Effect of calcium and kinase inhibitors on HAMLET-induced sensitization of pneumococci to gentamicin.

<p>(A) <i>S. pneumoniae</i> D39 were treated with a lethal concentration of HAMLET (12X MIC) in the absence of inhibitor (HL) or presence of 20 μM staurosporine (HL + Sts), or 30 µM ruthenium red (HL + RuR) for 1 hour at 37°C. The treated bacteria were diluted and plated on blood agar plates and viable CFU/ml were determined after overnight growth. (B and C) <i>S. pneumoniae</i> D39 were treated with 50 µg/mL HAMLET (HL), 50 µg/mL gentamicin (Gent), 20 µg/mL penicillin G (PcG), or a combination of gentamicin and HAMLET or penicillin G and HAMLET in the absence (HL + Gent, HL + PcG) or presence of 20 μM staurosporine (Sts), or 30 µM ruthenium red (RuR) for 1 hour at 37°C. The treated bacteria were diluted and plated on blood agar plates and viable CFU/ml were determined after overnight growth. The graph depicts the log₁₀ death induced by each treatment and showed that staurosporine and ruthenium red significantly reduced HAMLET-induced death (A) and also significantly blocked HAMLET's ability to sensitize pneumococci to gentamicin (B) and penicillin G (C). The results are based on three individual experiments with duplicate samples. Statistics was performed using the unpaired Student t-test. Significance was indicated as follows: *** = <i>P</i><0.001.