Demonstrating the Protective Efficacy of the Novel Fluoroquinolone Finafloxacin against an Inhalational Exposure to Burkholderia pseudomallei

Abstract

Burkholderia pseudomallei is the causative agent of melioidosis, a serious disease endemic in Southeast Asia and Northern Australia. Antibiotic treatment is lengthy and relapse often occurs. Finafloxacin is a novel fluoroquinolone with increased antibacterial activity in acidic conditions in contrast to other fluoroquinolones which demonstrate reduced activity at a lower pH. Therefore, finafloxacin may have improved efficacy against B. pseudomallei, which can survive within host cells where the local pH is acidic. In vitro analysis was performed using MICs, minimal bactericidal concentrations (MBCs), time-kill assays, persister cell assays, and macrophage assays. Finafloxacin showed increased bactericidal activity at pH 5 in comparison to pH 7 and ciprofloxacin at pH 5. In vivo studies in BALB/c mice included pharmacokinetic studies to inform an appropriate dosing regimen. Finafloxacin efficacy was evaluated in an inhalational murine model of melioidosis where antibiotic treatment was initiated at 6 or 24 h postchallenge and continued for 14 days, and mice were observed for 63 days. The survival of infected mice following 14 days of treatment was 80%, 60% or 0% for treatments initiated at 6 h and 60%, 30% or 0% for treatments initiated at 24 h for finafloxacin, co-trimoxazole, or ciprofloxacin, respectively. In summary, finafloxacin has increased bactericidal activity for B. pseudomallei under acidic conditions in vitro and improves survival in a murine model of melioidosis compared with those for ciprofloxacin. Furthermore, finafloxacin improves bacteriological clearance compared with that of co-trimoxazole, suggesting it may offer an effective postexposure prophylaxis against B. pseudomallei.

Keywords: Burkholderia; aerosol; finafloxacin; mouse model.

FIG 1

Chemical structure of finafloxacin.

FIG 2

Effect of pH on the efficacy of ciprofloxacin and finafloxacin against B. pseudomallei strains over a 24-h period. Time-kill assays were performed in L broth adjusted to pH 5 or pH 7 with finafloxacin (16 μg/ml) or ciprofloxacin (4 μg/ml). An untreated bacterial control was included. (A) Ciprofloxacin against B. pseudomallei strain K96243. (B) Ciprofloxacin against B. pseudomallei strain 576. (C) Finafloxacin against B. pseudomallei strain K96243. (D) Finafloxacin against B. pseudomallei strain 576. The error bars represent the standard errors of the mean (SEMs) from 3 biological replicates. ┈, 3-log₁₀ reduction in CFU/ml from the starting inoculum.

FIG 3

Fold difference change in finafloxacin- and ciprofloxacin-treated J774 macrophages infected with B. pseudomallei strain AI. Macrophages were infected and incubated with L 15 medium at 37°C containing finafloxacin (16 μg/ml or 4 μg/ml for 4× MIC or 1× MIC, respectively) or ciprofloxacin (4 μg/ml or 1 μg/ml for 4× MIC or 1× MIC, respectively) over a 24-h period. An untreated bacterial control was also included. The error bars represent the SEMs from 3 biological replicates. Statistical analysis was performed by 2-way ANOVA with experimental run used as a repeated measure, and fold change in bacterial numbers is graphed. *, P < 0.05.

FIG 4

Effects of finafloxacin and ciprofloxacin on the formation of B. pseudomallei persister cells. The percentages of B. pseudomallei persister cells produced when strain 576 (A) or strain K96243 (B) was incubated with 100× MIC of finafloxacin (400 μg/ml) or ciprofloxacin (100 μg/ml) for 24 h. These data were analyzed using repeated measures (accounting for experimental run) ANOVA. *, P < 0.05; ***, P < 0.001.

FIG 5

Percent survival of mice in each treatment group following a challenge with aerosolized B. pseudomallei K96243. Mice were challenged with approximately 128 CFU of B. pseudomallei K96243 by the inhalational route and treated with oral finafloxacin (37.5 mg/kg) every 8 h, intraperitoneal ciprofloxacin (30 mg/kg) every 12 h, or oral co-trimoxazole (240 mg/kg) every 12 h. Regimens were initiated at 6 h (A) or 24 h (B) postchallenge. Control animals received intraperitoneal PBS or oral diluent. Statistical analysis was performed using the Mantel-Haenszel log rank test. *, P = 0.043; **, P = 0.004; ****, P < 0.0001.

FIG 6

Bacterial loads in organs at 24 h following initiation of antibiotic treatment. Bacterial counts (CFU/g) in the spleens, livers, and lungs of 5 mice per group at 30 h (A) or 48 h (B) postchallenge. Each group received 24-h treatment with finafloxacin (37.5 mg/kg every 8 h orally), co-trimoxazole (240 mg/kg every 12 h orally), or ciprofloxacin (30 mg/kg every 12 h via intraperitoneal injection). Control animals received intraperitoneal PBS or oral diluent. Statistical analysis was performed using pairwise comparisons of Mood's median test. *, P < 0.05.

FIG 7

Bacterial loads in organs at the end of antibiotic treatment. Bacterial counts (CFU/g) in the spleens, livers, and lungs of 5 mice per group at 14 days posttreatment, when treatment commenced at 6 h (A) or 24 h (B) postchallenge. Each group received 14 days of treatment with finafloxacin (37.5 mg/kg every 8 h orally), co-trimoxazole (240 mg/kg every 12 h orally), or ciprofloxacin (30 mg/kg every 12 h via intraperitoneal injection). Statistical analysis was performed using pairwise comparisons of Mood's median test. *, P < 0.05.

FIG 8

Bacterial loads in organs at 63 days postchallenge with aerosolized B. pseudomallei. Bacterial counts (CFU/g) in the spleens, livers, and lungs of mice at day 63 postchallenge when treatment commenced at 6 h (A) or 24 h (B) postchallenge. Each group received 14 days of treatment with finafloxacin (37.5 mg/kg every 8 h orally), co-trimoxazole (240 mg/kg every 12 h orally), or ciprofloxacin (30 mg/kg every 12 h via intraperitoneal injection). Statistical analysis was performed using pairwise comparisons of Mood's median test. *, P < 0.05.