The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of TR against drug resistant clinical isolates and laboratory strains of Mtb were tested in-vitro.
The MIC of TR against individual Mtb clinical isolates and laboratory strain (H37Rv) was evaluated in comparison to the other known drugs, isoniazid, rifampicin, kanamycin, moxifloxacin, bedaquiline and delamanid. MIC determination was carried out using the Broth-microdilution method. The 1.0 McFarland matched suspension of Mtb culture that was grown on LJ slope was further diluted in a 1:19 ratio using 7H9 broth and used to estimate the MIC using the broth micro-dilution method as described elsewhere [13 (link)]. In brief, the TR and other standard drugs were taken in 96 well Microtiter plates in the specified range of concentrations. Each well was loaded with 100μl of diluted Mtb culture suspension to arrive at a final volume of 200μl. Separate culture controls and solvent controls were included to ensure the culture viability. Post 14 days of incubation at 37°C, the plates were observed under an inverted phase-contrast microscope for the Mtb characteristic serpentine cord formation. The least concentration of the drug that resulted in the visible absence of growth was accounted for as its MIC. All the tests were carried out in duplicates and the absence of microbial contamination was ensured by spotting the aliquots from the plate onto Brain Heart Infusion (BHI) agar plates.
To determine the MBC of TR, cell suspension of Mtb clinical isolates (MDR and XDR) and laboratory strain (H37Rv) equivalent to 1.0 McFarland standard was taken and further diluted in 1:19 ratio using 7H9 media. The cell suspension was taken in 96 well Microtiter plate and treated with different drug concentrations. After 3 days of incubation, 5μl cell suspension from each well was spotted onto 7H11 agar plate in triplicates and incubated until the growth appears. The minimum concentration with no growth in all of the triplicates was considered as MBC.
The MIC of TR against individual Mtb clinical isolates and laboratory strain (H37Rv) was evaluated in comparison to the other known drugs, isoniazid, rifampicin, kanamycin, moxifloxacin, bedaquiline and delamanid. MIC determination was carried out using the Broth-microdilution method. The 1.0 McFarland matched suspension of Mtb culture that was grown on LJ slope was further diluted in a 1:19 ratio using 7H9 broth and used to estimate the MIC using the broth micro-dilution method as described elsewhere [13 (link)]. In brief, the TR and other standard drugs were taken in 96 well Microtiter plates in the specified range of concentrations. Each well was loaded with 100μl of diluted Mtb culture suspension to arrive at a final volume of 200μl. Separate culture controls and solvent controls were included to ensure the culture viability. Post 14 days of incubation at 37°C, the plates were observed under an inverted phase-contrast microscope for the Mtb characteristic serpentine cord formation. The least concentration of the drug that resulted in the visible absence of growth was accounted for as its MIC. All the tests were carried out in duplicates and the absence of microbial contamination was ensured by spotting the aliquots from the plate onto Brain Heart Infusion (BHI) agar plates.
To determine the MBC of TR, cell suspension of Mtb clinical isolates (MDR and XDR) and laboratory strain (H37Rv) equivalent to 1.0 McFarland standard was taken and further diluted in 1:19 ratio using 7H9 media. The cell suspension was taken in 96 well Microtiter plate and treated with different drug concentrations. After 3 days of incubation, 5μl cell suspension from each well was spotted onto 7H11 agar plate in triplicates and incubated until the growth appears. The minimum concentration with no growth in all of the triplicates was considered as MBC.
Free full text: Click here
