03-0.5 μg/ml, EC50 of 0.12 μg/ml) [28]. Thus, the Type A Francisella tularensis SchuS4, F. this website novicida and F. philomiragia are all sensitive to Az in vitro. Type B Francisella LVS was also determined to be sensitive, but at a higher concentration of Az. Table 2 MIC Assay of Az for Francisella strains. Bacteria Ro 61-8048 ic50 Az MIC (μg/ml) Az EC50(μg/ml)

p-value Gent MIC (μg/ml) Gent EC50(μg/ml) F. tularensis LVS 25 17.34 —- 0.39 0.09 F. philomiragia 1.56 0.13 <0.001 0.39 0.22 F. novicida 0.78 0.16 <0.001 0.20 0.12 F. tularensis Schu S4 0.78 0.1453 0.004 n/a n/a The p-value is for comparisons of the EC50 values. Figure 2 MIC determination of Az for F. tularensis LVS, F. philomiragia, F. novicida , and F.tularensis Schu S4. Az MIC for F. tularensis LVS (circles) is higher than F. philomiragia (squares), F. novicida (up triangle), and F. tularensis Schu S4 (down triangle). Az MICs for F.

novicida and F. tularensis Schu S4 are 0.78 μg/ml with an EC50 of 0.16 μg/ml and 0.15 μg/ml respectively. F. philomiragia’s Az MIC is 1.56 μg/ml with an EC50 of 0.13 μg/ml, and F. tularensis LVS’s Az MIC is 25 μg/ml with an EC50 of 17.34 μg/ml. J774A.1 and A549 cells CX 5461 were infected with Francisella and treated with Az. The same multiplicity of infection (MOI = 500) was used, based on previous studies for Francisella infection [30]. Cells were lysed and bacteria were recovered and counted as colony forming units (CFU). Francisella-infected J774A.1 and A549 cells were found to have more than 105 CFU/ml of Francisella after 22 hours after infection. J774A.1 cells infected with Francisella and treated with Az had decreasing CFUs as the antibiotic concentration increased. In J774A.1 cells infected with F. philomiragia, no CFUs were recovered when treated with 0.1 μg/ml Az (less than the MIC). In J774A.1 cells infected with either F. novicida or F. tularensis LVS, bacterial concentrations decreased with the addition of Az. At 5 μg/ml Az, no CFUs were recovered (p-value < 0.005 compared

to 0 μg/ml Az) (Figure 3A). In this case, the Az concentration was less than the MIC for F. tularensis LVS. Francisella-infected PRKD3 A549 cells required higher concentrations of Az than J774A.1 cells, suggesting that epithelial cells are not able to concentrate Az in the same manner as macrophages. As before, intracellular F. novicida, F. philomiragia, and F. tularensis LVS CFU counts decreased when A549 cells were treated with Az. Recovered intracellular CFU counts for F. philomiragia and F. novicida remained approximately equal when treated with 0.1 and 5 μg/ml Az (p-value > 0.05), but strongly decreased at 25 μg/ml Az (p-value < 0.005 compared to 0 μg/ml Az). For these two organisms, the required external antibiotic concentration was higher than the in vitro MIC. F.