Relationship between Efflux Pump Expression and Antibiotic Susceptibility in an in-Vitro Evolved Drug Resistant Mycobacterium to Develop Better Drug Targeting Strategies.

Multidrug resistance poses a significant challenge in the treatment of tuberculosis. To address this epidemic, many approaches are currently being tried, such as repurposing of existing drugs and combination therapy by combining exiting drugs with adjuvants. Through many genomic studies, it is now clear that resistance is a multifaceted phenomenon, and in addition to mutations in the drug target genes, many mechanisms are at play in multidrug-resistant organisms. Understanding the alternate mechanisms of resistance will provide us with novel targets for the development of new bactericidal drugs.

In this work, we have investigated the contribution of efflux mediated resistance in multidrug resistance/ extremely drug resistance (MDR/XDR) Mycobacterium. Mycobacterium smegmatis mc² 155 (WT cells), our model organism was subjected to increasing concentrations of a Fluoroquinolone (FQ) – Norfloxacin, and a mutant was isolated, named as Nor^r Mutant. This mutant exhibited increased resistance towards many drugs, including FQ’s and other first and second-line drugs from the current tuberculosis regime. Interestingly, no mutations were identified in any of the drug target genes on sequencing such as gyrA, gyrB, katG, and rpoB. However, the mutant exhibited decreased intracellular drug uptake. Alteration in drug transport was also confirmed through drug transport studies using a fluorescent tracer (Ethidium bromide-EtBr). This observation suggested that either the uptake or efflux of the drug was affected in the mutant. Also, diffusivity across the membrane was measured using 1-N-Phenyl-Naphthylamine (NPN) dye, which was found to be similar to that of WT, which further indicated the role of drug efflux in imparting resistance. The expression level of many efflux pumps was measured that showed upregulation of the basal levels of multiple pumps in the mutant.

To investigate efflux-pump mediated resistance, we integrated another copy of selected efflux pump genes in the genome under an inducible promoter. Three efflux-pumps were selected from among those that were differentially expressed in the drug-resistant mutant. Over-expression of each of the pumps led to increased resistance that was proportional to the expression level of the efflux pump. Thus, a quantitative relationship between MIC of various drugs and the expression level of efflux pumps could be established. Next, to identify the regulator(s) leading to this enhanced expression of efflux pumps, the mutant was sequenced on the Illumina platform. While multiple mutations were observed, the mutation in a global regulator was prominent. The gene for the regulator was also expressed under an inducible promoter, resulting in increased susceptibility of the bacteria to multiple drugs, with differential regulation of multiple efflux-pumps. Targeting this global regulator could be an alternate strategy to counter this multidrug-resistant strain.

Thus, combined therapy targeting such transcriptional regulators along with efflux pump inhibitors can be a powerful tool to combat drug resistance in mycobacterium. Overall, this work established how non-target gene-based mechanisms, such as multiple efflux-pumps, contribute to the MDR/XDR phenotype of Mycobacteria. This knowledge will help us to develop alternate strategies to target resistant Mycobacteria.