tularensis Schu S4 (EC50 of 0.145 μg/ml), reflecting the altered shape of the MIC curve and indicating increased sensitivity. Only ΔacrB was statistically significantly different for EC50 when compared to the wild-type F. tularensis Schu S4 (p-value < 0.05).

Thus, F. tularensis Schu S4 ΔacrA and ΔacrB mutants had greater sensitivity to Az compared to F. novicida mutants, or the parental F. tularensis Schu S4 strain by disc inhibition assay and MIC. Az inhibition of intracellular Francisella mutant strains J774A.1 and A549 cells infected with F. novicida transposon LPS mutant wbtA and multidrug efflux mutants ftlC, tolC, acrA, and acrB had more than 104 CFU/ml 22 hours post-infection (Figure 5). ftlC generally had lower CFU counts, whereas the acrA and acrB had higher CFU counts in both cell lines. The CFU of F. novicida transposon mutants decreased as the Az concentration increased for each cell line (p-value < 0.005 for each Regorafenib datasheet Az treatment compared to 0 μg/ml Az). At 35 μg/ml Az treatment, the bacterial CFU count was near 0 CFU/ml in J774A.1 and A549 cells (Figure 5). Thus, wbtA and the RND mutants are capable of replication within J774A.1 and A549 cells, although the overall number of bacteria per cell was lower than for the parental F. novicida infection (1.76 × 105 ± 6.36 × 103 CFU/ml in J774A.1 and 1.80 × 105 ± 1.41 × 104 CFU/ml in A549 cells Vorinostat research buy at 0 μg/ml). Mutant trends

after Az treatments were significantly different from the wild-type F. novicida with a p-value < 0.05 (wild-type decreased to 0 CFU/ml at 5 μg/ml Az in J774A.1 cells and decreased to 0 CFU/ml at 25 μg/ml Az in A549 cells). Corresponding to the higher MICs identified in vitro, LPS mutants require more Az to eliminate the bacteria from infected cells. Figure 5 Az inhibition of intracellular F. novicida mutants. A) J774A.1 and B) A549 cells were infected with various mutants at an MOI 500. At 22 hours, the number selleckchem of CFUs/ml recovered from F. novicida multidrug efflux mutants ftlC, tolC, acrA, and acrB and LPS O-antigen mutant wbtA decreased as Az concentrations increased and was near 0 CFU/ml at 35 μg/ml

Az (p-value < 0.005 for all Az treatments compared to 0 μg/ml Az for each mutant). The recovery of mutant strains after Az treatments were significantly different from the wild-type F. novicida with a p-value < 0.05 (1.76 × 105 ± 6.36 × 103 CFU/ml in J774A.1 at 0 μg/ml Az which decreased to 0 CFU/ml at 5 μg/ml Az and 1.80 × 105 ± 1.41 × 104 CFU/ml in A549 cells at 0 μg/ml Az which decreased to 0 CFU/ml at 25 μg/ml Az). J774A.1 cells had higher bacterial counts than A549 cells. G. mellonella infection by Francisella and antibiotic treatment Francisella-infected G. mellonella was used as a model system [25] to study Az treatment. G. mellonella were infected with either 3 × 106 CFU bacteria/larva of F. novicida or F. tularensis LVS and then treated with a single dose of 10 μl injections PBS (no antibiotic), 20 μg/ml ciprofloxacin, or 25 μg/ml Az.