Contribution of efflux to the emergence of isoniazid and multidrug resistance in Mycobacterium tuberculosis

Abstract

Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment.

Figure 1. Schematic representation of exposure of strain H37Rv to 0.1 µg/ml INH using the BACTEC™ MGIT™ 960 and characterization assays performed at selected points.

For each strain, exposure to INH was done in duplicate, in two independent assays - processes A and B. INH(a): exposure process A; INH(b): exposure process B; INH: isoniazid; EI: efflux inhibitor. Vertical arrows represent transfer to new MGIT tubes containing 0.1 µg/ml INH. Seq: nucleotide sequence determination for specific fragments of the genes involved in the resistance to INH; AST: susceptibility testing to all first line antibiotics. MIC_INH: minimum inhibitory concentration determination of isoniazid. Note: This same procedure, here depicted as an example, was carried out for isoniazid exposure of each strain involved in this study.

Figure 2. Map of the region deleted in the M. tuberculosis H37Rv reference strain as a result of the exposure to isoniazid.

The region analyzed spans from positions 5′-2150314 to 5′-2159943 of the M. tuberculosis H37Rv genome sequence , adapted from Tuberculist, 2010, http://tuberculist.epfl.ch/. The area delimited corresponds to the fragment deleted in strain H37Rv_INH(a)3.

Figure 3. Accumulation of EtBr by the M. tuberculosis strains tested.

The figure shows the accumulation of EtBr by the strains from adaptation process A as an example. The values at bold type correspond to the higher concentration of EtBr that cells can handle without detectable accumulation. The dotted line corresponds to the assay run using the EtBr concentrations for which influx-efflux are at equilibrium, in the presence of the EI verapamil, at sub-inhibitory concentrations. Panel (A): Parental strains (passage #0); Panel (B) strains after first passage with INH and Panel (C); strains after 26 passages with INH. INH: isoniazid.