Abstract
The increasing incidence of antibiotic-resistant bacterial infections both in hospitals and in the community intensifies the need for new antibacterial strategies and targets. Although high-throughput screening against live bacteria allows rapid discovery of compounds with growth-inhibitory activities, these efforts have failed to fill the pipeline with the anticipated antibacterial compounds because target identification is often onerous. Recently, a strategy was reported that employs a bacteria growth inhibition assay readout using optical density measurements on paired strains - both a wildtype strain and a pathway-null mutant - to find inhibitors of wildtype bacterial growth that specifically target conditionally essential enzymes in the pathway of interest. Protocols are provided here for determining the robustness of an assay, screening in a high-throughput format and setting up dose-response curves in paired Staphylococcus aureus strains. However, the protocols can be used to screen for growth-inhibitory compounds in any bacterial strain of interest.