2.2. Anti-tubercular screening: resazurin microplate assay (REMA)

Anti-TB screening of test compounds 3a–3i (Table 1) was performed using the colorimetric REMA plate approach^19,67. In order to determine the minimum inhibitory concentration (MIC), test compounds (3a–3i) were assessed using the agar incorporation approach, which was performed three times, and targeted in H37Rv and MDR-TB strains (isoniazid, 0.2 µg/mL; rifampicin, >1.0 µg/mL). MIC determination was then carried out with some modifications⁶⁸. A Level II Biosafety Laboratory was used to carry out this experiment. MTB reference strain H37Rv (American Type Culture Collection [ATCC], Manassas, VA, USA: 25177) and MDR-TB were cultured in Middlebrook 7H11 medium for a total of 3 weeks⁶⁹. The strain was supplemented with OADC (0.005%, v/v, oleic acid; 0.2%, w/v, glucose; 0.085%, w/v, NaCl; 0.02%, v/v, catalase; and 0.5%, 171 w/v, bovine serum albumin [BSA]), and incubated at a temperature of 37 °C. Fresh cultures were used to prepare a standardised inoculum in a sterile tube (5 mm in diameter) containing 0.05% Tween 80 and 4.5 ml of phosphate buffer for vortexing. The bacterial supernatant was then standardised to McFarland Number 1 with water, resulting in a bacterial concentration ∼1 × 10⁷cfu/mL. The bacterial suspension was then diluted with water, after which a total of 100 µL of the dilution was placed onto Middlebrook 7H10 agar plates with drug doses ranging from 128–0.125 µg/mL (to begin, 8 µg/mL of the drug was dissolved in distilled water and diluted two fold to achieve the desired concentration prior to being added to the agar medium). The MICs of the drugs (i.e. the concentration that inhibited >1% of the organism’s growth when compared with controls) were obtained 3 weeks following the incubation. These compounds were also tested against previously well-characterised M(XDR) – TB clinical isolates. These isolates were characterised using both a gold standard agar proportion susceptible testing method and a genotyping PCR (MTBDRplus & MTBDRsl, Hain-Lifesciences, Germany). MDR TB clinical isolates were resistant to both isoniazid and rifampicin only whereas XDR TB clinical isolates were resistant to isoniazid, rifampicin, a fluoroquinolone, and an aminoglycoside/cyclic peptide.

In vitro anti-TB activity of ((5-chloro-2-((3-substituedphenyl-1,2,4-oxadiazol-5-yl)methoxy)phenyl) (phenyl)methanone molecules (3a–i) against H37Rv, MDR and XDR strains of M. tuberculosis.