The Fluoroquinolone Finafloxacin Protects BALB/c Mice Against an Intranasal Infection With Francisella tularensis Strain SchuS4

Abstract

The efficacy of the novel fluoroquinolone finafloxacin was evaluated as a potential therapeutic in vitro and in vivo, following an intranasal infection of Francisella tularensis strain SchuS4 in BALB/c mice. We demonstrated that short treatment courses of finafloxacin provide high levels of protection, with a single dose resulting in a significant increase in time to death when compared to ciprofloxacin. In addition, following investigation into the window of opportunity for treatment, we have shown that finafloxacin can provided protection when administered up to 96 h post-challenge. This is particularly encouraging since mice displayed severe signs of disease at this time point. In summary, finafloxacin may be a promising therapy for use in the event of exposure to F. tularensis, perhaps enabling the treatment regimen to be shortened or if therapy is delayed. The efficacy of finafloxacin against other biological threat agents also warrants investigation.

Keywords: Francisella tularensis; biothreat; finafloxacin; in vivo efficacy; therapeutic.

FIGURE 1

The activity of finafloxacin and ciprofloxacin against F. tularensis strain SchuS4 over 24 h. Time kill assays were performed in supplemented MCPH broth adjusted to pH 6 (A) or pH 7 (B), at 4 × MIC of finafloxacin (0.064 mg/L) or ciprofloxacin (0.12 μg/ml). An untreated bacterial culture was included. The dotted line represents a 3 log₁₀ reduction in CFU/ml from the starting inoculum. Statistics was performed using a two-way ANOVA with Tukey’s multiple comparison test. ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001. Each data point is the mean of 3 replicates and the error bars represent the standard error. LOD, limit of detection.

FIGURE 2

A graphical schema detailing the two in vivo studies discussed in the manuscript.

FIGURE 3

The percentage survival of mice following challenge with F. tularensis SchuS4 by the intranasal route. Mice were challenged with 100 CFU of F. tularensis by the intranasal route and treated with finafloxacin (37.5 mg/kg) by the oral route every 8 h or ciprofloxacin (30 mg/kg) by the intraperitoneal route every 12 h. Regimens were initiated at 24 h post-challenge, and continued for 1 (A), 3 (B), or 7 (C) days. Control animals received PBS by the IP route or diluent by the oral route. A group of animals were infected and received no treatment. Statistical analysis was performed using a Mantel-Haenszel log rank test. ^∗∗p < 0.01.

FIGURE 4

The bacterial load in organs at day 4 following challenge with F. tularensis SchuS4 by the intranasal route. Mice were challenged with 100 CFU of F. tularensis by the intranasal route and treated with finafloxacin (37.5 mg/kg) by the oral route every 8 h or ciprofloxacin (30 mg/kg) by the intraperitoneal route every 12 h until the control animals reached an average clinical score of 4 when 5 mice per group were culled and their organs harvested. Therapy was initiated at 24 h and continued for 1 day (A) or 3 days (B). Control animals received PBS by the IP route or diluent by the oral route. Statistical analysis was performed using pairwise comparisons of a Mood’s Median test. ^∗∗p < 0.01. The lines within the individual data points are the means of the log₁₀ transformed data for each group.

FIGURE 5

The percentage survival of mice following challenge with F. tularensis SchuS4 by the intranasal route. Mice were challenged with 101 CFU of F. tularensis by the intranasal route and treated with finafloxacin (37.5 mg/kg) by the oral route every 8 h or ciprofloxacin (30 mg/kg) by the intraperitoneal route every 12 h. Regimens were initiated at 24 or 48 h (A) or 72 or 96 h (B) post-challenge, and continued for 7 days. Control animals received PBS by the IP route or diluent by the oral route. A group of animals were infected and received no treatment. Survival data for the groups receiving therapy at 72 and 96 h have been combined (C). Statistical analysis was performed using the Mantel-Haenszel log rank test. ^∗p < 0.05.

FIGURE 6

The clinical scores of mice infected with F. tularensis and treated with finafloxacin and ciprofloxacin. Mice were challenged with 101 CFU of F. tularensis by the intranasal route and treated with finafloxacin (37.5 mg/kg) by the oral route every 8 h or ciprofloxacin (30 mg/kg) by the intraperitoneal route every 12 h. A group of animals were infected and received no treatment. Mice receiving the control substances displayed very similar patterns of clinical signs to the untreated control group (data not included in the Figure). The graphs show the median daily summed clinical scores per treatment group when antibiotics were initiated at 24 (A), 48 (B), 72 (C), or 96 h (D) post-challenge and continued for 7 days. The error bars are the interquartile ranges.

FIGURE 7

The percentage weight change following ciprofloxacin and finafloxacin therapy in mice challenged with F. tularensis SchuS4 by the intranasal route. Mice were challenged with 101 CFU of F. tularensis by the intranasal route and treated with finafloxacin (37.5 mg/kg) by the oral route every 8 h or ciprofloxacin (30 mg/kg) by the intraperitoneal route every 12 h. Control animals received PBS by the IP route or diluent by the oral route. A group of animals were infected and received no treatment. The graphs show the percentage change in weight over time when antibiotics were initiated at 24 (A), 48 (B), 72 (C), or 96 h (D) post-challenge and continued for 7 days. Each line represents an individual mouse.

FIGURE 8

The organ weight and bacterial burden in surviving animals. Mice were challenged with 101 CFU of F. tularensis by the intranasal route and treated with finafloxacin (37.5 mg/kg) by the oral route every 8 h or ciprofloxacin (30 mg/kg) by the intraperitoneal route every 12 h. Mice that survived until the end of the study were culled and organs removed and weighed (A) and processed for bacterial burden (B). Statistical analysis was performed using the Mood’s Median test with pairwise adjustment to analyze bacterial burden. Circles represent treatment initiated at 24 h, squares 48, triangles 72, and diamonds 96 h post-challenge. ^∗p < 0.05. For the organ weights, the lines represent the means for each group. For the bacterial loads, the lines represent the medians for each group. LOD – limit of detection.