Aerosol infection with M. tuberculosis.

All animal procedures were approved by the Animal Care and Use Committee of Johns Hopkins University. Six-week-old female BALB/c mice and immunodeficient athymic CD-1 nude mice (Charles River Laboratories, Wilmington, MA) were infected with M. tuberculosis H37Rv or the isogenic pncA or ddn mutant, using the inhalation exposure system (Glas-Col, Terre Haute, IN) and a fresh log-phase broth culture (optical density at 600 nm, 0.8 to 1.0), with the goal of implanting 4 log₁₀ CFU in the lungs of each mouse (4). Four or five mice were humanely killed 1 day after infection (D−13) and on the day of treatment initiation (D0) to determine the number of bacteria implanted in the lungs and at the start of treatment, respectively.

Female C3HeB/FeJ mice (Jackson Laboratory, Bar Harbor, ME), 10 weeks old, were aerosol infected with M. tuberculosis HN878 on two occasions spaced 10 days apart (with mice divided into 2 runs per occasion) in a repeated infection protocol intended to promote more advanced caseating lung lesions. On each occasion, a frozen stock culture was thawed and diluted with the intention to implant approximately 200 CFU per run. Treatment started at 4 weeks after the first infection (W−4). Six and nine mice (2 or 3 mice per run) were sacrificed for lung CFU counts at W−4 and D0 to determine the number of CFU implanted and the number present at the start of treatment, respectively.