Determination of the antibacterial activity of the HTS hits

The antimicrobial activity of the HTS hits was tested by the colorimetric method of the resazurin microplate assay³⁶ according to broth microdilution method guidelines (CLSI; Clinical and Laboratory Standards Institute). Serial 2-fold dilutions of the HTS hits were performed in BHI medium, in 96-well microtiter plates, with a final volume of 100 µl per well. Subsequently, liquid cultures of C. ammoniagenes ATCC 6872 in logarithmic phase were adjusted to 10⁶ CFU/ml in BHI broth, and 100 µl of this suspension were added to each well, making a final inoculum of 5 × 10⁵ CFU/ml. Plates were incubated 16 h at 37 °C. 30 µl of 0.1 mg/ml resazurin solution were then added to each well, and results were observed after 4 h of incubation at 37 °C. Resazurin (blue) is an indicator of bacterial growth, since metabolic activity of bacteria reduces it to resorufin (pink). The minimum inhibitory concentration (MIC) is the lowest concentration of compound that does not change the resazurin colour from blue to pink.

Similarly, the HTS hits were also assayed at 37 °C against M. tuberculosis ATCC 27,294 and S. pneumoniae ATCC 49619 cells. In these experiments the initial cell concentration was also 5 × 10⁵ CFU/ml, and plates were incubated for 10 h (S. pneumoniae), and 6 days (M. tuberculosis) before addition of resazurin. Results were observed after incubation with resazurin 4 h and two days for S. pneumoniae and M. tuberculosis, respectively. In these experiments, culture media were; Middlebrook 7H9 (Difco) supplemented with 10% ADC (0.2% dextrose, 0.5% V fraction BSA and 0.0003% bovine catalase) (BD Difco) and with 0.5% glycerol (Scharlau) for M. tuberculosis growth, and BHI supplemented with 4% FBS (Gibco) for S. pneumoniae growth.