Abstract
DC-159a yielded MICs of ≤1 μg/ml against 316 strains of both quinolone-susceptible and -resistant pneumococci (resistance was defined as a levofloxacin MIC ≥4 μg/ml). Although the MICs for DC-159a against quinolone-susceptible pneumococci were a few dilutions higher than those of gemifloxacin, the MICs of these two compounds against 28 quinolone-resistant pneumococci were identical. The DC-159a MICs against quinolone-resistant strains did not appear to depend on the number or the type of mutations in the quinolone resistance-determining region. DC-159a, as well as the other quinolones tested, was bactericidal after 24 h at 2× MIC against 11 of 12 strains tested. Two of the strains were additionally tested at 1 and 2 h, and DC-159a at 4× MIC showed significant killing as early as 2 h. Multistep resistance selection studies showed that even after 50 consecutive subcultures of 10 strains in the presence of sub-MICs, DC-159a produced only two mutants with maximum MICs of 1 μg/ml.
Penicillin-resistant strains of Streptococcus pneumoniae have been increasingly isolated from various parts of the world, including the United States, where the incidence from clinical specimens is over 20% (1-3, 13-16). There is an urgent need for an oral agent that can be used for the empirical treatment of community-acquired pneumonia, sinusitis, acute exacerbations of chronic bronchitis, and other respiratory tract infections caused by penicillin-resistant pneumococci (8, 9). These resistant clones have the capacity to spread from country to country and from continent to continent (19, 20).
A recent study performed by our group has documented a combined rate of penicillin intermediate and penicillin resistance of approximately 50% among pneumococci, with an overall macrolide resistance rate of approximately 33% (with higher rates of macrolide resistance among strains with raised penicillin G MICs) (16). Although quinolone resistance rates are still low, they may rise with the widespread use of broader-spectrum quinolones. Such quinolone-resistant strains have been reported at increased rates from Hong Kong (10), Canada (5), and Spain (18). Although the cases of meningitis and bacteremia caused by these drug-resistant pneumococcal strains will almost certainly disappear with the introduction of the new pediatric conjugate vaccine, the influence of the latter on community-acquired respiratory tract infections caused by these resistant pneumococcal strains is less certain.
Of the currently available quinolones, levofloxacin, moxifloxacin, and gemifloxacin have clinically applicable antipneumococcal activities. However, only gemifloxacin has free area under the concentration-time curve/MICs of ≥25, which point to clinical efficacy against many (but not all) quinolone-resistant strains, depending on the number of mutations in the quinolone resistance-determining region (QRDR) (6, 11, 17, 23, 27, 28).
DC-159a (12) is a new broad-spectrum oral fluoroquinolone being developed for the oral therapy of community-acquired respiratory tract infections. The present study was performed (i) to examine the susceptibilities of 316 pneumococci of various phenotypes and genotypes to DC-159a compared to those to ciprofloxacin, levofloxacin, moxifloxacin, gatifloxacin, gemifloxacin, telithromycin, azithromycin, amoxicillin-clavulanic acid, and penicillin G by the agar dilution MIC method; (ii) to test 12 pneumococcal strains by time-kill analysis with DC-159a, levofloxacin, moxifloxacin, gatifloxacin, gemifloxacin, and azithromycin; and (iii) to examine by multistep resistance selection studies the ability of DC-159a to select for resistance in 10 pneumococcal strains compared to that of levofloxacin, moxifloxacin, gatifloxacin, gemifloxacin, azithromycin, and amoxicillin-clavulanic acid.
MATERIALS AND METHODS
Drugs and organisms.
DC-159a powder was obtained from Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan, and the other drugs were obtained from their respective manufacturers. Of the 316 pneumococci tested by the agar dilution MIC method, 156 were azithromycin susceptible and 160 were azithromycin resistant. Of the isolates in the latter group, 66 strains carried the ermB gene, 62 strains carried mefA, 1 strain had both ermB and mefA, 4 strains were ermA positive, 23 strains had mutations in ribosomal protein L4, and 4 strains had mutations in 23S rRNA (26). Throughout this study strains with quinolone resistance are defined as those for which levofloxacin MICs are ≥4 μg/ml (thus including strains both intermediate and resistant by the current criteria) (22). Twenty-eight strains with known quinolone resistance mechanisms, each with two or more mutations in the QRDRs, were also included: 26 isolates had single mutations (S81F/Y/C, E85K) in GyrA. The majority of isolates (26 of 28) had single (S79F/Y, D83N) or double (S79F/Y and K137N or D83N and K137N) mutations in ParC. There were 23 isolates with single mutations in ParE (I460V/N, D435N). Efflux was tested by the reserpine method (4). Of the 316 strains, 110 were penicillin G susceptible, 101 were penicillin G intermediate, and 105 were penicillin G resistant. Twelve strains were tested by time-kill analysis: 4 penicillin-susceptible strains, 4 penicillin-intermediate strains, and 4 penicillin-resistant strains. Of these 12 strains, 7 were macrolide resistant (3 were ermB positive, 3 were mefA positive, and 1 had a mutation in L4), 8 were quinolone susceptible, and 4 were quinolone resistant and had defined mutations in the QRDRs. For multistep resistance selection, four erythromycin-susceptible strains, three ermB-positive strains, and three mefA-positive strains (including penicillin-susceptible, -intermediate, and -resistant strains and two quinolone-resistant strains) were tested.
Agar dilution MIC studies.
The 316 pneumococci were tested by the agar dilution method, according to CLSI (former NCCLS) guidelines (22), by using Mueller-Hinton agar supplemented with 5% defibrinated sheep blood. Suspensions equivalent to a 0.5 McFarland standard were made from blood agar plates and were diluted to obtain a final inoculum of 1 × 104/CFU per spot. The plates were incubated overnight in air at 35°C. Standard quality control strains, including S. pneumoniae ATCC 49619, were included in each run (22).
Time-kill studies.
For the time-kill experiments, glass tubes containing 5 ml cation-adjusted Mueller-Hinton broth (BBL) and 5% lysed horse blood with doubling antibiotic concentrations were inoculated with approximately 5 × 105 CFU/ml (5 × 105 to 5 × 106 CFU/ml) of the test organism and were incubated at 35°C in a shaking water bath. Antibiotic concentrations were chosen to comprise the MIC and 2 doubling dilutions above the MIC (27, 28).
Lysed horse blood was prepared by freezing and thawing horse blood (Cleveland Scientific, Bath, OH) six times. Equal volumes of lysed blood and sterile deionized water were then mixed and centrifuged at 12,000 × g for 20 min. Appropriate amounts of 50% lysed blood were then added to the cation-adjusted Mueller-Hinton broth to yield a final concentration of 5% lysed horse blood. The bacterial inoculum was prepared by scraping the growth from a Trypticase soy agar 5% sheep blood plate and creating a suspension equivalent to a 0.5 McFarland concentration in Mueller-Hinton broth. The dilutions required to obtain the correct inoculum (approximately 5 × 105 CFU/ml) were determined by prior viability studies with each strain (27, 28).
To inoculate each tube with serially diluted antibiotic, 50 μl of diluted inoculum was delivered by a pipette beneath the surface of the broth, and then the tube was vortexed and the contents were plated for viability count determinations (0 h). Only tubes containing an initial inoculum within the range of 5 × 105 to 5 × 106 CFU/ml were acceptable. Viability counts for the antibiotic-containing suspensions were performed at 0, 3, 6, 12, and 24 h (two strains, one quinolone susceptible and the other quinolone resistant, were also tested at 1 and 2 h) by plating 10-fold dilutions of 0.1-ml aliquots from each tube in sterile Mueller-Hinton broth onto Trypticase soy agar 5% sheep blood agar plates (BBL). The recovery plates were incubated for up to 48 h. The colony counts on the plates were determined from plates yielding 30 to 300 colonies (27, 28).
The time-kill assay results were analyzed by determining the number of strains which yielded changes in the log10 CFU/ml of −1, −2, and −3 at 0, 1, 2 (two strains only), 3, 6, 12, and 24 h compared to the counts at time zero. The antimicrobials were considered bactericidal if the lowest concentration tested reduced the original inoculum by ≥3 log10 CFU/ml (99.9%) at each of the time periods and bacteriostatic if the lowest concentration tested reduced the original inoculum by 0 to <3 log10 CFU/ml.
Multistep resistance selection.
Serial passages of each strain were performed daily in the presence of subinhibitory concentrations of all antimicrobials. In all cases, the broth medium used in each tube was 1 ml of cation-adjusted Mueller-Hinton broth (BBL) plus 5% lysed horse blood. For each subsequent daily passage, an inoculum (10 μl) was taken from the tube with a concentration 1 to 2 dilutions below the MIC that matched the turbidity of a growth control tube. This inoculum was used to determine the next MIC. Daily passages were performed until a significant increase in the MIC (eight or more times) was obtained. A minimum of 14 passages was performed in every case. The maximal number of passages was 50. For DC-159a, clones with raised MICs were subcultured for a maximum of 50 days. The stability of the acquired resistance was determined by MIC determinations after 10 daily passages of the mutants on blood agar without antibiotic (7, 21). The MICs of each compound for each resistant pneumococcal clone were determined by the macrodilution method (21). The identities of the mutants obtained and their respective parents were confirmed by pulsed-field gel electrophoresis at the end of the study (7, 21, 25).
Determination of quinolone resistance mechanism.
PCR, performed by using the primers and cycling conditions described previously (7, 21), was used to amplify the QRDRs in the gyrA, gyrB, parC, and parE genes. The template DNA for PCR was prepared by using the InstaGen matrix, as recommended by the manufacturer (Bio-Rad Laboratories, Hercules, CA). After amplification the PCR products were purified from excess primers and nucleotides by using the QIAquick PCR purification kit (Qiagen, Valencia, CA) and were sequenced directly by using the CEQ8000 genetic analysis system (Beckman Coulter, Fullerton, CA).
RESULTS
The agar dilution MICs are listed in Table 1. The DC-159a MICs ranged from 0.06 to 0.25 μg/ml against quinolone-susceptible strains and 0.25 to 1 μg/ml against quinolone-resistant strains. By comparison, gemifloxacin yielded lower MICs against quinolone-susceptible strains (0.008 to 0.25 μg/ml) but MICs similar to those of DC-159a (0.125 to 1 μg/ml) against quinolone-resistant strains. The potencies of the quinolones as well as those of other agents not only must be judged by the MIC but also must be interpreted together with their pharmacokinetic and pharmacodynamic properties (13-16). The results for the other quinolones tested are listed in Table 1. β-Lactam and macrolide MICs rose with those of penicillin G. Telithromycin yielded lower MICs compared to those of azithromycin against all groups of macrolide-resistant pneumococci tested (data not shown). However, the marketing of telithromycin has recently been either severely curtailed or discontinued due to toxicity and other factors.
TABLE 1.
MICs of drugs
| Drug and strain susceptibility (no. of isolates) | MIC (μg/ml)
|
||
|---|---|---|---|
| Range | 50% | 90% | |
| Penicillin G | |||
| Penicillin susceptible (110) | 0.016-0.06 | 0.03 | 0.06 |
| Penicillin intermediate (101) | 0.12-1 | 0.25 | 1 |
| Penicillin resistant (105) | 2-16 | 2 | 4 |
| Azithromycin susceptible (156) | 0.016-8 | 0.25 | 2 |
| Azithromycin resistant (160) | 0.016-16 | 0.5 | 4 |
| Quinolone susceptible (288) | 0.016-16 | 0.25 | 4 |
| Quinolone resistant (28) | 0.016-4 | 0.5 | 4 |
| DC-159a | |||
| Penicillin susceptible (110) | 0.06-1 | 0.12 | 0.25 |
| Penicillin intermediate (101) | 0.06-1 | 0.12 | 0.12 |
| Penicillin resistant (105) | 0.06-1 | 0.12 | 0.5 |
| Azithromycin susceptible (156) | 0.06-1 | 0.12 | 0.5 |
| Azithromycin resistant (160) | 0.06-1 | 0.12 | 0.12 |
| Quinolone susceptible (288) | 0.06-0.25 | 0.12 | 0.12 |
| Quinolone resistant (28) | 0.25-1 | 1 | 1 |
| Ciprofloxacin | |||
| Penicillin susceptible (110) | 0.5->32 | 2 | 16 |
| Penicillin intermediate (101) | 0.5->32 | 1 | 2 |
| Penicillin resistant (105) | 0.5->32 | 2 | 16 |
| Azithromycin susceptible (156) | 0.5->32 | 2 | 16 |
| Azithromycin resistant (160) | 0.5->32 | 1 | 2 |
| Quinolone susceptible (288) | 0.5-16 | 1 | 2 |
| Quinolone resistant (28) | 8->32 | 16 | >32 |
| Levofloxacin | |||
| Penicillin susceptible (110) | 0.5-32 | 1 | 4 |
| Penicillin intermediate (101) | 0.5-32 | 1 | 1 |
| Penicillin resistant (105) | 0.5-16 | 1 | 8 |
| Azithromycin susceptible (156) | 0.5-32 | 1 | 8 |
| Azithromycin resistant (160) | 0.5-32 | 1 | 2 |
| Quinolone susceptible (288) | 0.5-2 | 1 | 1 |
| Quinolone resistant (28) | 4-32 | 16 | 16 |
| Gatifloxacin | |||
| Penicillin susceptible (110) | 0.25-16 | 0.5 | 1 |
| Penicillin intermediate (101) | 0.25-8 | 0.5 | 0.5 |
| Penicillin resistant (105) | 0.25-16 | 0.5 | 4 |
| Azithromycin susceptible (156) | 0.25-16 | 0.5 | 4 |
| Azithromycin resistant (160) | 0.25-8 | 0.5 | 0.5 |
| Quinolone susceptible (288) | 0.25-1 | 0.5 | 0.5 |
| Quinolone resistant (28) | 1-16 | 8 | 8 |
| Moxifloxacin | |||
| Penicillin susceptible (110) | 0.12-4 | 0.25 | 0.5 |
| Penicillin intermediate (101) | 0.12-4 | 0.12 | 0.25 |
| Penicillin resistant (105) | 0.06-4 | 0.25 | 2 |
| Azithromycin susceptible (156) | 0.06-4 | 0.25 | 2 |
| Azithromycin resistant (160) | 0.12-4 | 0.25 | 0.25 |
| Quinolone susceptible (288) | 0.06-0.5 | 0.25 | 0.25 |
| Quinolone resistant (28) | 0.25-4 | 4 | 4 |
| Gemifloxacin | |||
| Penicillin susceptible (110) | 0.016-1 | 0.03 | 0.12 |
| Penicillin intermediate (101) | 0.008-1 | 0.03 | 0.06 |
| Penicillin resistant (105) | 0.016-1 | 0.03 | 0.25 |
| Azithromycin susceptible (156) | 0.016-1 | 0.03 | 0.25 |
| Azithromycin resistant (160) | 0.008-1 | 0.03 | 0.06 |
| Quinolone susceptible (288) | 0.008-0.25 | 0.03 | 0.06 |
| Quinolone resistant (28) | 0.12-1 | 0.25 | 1 |
| Telithromycin | |||
| Penicillin susceptible (110) | 0.008-1 | 0.03 | 0.12 |
| Penicillin intermediate (101) | 0.008-0.25 | 0.03 | 0.12 |
| Penicillin resistant (105) | 0.008-1 | 0.06 | 0.25 |
| Azithromycin susceptible (156) | 0.008-0.06 | 0.016 | 0.03 |
| Azithromycin resistant (160) | 0.008-1 | 0.06 | 0.25 |
| Quinolone susceptible (288) | 0.008-1 | 0.03 | 0.12 |
| Quinolone resistant (28) | 0.008-1 | 0.016 | 0.25 |
| Azithromycin | |||
| Penicillin susceptible (110) | 0.06->64 | 0.12 | >64 |
| Penicillin intermediate (101) | 0.03->64 | 0.12 | >64 |
| Penicillin resistant (105) | 0.03->64 | 2 | >64 |
| Azithromycin susceptible (156) | 0.03-0.5 | 0.12 | 0.12 |
| Azithromycin resistant (160) | 1->64 | >64 | >64 |
| Quinolone susceptible (288) | 0.03->64 | 1 | >64 |
| Quinolone resistant (28) | 0.03->64 | 0.06 | >64 |
| Amoxicillin-clavulanic acid | |||
| Penicillin susceptible (110) | ≤0.016-0.25 | 0.03 | 0.03 |
| Penicillin intermediate (101) | 0.03-2 | 0.25 | 1 |
| Penicillin resistant (105) | 0.5-16 | 2 | 8 |
| Azithromycin susceptible (156) | ≤0.016-4 | 0.12 | 2 |
| Azithromycin resistant (160) | ≤0.016-16 | 0.25 | 4 |
| Quinolone susceptible (288) | ≤0.016-16 | 0.25 | 4 |
| Quinolone resistant (28) | ≤0.016-4 | 0.5 | 4 |
When the MICs of the quinolone-resistant strains were analyzed separately (Table 2), the DC-159a MICs were similar irrespective of the number of mutations in the QRDRs. Only five strains had efflux present according to MIC testing with and without reserpine: two strains had efflux for ciprofloxacin and gemifloxacin; one strain had efflux for ciprofloxacin, levofloxacin, and gatifloxacin; one strain had efflux for ciprofloxacin and levofloxacin; and one strain had efflux for gemifloxacin. No strain had efflux for DC-159a.
TABLE 2.
Quinolone MICs for 28 strains with defined mutations in the QRDR
| Strain no. | HMC strain no. | MIC (μg/ml)e
|
Mutation detected in QRDR off:
|
|||||||
|---|---|---|---|---|---|---|---|---|---|---|
| DC-159a | CIPRO | LEVO | MOXI | GATI | GEMI | GyrA | ParC | ParE | ||
| 1 | 1149a | 0.25 | 16 | 4 | 0.25 | 1 | 0.25 | S81F | S79Y | |
| 2 | 1151a | 0.25 | 16 | 4 | 0.25 | 2 | 0.25 | D83N, K137N | I460V | |
| 3 | 1058 | 0.5 | 16 | 8 | 0.5 | 2 | 0.125 | S79F, K137N | I460V | |
| 4 | 1060 | 0.5 | 16 | 8 | 2 | 4 | 0.25 | S81F | S79F, K137N | I460V |
| 5 | 1066 | 0.5 | 16 | 8 | 2 | 4 | 0.25 | S81F | S79F | I460V |
| 6 | 1070 | 1 | 32 | 8 | 4 | 4 | 0.25 | S81F | D83N | I460V |
| 7 | 1073 | 1 | 8 | 8 | 2 | 4 | 0.125 | S81F | I460V | |
| 8 | 1076 | 0.5 | 16 | 8 | 2 | 4 | 0.5 | S81F | D83N | I460V |
| 9 | 1146 | 1 | >32 | 8 | 2 | 4 | 0.5 | S81F | D435N | |
| 10 | 1147b | 1 | >32 | 8 | 4 | 8 | 1 | S81F | S79F | I460V |
| 11 | 1150 | 0.5 | 16 | 8 | 1 | 4 | 0.25 | S81C | S79F | I460V |
| 12 | 2538c | 1 | 16 | 8 | 2 | 8 | 0.25 | S81Y | S79F, K137N | I460V |
| 13 | 5041 | 1 | 16 | 8 | 4 | 4 | 0.5 | S81F | S79F | |
| 14 | 1055 | 1 | 32 | 16 | 4 | 8 | 0.5 | S81F | S79F, K137N | I460V |
| 15 | 1059 | 1 | 32 | 16 | 4 | 8 | 0.5 | S81F | S79F, K137N | I460N |
| 16 | 1071 | 1 | 32 | 16 | 4 | 8 | 0.5 | S81F | S79F, K137N | |
| 17 | 1072 | 1 | >32 | 16 | 4 | 8 | 0.5 | S81F | S79Y | I460V |
| 18 | 1074 | 0.5 | 16 | 16 | 2 | 4 | 0.5 | S81F | S79F | I460V |
| 19 | 1077 | 1 | >32 | 16 | 4 | 8 | 0.25 | S81Y | S79F | |
| 20 | 1078 | 1 | >32 | 16 | 4 | 8 | 0.5 | S81F | S79F | I460V |
| 21 | 1139 | 1 | 32 | 16 | 4 | 8 | 0.25 | S81F | S79F, K137N | I460V |
| 22 | 1156 | 1 | >32 | 16 | 4 | 8 | 0.1 | S81F | S79F | |
| 23 | 1541 | 0.5 | 16 | 16 | 2 | 4 | 0.25 | S81Y | S79F, K137N | I460V |
| 24 | 2527 | 1 | 16 | 16 | 4 | 16 | 0.25 | S81Y | S79F, K137N | I460V |
| 25 | 2536 | 1 | 16 | 16 | 4 | 16 | 0.125 | S81Y | S79F, K137N | I460V |
| 26 | 4026 | 1 | >32 | 16 | 4 | 16 | 0.5 | S81Y | S79F, K137N | I460V |
| 27 | 1062 | 1 | >32 | 32 | 4 | 16 | 1 | S81F | S79F | I460V |
| 28 | 1068d | 1 | 32 | 32 | 4 | 8 | 0.5 | E85K | S79Y, K137N | I460V |
Has efflux for ciprofloxacin and gemifloxacin.
Has efflux for gemifloxacin.
Has efflux for ciprofloxacin, levofloxacin, and gatifloxacin.
Has efflux for ciprofloxacin and levofloxacin.
CIPRO, ciprofloxacin; LEVO, levofloxacin; MOXI, moxifloxacin; GATI, gatifloxacin; GEMI, gemifloxacin.
No mutation was detected in GyrB.
The MICs of the strains used for the time-kill assays are shown in Table 3. Among the 12 strains tested by time-kill analysis, DC-159a was bactericidal against 11 strains after 24 h at 2× MIC (Table 4). None of the quinolones tested achieved 99.9% killing of one strain after 24 h, but the strain was uniformly bacteriostatically inhibited at 2× MIC. The killing kinetics of the other quinolones tested at 24 h were similar, relative to the different MICs, to those of DC-159a. At earlier time periods, DC-159a was bactericidal against 11 of 12 strains at 4× MIC after 12 h, whereas the other quinolones were bactericidal against 8 or 9 of 12 strains. At 6 h, 99% killing of all 12 strains was observed with DC-159a at 4× MIC, whereas 99% killing of 7 to 9 strains was observed with the other quinolones; and at 3 h DC-159a showed 99% killing of 9 strains at 4× MIC, whereas the other quinolones showed 99% killing of 1 to 5 strains. DC-159a at 4× MIC achieved 90% killing of the two strains tested at 2 h (strains Hershey Medical Center [HMC] 1072 and HMC 100; Table 3), whereas moxifloxacin achieved 90% killing of one strain. Amoxicillin-clavulanic acid at 2× MIC was bactericidal against all 12 strains tested after 24 h, with significant killing at earlier time points. By comparison, azithromycin showed slower killing of susceptible strains.
TABLE 3.
MICs of time-kill study strains tested by broth macrodilution
| Drugj | MIC (μg/ml) for HMC strain:
|
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1072a,e,i | 63a,d,h | 1074a,d,i | 5055a,f,h | 100b,d,h | 99b,d,h | 441b,e,h | 5048b,f,h | 1058c,d,i | 228c,e,h | 3583c,f,h | 1541c,g,i | |
| DC-159a | 1 | 0.12 | 1 | 0.12 | 0.12 | 0.12 | 0.12 | 0.12 | 0.5 | 0.12 | 0.12 | 1 |
| LEVO | 16 | 1 | 16 | 1 | 1 | 1 | 0.5 | 1 | 8 | 1 | 1 | 16 |
| MOXI | 4 | 0.25 | 4 | 0.25 | 0.25 | 0.12 | 0.12 | 0.12 | 2 | 0.12 | 0.12 | 4 |
| GATI | 8 | 0.5 | 4 | 0.25 | 0.5 | 0.25 | 0.25 | 0.25 | 4 | 0.5 | 0.25 | 4 |
| GEMI | 0.5 | 0.06 | 0.25 | 0.03 | 0.06 | 0.06 | 0.016 | 0.03 | 0.12 | 0.06 | 0.03 | 0.5 |
| Amox/Clav | 0.016 | 0.016 | 0.03 | 0.03 | 0.25 | 0.25 | 0.12 | 0.5 | 2 | 2 | 2 | 4 |
| Azithro | >64 | 0.016 | 0.03 | 2 | 0.03 | 0.016 | >64 | 4 | 0.06 | >64 | 2 | >64 |
Penicillin susceptible.
Penicillin intermediate.
Penicillin resistant.
Macrolide susceptible.
ermB positive.
mefA positive.
L4 mutations.
Quinolone susceptible.
Quinolone resistant.
LEVO, levofloxacin; MOXI, moxifloxacin; GATI, gatifloxacin; GEMI, gemifloxacin; Amox/Clav, amoxicillin-clavulanic acid; Azithro, azithromycin.
TABLE 4.
Results of time-kill analysis of 12 strains
| Agent and multiple of MIC | No. of strains for which the killing was at the indicated level at the following timesa:
|
|||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 h
|
2 h
|
3 h
|
6 h
|
12 h
|
24 h
|
|||||||||||||
| −1 | −2 | −3 | −1 | −2 | −3 | −1 | −2 | −3 | −1 | −2 | −3 | −1 | −2 | −3 | −1 | −2 | −3 | |
| DC-159a | ||||||||||||||||||
| 4× MIC | 1 | 0 | 0 | 2 | 0 | 0 | 12 | 9 | 2 | 12 | 12 | 4 | 12 | 12 | 11 | 12 | 12 | 11 |
| 2× MIC | 1 | 0 | 0 | 1 | 0 | 0 | 12 | 6 | 1 | 12 | 9 | 3 | 12 | 12 | 9 | 12 | 12 | 11 |
| MIC | 0 | 0 | 0 | 1 | 0 | 0 | 8 | 1 | 0 | 8 | 7 | 1 | 12 | 8 | 6 | 10 | 10 | 7 |
| Levofloxacin | ||||||||||||||||||
| 4× MIC | 0 | 0 | 0 | 0 | 0 | 0 | 11 | 5 | 2 | 12 | 8 | 3 | 12 | 12 | 8 | 12 | 12 | 11 |
| 2× MIC | 0 | 0 | 0 | 0 | 0 | 0 | 11 | 3 | 0 | 12 | 7 | 2 | 12 | 12 | 8 | 12 | 12 | 11 |
| MIC | 0 | 0 | 0 | 0 | 0 | 0 | 5 | 0 | 0 | 10 | 2 | 1 | 12 | 9 | 5 | 9 | 8 | 5 |
| Moxifloxacin | ||||||||||||||||||
| 4× MIC | 0 | 0 | 0 | 1 | 0 | 0 | 11 | 5 | 1 | 12 | 9 | 3 | 12 | 12 | 9 | 12 | 12 | 11 |
| 2× MIC | 0 | 0 | 0 | 1 | 0 | 0 | 10 | 2 | 0 | 12 | 8 | 2 | 12 | 12 | 9 | 12 | 12 | 11 |
| MIC | 0 | 0 | 0 | 0 | 0 | 0 | 7 | 0 | 0 | 10 | 5 | 0 | 11 | 9 | 6 | 10 | 9 | 6 |
| Gatifloxacin | ||||||||||||||||||
| 4× MIC | 0 | 0 | 0 | 0 | 0 | 0 | 12 | 3 | 1 | 12 | 9 | 3 | 12 | 12 | 9 | 12 | 12 | 11 |
| 2× MIC | 0 | 0 | 0 | 0 | 0 | 0 | 10 | 1 | 0 | 12 | 9 | 1 | 12 | 11 | 8 | 12 | 12 | 11 |
| MIC | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 0 | 0 | 10 | 4 | 0 | 11 | 6 | 4 | 8 | 7 | 5 |
| Gemifloxacin | ||||||||||||||||||
| 4× MIC | 0 | 0 | 0 | 0 | 0 | 0 | 10 | 1 | 0 | 12 | 7 | 1 | 12 | 12 | 8 | 12 | 12 | 11 |
| 2× MIC | 0 | 0 | 0 | 0 | 0 | 0 | 9 | 0 | 0 | 12 | 7 | 0 | 12 | 12 | 8 | 12 | 12 | 11 |
| MIC | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 0 | 0 | 9 | 2 | 0 | 12 | 10 | 3 | 11 | 9 | 5 |
| Amoxicillin-clavulanic acid | ||||||||||||||||||
| 4× MIC | 1 | 0 | 0 | 1 | 0 | 0 | 11 | 4 | 0 | 12 | 9 | 2 | 12 | 12 | 11 | 12 | 12 | 12 |
| 2× MIC | 1 | 0 | 0 | 1 | 0 | 0 | 10 | 2 | 0 | 12 | 9 | 0 | 12 | 12 | 10 | 12 | 12 | 12 |
| MIC | 0 | 0 | 0 | 1 | 0 | 0 | 8 | 0 | 0 | 12 | 7 | 0 | 12 | 11 | 9 | 11 | 9 | 7 |
| Azithromycinb | ||||||||||||||||||
| 4× MIC | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 3 | 2 | 1 | 8 | 4 | 3 | 8 | 8 | 5 |
| 2× MIC | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 2 | 2 | 1 | 7 | 3 | 2 | 8 | 7 | 5 |
| MIC | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 2 | 2 | 0 | 7 | 3 | 2 | 6 | 5 | 3 |
−1, 90% killing; −2, 99% killing; −3, 99.9% killing. Only two strains were tested at 1 and 2 h.
Four azithromycin-resistant strains (MICs > 64 μg/ml) were not tested.
The results of multistep resistance selection can be seen in Table 5. The pulsed-field patterns of all resistant clones were identical to those of the parent strains. DC-159a yielded clones of 2 of 10 strains with increased MICs after 50 days of continuous selection. The DC-159a MICs of all 10 parents ranged from 0.06 to 1 μg/ml. The MICs of the parents of two strains that yielded clones with increased MICs were both 0.125 μg/ml; the MICs of resistant clones rose to 1.0 μg/ml after 28 and 49 days. The MICs of one clone did not rise after a maximum of 50 days of subculture. Both DC-159a-resistant clones yielded raised MICs against levofloxacin (8 to 16 μg/ml), moxifloxacin (2 μg/ml), gatifloxacin (4 μg/ml), and gemifloxacin (0.25 to 1 μg/ml). By comparison, levofloxacin yielded resistant clones of two strains, with the MICs rising from 1 μg/ml (for the parent strains) to 8 to 32 μg/ml after 44 and 42 days, respectively. The DC-159a MICs of both of these resistant clones were 1 μg/ml. Moxifloxacin yielded resistant clones of three strains after 22 to 41 days, with the MICs being between 2 and 4 μg/ml (parent strain MICs, 0.125 to 0.25 μg/ml). Gatifloxacin yielded resistant clones, with the MICs being 4 μg/ml (parent strain MIC, 0.5 μg/ml) after 38 to 46 days. Gemifloxacin yielded resistant clones of four strains after 27 to 41 days, with the MICs being between 0.25 and 4 μg/ml (parent strain MICs, 0.03 to 0.5 μg/ml). One strain (HMC 1074) with resistance selected by gemifloxacin had a DC-159a MIC of 4 μg/ml and levofloxacin, gatifloxacin, moxifloxacin, and gemifloxacin MICs of 64, 16, 16, and 2 μg/ml, respectively. The seven strains with azithromycin MICs of 0.03 to 8 μg/ml did not yield resistant mutants after 50 days, and none of the 10 strains yielded mutants resistant to amoxicillin-clavulanic acid after the same period of time. Cross-reacting strains (increase in the MIC of eight times or greater) are designated in boldface in Table 5.
TABLE 5.
S. pneumoniae multistep resistance selection results
| Strain | Agent | Initial MIC (μg/ml) | Resistance selection
|
Retest MIC (μg/ml) after 10 drug-free subculturesa
|
Mutation(s) in:
|
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MIC (μg/ml) | No. of days | DC-159a | LEVO | MOXI | GATI | GEMI | Azithro | Amox/ Clav | GyrA | GyrB | ParC | ParE | |||
| HMC 5055 | DC-159a | 0.125 | 0.125 | 50 | |||||||||||
| Levofloxacin | 1 | 2 | 50 | ||||||||||||
| Moxifloxacin | 0.125 | 0.125 | 50 | ||||||||||||
| Gatifloxacin | 0.25 | 0.5 | 50 | ||||||||||||
| Gemifloxacin | 0.03 | 0.06 | 50 | ||||||||||||
| Azithromycin | 8 | 8 | 50 | ||||||||||||
| Amox/Clav | 0.03 | 0.03 | 50 | ||||||||||||
| HMC 63 | DC-159a | 0.125 | 0.125 | 50 | |||||||||||
| Levofloxacin | 1 | 1 | 50 | ||||||||||||
| Moxifloxacin | 0.25 | 0.25 | 50 | ||||||||||||
| Gatifloxacin | 0.5 | 1 | 50 | ||||||||||||
| Gemifloxacin | 0.03 | 0.5 | 34 | 1 | 16 | 4 | 8 | 0.5 | 0.03 | 0.03 | S81F | D83Y | I460Vb | ||
| Azithromycin | 0.03 | 0.06 | 50 | ||||||||||||
| Amox/Clav | 0.016 | 0.03 | 50 | ||||||||||||
| HMC 5048 | DC-159a | 0.06 | 0.125 | 50 | |||||||||||
| Levofloxacin | 0.5 | 0.5 | 50 | ||||||||||||
| Moxifloxacin | 0.06 | 0.125 | 50 | ||||||||||||
| Gatifloxacin | 0.125 | 0.5 | 50 | ||||||||||||
| Gemifloxacin | 0.03 | 0.125 | 50 | ||||||||||||
| Azithromycin | 4 | 8 | 50 | ||||||||||||
| Amox/Clav | 0.5 | 1 | 50 | ||||||||||||
| HMC 99 | DC-159a | 0.125 | 0.125 | 50 | |||||||||||
| Levofloxacin | 0.5 | 2 | 50 | ||||||||||||
| Moxifloxacin | 0.125 | 4 | 41 | 1 | 32 | 4 | 16 | 1 | 0.016 | 0.25 | S81Y | S79Y | |||
| Gatifloxacin | 0.25 | 0.5 | 50 | ||||||||||||
| Gemifloxacin | 0.03 | 0.125 | 50 | ||||||||||||
| Azithromycin | 0.03 | 0.06 | 50 | ||||||||||||
| Amox/Clav | 0.25 | 0.5 | 50 | ||||||||||||
| HMC 100 | DC-159a | 0.125 | 0.5 | 50 | |||||||||||
| Levofloxacin | 1 | 32 | 42 | 1 | 32 | 4 | 8 | 0.5 | 0.03 | 0.25 | S81Y | S79Y | |||
| Moxifloxacin | 0.25 | 2 | 22 | 0.5 | 8 | 1 | 4 | 0.25 | 0.03 | 0.25 | S81Y | ||||
| Gatifloxacin | 0.5 | 0.5 | 50 | ||||||||||||
| Gemifloxacin | 0.06 | 1 | 34 | 0.5 | 16 | 2 | 4 | 1 | 0.06 | 0.25 | S79Y | ||||
| Azithromycin | 0.03 | 0.03 | 50 | ||||||||||||
| Amox/Clav | 0.125 | 0.25 | 50 | ||||||||||||
| HMC 3041 | DC-159a | 0.06 | 0.125 | 50 | |||||||||||
| Levofloxacin | 0.5 | 1 | 50 | ||||||||||||
| Moxifloxacin | 0.06 | 0.25 | 50 | ||||||||||||
| Gatifloxacin | 0.5 | 0.5 | 50 | ||||||||||||
| Gemifloxacin | 0.03 | 0.03 | 50 | ||||||||||||
| Azithromycin | >64 | NTc | NT | ||||||||||||
| Amox/Clav | 0.25 | 0.25 | 50 | ||||||||||||
| HMC 228 | DC-159a | 0.125 | 1 | 28 | 1 | 16 | 2 | 4 | 1 | >64 | 2 | S81F | D83N | I460Vb | |
| Levofloxacin | 1 | 2 | 50 | ||||||||||||
| Moxifloxacin | 0.125 | 2 | 36 | 1 | 32 | 2 | 16 | 2 | >64 | 2 | S81F | S79Y | I460Vb | ||
| Gatifloxacin | 0.5 | 4 | 38 | 1 | 16 | 4 | 4 | 1 | >64 | 2 | S81F | D83N | I460Vb | ||
| Gemifloxacin | 0.06 | 0.25 | 50 | ||||||||||||
| Azithromycin | >64 | NT | NT | ||||||||||||
| Amox/Clav | 2 | 2 | 50 | ||||||||||||
| HMC 3583 | DC-159a | 0.125 | 1 | 49 | 1 | 8 | 2 | 4 | 0.25 | 2 | 2 | S81F | D83N | I460Vb | |
| Levofloxacin | 1 | 8 | 44 | 1 | 8 | 2 | 4 | 0.25 | 2 | 2 | S81F | D83N | I460Vb | ||
| Moxifloxacin | 0.125 | 0.5 | 50 | ||||||||||||
| Gatifloxacin | 0.5 | 4 | 46 | 1 | 16 | 4 | 8 | 0.25 | 1 | 2 | S81F | I460V,b D435N | |||
| Gemifloxacin | 0.03 | 0.25 | 41 | 1 | 4 | 0.5 | 2 | 0.25 | 1 | 2 | I460Vb | ||||
| Azithromycin | 2 | 4 | 50 | ||||||||||||
| Amox/Clav | 1 | 2 | 50 | ||||||||||||
| HMC 1072 | DC-159a | 1 | 1 | 50 | |||||||||||
| Levofloxacin | 16 | 32 | 50 | ||||||||||||
| Moxifloxacin | 4 | 4 | 50 | ||||||||||||
| Gatifloxacin | 8 | 8 | 50 | ||||||||||||
| Gemifloxacin | 0.5 | 1 | 50 | ||||||||||||
| Azithromycin | >64 | NT | NT | ||||||||||||
| Amox/Clav | 0.016 | 0.016 | 50 | ||||||||||||
| HMC 1074 | DC-159a | 1 | 1 | 50 | |||||||||||
| Levofloxacin | 16 | 16 | 50 | ||||||||||||
| Moxifloxacin | 4 | 8 | 50 | ||||||||||||
| Gatifloxacin | 4 | 8 | 50 | ||||||||||||
| Gemifloxacin | 0.5 | 4 | 27 | 4 | 64 | 16 | 16 | 2 | 0.016 | 0.03 | S81F,b E85K | S79Fb | I460Vb | ||
| Azithromycin | 0.03 | 0.03 | 50 | ||||||||||||
| Amox/Clav | 0.03 | 0.03 | 50 | ||||||||||||
Cross-reacting strains are designated in boldface (increase in the MIC of eight times or greater). LEVO, levofloxacin; MOXI, moxifloxacin; GATI, gatifloxacin; GEMI, gemifloxacin; Azithro, azithromycin; Amox/Clav, amoxicillin-clavulanic acid.
This mutation was present in the parent strain prior to resistance selection.
NT, not tested.
The mutations identified in the QRDR nucleotide sequences of the gyrA, gyrB, parC, and parE genes of all mutant strains are summarized in Table 5. Prior to resistance selection, one parent strain (HMC 1074) had a mutation in GyrA (S81F) and four parent strains (HMC 63, HMC 228, HMC 3583, HMC 1074) had a mutation in ParE (I460V). Additionally, parent strain HMC 1074 had a mutation in ParC. All mutants except the gemifloxacin-resistant mutant of parent HMC 3583 acquired mutations in either or both GyrA and ParC: nine clones had mutations in both GyrA (S81Y/F, E85K) and ParC (S79Y or D83N/Y), two clones (a moxifloxacin-selected mutant of parent strain HMC 100 and a gatifloxacin-selected mutant of parent strain HMC 3583) had a single mutation in GyrA (S81Y/F; the latter clone also acquired the D435N mutation in ParE), and one clone (a gemifloxacin-selected mutant of parent HMC 100) developed a mutation in ParC (S79Y). A gemifloxacin-selected clone of parent HMC 1074 with the highest DC-159a MIC (4 μg/ml) acquired a second mutation (E85K) in GyrA, in addition to the original S81F mutation, as well as a mutation in ParC (S79F).
DISCUSSION
DC-159a is a new 8-methoxy fluoroquinolone (12) with a broad spectrum of activity against gram-positive and -negative bacteria, especially streptococci and staphylococci from community-acquired infections. Hoshino and coworkers showed that DC-159a has an MIC90 of 0.125 μg/ml and an MIC90 of 1 μg/ml for levofloxacin-intermediate and -resistant strains (12). DC-159a was also active against quinolone-susceptible staphylococci, Haemophilus influenzae, and Moraxella catarrhalis and was more potent than levofloxacin against Mycoplasma pneumoniae and Chlamydophila pneumoniae. DC-159a also demonstrated rapid bactericidal activity against quinolone-resistant S. pneumoniae strains both in vitro and in vivo and had bactericidal activity superior to that of moxifloxacin in a murine muscle infection model (12).
Similar to the findings noted above by other workers (12), our studies showed that DC-159a yielded low MICs against all pneumococcal strains tested, irrespective of their quinolone susceptibilities. The DC-159a MICs ranged from 0.06 to 0.25 μg/ml for quinolone-susceptible strains and from 0.25 to 1 μg/ml for quinolone-resistant strains, irrespective of the strains' resistance genotypes. It is interesting to note that although the DC-159a MICs against quinolone-susceptible pneumococci were a few dilutions higher than those of gemifloxacin, the MICs of the two quinolones against quinolone-resistant strains were similar.
DC-159a killed 12 strains at rates similar to those of the other quinolones tested at 24 h, relative to the different MICs, but had better killing than all other quinolones tested at earlier time points (12). The one strain (HMC 228; Table 3) for which DC-159a and all other quinolones tested were not bactericidal at 4× MIC at 24 h was penicillin resistant, macrolide resistant (ermB positive), and quinolone susceptible.
Resistance selection studies showed a maximal DC-159a MIC of 1 μg/ml (i.e., the same as that against clinical quinolone-resistant isolates) in laboratory clones selected with DC-159a. Selection with gemifloxacin resulted in a clone with a DC-159a MIC of 4 μg/ml due to a second mutation in GyrA (E85K). With the exception of the latter clone, the mutations found in the QRDRs of the clones with raised DC-159a MICs were similar to those found in the QRDRs of quinolone-resistant clinical isolates.
The results for drugs other than DC-159a reflect previous findings. We have no explanation for the lack of selection of azithromycin-resistant clones, which is in contrast to the results of previous studies which were able to select azithromycin-resistant clones (6, 7, 11, 15, 16, 21, 23-25, 27, 28).
All of the results presented above demonstrate the potential usefulness of DC-159a as an oral drug for the treatment of community-acquired respiratory tract infections. If the human free area under the concentration-time curve/MIC for this compound is found to be ≥25 at an MIC of at least 1 μg/ml and toxicity studies do not yield problems, DC-159a will represent an extremely promising new oral quinolone.
Acknowledgments
This study was supported by a grant from Daiichi Pharmaceutical, Co., Ltd.
Footnotes
Published ahead of print on 15 October 2007.
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