Raw sequence reads from Mycobacterium tuberculosis OH190 resistant isolates

OH190 is a synthetic analogue of pyridomycin, whose anti-tuberculosis activity appeared unchanged by episomal overexpression of the pyridomycin target, the enoyl-reductase InhA. In search of alternative/additional protein targets, OH190 and pyridomycin resistant H37Rv isolates were selected on plates containing 10 mg/L and 20 mg/L OH190 and pyridomycin, respectively. For both antibiotics, no colonies were isolated at 40 mg/L. Three OH190 resistant clones randomly selected (from 122 resistant clones) were confirmed to have low level resistance (2-fold) to OH190 and to pyridomycin, while the three selected pyridomycin resistant isolates were >16-fold more resistant to pyridomycin and 8-fold more resistant to OH190. Sanger sequencing of InhA and its promoter confirmed that all three OH190 resistant isolates were wild-type for this DNA region, while all three pyridomycin resistant strains carried a previously undescribed mutation in InhA, namely, a481c (M161L). Whole genome sequencing and variant analysis of the three OH190 resistant isolates and the parental H37Rv wild-type strain, found that 2 of the resistant isolates (named OH-RC-10.4, and OH-RC-20.1) carried a unique non-synonymous SNP in Rv0678 (c212a [A71D] and a208g [N70D] respectively, both located in the helix-turn-helix DNA binding domain). No variants (SNP or small In/Del) were found in the third OH190 resistant isolate (named OH-RC-10.5). Due to the mutations identified in Rv0678, the sequence reads of OH-RC-10.5 mapped to this region were scrutinised. The read depth between bases 28-32 of Rv0678 was found disproportionately higher, and the reads only partially mapped to the reference gene. The unmapped portion of the partially mapped reads was found to align to the extremities of an IS6110, clearly indicating its intragenic insertion in this resistant clone. This finding was confirmed by an increase in the size of a PCR product for Rv0678 and subsequent Sanger sequencing. Rv0678 codes for a transcriptional repressor whose loss of function leads to overexpression of the efflux pump mmpL5/S5, which in turn results in low level resistance to the anti-tuberculosis drugs bedaquiline and clofazimine. The identification of this IS6110 mediated inactivation of Rv0678 and consequential resistance to OH190, led us to question if such IS6110 mediated resistance is also observed clinically.