Fig 2. Time kill and point of resistance assays.

NCL195 was prepared at 2× and 4× MIC (using 4× MIC of ampicillin (or 4× MIC of daptomycin) and normal saline as controls) in either LB broth supplemented with 3% lysed horse blood and 5% horse serum for S. pneumoniae D39 (A) or LB broth without supplementation for S. aureus Xen29 (B and C). Cultures were incubated statically at 37°C, 5% CO₂ (for S. pneumoniae D39) or at 37°C with agitation at 200 rpm (for S. aureus Xen29). Samples withdrawn at indicated times and plated on HBA overnight at 37°C, 5% CO₂ (for S. pneumoniae D39) or at 37°C with aeration (for S. aureus Xen29) for bacterial enumeration. For (D), S. aureus Xen29 was grown in 2 ml LB broth the presence of 0.5×MIC, 0.75×MIC, 1×MIC, 1.5×MIC, 2×MIC, and 4×MIC of NCL195, using 1×MIC, 4×MIC and 8×MIC of daptomycin as control.