Investigation of a combination therapy approach for the treatment of melioidosis

Abstract

The efficacy of finafloxacin as a component of a layered defense treatment regimen was determined in vitro and in vivo against an infection with Burkholderia pseudomallei. Doxycycline was down-selected from a panel of antibiotics evaluated in vitro and used in combination with finafloxacin in a Balb/c mouse model of inhalational melioidosis. When treatment was initiated at 24 h post-infection with B. pseudomallei, there were no differences in the level of protection offered by finafloxacin or doxycycline (as monotherapies) when compared to the combination therapy. There was evidence for improved bacterial control in the groups treated with finafloxacin (as monotherapies or in combination with doxycycline) when compared to mice treated with doxycycline. Survival comparisons of finafloxacin and doxycycline (as monotherapies) or in combination initiated at 36 h post-infection indicated that finafloxacin was superior to doxycycline. Doxycycline was also unable to control the levels of bacteria within tissues to the extent that doxycycline and finafloxacin used in combination or finafloxacin (as a sole therapy) could. In summary, finafloxacin is a promising therapy for use in the event of exposure to B. pseudomallei.

Keywords: Burkholderia pseudomallei; antibiotics; melioidosis; mice; therapy.

FIGURE 1

The time-kill assays for Burkholderia pseudomallei grown in the presence of antibiotics. These assays were performed in CAMBH adjusted to pH 5 or pH 7. An untreated bacterial control was included. (A) pH 5, finafloxacin (1 μg/ml), doxycycline (1 μg/ml), and co-trimoxazole (16 μg/ml). (B) pH 7, finafloxacin (4 μg/ml), doxycycline (0.5 μg/ml), and co-trimoxazole (16 μg/ml). (C) pH 5, finafloxacin (2 μg/ml), doxycycline (2 μg/ml), and co-trimoxazole (32 μg/ml). (D) pH 7, finafloxacin (8 μg/ml), doxycycline (1 μg/ml), and co-trimoxazole (32 μg/ml). The error bars represent the SEM of two biological replicates. *p < 0.05, **p < 0.01, ****p < 0.0001.

FIGURE 2

The concentration of B. pseudomallei and the weight of organs before treatment was initiated. Bacterial counts (CFU/g or CFU/mL) (A) and organ weights (B) were determined for a panel of organs at 24 or 36 h post-infection. Five mice were challenged and culled at either 24 or 36 h post-infection. LoD, limit of detection.

FIGURE 3

The percentage survival of mice in each treatment group following challenge with aerosolized B. pseudomallei. Mice were challenged with a mean retained dose of 106 CFU of B. pseudomallei by the inhalational route and received either finafloxacin (23.1 mg/kg), doxycycline monohydrate (100 mg/kg), or both antibiotics in combination, by the oral route every 8 h. Control animals received the vehicle by the oral route. Regimens were initiated at 24 (A) or 36 (B) h post-infection with mice receiving 14 days of therapy. Survival curves were compared using log-rank (Mantel-Cox) tests. *p < 0.05.

FIGURE 4

The bacterial load in a panel of organs at different time points throughout the B. pseudomallei study. Bacterial counts (CFU/g of tissue or CFU/mL of blood) in organs from mice treated from 24 h post-infection and culled at the end of the treatment regimen (day 15 post-infection) (A), from mice treated from 36 h post-infection and culled at the end of the treatment regimen (day 15 post-infection) (B), from mice treated from 24 h post-infection and culled at the end of the study (day 42 post-infection) (C), and from mice treated from 36 h post-infection and culled at the end of the study (day 43 post-infection) (D). LoD, limit of detection. The colored lines in the columns represent the mean of the group. The data associated with this figure was analyzed in the model used to generate Figure 5.

FIGURE 5

The bacterial load in a panel of organs at different time points throughout the B. pseudomallei study. This analysis investigated the concentration of bacteria in tissues at both time points (day 15 and 42/3 post-infection), the different treatments, and treatment initiation time, 24 h (A) and 36 h post-infection (B). The significance markers are derived from indicative of Bonferroni’s corrected negative binomial generalized linear model Wald tests. Each dot represents an individual organ. These graphs use a representation of a log10 scale that allows the visualization of zero bacteria (marked 0) and one single bacterium (at 10⁰). The lines are the median values for each group and the error bars are the interquartile range. This is a representation of a key interaction of a statistical model. ^***p < 0.001, ^**p < 0.01, *p < 0.05. ns, not significant.

FIGURE 6

The clinical scores recorded throughout the study. Mice were challenged with a mean retained dose of 106 CFU of B. pseudomallei by the inhalational route and received either finafloxacin (23.1 mg/kg), doxycycline monohydrate (100 mg/kg), or both antibiotics in combination, by the oral route every 8 h. Control animals received the vehicle by the oral route. Regimens were initiated at 24 (A) or 36 (B) h post-infection with mice receiving 14 days of therapy. Clinical scores were recorded a minimum of two times daily. The data are shown as the median value for each group at each time point, and the error is the interquartile range. When mice reached their lethal endpoint, an arbitrary value of 9 (one greater than the maximum) was assigned for all subsequent time points. Scores of zero have been offset between groups to help with visualization.

FIGURE 7

The weight loss observed in mice infected with B. pseudomallei and treated with antibiotics. Mice were challenged with a mean retained dose of 106 CFU of B. pseudomallei by the inhalational route and received either finafloxacin (23.1 mg/kg), doxycycline monohydrate (100 mg/kg), or both antibiotics in combination, by the oral route every 8 h, initiated at 24 and 36 h post-infection. Control animals received the vehicle by the oral route. Mice were weighed daily. The data show the mean of multiple mice (n given before and after the cull at the end of the treatment) and the 95% confidence interval generated by the model. The dotted line represents the scheduled cull point at the end of the treatment regimen. All mice (treated from 36 and 48 h post-infection) with all treatments are included in this figure.