FIG 4.

Effect of coculture of mycobacterial strains within amoebae on virulence in mice. (A) BALB/c mice were aerosolized with the CIP-S (white bars), CIP-S plc KO (black bars), and CIP-S plc KO plc-complemented (gray bars) strains cultivated in 7H9 medium. Mice were challenged with 10⁸ mycobacteria and sacrificed at days 1, 7, 14, 21, and 28. Lungs, livers, and spleens were collected, homogenized, diluted (1/1, 1/5, 1/25, and 1/125), and cultured on VCA3 agar plates. CFU were counted after 5 days of growth. Twenty-five mice per group were challenged. (B) BALB/c mice were aerosolized with CIP-S cultivated in 7H9 medium (○) or obtained after 3 days of coculture in amoebae (■). Mice were sacrificed at days 3 and 7. Lungs were collected, diluted (1/1, 1/5, 1/25, and 1/125), and cultured on VCA3 agar plates. CFU were counted for the different dilutions after 5 days of growth. Ten mice per group were challenged. (****, P < 0.0001). (C) BALB/c mice were aerosolized with the CIP-S (empty bars) or CIP-S plc KO (black bars) strain cultivated in 7H9 medium or obtained after 3 days of coculture within amoebae. Mice were sacrificed at days 3 and 7. Lungs were collected, diluted (1/1, 1/5, 1/25, and 1/125), and cultured on VCA3 agar plates. CFU were counted after 5 days of growth. Ten mice per group were challenged. (**, P < 0.01; ***, P < 0.001).