Abstract
Time-kill studies compared the activities of grepafloxacin with those of ciprofloxacin, levofloxacin, sparfloxacin, amoxicillin-clavulanate, and clarithromycin against 12 pneumococcal strains. Grepafloxacin was bactericidal after 24 h against all strains at a concentration of ≤0.5 μg/ml, while sparfloxacin, levofloxacin, and ciprofloxacin were bactericidal at concentrations of ≤1.0, ≤2.0, and ≤8.0 μg/ml, respectively. Amoxicillin-clavulanate and clarithromycin were bactericidal at 2× the MIC after 24 h against 12 of 12 strains and against all 8 macrolide-susceptible strains, respectively.
The incidence of Streptococcus pneumoniae strains resistant to β-lactam and non-β-lactam compounds has increased worldwide at an alarming rate (1, 9, 10). A recent survey from the United States has reported that the MICs of penicillin G for 23.6% of 1,527 clinically significant pneumococcal isolates were ≥0.125 mg/ml (7).
There is an urgent need for new oral antibiotics to treat otitis media, sinusitis, bronchitis, and community-acquired pneumonia caused by penicillin-susceptible and -resistant pneumococci (10). Available quinolones, such as ciprofloxacin and ofloxacin, have marginal antipneumococcal activity (16, 19). There is, thus, a need for a quinolone with expanded antipneumococcal activity for use in treatment of the above-mentioned infections.
Grepafloxacin (OPC 17116) is a broad-spectrum, 5-methyl-substituted quinolone with expanded activity against gram-positive and -negative pathogens, including pneumococci (2, 8, 11, 14, 20–22). The present study employed microbroth and time-kill methodology to examine the activities of grepafloxacin, sparfloxacin, levofloxacin, ciprofloxacin, amoxicillin-clavulanate, and clarithromycin against 12 penicillin-susceptible and -resistant pneumococci.
Pneumococci consisted of four penicillin-susceptible (MICs, ≤0.016 μg/ml), four penicillin-intermediate (MICs, 0.25 μg/ml), and four penicillin-resistant (MICs, 2.0 to 4.0 μg/ml) strains. Microdilution MICs were determined according to recommendations by the National Committee for Clinical Laboratory Standards (13) with cation-adjusted Mueller-Hinton broth with 5% lysed defibrinated horse blood and inocula of 5 × 105 CFU/ml. Clavulanate was combined with amoxicillin at a ratio of 1:2. Standard quality control strains, including S. pneumoniae ATCC 49619, were included with each assay (13).
Time-kill studies were performed with Mueller-Hinton broth plus 5% lysed horse blood as described previously. Dilutions required to obtain the correct inoculum (5 × 105 to 5 × 106 CFU/ml) were determined by prior viability studies with each strain (15, 18).
Viability counts of antibiotic-containing suspensions were performed at 0, 3, 6, 12, and 24 h. The lower limit of sensitivity of colony counts was 300 CFU/ml (15, 18). Time-kill values were analyzed by determining the number of strains which yielded Δlog10s of CFU/ml of −1, −2, and −3 at 0, 3, 6, 12, and 24 h, compared with counts at time 0 h. Antimicrobials were considered bactericidal at the lowest concentration that reduced the original inoculum by ≥3 log10 of CFU/ml (99.9%) at each of the time periods and were considered bacteriostatic when the inoculum was reduced by 0 to 3 log10 of CFU/ml. With the sensitivity threshold and inocula used in these studies, no problems were encountered in delineating 99.9% killing when it was present. The problem of bacterial carryover was addressed as described previously (15, 18). Clarithromycin time-kill assays were not performed with the four strains for which the clarithromycin MICs were >64.0 μg/ml.
For the two strains which did not yield 99.9% killing by grepafloxacin at 2× the MIC after 24 h, the pH of broths used in time-kill studies was adjusted to 7.6. Previous studies (data not shown) found no significant change in viability counts between broths at pH 7.6 and at pH 7.1 (the latter being the pH of the blood-containing Mueller-Hinton broth used in our routine pneumococcal time-kill experiments).
The results of microdilution MIC testing are presented in Table 1. Quinolone MICs were independent of those of penicillin G, with grepafloxacin, sparfloxacin, levofloxacin, and ciprofloxacin inhibiting all pneumococcal strains at MICs (in micrograms per milliliter) of 0.25, 0.25, 1, and 4, respectively. The MICs of the amoxicillin component of amoxicillin-clavulanate were higher for penicillin-intermediate (0.03 to 0.25 μg/ml) and -resistant (1 to 2 μg/ml) strains compared to those for penicillin-susceptible (0.008 to 0.016 μg/ml) strains, while strains highly resistant to clarithromycin were found mostly in penicillin-resistant strains.
TABLE 1.
Broth microdilution MICs of agents against 12 pneumococci
| Drug | MIC (μg/ml)a
|
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 (S) | 2 (S) | 3 (S) | 4 (S) | 5 (I) | 6 (I) | 7 (I) | 8 (I) | 9 (R) | 10 (R) | 11 (R) | 12 (R) | |
| Penicillin | 0.016 | 0.016 | 0.016 | 0.016 | 0.25 | 0.25 | 0.25 | 0.25 | 4.0 | 2.0 | 4.0 | 4.0 |
| Grepafloxacin | 0.125 | 0.25 | 0.125 | 0.25 | 0.125 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.06 | 0.125 |
| Ciprofloxacin | 1.0 | 1.0 | 2.0 | 1.0 | 1.0 | 2.0 | 2.0 | 1.0 | 1.0 | 1.0 | 4.0 | 2.0 |
| Levofloxacin | 1.0 | 1.0 | 1.0 | 0.5 | 1.0 | 0.5 | 0.5 | 0.5 | 1.0 | 1.0 | 0.5 | 1.0 |
| Sparfloxacin | 0.125 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
| Amoxicillin-clavulanate | 0.008 | 0.016 | 0.016 | 0.016 | 0.03 | 0.25 | 0.25 | 0.03 | 2.0 | 1.0 | 2.0 | 2.0 |
| Clarithromycin | >64.0 | 0.03 | 0.03 | 0.03 | 0.016 | 0.06 | 0.03 | 0.06 | >64.0 | >64.0 | 0.03 | >64.0 |
Pneumococcal strains are indicated by numbers (1 to 12). S, penicillin susceptible; I, penicillin intermediate; R, penicillin resistant.
The results of time-kill experiments are presented in Table 2. As can be seen, grepafloxacin at 2× the MIC after 24 h was bactericidal (99.9% killing) against 10 of 12 strains, with 99% killing of 11 of 12 strains. For the two strains which did not show 99.9% killing by grepafloxacin at 2× the MIC after 24 h, the grepafloxacin MICs were 0.064 and 0.125 μg/ml. These strains showed 99.9% killing at 4× the MIC. By comparison, ciprofloxacin was bactericidal against 11 of 12 strains at 2× the MIC after 24 h, with 99% killing of 11 of 12 strains at 2× the MIC after 12 h. Levofloxacin yielded 99.9% killing of all 12 strains at 2× the MIC after 24 h and 99% killing after 12 h. Sparfloxacin yielded bactericidal activity against 9 of 12 strains at 2× the MIC after 24 h, with 99% killing of all strains at 2× the MIC after 24 h. Lower kill rates were seen with sparfloxacin relative to those for levofloxacin and ciprofloxacin at earlier time periods. Amoxicillin-clavulanate showed 99.9% killing of all strains at 2× the MIC after 24 h and 99% killing of all strains after 12 h. The eight strains for which the clarithromycin MICs were ≤0.25 μg/ml showed 99.9% killing at 2× the MIC after 24 h; the four strains for which the MICs were >64.0 μg/ml were not tested. At earlier time periods of 3 to 6 h, some degree of killing in the 90 to 99% range was observed for all compounds (Table 2).
TABLE 2.
Time-kill results
| Drug and MIC level | No. of strains killed by the following times at the indicated Δlog10 of CFU/ml valuesa:
|
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 3 h
|
6 h
|
12 h
|
24 h
|
|||||||||
| −1 | −2 | −3 | −1 | −2 | −3 | −1 | −2 | −3 | −1 | −2 | −3 | |
| Grepafloxacin | ||||||||||||
| 8× MIC | 8 | 3 | 0 | 12 | 4 | 2 | 12 | 12 | 10 | 12 | 12 | 12 |
| 4× MIC | 4 | 1 | 0 | 11 | 2 | 0 | 12 | 11 | 10 | 12 | 12 | 12 |
| 2× MIC | 3 | 0 | 0 | 10 | 4 | 0 | 12 | 11 | 8 | 12 | 11 | 10 |
| MIC | 0 | 0 | 0 | 3 | 0 | 0 | 8 | 7 | 0 | 7 | 6 | 5 |
| 0.5× MIC | 0 | 0 | 0 | 1 | 0 | 0 | 1 | 1 | 0 | 2 | 2 | 2 |
| Ciprofloxacin | ||||||||||||
| 8× MIC | 8 | 3 | 0 | 12 | 10 | 4 | 12 | 12 | 9 | 12 | 12 | 12 |
| 4× MIC | 8 | 2 | 0 | 12 | 8 | 3 | 12 | 12 | 9 | 12 | 12 | 12 |
| 2× MIC | 7 | 2 | 0 | 12 | 7 | 3 | 12 | 11 | 7 | 12 | 12 | 11 |
| MIC | 2 | 0 | 0 | 7 | 3 | 1 | 10 | 9 | 4 | 10 | 10 | 5 |
| 0.5× MIC | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 2 | 0 | 0 |
| Levofloxacin | ||||||||||||
| 8× MIC | 10 | 4 | 1 | 12 | 12 | 5 | 12 | 12 | 10 | 12 | 12 | 12 |
| 4× MIC | 9 | 4 | 1 | 12 | 9 | 4 | 12 | 12 | 10 | 12 | 12 | 12 |
| 2× MIC | 9 | 4 | 0 | 12 | 8 | 1 | 12 | 12 | 10 | 12 | 12 | 12 |
| MIC | 3 | 2 | 0 | 8 | 1 | 1 | 8 | 8 | 4 | 8 | 7 | 6 |
| 0.5× MIC | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 1 |
| Sparfloxacin | ||||||||||||
| 8× MIC | 8 | 3 | 0 | 12 | 6 | 3 | 12 | 12 | 9 | 12 | 12 | 12 |
| 4× MIC | 4 | 1 | 0 | 12 | 6 | 2 | 12 | 10 | 7 | 12 | 12 | 12 |
| 2× MIC | 4 | 0 | 0 | 11 | 4 | 1 | 12 | 8 | 6 | 12 | 12 | 9 |
| MIC | 2 | 0 | 0 | 7 | 2 | 1 | 6 | 5 | 1 | 6 | 4 | 2 |
| 0.5× MIC | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Amoxicillin-clavulanate | ||||||||||||
| 8× MIC | 11 | 6 | 0 | 12 | 10 | 4 | 12 | 12 | 9 | 12 | 12 | 12 |
| 4× MIC | 8 | 3 | 0 | 11 | 9 | 2 | 12 | 12 | 9 | 12 | 12 | 12 |
| 2× MIC | 8 | 2 | 0 | 10 | 9 | 2 | 12 | 12 | 9 | 12 | 12 | 12 |
| MIC | 6 | 1 | 0 | 8 | 4 | 0 | 11 | 8 | 6 | 10 | 8 | 8 |
| 0.5× MIC | 0 | 0 | 0 | 1 | 0 | 0 | 2 | 1 | 0 | 5 | 3 | 2 |
| Clarithromycinb | ||||||||||||
| 8× MIC | 5 | 2 | 0 | 8 | 3 | 2 | 7 | 6 | 5 | 8 | 8 | 8 |
| 4× MIC | 4 | 1 | 0 | 7 | 3 | 2 | 7 | 6 | 5 | 8 | 8 | 8 |
| 2× MIC | 4 | 1 | 0 | 6 | 3 | 2 | 7 | 6 | 5 | 8 | 8 | 8 |
| MIC | 3 | 1 | 0 | 4 | 3 | 0 | 7 | 6 | 3 | 8 | 8 | 7 |
| 0.5× MIC | 0 | 0 | 0 | 4 | 1 | 0 | 3 | 3 | 3 | 5 | 4 | 2 |
Δlog10 of CFU/ml values that were lower than those at 0 h.
Time-kill studies were not performed for four strains for which the clarithromycin MICs were >64 μg/ml.
When time-kill rates and microdilution MICs were considered together, grepafloxacin, sparfloxacin, levofloxacin, and ciprofloxacin were bactericidal after 24 h against all strains at ≤0.5, ≤1.0, ≤2.0, and ≤8.0 μg/ml, respectively. Quinolone time-kill rates in the blood-containing Mueller-Hinton broth (pH 7.6) did not differ from those obtained with the medium at pH 7.2. An agar dilution study performed in our laboratory (16) has confirmed the excellent antipneumococcal activity of grepafloxacin irrespective of the penicillin susceptibilities of the strains, with a MIC at which 50% of the strains are inhibited (MIC50) of 0.25 μg/ml and a MIC90 of 0.5 μg/ml.
Microdilution MIC results in the present study reflect previous findings by our group (15, 16, 18, 19), with grepafloxacin and sparfloxacin having similarly low MICs, followed by those of levofloxacin and ciprofloxacin. Although the results of time-kill experiments in the present study document slightly more rapid killing by levofloxacin compared to those by other quinolones (19), they show that grepafloxacin was uniformly bactericidal after 24 h at ≤0.5 μg/ml, followed by sparfloxacin at ≤1.0 μg/ml, levofloxacin at ≤2.0 μg/ml, and ciprofloxacin at ≤8.0 μg/ml. The time-kill results of amoxicillin-clavulanate must be interpreted together with the MICs, which increased in a manner parallel to that for the MICs of penicillin G (17), while clarithromycin was slowly bactericidal at 24 h for macrolide-susceptible strains only.
Child et al. (3) have documented a mean peak concentration of grepafloxacin in plasma of 1.5 μg/ml 2 h after a single oral dose of 400 mg in humans. Cook et al. (6) have shown that grepafloxacin concentrations are significantly increased in bronchial mucosae, epithelial lining fluids, and macrophages compared to serum concentrations. Penetrations of these compartments considerably exceeded those reported for ciprofloxacin and temafloxacin, and concentrations in all tissues were higher than the previously reported MIC90s for a variety of bacteria, including those for S. pneumoniae. Grepafloxacin has also been shown to be effective in the treatment of respiratory tract infections in mice (8) and humans (5).
The MICs and bactericidal concentrations of sparfloxacin and levofloxacin obtained in this study are also well within achievable concentrations in serum (4, 12), since both of these compounds, like grepafloxacin, are active against respiratory pathogens other than pneumococci. Clinical studies will determine the place of these new quinolones in the treatment of upper- and lower-respiratory-tract infections such as community-acquired pneumonia, acute bacterial exacerbations of chronic bronchitis, and sinusitis.
Acknowledgments
This study was supported by a grant from Glaxo Wellcome, Inc., Research Triangle Park, N.C.
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