Antimicrobial Susceptibility Testing of Mycobacteria

MIC was determined using the broth microdilution method in 7H9 as described previously with minor modifications (25 (link)). SPR719 and comparator antibiotics were serially diluted 2-fold in 100 μL volume of broth up to 10 concentration points in a 96-well plate format (flat bottom, Corning). Bacteria were grown to mid-log phase (OD₆₀₀ 0.4 to 0.6) and the cultures were diluted to OD₆₀₀ = 0.1. 100 μL of bacterial suspensions were dispensed into the wells, resulting in a final volume of 200 μL and a seeding density of OD₆₀₀ = 0.05. After reading at day 0, OD₆₀₀, plates were sealed with Nunc sealing tape (Thermo Scientific), placed in a humidified plastic box (Sterilite), and incubated for 4 days (M. avium) or 3 days (M. abscessus) with shaking at 90 rpm at 37°C. Growth inhibition was measured by reading optical density (OD₆₀₀) using TECAN infinite M200Pro microplate reader (TECAN). MIC values, defined as the concentration that inhibited 90% of bacterial growth compared with drug-free control unless stated otherwise, were deduced from the resulting dose response curves. Growth curves were determined in broth via OD₆₀₀ measurement of cultures growing in 1-L roller bottles (Corning) at 37°C (starting OD₆₀₀ = 0.05) using Ultrospec 10 cell density meter (Biochrom, Holliston, MA, USA). GraphPad Prism 8 (GraphPad Software, Inc.) was used to determine the dose response and growth curves.