Differential expression of efflux pump genes of Mycobacterium tuberculosis in response to varied subinhibitory concentrations of antituberculosis agents - 13/03/15

Doi : 10.1016/j.tube.2015.01.005

Kushal Garima ^a, Rakesh Pathak ^a,^{^{1
Present address: Department of Biological Science, Oakland University, MI, USA.}}, Rashmi Tandon ^b, Nisha Rathor ^a, Rajesh Sinha ^a,^{^{2
Present address: Biomedical Sciences, University of Central Florida, Orlando, FL, USA.}}, Mridula Bose ^a, Mandira Varma-Basil ^a,^⁎
^a Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India
^b Department of Biochemistry, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India

^∗Corresponding author. Department of Microbiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110007, India. Tel.: +91 11 27402417; fax: +91 11 27666549.

Summary

Several reports have elaborated on the role of efflux pumps in drug resistance in Mycobacterium tuberculosis by analysing the mRNA expression profiles. However, there is no uniformity in the subinhibitory concentrations of drugs chosen in these studies. Some investigators studied the expression of efflux pumps under a drug concentration of 1/2 minimum inhibitory concentration (MIC), while others used 1/3, 1/4 or 1/8MIC. The present study was planned to understand the effect of different concentrations of antituberculosis drugs on the expression of efflux pump genes. Log phase culture of the laboratory strain M. tuberculosis H37Rv was exposed to rifampicin (RIF), isoniazid (INH), streptomycin (SM) and ethambutol (EMB) at different drug concentrations (1/2MIC, 1/3MIC and 1/4MIC). The expression of 10 putative efflux pump genes was studied using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). We observed an optimal expression of efflux pumps at higher concentrations of INH; and at lower concentrations of RIF and EMB. However, in the presence of SM, a decreased expression of efflux genes with increasing concentrations of the drug was confounded by a significant reduction in Colony Forming Units (CFU).

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Keywords : Mycobacterium tuberculosis, Antituberculosis agents, Efflux pumps

Plan

Introduction

Materials and methods

Selection of putative efflux genes

Designing and synthesis of primers

Bacterial strain, growth conditions and reagents

MIC determination by Microplate Alamar Blue Assay (MABA)

In vitro drug exposure to M. tuberculosis H37Rv strain and expression study of putative efflux pump genes by qRT-PCR

Colony forming unit (CFU) analysis under different concentrations of drug pressure

Statistical analysis

Results

Selection of efflux pump genes

MIC determination for M. tuberculosis H37Rv

In vitro expression profile of drug efflux genes

Efflux pumps display multiple substrate specificities

CFU enumeration after exposure to different subinhibitory concentration of drugs

Discussion

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Vol 95 - N° 2

P. 155-161 - mars 2015 Retour au numéro

Article précédent

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Differential expression of efflux pump genes of Mycobacterium tuberculosis in response to varied subinhibitory concentrations of antituberculosis agents - 13/03/15

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