Figure 2
From: Adaption of the ex vivo mycobacterial growth inhibition assay for use with murine lung cells
Residual BCG in murine lung cells following intranasal vaccination in the ex vivo MGIA. (A) C57BL/6 mice (n = 6/group) received s.c. or i.n. BCG Pasteur Aeras at week 0 or no treatment (control). At six weeks, 1 × 106 lung cells were co-cultured with 100 CFU MTB Erdman in 48-well plates. At 96 hours, samples were transferred to the BACTEC system until TTP values were generated. TTP values were converted to log10 CFU based on a standard curve. Data points (n = 4/group) represent samples generated from pooled cells from six mice. Statistical significance was tested by one-way ANOVA followed by Tukey’s multiple comparison test. *p < 0.05 (adjusted). (B) Residual BCG in 1 × 106 murine lung cells six weeks following immunisation with i.n BCG Pasteur Aeras was quantified using the BACTEC MGIT system. Data points represent four pools of cells from four individual mouse experiments (run as duplicate or single). (C and D) 100 CFU BCG Pasteur Aeras or MTB Erdman, or 100 CFU BCG Pasteur Aeras and 100 CFU MTB Erdman were: (C) plated on 7H11 agar plates in the presence or absence of 2 μg/mL TCH to confirm the inhibitory properties of TCH on BCG only (n = 2/condition); (D) added to MGITs in the absence or presence TCH (five-fold serial dilutions of 2 μg/mL stock: 0.4, 0.08, 0.016 μg/mL) to determine the optimal concentration of TCH to inhibit BCG growth only (n = 1/condition) in the BACTEC system. A TTP value of 1,008 hours (42 days) indicates a negative result (no bacteria detected). Dotted lines indicate mycobacterial input. Error bars represent mean ± standard deviation. ANOVA, analysis of variance; BCG, Bacille Calmette-Guérin; CFU, colony-forming units; i.n., intranasal; MGIA, mycobacterial growth inhibition assay; MGIT, mycobacterial growth indicator tube; s.c., subcutaneous; TCH, 2-thiophenecarboxylic acid hydrazide; TTP, time to positivity.
