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Resistance to multiple drugs is one of the significant barriers in the treatment of tuberculosis (TB). Knowledge of mechanisms of resistance is important to design effective treatment strategies. While mutations in genes coding for drug targets are thought to be the primary source of drug resistance, absence of mutations in these genes in many clinical strains suggests additional mechanisms of resistance. In this study, we employ adaptive laboratory evolution ofMycobacterium smegmatisto understand alternate mechanisms of drug resistance to norfloxacin, a fluoroquinolone (FQ). Results show that, in addition to fluoroquinolones, the evolved strain, Norr, is resistant to first-line drugs, rifampicin and isoniazid, and a second-line drug (amikacin), exhibiting extreme drug resistance phenotype. However, mutations were absent in any of the drug target genes. Drug uptake studies revealed that resistance is an attribute of decreased intracellular accumulation, primarily due to increased efflux. Further, drug transport kinetics demonstrate the involvement of efflux mediated resistance, which was found to be reversed in the presence of efflux pump inhibitors (EPIs). Gene transcript analysis suggests differential upregulation of multiple efflux pumps across the genome of the mutant. Overexpression of one of the upregulated efflux pumpsMsmeg_5659-5661, partially explains the XDR phenotype of the mutant, while also suggesting that the contribution of other efflux pumps is significant. Whole-genome sequencing (WGS) of Norrreveals that a mutation insoxR, a transcriptional regulator, could be responsible for the upregulation of theMsmeg_5659-5661 efflux pump by direct regulation, and other efflux pumps via indirect regulation. Thus, the present work demonstrates that high resistance to multiple drugs can arise even when theMycobacteriumwas subjected to a single selection pressure. Further, alterations in drug transport is an important mechanism that leads to resistance to multiple drugs simultaneously. |
