In Vitro Pharmacodynamic Activities of ABT-492, a Novel Quinolone, Compared to Those of Levofloxacin against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis

Bacteria and antimicrobial agents.Two strains of penicillin-sensitive (MIC, <0.1 μg/ml) and two strains of penicillin-resistant (MIC, 2 μg/ml) S. pneumoniae, two strains of β-lactamase-positive and two strains of β-lactamase-negative Haemophilus influenzae, and two strains of β-lactamase-positive and one strain of β-lactamase-negative Moraxella catarrhalis were obtained from clinical respiratory specimens from the microbiology laboratories at the University of Illinois at Chicago Medical Center, Chicago. Isolates were stored at −70°C in double-strength skim milk (Remel, Lenexa, Kans.) and were subcultured three times prior to use. In addition, one quinolone-resistant S. pneumoniae strain was obtained from The Jones Group (JMI Laboratories, North Liberty, Iowa) and stored similarly. The ABT-492 and levofloxacin antibiotics for the MIC determination and time-kill kinetic studies were supplied as laboratory powders of known potency from Abbott Laboratories, Abbott Park, Ill. Antibiotic stock solutions were prepared according to the manufacturer's recommendations, diluted to concentrations ranging from 1,000 to 10,000 μg/ml, stored in unit-of-use vials, and frozen at −70°C until needed. The media used for MIC and time-kill kinetic studies included cation-adjusted Mueller-Hinton broth (Becton Dickinson, Sparks, Md.) with 5% lysed horse blood (Remel) (11) for the S. pneumoniae isolates and Haemophilus test medium (Dade Behring, West Sacramento, Calif.) for the H. influenzae and M. catarrhalis isolates.

MIC determination.The MIC for each isolate was determined by broth microdilution techniques as outlined by the National Committee for Clinical Laboratory Standards, and testing was performed in duplicate (10). Control strains (S. pneumoniae ATCC 49619 and H. influenzae ATCC 49247) were used to validate the MIC results. The inoculum was prepared by suspending S. pneumoniae organisms grown on blood agar plates or H. influenzae and M. catarrhalis organisms grown on chocolate agar plates, which had been incubated for a full 24 h, in 2 ml of sterile saline. Suspensions were adjusted to a 0.5 McFarland turbidity standard by using a spectrophotometer and diluted in broth to obtain a final inoculum of approximately 5 × 10⁵ CFU/ml for each well. Inoculum checks were performed via colony counts. The microtiter plates were incubated overnight at 35°C in humidified air, and the results were read at 24 h. The lowest concentration of antibiotic in the wells showing no visible growth was defined as the MIC.

Time-kill assays.Prior to time-kill kinetic studies, a carryover study was performed for each isolate as outlined by the National Committee for Clinical Laboratory Standards guidelines (9). The time-kill studies were performed with antibiotic concentrations selected to comprise four doubling dilutions below and three doubling dilutions above the MIC for each test isolate, as previously described (3, 11, 14). A growth control (no antibiotic) was included for each isolate. Briefly, glass tubes containing 4.950 ml of the appropriate medium and antibiotic were inoculated with 50 μl of a diluted bacterial suspension with a pipette below the surface of the broth. S. pneumoniae was incubated in broth medium (described above) for 16 h prior to use, and H. influenzae and M. catarrhalis were taken directly from a 24-h culture plate. Inoculated tubes containing a concentration of 5 × 10⁵ to 5 × 10⁶ CFU/ml were incubated at 35°C on a rotator for 24 h.

Counts of viable cells in the antibiotic-containing suspensions and the growth controls were performed at 0, 2, 4, 6, 12, and 24 h by plating 10-fold dilutions of 0.1-ml aliquots from each tube on blood agar plates for S. pneumoniae and on chocolate agar plates for H. influenzae and M. catarrhalis. Recovery plates were incubated at 35°C for 24 to 48 h. Colony counts at 0 h were used to validate the starting inoculum. For undiluted inocula, carryover was addressed by immediately spreading the broth over the agar plate until it was dry (11, 14). The lower limit of sensitivity for colony counts was 300 CFU/ml.

Colony counts (log₁₀ numbers of CFU per milliliter) from duplicate time-kill runs performed on different days were averaged and plotted versus time for each isolate. Bacteriostatic activity was defined as a 0- to <3-log₁₀ reduction, and bactericidal activity was defined as a ≥99.9% (a ≥3-log₁₀) reduction in the number of CFU per milliliter from that of the starting bacterial concentration. Composite concentration-effect graphs were constructed by plotting the change in log₁₀ numbers of CFU per milliliter from the starting inoculum at each time point for all isolates versus the numbers in cultures with antibiotic concentrations standardized to multiples of the MIC.

Dose-effect response.To compare the concentration-effect relationships at 2, 4, 6, 12, and 24 h of ABT-492 and levofloxacin against S. pneumoniae, H. influenzae, and M. catarrhalis, the mean time-kill data at each time point for each bacterial isolate were combined according to bacterial species. The net change (log₁₀ numbers of CFU per milliliter) in bacterial density at each time point for each concentration was fitted by multivariate nonlinear-regression analysis with a four-parameter sigmoidal Hill (E_max) model by using SigmaPlot 2000 for Windows (version 6.0; SPSS Inc.) (6, 8). In this model, effect (the net change in the log₁₀ number of CFU per milliliter) is equal to E₀ − (E_max × Cⁿ)/(EC₅₀ⁿ + Cⁿ), where E₀ is the baseline effect (bacterial growth for the control), E_max is the maximal bacterial-kill effect, C is the concentration of interest (a multiple of the MIC), EC₅₀ is the antibacterial concentration that produced 50% of the maximal effect, and n is the sigmoidicity factor that gives flexibility to the shape of the curve. E₀, EC₅₀, E_max, and n were given in the regression output. The concentration that produced the EC₉₀ between E₀ and E_max was calculated.

Antibacterial susceptibility results.The MICs of each agent for each isolate are listed in Table 1. All 12 strains, except the quinolone-resistant S. pneumoniae strain (MIC = 32 μg/ml), were sensitive to levofloxacin. The breakpoints for ABT-492 have not been established; however, ABT-492 demonstrated potent activity against all bacterial isolates for which the MICs were ≤0.125 μg/ml. Against S. pneumoniae, the MICs of ABT-492 ranged from 0.0078 to 0.125 μg/ml compared to 1 to 32 μg/ml for levofloxacin. For H. influenzae and M. catarrhalis, the MICs of ABT-492 were much lower and ranged from 0.000625 to 0.0025 μg/ml compared to 0.0156 to 0.0625 μg/ml for levofloxacin.

Antibacterial carryover.A carryover effect was seen in all S. pneumoniae isolates at eight times the MIC, regardless of the tested quinolone. At four times the MIC, all carryover was eliminated, except that a slight carryover was noted at four times the MIC of ABT-492 for the quinolone-resistant S. pneumoniae isolate. A potential for carryover existed when direct sampling of the inoculated test tubes was needed at higher multiples of the MIC; however, the 0.1-ml aliquots were immediately spread over the agar plates, which contained 25 ml of medium. This reduced the carryover by causing a 1:250 dilution of the antibiotic (11, 14). Carryover was not observed with any of the H. influenzae and M. catarrhalis isolates.

Concentration-effect response.The mean starting inoculum for all concentrations in the time-kill curves was 5.813 log₁₀ CFU/ml (standard deviation, 0.470). When all 12 isolates were compared based on the extent of the bacterial kill, ABT-492 and levofloxacin at eight times the MIC demonstrated bactericidal activity against all isolates. ABT-492 and levofloxacin at four times the MIC were bactericidal against 83% (10 of 12) and 100% (12 of 12) of the isolates, respectively. A bacteriostatic effect was observed between one and two times the MIC for many isolates. A delay in the bacterial growth compared to that of the control was observed at 0.25 and 0.5 times the MIC. When time-kill data were separated according to bacterial species, ABT-492 failed to produce bactericidal effects at four times the MIC for the quinolone-resistant isolate and one penicillin-resistant S. pneumoniae isolate. At two times the MIC, ABT-492 was bactericidal against 20% (one of five) of the S. pneumoniae isolates compared to 100% (five of five) with levofloxacin. Regrowth (defined as an increase in growth of ≥2 log₁₀ CFU/ml after 6 h) (9) was not seen in the ABT-492-tested isolates but occurred in one levofloxacin-tested penicillin-sensitive S. pneumoniae. The time-kill kinetic data for H. influenzae and M. catarrhalis were similar for both ABT-492 and levofloxacin. Concentrations at four and eight times the MIC produced bactericidal activity in all strains. At two times the MIC, bactericidal activity was observed in 29% (two of seven) of the H. influenzae and M. catarrhalis isolates with ABT-492 compared to 71% (five of seven) with levofloxacin.

A time-kill plot of the activities of ABT-492 and levofloxacin against each bacterial species plus the quinolone-resistant S. pneumoniae isolate is shown in Fig. 1. The plots of the log₁₀ numbers of CFU per milliliter versus time for all test isolates demonstrate the concentration-dependent bactericidal activity. Increases in the concentrations of both ABT-492 and levofloxacin consistently led to an increase in the rate and extent of bactericidal activity. The results of sigmoidal E_max models, plotted as the antibacterial activities (the net change in numbers of CFU per milliliter) of both quinolones relative to the concentrations used (multiples of the MIC) after 4, 6, and 12 h of exposure, for the quinolone-sensitive S. pneumoniae isolates are presented in Fig. 2. The quinolone-resistant S. pneumoniae isolate was not included in the S. pneumoniae analysis because the results from time-kill data significantly differed from the data for quinolone-sensitive isolates. The results of sigmoidal E_max models for H. influenzae and M. catarrhalis were similar to those for S. pneumoniae and are thus not depicted.

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FIG. 1.

Time-kill curves for four clinical isolates. Curves on the left represent the activity of ABT-492, and the curves on the right represent the activity of levofloxacin against the same isolate. ▴, control; ⋄, 0.0625 times the MIC; ♦, 0.125 times the MIC; □, 0.25 times the MIC; ▪, 0.5 times the MIC; ▿, 1 times the MIC; ▾, 2 times the MIC; ○, 4 times the MIC; •, 8 times the MIC.

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FIG. 2.

Composite concentration-response curves for penicillin-sensitive S. pneumoniae (SPS) and penicillin-resistant S. pneumoniae (SPR) isolates following antibiotic exposure to ABT-492 (left-hand side) and levofloxacin (right-hand side) at 4, 6, and 12 h.

The EC₅₀, EC₉₀, and E_max data obtained from all plots are presented in Table 2. The EC₅₀s for all isolates ranged from 0.884 (standard error, 0.086) to 1.96 (standard error, 0.125) times the MIC. The EC₉₀s for all isolates ranged from 1.270 to 6.383 times the MIC. With increased time exposure, the maximal bacterial-killing effect increased and the multiple of the MIC needed to achieve the EC₉₀ decreased, except at 24 h. When the EC₅₀s of ABT-492 were compared to those of levofloxacin, the values fell within the standard errors of each other at 2 and 4 h for S. pneumoniae and H. influenzae, respectively. At the 12- and 24-h antibacterial exposures, the EC₅₀ of levofloxacin was lower than that of ABT-492, except in the case of H. influenzae.

Conclusion.This study demonstrates that ABT-492 is a potent concentration-dependent antibiotic with in vitro activity similar to that of levofloxacin. The compound's very low MICs for gram-positive, gram-negative, and levofloxacin-resistant S. pneumoniae respiratory pathogens warrant further investigation. Sigmoidal E_max models were constructed to validate the time-kill pharmacodynamic analysis. The relationship between EC₅₀ and EC₉₀ became closer over time, supporting concentration-dependent pharmacodynamic activity. The models demonstrated that 50% maximal activity occurred between one and two times the MIC, and 90% maximal activity was observed at one to six times the MIC. Knowledge of the concentrations that are needed to achieve near-maximal activity of an antibiotic can help determine a goal for free antibiotic concentrations in serum in vivo.