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.
Highlights
Tuberculosis (TB) remains a serious public health threat around the world, and according to the World Health Organization, nearly two billion people are infected with Mycobacterium tuberculosis, with about 8.8 million of new TB cases and 1.3 million deaths in 2010 [1]
We further demonstrate that this isoniazid induced resistance can be reverted by efflux inhibitors, supporting their role as adjuvants in anti-tuberculosis therapy and prevention of Multidrug resistant (MDR)-TB emergence
Exposure to isoniazid Two M. tuberculosis strains susceptible to the first-line antibiotics and two clinical strains monoresistant to rifampicin were constantly exposed to the critical concentration of isoniazid, 0.1 mg/ml, during an extended period of time – see Figure 1
Summary
Tuberculosis (TB) remains a serious public health threat around the world, and according to the World Health Organization, nearly two billion people are infected with Mycobacterium tuberculosis, with about 8.8 million of new TB cases and 1.3 million deaths in 2010 [1]. Chromosomal gene mutation has been considered the single cause for antibiotic resistance in M. tuberculosis, with multidrug resistance arising as a consequence of sequential accumulation of spontaneous mutations in target genes [2]. The main mechanism of resistance to isoniazid is the occurrence of mutations in its activator, KatG [6,8], whereas mutations in the inhA gene represent the second most common mechanism. Together, mutations in these two genes are responsible for approximately 75% of the cases of M. tuberculosis resistance to isoniazid in the clinical setting [9]. Resistance to isoniazid has been associated with mutations in several other genes (e.g. ndh, kasA and oxyR–ahpC intergenic region) [10], but its direct association with resistance is still unclear
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