The use of resazurin as a novel antimicrobial agent against Francisella tularensis

Abstract

The highly infectious and deadly pathogen, Francisella tularensis, is classified by the CDC as a Category A bioterrorism agent. Inhalation of a single bacterium results in an acute pneumonia with a 30-60% mortality rate without treatment. Due to the prevalence of antibiotic resistance, there is a strong need for new types of antibacterial drugs. Resazurin is commonly used to measure bacterial and eukaryotic cell viability through its reduction to the fluorescent product resorufin. When tested on various bacterial taxa at the recommended concentration of 44 μM, a potent bactericidal effect was observed against various Francisella and Neisseria species, including the human pathogens type A F. tularensis (Schu S4) and N. gonorrhoeae. As low as 4.4 μM resazurin was sufficient for a 10-fold reduction in F. tularensis growth. In broth culture, resazurin was reduced to resorufin by F. tularensis. Resorufin also suppressed the growth of F. tularensis suggesting that this compound is the biologically active form responsible for decreasing the viability of F. tularensis LVS bacteria. Replication of F. tularensis in primary human macrophages and non-phagocytic cells was abolished following treatment with 44 μM resazurin indicating this compound could be an effective therapy for tularemia in vivo.

Keywords: Francisella; Neisseria; antibacterial; antibiotic; macrophages; resazurin; resorufin; tularemia.

Figure 1

The reduction of resazurin to resorufin.

Figure 2

Resazurin has an antimicrobial effect on F. tularensis LVS. Bacteria were cultivated in tryptic soy broth supplemented with 0.1% cysteine HCl (TSBc) in the presence or absence of resazurin at the designated concentrations for 24 h. Cultures were then diluted and plated to determine the number of viable F. tularensis LVS bacteria 24 h post inoculation. Data shown are mean ± s.e.m. from three individual experiments. The limit of detection was 100 CFU per ml. Statistically significant differences in growth post-inoculation were determined by One-Way ANOVA followed by Dunnett's post-hoc test (^***p < 0.001 compared to 0 μM resazurin).

Figure 3

F. tularensis LVS reduces resazurin to resorufin which also inhibits bacterial growth. F. tularensis LVS bacteria were cultivated in TSBc that was treated with resazurin, resorufin, or the solvent vehicle (DMSO). (A) Conversion of resazurin (absorbance at 600 nm) to resorufin (absorbance at 570 nm) in F. tularensis LVS cultures over 24 h. (B,C) F. tularensis LVS cultures were diluted and plated to determine the number of viable bacteria at 24 h (B) or the indicated timepoints (C). Data shown are mean ± s.e.m. from three individual experiments. The limit of detection was 100 CFU per ml. Statistically significant differences in growth post-inoculation were determined by (B) One-Way ANOVA followed by Dunnett's post-hoc test (^**p < 0.01 compared to TSBc) or (C) Two-Way ANOVA followed by Bonferroni comparison of means (p < 0.05 for Control vs. Resazurin and Control vs. Resorufin for 12–24 h).

Figure 4

Resazurin exhibits antimicrobial activity on intracellular F. tularensis LVS. Primary human macrophages (A) or HEK293 (B) cells were infected with F. tularensis LVS bacteria using a gentamicin protection assay. Cells were treated with 44 μM resazurin during the entire assay (+ Rz), at 2 h post-infection (+ 2 h Rz), or were untreated. Data shown are mean ± s.e.m. and representative of three independent experiments. The limit of detection was 100 CFU. Statistically significant differences in growth at 24 h post-infection were determined by Two-Way ANOVA with Dunnett's post-hoc test (^***p < 0.001).

Figure 5

Chemical structures of resazurin and acridine.