Figure 1.

Generation of resuscitation-promoting factor (Rpf)-dependent Mycobacterium bovis (BCG) in murine lungs. (A and B) Nine-week-old BALB/c mice were infected intranasally with 2 × 10⁵ bacterial colony-forming units (CFUs) per animal. (A) Homogenized tissue samples were used for assessment of CFU counts (circles) or most-probable number assay (MPN) counts in liquid 7H9 medium (squares) or Rpf-dependent mycobacteria (RDM) counts in 7H9 medium supplemented with culture supernatant (triangles). A PANTA antimicrobial mixture (Becton, Dickinson and Co., Franklin Lakes, NJ) was added to all cultivation media. Average values for four independent animals are presented; error bars indicate standard deviations. **RDM values were significantly different from CFU and MPN counts (P < 0.01, t test); ***RDM values were significantly different from CFU counts (P < 0.001, t test). (B) Control experiments. CFU and MPN counts were determined in lung homogenates from mice 3 weeks postinfection. The Rpf inhibitors NTB (3-nitro-4-thiocyanato-benzonitrile), designated as I₁, and NTPPM [(3-nitro-4-thiocyanato-phenyl)-phenyl-methanone], designated as I₂, did not inhibit growth of active M. bovis BCG in vitro at the concentration used in these experiments (5 μg/ml). SN = culture supernatant. (C) Effect of murine serum on M. bovis BCG viability. Bacteria from the logarithmic phase were exposed to 20% (vol/vol) murine serum in phosphate-buffered saline. CFU and RDM counts were determined after 1 and 3 days of exposure.