Single nucleotide polymorphisms in efflux pumps genes in extensively drug resistant Mycobacterium tuberculosis isolates from Pakistan.

It is challenging to understand mechanisms of drug resistance in Mycobacterium tuberculosis (MTB) due to the large variability in resistance associated genes. Efflux pump genes contribute to drug resistance and thus add to this complexity. Efflux pump gene protein superfamilies have been characterized by genome analysis of drug resistant strains and through in vitro transcriptional studies. However, there is limited information regarding efflux pump genes in extensively drug resistant (XDR) tuberculosis (TB) isolates. Whole genome sequencing (WGS) based analysis of 37 extensively drug resistant (XDR) and five drug sensitive (DS) MTB clinical isolates was performed. Single nucleotide polymorphisms (SNPs) in efflux pump genes Rv0194, Rv1217, Rv1218, drrA, drrB, Rv1258, Rv1634, Rv2688, Rv1273, Rv1819, Rv1458, Rv1877 and Rv1250 were determined in the clinical isolates as compared with the H37Rv reference strain. Allele frequencies of SNPs identified in XDR strains were compared with DS strains. Gene expression of Rv0194, Rv2688, Rv1634, drrA and drrB was determined in XDR -TB isolates (n = 9), DS-TB strains (n = 4) and H37Rv. We identified SNPs in XDR-TB isolates which were either unique or present at very low frequencies in DS strains; Rv0194 G170V; Rv1217 L151R; Rv1258 P369T and G391R; Rv1273 S118G and I175T; Rv1877 I534T; Rv1250 V318X/A and S333A, and Rv2688 P156T. The expression of Rv2688 and drrB was found to be raised in XDR-TB as compared with DS-TB strains. We identified unique SNPs in efflux pump genes which may be associated with increased drug resistance in the isolates. Increased levels of Rv2688 and drrB efflux pump gene expression observed in XDR strains even in the absence of antibiotics suggests that these clinical isolates may be more refractory to treatment. Further studies are required to directly associate these mutations with increased resistance in MTB