Recombinant BCG Strains for Imaging

Construction of the recombinant strains rBCG::noPGL, rBCG::PGL-b, rBCG::PGL-I have been described previously (17 (link), 21 (link)). The various recombinant strains were transformed with plasmid pWM124, a mycobacterial replicative plasmid carrying the gfp gene under the control of the pblaF^* promotor (17 (link)). Strains were cultured in Middlebrook 7H9 broth (Invitrogen, Cergy-Pontoise, France) containing 0.05% Tween 80 (Sigma-Aldrich, St. Louis, USA) and ADC (5% BSA fraction V, 2% dextrose, 0.003% beef catalase and 0.85% NaCl; BD Microbiology Systems) and supplemented with 40 μg/ml of Kanamycin sulfate (Sigma-Aldrich, St. Louis, USA) or 50 μg/ml of Hygromycin B (Sigma-Aldrich, St. Louis, USA) for the fluorescent strains. Ten days before infection, bacteria were inoculated into 7H9 with ADC without Tween 80. Bacteria were pelleted at 3,000× g 10 min, washed and suspended in PBS. Clumps were dispersed by vortex with 4 mm diameter glass beads. Bacteria were centrifuged (200× g) for 5 min and concentration of bacterial suspensions was measured by OD 600 nm (1 OD = 10⁸ bacilli/ml). To assess CFUs, serial dilutions were plated on Middlebrook 7H11 agar plates supplemented with OADC (ADC supplemented with 0.05% oleic acid).