Generation of resistant mutants and whole genome sequencing
Mtb H37Rv ATCC27294 was grown to OD650nm of 0.5 in 7H9/ADC/Tw, harvested and resuspended at 1010, 109 and 108 CFU/ml. Aliquots (0.1 ml) were spread on solid medium consisting of Middlebrook 7H11 (Becton Dickinson) supplemented with OADC [final concentration of 0.5% bovine serum albumin fraction V, 0.08% NaCl, 0.2% glucose, 0.2% glycerol, 0.06% oleic acid] containing 10X MIC concentration of TUN-8,8 or TUN-10,10. Colonies (1 for Tun-8,8 and 1 for TUN-10,10) that grew after 4-5 weeks of incubation were picked, grown up in 7H9/ADC/Tw liquid medium and resistance confirmed by MIC determination as described above. The observed frequency of resistance was 10-9 with 16-fold level of resistance recorded to the TUN-analogues. Genomic DNA was purified by the CTAB method (van Soolingen et al., 1991) and whole genome sequencing by Illumina MiSeq and assembly of paired-end reads by SPAdes performed as previously described (Weingarten et al., 2018). A resistant mutant raised against TUN-10,10 had a SNP at position 842361 (C to G) resulting in an Ala to Gly mutation in Rv0751c. A resistant mutant raised to TUN-8,8 had a T to A SNP at position 3336597 which is in the intergenic region between Rv2980 and Rv2981c.