Abstract
Activities of HMR 3787, a new 2-fluoroketolide, and its (des)-fluor derivative, RU 64399, were tested against 111 Haemophilus influenzae and 26 H. parainfluenzae strains and compared with those of telithromycin, erythromycin, azithromycin, and clarithromycin. HMR 3787 and RU 64399 MICs were comparable with those of azithromycin but were less affected by incubation in CO2. Time-kill studies of 12 strains showed that HMR 3787, RU 64399, and telithromycin were bactericidal against all strains after 24 h at two times the MIC.
Haemophilus influenzae remains an important cause of respiratory tract infections. H. parainfluenzae may play a role in acute exacerbation of chronic bronchitis (8). The major mechanism of antibiotic resistance in H. influenzae is the production of β-lactamases; approximately 40% of H. influenzae isolates in the United States are β-lactamase positive (4). Among the members of the macrolide, azalide, and ketolide groups, azithromycin has the lowest MICs against these organisms, followed by telithromycin, erythromycin A, and clarithromycin (1, 2, 5). HMR 3787 is a new 2-fluoroketolide, and RU 64399 is its (des)-fluor derivative (H. Drugeon, A. Bryskier, and P. Bemer-Melchior, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1818, 2000). These two new antibiotics have activity similar to that of telithromycin against pneumococci (6).
In this study, the activities of HMR 3787 and RU 64399 against 111 H. influenzae and 26 H. parainfluenzae strains were established by determining their MICs with the microdilution method (7) and compared with the activities of telithromycin, erythromycin, azithromycin, and clarithromycin.
A total of 111 H. influenzae and 26 H. parainfluenzae strains from our collection were used for this study. Organisms were all recent clinical isolates. Of the 111 H. influenzae strains, 6 were type b and the remainder were nontypeable. β-Lactamase testing was performed by the nitrocefin disk method (Cefinase; BBL Microbiology Systems, Cockeysville, Md.). HMR 3787, RU 64399, and telithromycin were obtained from the Aventis-Pharma Infectious Disease Group, Romainville, France, and other compounds were obtained from their respective manufacturers.
MIC determinations were performed by the NCCLS (7) microdilution method using commercially prepared frozen panels (Dade MicroScan, Inc., Sacramento, Calif.) with freshly prepared Haemophilus test medium (3, 4). Inoculum was prepared from chocolate agar plates incubated for a full 24 h by the direct colony suspension method as recommended by the manufacturer. The final well concentration was approximately 5 × 105 CFU/ml. The standard quality control strains H. influenzae ATCC 49766 and H. influenzae ATCC 49247 were used as controls on each day of testing. Inoculum checks were done, and only suspensions yielding 3 × 105 to 7 × 105 CFU/ml were used. Trays were covered and incubated at 35°C. Two trays were used for each strain, one incubated in ambient air and the other in 5% CO2.
Time-kill studies were done as described previously (3). Glass tubes containing 5 ml of freshly made Haemophilus test medium with doubling antibiotic concentrations were inoculated with approximately 5 × 105 CFU of the organism/ml and incubated at 35°C in a shaking water bath. Viability counts of antibiotic-containing suspensions were performed at 0, 3, 6, 12, and 24 h by plating 10-fold dilutions of 0.1-ml aliquots from each tube in sterile Haemophilus test medium onto chocolate agar plates. Recovery plates were incubated for up to 48 h. Colony counts were performed on plates, yielding counts of 30 to 300 colonies (3). The lower limit of sensitivity of colony counts was 300 CFU/ml. Results were analyzed by determining the number of strains which yielded values of −1, −2, and −3 Δlog10 CFU per milliliter at 3, 6, 12, and 24 h compared to counts at 0 h. Antibacterials were considered bactericidal at the lowest concentration that reduced the original inoculum by ≥3 log10 CFU/ml (99.9%) at each of the time points and were considered bacteriostatic when the inoculum was reduced by <3 log10 CFU/ml.
H. influenzae isolates were from <1- to 81-year-old patients (mean age = 14 years). Of the 111 H. influenzae strains tested, 53 (47.7%) produced β-lactamase, and of the 26 H. parainfluenzae strains tested, 7 (26.9%) produced β-lactamase. Eleven of 58 β-lactamase-negative H. influenzae strains were resistant to ampicillin (MICs ≥ 1 μg/ml) and were classified as β-lactamase-negative ampicillin resistant (9). Microdilution MICs are presented in Table 1. There were no significant differences in antibiotic MICs for strains on the basis of β-lactamase production, ampicillin resistance, or serotype. When incubated in ambient air, HMR 3787, RU 64399, and azithromycin had lower MICs against H. influenzae than telithromycin, erythromycin, and clarithromycin. The MICs at which 50% of the isolates tested were inhibited (MIC50s) and MIC90s in ambient air were 0.5 and 1 μg/ml, respectively, for HMR 3787, RU 64399, and azithromycin, 1 and 2 μg/ml for telithromycin, 2 and 4 μg/ml for erythromycin, and 4 and 8 μg/ml for clarithromycin (Table 1). Azithromycin had the lowest MIC90s against H. parainfluenzae (0.5 μg/ml), followed by HMR 3787 and RU 64399 (2 μg/ml), telithromycin and erythromycin (4 μg/ml), and clarithromycin (8 μg/ml). HMR 3787 and RU 64399 were more potent than the other agents against both H. influenzae and H. parainfluenzae when trays were incubated in 5% CO2. Incubation in 5% CO2 caused 2- to 16-fold increases in the MICs for all antimicrobials tested (Table 1). Azithromycin was the antimicrobial most affected by incubation in CO2, followed by erythromycin, clarithromycin, telithromycin, HMR 3787, and RU 64399.
TABLE 1.
MICs of various antibacterial agents against 111 H. influenzae and 26 H. parainfluenzae strains when incubated in ambient air or 5% CO2
| Antibiotic and microorganism (incubation condition) | MIC range (μg/ml) | MIC50 (μg/ml) | MIC90 (μg/ml) |
|---|---|---|---|
| HMR 3787 | |||
| H. influenzae | |||
| Ambient air | 0.12-1 | 0.5 | 1 |
| CO2 | 0.25-4 | 1 | 2 |
| H. parainfluenzae | |||
| Ambient air | 0.12-4 | 1 | 2 |
| CO2 | 0.5-8 | 2 | 4 |
| RU 64399 | |||
| H. influenzae | |||
| Ambient air | 0.25-2 | 0.5 | 1 |
| CO2 | 0.25-4 | 1 | 2 |
| H. parainfluenzae | |||
| Ambient air | 0.25-4 | 1 | 2 |
| CO2 | 0.5-8 | 2 | 4 |
| Telithromycin | |||
| H. influenzae | |||
| Ambient air | 0.25-2 | 1 | 2 |
| CO2 | 0.5-8 | 2 | 4 |
| H. parainfluenzae | |||
| Ambient air | 0.25-8 | 1 | 4 |
| CO2 | 1-16 | 4 | 8 |
| Erythromycin A | |||
| H. influenzae | |||
| Ambient air | 1-16 | 2 | 4 |
| CO2 | 1-32 | 4 | 8 |
| H. parainfluenzae | |||
| Ambient air | 0.5-4 | 2 | 4 |
| CO2 | 2-32 | 8 | 16 |
| Azithromycin | |||
| H. influenzae | |||
| Ambient air | 0.12-2 | 0.5 | 1 |
| CO2 | 0.5-8 | 1 | 4 |
| H. parainfluenzae | |||
| Ambient air | 0.12-1 | 0.25 | 0.5 |
| CO2 | 0.25-8 | 8 | 8 |
| Clarithromycin | |||
| H. influenzae | |||
| Ambient air | 1-16 | 4 | 8 |
| CO2 | 4-32 | 8 | 16 |
| H. parainfluenzae | |||
| Ambient air | 1-32 | 4 | 8 |
| CO2 | 4-32 | 16 | 32 |
The MIC ranges of the drugs for the strains tested by time-kill methods were as follows: HMR 3787, 1.0 to 2.0 μg/ml; RU 64399, 1.0 to 2.0 μg/ml; erythromycin, 4.0 to 32.0 μg/ml; azithromycin, 0.5 to 2 μg/ml; and clarithromycin, 4.0 to 16.0 μg/ml. The results of the time-kill studies are shown in Table 2. HMR 3787, RU 64399, and telithromycin were bactericidal (99.9% killing) against all 12 strain tested after 24 h of incubation at two times the MIC. Erythromycin, azithromycin, and clarithromycin were bactericidal for 11 of the 12 strains tested. Only HMR 3787 was bactericidal after 12 h at two times the MIC against all 12 strains. The kill kinetics of all compounds tested relative to their MICs were similar. No differences were observed in the bactericidal activities of the antibacterials tested based on β-lactamase production or β-lactamase-negative ampicillin-resistant phenotype.
TABLE 2.
Results of kill kinetics studies for 12 H. influenzae strains
| Drug and concentration | No. of strains demonstrating −1, −2, or −3 log10 killinga
|
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 3 h
|
6 h
|
12 h
|
24 h
|
|||||||||
| −1 | −2 | −3 | −1 | −2 | −3 | −1 | −2 | −3 | −1 | −2 | −3 | |
| HMR 3787 | ||||||||||||
| 4× MIC | 10 | 10 | 7 | 12 | 12 | 11 | 12 | 12 | 12 | 12 | 12 | 12 |
| 2× MIC | 8 | 3 | 1 | 12 | 9 | 6 | 12 | 12 | 12 | 12 | 12 | 12 |
| MIC | 1 | 1 | 0 | 4 | 2 | 1 | 10 | 8 | 6 | 10 | 10 | 10 |
| RU64399 | ||||||||||||
| 4× MIC | 9 | 3 | 0 | 11 | 9 | 5 | 12 | 12 | 12 | 12 | 12 | 12 |
| 2× MIC | 4 | 0 | 0 | 10 | 4 | 0 | 12 | 11 | 10 | 12 | 12 | 12 |
| MIC | 1 | 0 | 0 | 2 | 0 | 0 | 9 | 4 | 1 | 12 | 9 | 8 |
| Telithromycin | ||||||||||||
| 4× MIC | 7 | 5 | 0 | 10 | 8 | 2 | 12 | 12 | 10 | 12 | 12 | 12 |
| 2× MIC | 5 | 1 | 0 | 9 | 4 | 1 | 12 | 11 | 8 | 12 | 12 | 12 |
| MIC | 2 | 0 | 0 | 4 | 2 | 0 | 10 | 7 | 2 | 10 | 10 | 9 |
| Erythromycin A | ||||||||||||
| 4× MIC | 9 | 3 | 0 | 10 | 6 | 2 | 12 | 12 | 11 | 12 | 12 | 12 |
| 2× MIC | 4 | 0 | 0 | 8 | 1 | 0 | 12 | 11 | 8 | 12 | 12 | 11 |
| MIC | 0 | 0 | 0 | 2 | 0 | 0 | 4 | 3 | 1 | 8 | 3 | 2 |
| Azithromycin | ||||||||||||
| 4× MIC | 8 | 5 | 1 | 12 | 11 | 7 | 12 | 12 | 12 | 12 | 12 | 12 |
| 2× MIC | 4 | 1 | 1 | 10 | 7 | 3 | 11 | 11 | 11 | 12 | 12 | 11 |
| MIC | 2 | 1 | 0 | 3 | 2 | 0 | 9 | 4 | 3 | 10 | 9 | 9 |
| Clarithromycin | ||||||||||||
| 4× MIC | 8 | 3 | 1 | 10 | 6 | 3 | 12 | 12 | 11 | 12 | 12 | 12 |
| 2× MIC | 6 | 2 | 0 | 10 | 5 | 0 | 12 | 10 | 8 | 12 | 12 | 11 |
| MIC | 0 | 0 | 0 | 0 | 0 | 0 | 6 | 3 | 2 | 8 | 6 | 3 |
ΔLog10 CFU per ml lower than at 0 h: −1 log10 CFU/ml = 90% killing; −2 log10 CFU/ml = 99% killing; −3 log10 CFU/ml = 99.9% killing.
The activities of HMR 3787 and RU 64399 were similar to those of azithromycin and higher than those of telithromycin, erythromycin, and clarithromycin against H. influenzae and H. parainfluenzae strains, irrespective of β-lactamase production or serotype. Fluoroketolide MICs were less affected by CO2 incubation than those of the other compounds tested. Azithromycin was the most affected by CO2 incubation. HMR 3787 had the best bactericidal activity among the antibacterials tested. The clinical relevance of these results depends on the pharmacokinetic and pharmacodynamic properties of these agents.
Acknowledgments
This study was supported by a grant from Aventis-Pharma, Romainville, France.
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