Susceptibility study results
The MICs for P. aeruginosa ATCC27853 were 0.5 μg/mL for levofloxacin and 1.0 μg/mL for ceftazidime. The MPCs for P. aeruginosa ATCC27853 were 5.1 μg/mL for levofloxacin and 82 μg/mL for ceftazidime. The FIC value of levofloxacin combined with ceftazidime was measured to be 1.0 (concentrations of levofloxacin and ceftazidime were 0.25 μg/mL and 0.5 μg/mL, respectively).
High-performance liquid chromatography (HPLC) studies
The retention time for levofloxacin was 6–8 min and ceftazidime was 8–10 min. The assay of levofloxacin was linear over a range of 1.5625–50 μg/mL (y=59.97x-24.055, r2=0.9998) and the assay of ceftazidime was linear over a range of 10–320 μg/mL (y=0.4659x+2.9403, r2=0.9999). The inter-day coefficients of variation for the quality control samples analyzed in triplicate at 3 concentrations on each analysis day ranged from 0.86–2.36% for levofloxacin and 0.67–1.61% for ceftazidime. The intra-day coefficients of variation ranged from 0.94–3.75% for levofloxacin and 0.33–1.46% for ceftazidime. Accuracies of levofloxacin and ceftazidime were 99.17–102.88% and 98.97–101.58%, respectively.
Resistant mutation frequency
For P. aeruginosa ATCC27853, the average density of resistant subpopulation at 5 × and 10 × MIC of levofloxacin value were – 4.35 and – 3.48 log CFU, respectively. The average density of resistant subpopulation at 5 × and 10 × MIC of ceftazidime were – 5.01 and – 4.42 log CFU, respectively. At 1 × and 2 × FIC value, the average densities of resistant subpopulation were –4.21 and – 2.96 log CFU, respectively.
Pharmacokinetic (PK) results
Typical concentration-time curves for levofloxacin and ceftazidime are shown in Fig. 1a and b. Data for all regimens are available on request.
(Fig. 1 Various PK simulations in the study. T1/2, elimination half-life; AUC0-24h, area under the concentration-curve from time 0 to 24 hours; Cmax, maximum drug concentration; Cmin, minimum drug concentration; T>MIC is the percentage of administration interval.)
Pharmacodynamic (PD) studies of monotherapy regimens
The monotherapy effects on total and resistant population burdens within 7 days are shown in Fig. 2 (control and normal renal function) and Fig.3 (abnormal renal function). The bacteria grew well in the untreated growth control arms and reached a bacterial density of 109 to 1010 CFU/mL by Day 2 (Fig. 2a). The effects of the levofloxacin and ceftazidime monotherapies for patients with normal renal function on the total and resistant population burdens for P. aeruginosa ATCC27853 are shown in Fig. 2b to d. The levofloxacin and ceftazidime control arms produced around a 0.5-log-CFU/mL reduction in the total population before 6 or 8 h; however, resistance was seen after 6 h for levofloxacin regimens and after 0 h for ceftazidime. Fig. 3 displays the total and resistant population burdens for the levofloxacin and ceftazidime monotherapies for patients with abnormal renal function. The levofloxacin and ceftazidime control arms produced less than a 0.5-log-CFU/mL reduction in the total population before 6 or 8 h, and 187.5 mg levofloxacin showed a trend of reducing the total population until 72 h. However, rapid resistance emergence at 0 h was seen in the 3 regimens. Regeneration in the total number of the 6 monotherapy regimens shown in Fig. 2 and Fig. 3 can be explained by the presence of resistance.
(Fig. 2 Observed time-sterilization curve of monotherapy regimens for patients with normal renal function. Data is expressed as the means ± SD of bacterial burdens.)
(Fig. 3 Observed time-sterilization curve of monotherapy regimens for patients with abnormal renal function. Data is expressed as the means ± SD of bacterial burdens.)
Pharmacodynamic (PD) studies of combination therapy regimens
Fig. 4 displays the efficacy of combination therapy on P. aeruginosa ATCC27853. There was a major effect of combination therapy, which resulted in a 2- to 3-log-CFU/mL bacterial kill on the total population. The growth of resistant bacteria was seen after 16 h in the patients with normal renal function, and at 0 h in the patients with abnormal renal function. But there was a downward trend after 8 h in the patients with abnormal renal function. Combination therapy regimens were able to suppress the resistant population amplification.
(Fig. 4 Observed time-sterilization curve of combination therapy regimens. Data is expressed as the means ± SD of bacterial burdens.)
Comparison of all regimens on total population burdens
Fig. 5a shows the comparison of total population burdens in patients with normal renal function, and Fig. 5b shows the same for patients with abnormal renal function. Combination therapy resulted in a 2-log-CFU/mL bacterial kill in patients with normal renal function, and a 3-log-CFU/mL bacterial kill in patients with abnormal renal function. The results were clearly different from those seen with monotherapy (p<0.01, analysis of variance). The comparison also showed that combination therapy was superior to monotherapy in resistance inhibition, because the regrowth in the total population for all regimens was expounded by resistant emergence.
(Fig. 5 Comparison of total population burdens on all regimens. Data is expressed as the means ± SD of bacterial burdens.)