Emergence of antibiotic resistance is a major problem especially in the control of tuberculosis (TB) globally. Mycobacterium tuberculosis, the causative agent of TB develops resistance to multiple drugs simultaneously. Strains resistant to first-line anti-TB drugs, isoniazid and rifampicin, are referred to as multidrug-resistant TB (MDR-TB) and those resistant to fluoroquinolones and any one of the second-line injectable drugs in addition to isoniazid and rifampicin resistance are referred to as extensively drug-resistant TB (XDR-TB). In order to develop strategies to counter multiple drug resistance it is important to have in-depth knowledge of various mechanisms that makes the organism resistant to multiple drugs. In the current study, we use a combination of gene sequencing, expression analysis, biochemical studies and microscopy to understand the varied survival mechanisms that makes the mycobacteria tolerant to various anti-TB drugs.
For this purpose, we have created three in vitro random mutants in Mycobacterium smegmatis, a model mycobacteria, which can sustain a very high drug concentration in comparison to the wild type (WT) strain. The mutants were selected by exposing the wild type strain to any one of the three drugs, rifampicin, isoniazid and norfloxacin as selection pressure. Interesting, two of the finally selected strains were MDR strains with resistance to both rifampicin and isoniazid while the third mutant exhibited characteristics of XDR strain and was resistant to rifampicin, isoniazid, amikacin and many fluoroquinolones.
To understand the mechanism of drug resistance in these mutants, we sequenced the drug targets to check for mutations that may lead to drug insensitivity. Further, measurement of intracellular drug levels along with extensive transport studies using fluorescent tracer confirmed that the uptake or efflux of drugs in the mutants was effected either constitutively or induced in the presence of drug. Moreover, these results were confirmed at molecular level by performing the gene sequencing of major efflux pumps and porins, along with real-time PCR studies. The morphology of mutants was also altered, from rods to coccid or elongated rods, in response to high drug concentrations, indicating that alteration of cell morphology could be one of the mechanisms involved in multi-drug or extensively-drug resistant mycobacteria.
Thus, this study demonstrates the involvement of multiple mechanisms in development of multi-drug and extensively-drug resistant tuberculosis.