P02

As we experienced insufficient growth with the resazurin-microtiter (REMA) assay with read-out at 7 days for lineage (L) 5 and L6 isolates, this project aimed to optimize growth conditions for fastidious lineages (L) of the Mycobacterium tuberculosis complex, to improve broth dilutions drug-susceptibility testing.

Based on previous studies, 4 potential growth stimulators were selected: sodium pyruvate, oxaloacetic acid, pancreatic digest of casein, and vitamin B12. Ten strains from L5 and L6 were tested, together with one strain each from L7, L8 and L9, and H37Rv (L4) as reference. For each strain, two 96-well plates were prepared by alternately adding 7H9 medium with and without glycerol to the rows, with serial dilutions of the 4 compounds. The 2 plates were placed in standard O₂ and 5-10% CO₂ incubators, with visual reading of the plates on days 7, 10, 14, 21 and 28. Growth in the compound-containing wells was compared to the growth controls (100% and 1% bacterial suspensions) without compounds.

Extending the incubation period to 21 days had a positive effect on all growth controls. CO₂ incubation, addition of glycerol, or oxaloacetic acid did not alter growth, whereas the other 3 compounds enhanced growth. The best performing concentrations of these compounds were selected for a new experiment comparing pairwise combinations of the compounds with added glycerol and O₂ incubation, and including more strains of all lineages. Results suggest that the combination of pancreatic digest of casein and vitamin B12 best enhances growth.