Characterization of efflux pumps genes involved in second-line drug resistance of tuberculosis

Thesis (PhD)--University of Pretoria, 2017.In this study, we used molecular and proteomic methods to detect novel changes within drug-resistant tuberculosis (TB) strains. Our goal was to detect changes within efflux pump (EP) genes of extensively drug resistant (XDR-TB) strains using genomic and transcriptomic methods. We firstly sequenced multiple genes in discordant samples that lacked molecular markers present on the GenoType® MTBDRsl assay. Further analysis by whole genome sequencing was done on XDR-TB strains. Transcriptomic changes of EP genes were detected by RNA sequencing strains. The minimum inhibitory concentration (MIC) of second-line drugs in the presence and absence of efflux pump inhibitors was also measured. Proteomic expression by cloning and expression of three efflux pump genes (Rv1258c, Rv1634 and Rv0194) was done and effect on MICs of second-line drugs measured in the presence and absence of efflux pump inhibitors. We analysed molecular markers responsible for resistance to second-line drugs of ofloxacin (OFX), amikacin (AMK), kanamycin (KAN) and capreomycin (CAP) in 636 drug-resistant strains using GenoType® MTBDRsl. We compared GenoType® MTBDRsl with phenotypic second-line drug susceptibility testing. After comparison, 102 (14.8%) strains were discordant between the two methods. In the discordant population, genetic regions of gyrA, gyrB, rrs, eis, tlyA and EP genes (Rv1634, Rv1258c and Rv0194) were sequenced and analysed in search of mutations. Combining sequencing and GenoType® MTBDRsl significantly improved the diagnosis of XDR-TB and second-line drugs. The Rv0194 belongs to the ATP (adenosine triphosphate) binding cassette (ABC) family while Rv1258c and Rv1634 belong to the major facilitator superfamily (MFS) transporters. Since these genes are implicated in multiple drug resistance, our hypothesis was that possible mutations in these genes could confer cross-resistance. Our analysis revealed the appearance of Rv1258c and Rv0194 mutations in strains with cross-resistance to second-line injectable drugs. Further analysis revealed rrs G878A mutation that was specific to EuroAmerican X3 lineage (P