Abstract
The activity of the ketolide ABT-773 against Haemophilus and Moraxella was compared to those of 11 other agents. Against 210 Haemophilus influenzae strains (39.0% β-lactamase positive), microbroth dilution tests showed that azithromycin and ABT-773 had the lowest MICs (0.5 to 4.0 and 1.0 to 8.0 μg/ml, respectively), followed by clarithromycin and roxithromycin (4.0 to >32.0 μg/ml). Of the β-lactams, ceftriaxone had the lowest MICs (≤0.004 to 0.016 μg/ml), followed by cefixime and cefpodoxime (0.008 to 0.125 and ≤0.125 to 0.25 μg/ml, respectively), amoxicillin-clavulanate (0.125 to 4.0 μg/ml), and cefuroxime (0.25 to 8.0 μg/ml). Amoxicillin was only active against β-lactamase-negative strains, and cefprozil had the highest MICs of all oral cephalosporins tested (0.5 to >32.0 μg/ml). Against 50 Moraxella catarrhalis strains, all of the compounds except amoxicillin and cefprozil were active. Time-kill studies against 10 H. influenzae strains showed that ABT-773, at two times the MIC, was bactericidal against 9 of 10 strains, with 99% killing of all strains at the MIC after 24 h; at 12 h, ABT-773 gave 90% killing of all strains at two times the MIC. At 3 and 6 h, killing by ABT-773 was slower, with 99.9% killing of four strains at two times the MIC after 6 h. Similar results were found for azithromycin, with slightly slower killing by erythromycin, clarithromycin, and roxithromycin, especially at earlier times. β-Lactams were bactericidal against 8 to 10 strains at two times the MIC after 24 h, with slower killing at earlier time periods. Most compounds gave good killing of five M. catarrhalis strains, with β-lactams killing more rapidly than other drugs. ABT-773 and azithromycin gave the longest postantibiotic effects (PAEs) of the ketolide-macrolide-azalide group tested (4.4 to >8.0 h), followed by clarithromycin, erythromycin, and roxithromycin. β-Lactam PAEs were similar and shorter than those of the ketolide-macrolide-azalide group for all strains tested.
Although development of an effective vaccine against Haemophilus influenzae type b has led to the disappearance of the organism in many parts of the world, its place has been taken by untypeable H. influenzae strains. These organisms (followed by Streptococcus pneumoniae and Moraxella catarrhalis) are now considered to be the leading cause of acute exacerbations of chronic bronchitis and an important cause, together with S. pneumoniae and M. catarrhalis, of acute otitis media, sinusitis, and community-acquired respiratory tract infections (1, 8, 10, 12, 14, 23).
Current recommendations by the National Committee for Clinical Laboratory Standards (NCCLS) for use of Haemophilus test medium (HTM) for Haemophilus susceptibility testing (13) have been complicated by difficulty in commercial manufacture of this medium and its short half-life when made in house. Reliable Haemophilus susceptibility testing with HTM requires the use of freshly made medium within 3 weeks of manufacture (11, 22).
ABT-773 is a new ketolide (2; A. M. Nilius, M. Bui, L. Almer, D. Hensey, J. Boor, Z. Ma, Y. S. Ar, and R. Flamm, Abstr. 9th Eur. Congr. Clin. Microbiol. Infect. Dis., abstr. P-177, 1999; Z. Ma, R. F. Clark, and Y. Or, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2133, 1999; Z. Cao, R. Hammond, S. Pratt, A. Saiki, C. Lerner, and P. Zhong, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2135, 1999). Previous preliminary studies have shown that this compound has low MICs against respiratory pathogens, including Haemophilus and Moraxella (2; D. Shortridge, N. C. Ramer, J. Boor, Z. Ma, Y. Or, and R. K. Flamm, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2136, 1999). This study further examined activity of ABT-773 against Haemophilus and Moraxella by (i) using NCCLS microdilution MIC methodology to test the activity of ABT-773 compared to those of erythromycin, azithromycin, clarithromycin, roxithromycin, amoxicillin, amoxicillin-clavulanate, cefuroxime, cefixime, cefpodoxime, cefprozil, and ceftriaxone against 210 H. influenzae and 50 M. catarrhalis strains; (ii) testing the kill kinetics of the above-mentioned compounds against 10 H. influenzae and 5 M. catarrhalis strains; and (iii) testing the postantibiotic effects (PAEs) of the above-mentioned compounds against 5 H. influenzae strains.
MATERIALS AND METHODS
Bacteria and antimicrobials.
Strains (210 H. influenzae and 50 M. catarrhalis) were isolated from clinical specimens within the past 2 years and stored at −70°C in double-strength skim milk (Difco Laboratories, Detroit, Mich.) prior to use. ABT-773 susceptibility powder was obtained from Abbott Laboratories, Chicago, Ill. Other drugs were obtained from their respective manufacturers.
MIC determination.
Microdilution MIC tests were performed by the NCCLS microdilution method (13). H. influenzae strains were all untypeable organisms. Inocula were prepared from chocolate agar plates incubated for a full 24 h by the direct colony suspension method as follows. In a tube of Mueller-Hinton broth (Difco), an organism suspension was made to a density of a 0.5 McFarland standard (108 CFU/ml). The inoculum was diluted in sterile saline such that final organism suspensions in trays yielded colony counts of 3 × 105 to 8 × 105 CFU/ml (11).
Frozen microdilution trays were obtained from MicroMedia Systems, Inc. (Cleveland, Ohio). Each tray contained all antimicrobials prepared in freshly made HTM. The wells were inoculated with 5 × 105 CFU/ml and incubated in ambient air at 35°C for 20 to 24 h. The lowest drug concentration showing no growth was read as the MIC. Clavulanate was added to amoxicillin at a ratio of 1 to 2. Standard quality control strains, including H. influenzae ATCC 49766, H. influenzae ATCC 49247, Staphylococcus aureus ATCC 29213, and Escherichia coli ATCC 25922 were included with each run.
Time-kill studies.
Glass tubes containing 5 ml of HTM (freshly made, as described above) with doubling antibiotic concentrations were inoculated with approximately 5 × 105 CFU (5 × 105 to 5 × 106 CFU) of organism/ml and incubated at 35°C in a shaking water bath. Antibiotic concentrations were chosen to comprise 3 doubling dilutions above and 3 dilutions below the MIC. Freshly made batches of HTM were used for all tests. The dilutions required to obtain the correct inoculum (approximately 5 × 105 CFU/ml) were determined by prior viability studies using each strain (17–20).
To inoculate each tube of serially diluted antibiotic, 50 μl of diluted inoculum was delivered by pipette beneath the surface of the broth and then vortexed and plated for viability counts (zero hour). Only tubes containing an initial inoculum within the range of 5 × 105 to 5 × 106 CFU/ml were acceptable (17–20).
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 HTM onto chocolate agar plates. Recovery plates were incubated for up to 48 h. Colony counts were performed on plates yielding 30 to 300 colonies (17–20).
The lower limit of sensitivity of colony counts was 300 CFU/ml.
Time-kills were analyzed by determining the number of strains which yielded a Δlog10 CFU per milliliter of −1, −2, and −3 at 0, 3, 6, 12, and 24 h compared to counts at 0 h. Antimicrobials 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 if the inoculum was reduced by 0 to 3 log10 CFU/ml. With the sensitivity threshold and inocula used in these studies, no problems were encountered in delineating 99.9% killing, when present. The problem of bacterial carryover was addressed as described previously (17–20).
Measurement of PAE.
PAE was determined by the viable plate count method (4) using freshly made HTM (7, 11). The bacterial inoculum was prepared by suspending growth from an overnight chocolate agar plate in broth. The broth was incubated at 35°C for 2 to 4 h in a shaking water bath until the turbidity matched a no. 1 MacFarland standard (approximately 5 × 108 CFU/ml).
For PAE experiments, 5-ml tubes of broth containing the antibiotic concentrations to be tested at 2 times the MIC (cefprozil), 4 times the MIC (ABT-773, erythromycin, azithromycin, clarithromycin, roxithromycin, amoxicillin, and amoxicillin-clavulanate), and 10 times the MIC (cefuroxime, cefixime, cefpodoxime, and ceftriaxone) (concentrations are based upon pharmacokinetics) were inoculated with 50 μl of inoculum to provide 5 × 106 CFU/ml. The tubes were then vortexed and plated for viability counts. Growth controls with inoculum but no antibiotic were included with each experiment. The inoculated test tubes were then placed in a shaking water bath at 35°C for an exposure period of 1 h. At the end of the exposure period, the cultures were diluted 1:1,000 in prewarmed broth to remove the antibiotic. An additional control culture containing bacteria and antibiotic at a concentration of 0.01 times the MIC was prepared to confirm that after dilution the antibiotic was no longer bacteriostatic (4, 7).
Viability counts were determined before exposure and immediately after dilution (zero hour) and then every 2 h until the turbidity of the tube reached a no. 1 MacFarland standard. Viability counts were performed by preparing 10-fold dilutions of 0.1-ml aliquots from each tube in HTM and plating 0.1-ml volumes onto chocolate agar plates. Recovery plates were inoculated for at least 72 h, and colony counts were performed on plates yielding 30 to 300 colonies.
The PAE was defined according to Craig and Gudmundsson (4) as PAE = T − C, where T is the time required for viability counts of an antibiotic-exposed culture to increase by 1 log10 unit above the counts observed immediately after dilution and C is the corresponding time for the growth control.
For each experiment, viability counts, expressed as log10 CFU per milliliter, were plotted against time. The results were expressed as the mean of two separate assays.
RESULTS
The results of MIC testing of H. influenzae are presented in Table 1. Against 210 H. influenzae strains (39.0% β-lactamase positive), microbroth dilution tests showed that azithromycin and ABT-773 had the lowest MICs (0.5 to 4.0 and 1.0 to 8.0 μg/ml, respectively), followed by clarithromycin and roxithromycin (4.0 to >32.0 μg/ml). Of the β-lactams, ceftriaxone had the lowest MICs (≤0.004 to 0.016 μg/ml), followed by cefixime and cefpodoxime (0.008 to 0.125 and ≤0.125 to 0.25 μg/ml, respectively), amoxicillin-clavulanate (0.125 to 4.0 μg/ml) and cefuroxime (0.25 to 8.0 μg/ml). Amoxicillin was only active against β-lactamase-negative strains, and cefprozil had the highest MICs of all oral cephalosporins tested (0.5 to >32.0 μg/ml). Against 50 M. catarrhalis strains, all compounds except amoxicillin and cefprozil were active (Table 1).
TABLE 1.
MICs of 210 H. influenzae and 50 M. catarrhalis strains
| Drug | β-lactamase-positive H. influenzae (82)a
|
β-lactamase-negative H. influenzae (128)
|
All H. influenzae (210)
|
M. catarrhalis (50)
|
||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Range | MIC50 | MIC90 | Range | MIC50 | MIC90 | Range | MIC50 | MIC90 | Range | MIC50 | MIC90 | |
| ABT-773 | 1.0–8.0 | 4.0 | 4.0 | 1.0–8.0 | 4.0 | 4.0 | 1.0–8.0 | 4.0 | 4.0 | 0.125–0.5 | 0.125 | 0.25 |
| Erythromycin | 2.0–16.0 | 8.0 | 16.0 | 2.0–>16.0 | 8.0 | 16.0 | 2.0–>16.0 | 8.0 | 16.0 | 0.25–1.0 | 0.5 | 0.5 |
| Azithromycin | 1.0–4.0 | 2.0 | 4.0 | 0.5–4.0 | 2.0 | 4.0 | 0.5–4.0 | 2.0 | 4.0 | 0.06–0.25 | 0.125 | 0.125 |
| Clarithromycin | 4.0–16.0 | 16.0 | 16.0 | 4.0–>32.0 | 8.0 | 16.0 | 4.0–>32.0 | 8.0 | 16.0 | 0.25–0.5 | ≤0.25 | ≤0.25 |
| Roxithromycin | 4.0–32.0 | 16.0 | 32.0 | 8.0–>32.0 | 16.0 | 32.0 | 4.0–>32.0 | 16.0 | 32.0 | 0.5–2.0 | 1.0 | 1.0 |
| Amoxicillin | 4.0–>16.0 | >16.0 | >16.0 | 0.125–2.0 | 0.5 | 1.0 | 0.125–>16.0 | 1.0 | >16.0 | 2.0–>16.0 | 8.0 | >16.0 |
| Amoxicillin-clavulanate | 0.5–4.0 | 1.0 | 2.0 | 0.125–2.0 | 0.5 | 1.0 | 0.125–4.0 | 1.0 | 2.0 | 0.125–0.5 | ≤0.125 | 0.25 |
| Cefuroxime | 0.25–8.0 | 0.5 | 1.0 | 0.25–4.0 | 0.5 | 1.0 | 0.25–8.0 | 0.5 | 1.0 | 0.25–8.0 | 2.0 | 4.0 |
| Cefixime | 0.008–0.06 | 0.03 | 0.06 | 0.008–0.125 | 0.03 | 0.03 | 0.008–0.125 | 0.03 | 0.06 | 0.008–1.0 | 0.25 | 0.5 |
| Cefpodoxime | ≤0.125–0.25 | ≤0.125 | ≤0.125 | ≤0.125–0.25 | ≤0.125 | ≤0.125 | ≤0.125–0.25 | ≤0.125 | ≤0.125 | 0.25–4.0 | 1.0 | 2.0 |
| Cefprozil | 1.0–>32.0 | 4.0 | 16.0 | 0.5–16.0 | 2.0 | 4.0 | 0.5–>32.0 | 4.0 | 8.0 | 0.25–>32.0 | 4.0 | 8.0 |
| Ceftriaxone | ≤0.004–0.016 | ≤0.004 | 0.008 | ≤0.004–0.016 | ≤0.004 | ≤0.004 | ≤0.004–0.016 | ≤0.004 | ≤0.004 | 0.016–2.0 | 0.5 | 2.0 |
Number of strains.
The MICs of the 15 strains tested by time-kill were similar to those listed in Table 1. Kill kinetics results of the 10 H. influenzae strains are shown in Table 2, and those of the 5 M. catarrhalis strains are shown in Table 3. Two H. influenzae and all five M. catarrhalis strains were β-lactamase positive and were not tested by time-kill with amoxicillin. As can be seen, ABT-773, at two times the MIC, was bactericidal (99.9% killing) against 9 of 10 strains, with 99% killing of all strains at the MIC after 24 h; at 12 h, ABT-773 gave 90% killing of all strains at two times the MIC. At 3 and 6 h, killing by ABT-773 was slower, with 99.9% killing of four strains at two times the MIC after 6 h. Similar results were found for azithromycin, with slightly slower killing by erythromycin, clarithromycin, and roxithromycin, especially at earlier time periods. β-Lactams were bactericidal against 8 to 10 strains at two times the MIC after 24 h, with slower killing at earlier time periods.
TABLE 2.
Results of kill kinetics studies for 10 H. influenzae strains
| Drug and concn | No. of strains
|
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 3 h
|
6 h
|
12 h
|
24 h
|
|||||||||
| −1a | −2 | −3 | −1 | −2 | −3 | −1 | −2 | −3 | −1 | −2 | −3 | |
| ABT-773 | ||||||||||||
| 4 × MIC | 7 | 5 | 2 | 8 | 5 | 5 | 10 | 8 | 5 | 10 | 10 | 10 |
| 2 × MIC | 6 | 2 | 1 | 8 | 5 | 4 | 10 | 7 | 5 | 10 | 10 | 9 |
| MIC | 2 | 1 | 0 | 4 | 2 | 0 | 8 | 4 | 4 | 10 | 10 | 6 |
| 0.5 × MIC | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Erythromycin | ||||||||||||
| 4 × MIC | 5 | 2 | 1 | 7 | 5 | 2 | 10 | 6 | 5 | 10 | 10 | 9 |
| 2 × MIC | 5 | 1 | 1 | 6 | 4 | 1 | 10 | 6 | 4 | 10 | 10 | 8 |
| MIC | 2 | 0 | 0 | 4 | 0 | 0 | 8 | 2 | 1 | 9 | 8 | 5 |
| 0.5 × MIC | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 |
| Azithromycin | ||||||||||||
| 4 × MIC | 7 | 4 | 3 | 10 | 6 | 5 | 10 | 10 | 9 | 10 | 10 | 10 |
| 2 × MIC | 4 | 3 | 1 | 9 | 5 | 3 | 10 | 10 | 7 | 10 | 10 | 10 |
| MIC | 3 | 1 | 0 | 6 | 2 | 0 | 9 | 4 | 3 | 9 | 9 | 7 |
| 0.5 × MIC | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 0 | 1 | 1 | 0 |
| Clarithromycin | ||||||||||||
| 4 × MIC | 5 | 2 | 0 | 8 | 5 | 2 | 10 | 6 | 5 | 10 | 10 | 9 |
| 2 × MIC | 4 | 1 | 0 | 7 | 4 | 2 | 10 | 5 | 4 | 10 | 10 | 9 |
| MIC | 1 | 0 | 0 | 2 | 0 | 0 | 8 | 2 | 0 | 7 | 7 | 3 |
| 0.5 × MIC | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Roxithromycin | ||||||||||||
| 4 × MIC | 5 | 3 | 1 | 8 | 5 | 2 | 10 | 6 | 5 | 10 | 10 | 9 |
| 2 × MIC | 4 | 2 | 1 | 7 | 4 | 2 | 9 | 6 | 4 | 10 | 10 | 9 |
| MIC | 2 | 0 | 0 | 3 | 1 | 0 | 6 | 4 | 3 | 7 | 7 | 3 |
| 0.5 × MIC | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Amoxicillin | ||||||||||||
| 4 × MIC | 6b | 0 | 0 | 8 | 2 | 0 | 8 | 7 | 3 | 8 | 8 | 8 |
| 2 × MIC | 4 | 0 | 0 | 7 | 0 | 0 | 8 | 5 | 1 | 8 | 8 | 7 |
| MIC | 3 | 0 | 0 | 5 | 0 | 0 | 5 | 3 | 0 | 6 | 4 | 3 |
| 0.5 × MIC | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 0 |
| Amoxicillin-clavulanate | ||||||||||||
| 4 × MIC | 4 | 1 | 0 | 9 | 3 | 0 | 10 | 9 | 3 | 10 | 10 | 9 |
| 2 × MIC | 3 | 0 | 0 | 9 | 3 | 0 | 10 | 9 | 3 | 10 | 10 | 9 |
| MIC | 3 | 0 | 0 | 7 | 2 | 0 | 10 | 4 | 2 | 10 | 7 | 5 |
| 0.5 × MIC | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 0 |
| Cefuroxime | ||||||||||||
| 4 × MIC | 5 | 0 | 0 | 10 | 2 | 0 | 10 | 9 | 4 | 10 | 10 | 10 |
| 2 × MIC | 4 | 0 | 0 | 9 | 2 | 0 | 10 | 8 | 3 | 10 | 10 | 8 |
| MIC | 2 | 0 | 0 | 5 | 0 | 0 | 8 | 4 | 1 | 8 | 8 | 6 |
| 0.5 × MIC | 0 | 0 | 0 | 1 | 0 | 0 | 2 | 0 | 0 | 1 | 0 | 0 |
| Cefixime | ||||||||||||
| 4 × MIC | 4 | 1 | 0 | 8 | 5 | 1 | 9 | 7 | 5 | 10 | 10 | 10 |
| 2 × MIC | 4 | 0 | 0 | 8 | 5 | 1 | 10 | 8 | 3 | 10 | 10 | 9 |
| MIC | 3 | 0 | 0 | 8 | 4 | 0 | 10 | 8 | 3 | 10 | 10 | 8 |
| 0.5 × MIC | 0 | 0 | 0 | 1 | 0 | 0 | 2 | 1 | 0 | 2 | 0 | 0 |
| Cefpodoxime | ||||||||||||
| 4 × MIC | 5 | 1 | 0 | 8 | 4 | 0 | 10 | 7 | 2 | 10 | 10 | 9 |
| 2 × MIC | 3 | 0 | 0 | 8 | 5 | 0 | 10 | 8 | 4 | 10 | 10 | 9 |
| MIC | 3 | 0 | 0 | 7 | 4 | 0 | 9 | 6 | 3 | 10 | 9 | 7 |
| 0.5 × MIC | 1 | 0 | 0 | 1 | 0 | 0 | 3 | 0 | 0 | 0 | 0 | 0 |
| Cefprozil | ||||||||||||
| 4 × MIC | 5 | 0 | 0 | 10 | 6 | 0 | 10 | 10 | 6 | 10 | 10 | 10 |
| 2 × MIC | 4 | 0 | 0 | 10 | 4 | 0 | 10 | 8 | 5 | 10 | 10 | 10 |
| MIC | 1 | 0 | 0 | 5 | 2 | 0 | 9 | 3 | 2 | 9 | 8 | 6 |
| 0.5 × MIC | 0 | 0 | 0 | 2 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 |
| Ceftriaxone | ||||||||||||
| 4 × MIC | 3 | 0 | 0 | 8 | 6 | 2 | 10 | 8 | 6 | 10 | 10 | 9 |
| 2 × MIC | 3 | 0 | 0 | 8 | 5 | 1 | 10 | 8 | 6 | 10 | 10 | 9 |
| MIC | 2 | 0 | 0 | 8 | 3 | 0 | 10 | 8 | 3 | 10 | 9 | 8 |
| 0.5 × MIC | 0 | 0 | 0 | 0 | 0 | 0 | 3 | 0 | 0 | 0 | 0 | 0 |
ΔLog10 CFU per milliliter lower than at 0 h.
Only tested against β-lactamase-negative strains.
TABLE 3.
Results of kill kinetics studies for five M. catarrhalis strainsa
| Drug and concn | No. of strains
|
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 3 h
|
6 h
|
12 h
|
24 h
|
|||||||||
| −1b | −2 | −3 | −1 | −2 | −3 | −1 | −2 | −3 | −1 | −2 | −3 | |
| ABT-773 | ||||||||||||
| 4 × MIC | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 3 | 0 | 5 | 5 | 5 |
| 2 × MIC | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 1 | 0 | 5 | 5 | 4 |
| MIC | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 5 | 5 | 3 |
| 0.5 × MIC | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 1 |
| Erythromycin | ||||||||||||
| 4 × MIC | 0 | 0 | 0 | 1 | 0 | 0 | 5 | 3 | 1 | 5 | 5 | 5 |
| 2 × MIC | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 2 | 0 | 5 | 5 | 5 |
| MIC | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 0 | 0 | 5 | 5 | 5 |
| 0.5 × MIC | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 |
| Azithromycin | ||||||||||||
| 4 × MIC | 0 | 0 | 0 | 4 | 0 | 0 | 5 | 5 | 3 | 5 | 5 | 5 |
| 2 × MIC | 0 | 0 | 0 | 1 | 0 | 0 | 5 | 5 | 2 | 5 | 5 | 5 |
| MIC | 0 | 0 | 0 | 0 | 0 | 0 | 5 | 0 | 0 | 5 | 5 | 5 |
| 0.5 × MIC | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 |
| Clarithromycin | ||||||||||||
| 4 × MIC | 0 | 0 | 0 | 1 | 0 | 0 | 5 | 2 | 1 | 5 | 5 | 4 |
| 2 × MIC | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 2 | 0 | 5 | 5 | 4 |
| MIC | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 5 | 5 | 3 |
| 0.5 × MIC | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 0 |
| Roxithromycin | ||||||||||||
| 4 × MIC | 0 | 0 | 0 | 2 | 0 | 0 | 4 | 3 | 0 | 5 | 5 | 5 |
| 2 × MIC | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 1 | 0 | 5 | 5 | 5 |
| MIC | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 5 | 4 | 3 |
| 0.5 × MIC | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Amoxicillin-clavulanate | ||||||||||||
| 4 × MIC | 4 | 1 | 0 | 5 | 5 | 1 | 5 | 5 | 5 | 5 | 5 | 5 |
| 2 × MIC | 4 | 1 | 0 | 5 | 5 | 1 | 5 | 4 | 3 | 5 | 5 | 5 |
| MIC | 3 | 0 | 0 | 5 | 3 | 0 | 5 | 3 | 1 | 5 | 4 | 1 |
| 0.5 × MIC | 1 | 0 | 0 | 4 | 1 | 0 | 1 | 1 | 1 | 1 | 0 | 0 |
| Cefuroxime | ||||||||||||
| 4 × MIC | 5 | 3 | 0 | 5 | 5 | 3 | 5 | 5 | 3 | 5 | 5 | 5 |
| 2 × MIC | 5 | 3 | 0 | 5 | 5 | 3 | 5 | 4 | 2 | 5 | 5 | 4 |
| MIC | 5 | 1 | 0 | 5 | 4 | 1 | 5 | 2 | 1 | 3 | 0 | 0 |
| 0.5 × MIC | 1 | 1 | 0 | 3 | 1 | 0 | 3 | 0 | 0 | 0 | 0 | 0 |
| Cefixime | ||||||||||||
| 4 × MIC | 3 | 2 | 0 | 4 | 3 | 2 | 5 | 5 | 3 | 5 | 5 | 5 |
| 2 × MIC | 3 | 0 | 0 | 4 | 3 | 1 | 5 | 5 | 2 | 5 | 4 | 2 |
| MIC | 2 | 0 | 0 | 3 | 2 | 0 | 4 | 2 | 0 | 3 | 2 | 0 |
| 0.5 × MIC | 0 | 0 | 0 | 1 | 0 | 0 | 2 | 0 | 0 | 0 | 0 | 0 |
| Cefpodoxime | ||||||||||||
| 4 × MIC | 5 | 5 | 0 | 5 | 5 | 4 | 5 | 5 | 5 | 5 | 5 | 4 |
| 2 × MIC | 5 | 1 | 0 | 5 | 5 | 2 | 5 | 5 | 3 | 5 | 3 | 2 |
| MIC | 3 | 0 | 0 | 5 | 3 | 1 | 5 | 3 | 1 | 3 | 2 | 1 |
| 0.5 × MIC | 1 | 0 | 0 | 3 | 1 | 1 | 2 | 1 | 0 | 0 | 0 | 0 |
| Cefprozil | ||||||||||||
| 4 × MIC | 5 | 4 | 1 | 5 | 5 | 3 | 5 | 5 | 3 | 5 | 4 | 2 |
| 2 × MIC | 5 | 4 | 0 | 5 | 5 | 3 | 5 | 4 | 2 | 2 | 0 | 0 |
| MIC | 5 | 3 | 0 | 5 | 5 | 3 | 5 | 4 | 0 | 0 | 0 | 0 |
| 0.5 × MIC | 5 | 2 | 0 | 4 | 3 | 2 | 4 | 1 | 0 | 0 | 0 | 0 |
| Ceftriaxone | ||||||||||||
| 4 × MIC | 5 | 1 | 0 | 5 | 5 | 1 | 5 | 5 | 3 | 5 | 4 | 1 |
| 2 × MIC | 4 | 0 | 0 | 5 | 4 | 0 | 5 | 5 | 2 | 5 | 2 | 0 |
| MIC | 4 | 0 | 0 | 5 | 1 | 0 | 5 | 4 | 1 | 2 | 0 | 0 |
| 0.5 × MIC | 0 | 0 | 0 | 4 | 0 | 0 | 4 | 0 | 0 | 0 | 0 | 0 |
Amoxicillin was not tested (all strains are β-lactamase positive).
ΔLog10 CFU per milliliter lower than at 0 h.
Time-kill studies for the five M. catarrhalis strains (Table 3) showed that all compounds except cefprozil and ceftriaxone were bactericidal at or above the MIC after 24 h, with other β-lactams showing more rapid killing at earlier time periods.
PAEs are presented in Table 4. As can be seen, ABT-773 and azithromycin gave the longest PAEs of the ketolide-macrolide-azalide group tested (4.4 to >8.0 h), followed by clarithromycin, erythromycin, and roxithromycin. β-Lactam PAEs were all similar and shorter than those of the ketolide-macrolide-azalide group for all strains tested.
TABLE 4.
MICs and PAEs of five H. influenzae strains
| Drug | MIC range (μg/ml) | PAE (h)a |
|---|---|---|
| ABT-773b | 1.0–8.0 | ≥6.1 (4.9–>8.0) |
| Erythromycinb | 4.0–16.0 | ≥3.8 (0.9–>6.7) |
| Azithromycinb | 2.0–8.0 | ≥6.1 (4.4–>7.4) |
| Clarithromycinb | 4.0–16.0 | ≥3.3 (1.6–>6.7) |
| Roxithromycinb | 8.0–32.0 | ≥3.3 (1.9–>6.7) |
| Amoxicillinb | 0.5–4.0 | 0.5 (0–2.6) |
| Amoxicillin-clavulanateb | 0.5–1.0 | 0.7 (0–3.4) |
| Cefuroximec | 0.5–1.0 | 1.0 (0–5.0) |
| Cefiximec | 0.016–0.125 | 0.7 (0–3.7) |
| Cefpodoximec | 0.125–0.25 | 0.7 (0–3.5) |
| Cefprozild | 4.0–16.0 | 0.7 (0–3.3) |
| Ceftriaxonec | 0.004–0.008 | 0.6 (0–3.2) |
Mean (range).
Exposure at 4 times MIC for 1 h.
Exposure at 10 times MIC for 1 h.
Exposure at 2 times MIC for 1 h.
DISCUSSION
ABT-773 is a new ketolide (Ma et al., Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., 1999; Cao et al., Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., 1999) which, in preliminary studies, has been reported to be more potent in vitro than the macrolides against H. influenzae, M. catarrhalis, Legionella spp., Neisseria gonorrhoeae, and Listeria monocytogenes. ABT-773 was also more potent against macrolide-susceptible strains of S. pneumoniae, Streptococcus pyogenes, S. aureus, Staphylococcus epidermidis, enterococci, Helicobacter pylori, and Mycobacterium avium complex and also against Corynebacterium spp., Mycoplasma pneumoniae, Chlamydia trachomatis, Borrelia burgdorferi, and Toxoplasma gondii. ABT-773 had potent activity against macrolide-resistant streptococci and enterococci irrespective of their macrolide resistance mechanisms but had little detectable activity against constitutively macrolide-resistant staphylococci and macrolide-resistant H. pylori and M. avium complex (2; Shortridge et al., Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., 1999; M. M. Neuhauser, J. L. Prause, R. Jung, N. Boyea, J. M. Hackleman, L. H. Danziger, and S. L. Pendland, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr 2139, 1999; F. Goldstein, M. D. Kitzis, M. Miegi, and J. F. Acar, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2142, 1999; A. L. Barry, P. C. Fuchs, and S. D. Brown, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2144, 1999; S. L. Pendland, J. L. Prause, M. M. Neuhauser, N. Boyea, J. M. Hackleman, and L. H. Danziger, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2145, 1999; R. Jung, D. H. Li, S. L. Pendland, and L. H. Danziger, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2146, 1999; A. A. Khan, F. G. Araujo, J. C. Craft, and J. S. Remington, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2147, 1999). ABT-773 has been shown to be effective against S. pneumoniae in a rat lung model (J. Meulbroek, M. Mitten, K. W. Mollison, P. Ewing, J. Alder, A. M. Nilius, R. K. Flamm, Z. Ma, and Y. Or, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2151, 1999).
In our study, the MICs of ABT-773 against H. influenzae and M. catarrhalis were similar to those recently reported by others (2; J. Dubois, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2163, 2000; T. Fujikawa, S. Miyazaki, T. Matsumoto, A. Ohno, N. Furuya, Y. Ishii, K. Tateda, and K. Yamaguchi, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2166, 2000), with MICs at which 90% of the strains are inhibited (MIC90s) of 4.0 μg/ml against H. influenzae and 0.06 to 0.25 μg/ml for M. catarrhalis. A recent study (V. Shortridge, N. Ramer, D. McDaniel, P. Johnson, and R. K. Flamm, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2137, 2000) has documented that, similar to our findings, ABT-773 was bactericidal against H. influenzae at four and eight times the MIC, with more rapid killing than erythromycin. ABT-773 was also found to have a longer PAE than erythromycin.
Previous studies have shown, similar to the findings reported here, that azithromycin was the most potent member of the macrolide-azalide-ketolide group by MIC and time-kill against H. influenzae strains, followed by the ketolides telithromycin, clarithromycin, and roxithromycin (9, 15–17, 21). In the present study, ABT-773 had kill kinetics against H. influenzae and M. catarrhalis comparable to that of azithromycin, the macrolide with the greatest overall in vitro activity against this group (9, 17), and also had the longest PAE of all compounds tested.
The clinical application of macrolide MICs against H. influenzae is a complex problem. Macrolides, azalides, and ketolides all exhibit a unimodal MIC distribution against this species, and macrolide resistance mechanisms similar to ribosomal methylase and efflux in gram-positive species have not been clearly defined. Also, there is a question concerning the validity of established breakpoints for macrolides and azalides against H. influenzae. Craig (3) has suggested that breakpoints for azithromycin and clarithromycin against Haemophilus are considerably lower than currently approved values in light of pharmacokinetic and pharmacodynamic parameters (11) and bacteriological outcome studies in otitis media (5, 6). Andes and Craig (Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2139, 2000) have demonstrated that ABT-773, like azithromycin and telithromycin, exhibits pharmacodynamic properties which correlate best with the area under the concentration-time curve/MIC ratio for S. aureus and S. pneumoniae. More detailed data on the free area under the concentration-time curve and MIC, as well as clinical studies, will be necessary to test the clinical validity of the above in vitro data. Long PAEs support once-daily dosing with ABT-773, similar to macrolides and azalides.
A tentative ABT-773 susceptibility breakpoint of ≤4.0 μg/ml against H. influenzae has been proposed (G. Stone, A. Nilius, D. Hensey-Rudloff, L. Almer, J. Beyer, and R. Flamm, abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2164, 2000). Other factors, such as the increased concentration of compounds like clarithromycin in epithelial lining fluid (11) and the known anti-inflammatory effect of this group of agents may also play a role. Whether this phenomenon plays a role with ABT-773 remains to be established. Because of their low MICs, good kill kinetics, and long PAEs, azithromycin and ABT-773 appear to be the most potent agents of this group against H. influenzae on the basis of in vitro results.
ACKNOWLEDGMENT
This study was supported by a grant from Abbott Laboratories, Chicago, Ill.
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