic. When the MBC was ≤2 dilutions greater than the MIC, it was considered bactericidal. This work was performed with approval from the UAB Institutional Review Board for Human Use.
MIC data are summarized in Table 2. Levonadifloxacin was active against all 8 M. genitalium isolates (MIC range, 0.125 to 0.5 μg/ml). Its overall activity was similar to that of moxifloxacin (MIC range, 0.063 to 0.125 μg/ml). The levofloxacin and tetracycline MIC ranges were 0.25 to 2 and 0.125 to 2 μg/ml, respectively. Azithromycin had the lowest MIC (0.0005 μg/ml) against M. genitalium. These data for M. genitalium have limitations because the numbers of isolates of M. genitalium available for testing were very limited, and all were obtained before macrolide and fluoroquinolone resistances emerged in this species. However, because levonadifloxacin MICs for the other species that were resistant to macrolides were comparable to those obtained for susceptible isolates, it seems reasonable to expect that this drug should maintain activity in vitro against strains of M. genitalium demonstrating similar resistance mechanisms.
TABLE 2.
MIC Summary for levonadifloxacin (WCK 771) and comparator agents
| Antimicrobial agent, by species | MIC (μg/ml) |
||
|---|---|---|---|
| Range | MIC50 | MIC90 | |
| M. genitalium (n = 8) | |||
| Levonadifloxacin | 0.125–0.5 | ||
| Levofloxacin | 0.25–2 | ||
| Moxifloxacin | 0.063–0.125 | ||
| Azithromycin | 0.0005 | ||
| Tetracycline | 0.125–2 | ||
| M. hominis (n = 20)a | |||
| Levonadifloxacin | 0.125–16 | 0.25 | 1 |
| Levofloxacin | 0.25–16 | 0.5 | 1 |
| Moxifloxacin | 0.032–8 | 0.125 | 1 |
| Clindamycin | 0.016–0.25 | 0.063 | 0.063 |
| Tetracycline | 0.063–32 | 0.25 | 16 |
| M. pneumoniae (n = 20)b | |||
| Levonadifloxacin | 0.125 | 0.125 | 0.125 |
| Levofloxacin | 0.5–1 | 1 | 1 |
| Moxifloxacin | 0.125 | 0.125 | 0.125 |
| Azithromycin | 0.0005–32 | 0.001 | 32 |
| Tetracycline | 0.125–0.5 | 0.25 | 0.5 |
| Ureaplasma species (n = 20)c | |||
| Levonadifloxacin | 0.063–4 | 0.125 | 2 |
| Levofloxacin | 0.5–16 | 1 | 4 |
| Moxifloxacin | 0.25–4 | 0.5 | 2 |
| Azithromycin | 0.25–32 | 1 | 4 |
| Tetracycline | 0.063–32 | 0.5 | 16 |
Includes 3 tetracycline-resistant and 2 fluoroquinolone-resistant isolates.
Includes 7 macrolide-resistant isolates.
Includes 1 macrolide-resistant; 1 tetracycline-resistant; 2 fluoroquinolone-resistant; 1 tetracycline- and fluoroquinolone-resistant; and 1 tetracycline-, fluoroquinolone-, and macrolide-resistant isolate.
MIC50 of levonadifloxacin against 20 M. hominis isolates (0.25 μg/ml) was 1 dilution greater than that of moxifloxacin (0.125 μg/ml) and 1 dilution less than that of levofloxacin (0.5 μg/ml). Against 1 fluoroquinolone-resistant M. hominis, the levonadifloxacin MIC was 4 and 8 times more potent than moxifloxacin and levofloxacin, respectively; but for the other fluoroquinolone-resistant M. hominis, the levonadifloxacin MIC was equivalent to that of levofloxacin and 1 dilution greater than that of moxifloxacin (Table 1). Levonadifloxacin MICs for 3 M. hominis isolates containing tet(M) were all ≤1 μg/ml, whereas the corresponding tetracycline MICs were 16 to 32 μg/ml. Clindamycin had the lowest MICs against M. hominis (range, 0.016 to 0.25 μg/ml; MIC50, 0.063 μg/ml).
Levonadifloxacin (MIC50, 0.125 μg/ml) was consistently active against all 20 M. pneumoniae isolates and comparable to moxifloxacin (MIC50, 0.125 μg/ml), 8-fold more potent than levofloxacin (MIC50, 1 μg/ml), and 2-fold more potent than tetracycline (MIC50, 0.25 μg/ml). Azithromycin had the lowest MICs for macrolide-susceptible organisms (≤0.001 μg/ml). However, levonadifloxacin MICs were not affected by high-level macrolide resistance (azithromycin MICs, 16 to 32 μg/ml) in 7 M. pneumoniae isolates (Table 1).
Levonadifloxacin MIC50 for 20 Ureaplasma species (0.125 μg/ml) was 4-fold lower than that of moxifloxacin (MIC50, 0.5 μg/ml) and 8-fold lower than that of levofloxacin (MIC50, 1 μg/ml). As observed with M. hominis, levonadifloxacin MICs were up to severalfold higher for isolates known to contain gene mutations that confer fluoroquinolone resistance than for those that were fully susceptible (Table 1). For 2 out of 4 isolates with elevated fluoroquinolone MICs, levonadifloxacin MICs were 2- to 4-fold lower than those of moxifloxacin and comparable to those in the other two organisms. Levonadifloxacin was active against tetracycline-susceptible isolates and isolates containing tet(M), for which tetracycline MICs were 16 to 32 μg/ml. For 1 clinical strain with azithromycin resistance (MIC, 32 μg/ml), the levonadifloxacin MIC was 0.125 μg/ml. No MIC differences were apparent between U. parvum and U. urealyticum isolates.
All MBCs for levonadifloxacin were ≤2 dilutions lower than the corresponding MICs, indicating a bactericidal effect against Mycoplasma and Ureaplasma spp. (data not shown). This observation is consistent with findings for other fluoroquinolones against these organisms (5).
This study demonstrates the in vitro activity of levonadifloxacin against Mycoplasma and Ureaplasma spp. of the respiratory and urogenital tracts, including organisms with well-documented high-level resistance to tetracyclines and macrolides, which are major drug classes currently used to treat infections caused by these pathogens. Levonadifloxacin activity was comparable to that of other fluoroquinolones, e.g., levofloxacin and moxifloxacin, except for levofloxacin-resistant Ureaplasma spp., in which levonadifloxacin was 2 to 16 times more active than levofloxacin. Its bactericidal activity may be beneficial in treatment of systemic infections in immunocompromised hosts. As with other quinolones, the presence of gene mutations associated with fluoroquinolone resistance resulted in elevation of levonadifloxacin MICs.
However, taking into account the proposed pharmacokinetic-pharmacodynamic breakpoint of ≤2 μg/ml for Staphylococcus aureus, levonadifloxacin has the potential to provide comprehensive coverage of these pathogens, including levofloxacin-resistant Ureaplasma strains (8). Furthermore, levonadifloxacin offers best-in-class unbound plasma and epithelial lining fluid (ELF) area under the concentration-time curve from 0 to 24 hours (AUC0–24) (unbound plasma AUC0–24, 45.0 μg · h/ml; ELF AUC0–24, 345.2 μg · h/ml) among quinolones, such as moxifloxacin (unbound plasma AUC0–24, 28.8 μg · h/ml; ELF AUC0–24, 192.6 μg · h/ml), levofloxacin (unbound plasma AUC0–24, 31.1 μg · h/ml; ELF AUC0–24, 115.6 μg · h/ml), and ciprofloxacin (unbound plasma AUC0–24, 19.2 μg · h/ml; ELF AUC0–24, 22.8 μg · h/ml) (9–15). These properties make levonadifloxacin a promising therapeutic option for the treatment of infections caused by ureaplasmas and mycoplasmas.
ACKNOWLEDGMENT
Financial support for this project was provided by Wockhardt, Bio AG, Switzerland.
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