Fig 1.
The PrestoBlue assay shows discrimination between the numbers of surviving cells.
A B. thailandensis culture was harvested, resuspended, and diluted in M9 media supplemented with 730 μM ceftazidime, to provide a series of cell densities at two-fold intervals. Samples were incubated statically at 28°C in 96 well plates. PrestoBlue was added following 20 hours of incubation and the fluorescence read gain optimized for the highest bacterial concentration. The results show reliable discrimination of two-fold differences in cell numbers when compared to a heat killed cell negative control. * All show Z’ > 0.5 when compared to the controls. Positive control: Cells resuspended in M9 media without ceftazidime. Data shows biological triplicates, whiskers indicate the minimum and maximum results, the box the 25th to 75th percentiles and the central line indicates the median.
Fig 2.
Inhibitory activity of test compounds screened with a phenotypic assay.
A B. thailandensis culture was harvested and resuspended in M9 media supplemented with 730 μM ceftazidime and 30 μM of each of the compounds. Cells were grown at 28°C for 20 h, and PrestoBlue added. Inhibition was calculated by comparisons to the controls. The distribution shows the percentage inhibition grouped in 5% windows for the HTS of 61,250 compounds. The median activity is 5.3%. The standard deviation of the positive tail is 9.65%, giving a statistical cut-off for activity of 34.3%. The red arrow indicates the selected pragmatic threshold at 45%. 345 compounds were identified as ‘hits’ according to this criterion.
Fig 3.
pIC50 determination of six candidate compounds using the PrestoBlue cell viability assay.
A: A B. thailandensis culture was harvested and resuspended to a concentration of 8x108 CFU/mL in M9 media supplemented with 730 μM ceftazidime. This was added to a 96 well plate containing two-fold dilutions of compounds in DMSO from 500 μM. Plates were incubated for 24 hours at 37°C before the addition of the PrestoBlue cell viability reagent and the fluorescence read. Results show three biological replicates with error bars indicating standard error. The derived IC50 values are detailed in Table 1. B: Structure of chloroxine.
Table 1.
Analysis of the concentration dependent killing data shown in Fig 3.
Fig 4.
A secondary assay evaluating the number of culturable cells remaining following treatment with ceftazidime and chloroxine.
A culture of B. thailandensis was treated with 730 μM ceftazidime hydrate, 30 μM chloroxine, both, or neither. Samples were incubated at 37°C with shaking. Samples were taken at time intervals, cells harvested and resuspended in LB broth before serial dilution and enumerating on agar. Error indicates standard error of serial dilution and CFU count. n = 6. Differences between the ceftazidime alone, chloroxine alone, and ceftazidime and chloroxine samples were analyzed using a Kruskal-Wallis test with Dunn post-hoc comparison using Graphpad v. 7.03. *—p < 0.01 between chloroxine alone, and ceftazidime with chloroxine. **—p < 0.05 between both chloroxine alone and ceftazidime alone, and both compounds.
Fig 5.
Hit expansion around chloroxine.
A B. thailandensis culture was harvested and resuspended to a concentration of 8x108 CFU/mL in M9 media supplemented with 730 μM ceftazidime. This was added to a 96 well plate containing two-fold dilutions of compounds in DMSO from a starting concentration of 1 mM. Plates were incubated for 24 hours at 37°C before addition of the PrestoBlue cell viability reagent and the fluorescence read. Results show three biological replicates with error bars indicating standard deviation. These experiments are equivalent to those in Fig 3 and can be compared to chloroxine in Fig 3. 7-amino-5-chloro-8-quinolinol differs to chloroxine through substitution of an amino group for a chlorine at position 7 (A). This modification causes a significant decrease in this compound’s activity as a co-treatment with ceftazidime, with a pIC50 ≈ 3.4. 5,7-dichloro-2-methyl-8-quinolinol differs from chloroxine by addition of a methyl group in the 2-position (B). This addition abolishes this compound’s activity as a co-treatment with ceftazidime at the concentrations tested. 7-Bromo-8-quinolinol differs from chloroxine by the removal of chlorine at the 5-position, and replacement of chlorine by bromine at the 7-position (C). This compound retains activity as a co-treatment with ceftazidime that is comparable with the parent compound (pIC50 = 5.1, 5.5 for chloroxine).
Fig 6.
pIC50 determination using the PrestoBlue cell viability assay to compare the concentration dependent killing for ceftazidime used in combination with chloroxine to treat B. thailandensis and B. pseudomallei.
B. thailandensis and B. pseudomallei cultures were harvested and resuspended to a concentration of 8x108 CFU/mL in M9 media supplemented with 730 μM ceftazidime. These were added to a 96 well plate containing two-fold dilutions of chloroxine in DMSO. Plates were incubated for 24 hours at 37°C before the addition of the PrestoBlue cell viability reagent and determination of fluorescence. Results show three biological replicates with error bars indicating standard deviation. pIC50 for B. thailandensis = 5.7; pIC50 for B. pseudomallei = 5.0.