INVESTIGATIONS ON THE ROLES OF EFFLUX PUMP INHIBITORS ON THE ANTIBIOTIC TOLERANCE OF NON-REPLICATING MYCOBACTERIUM SMEGMATIS

Normal healthy people are not susceptible to tuberculosis (TB) but immunocompromised and HIV positive patients are at high risk of TB. The treatment regimen (rifampin, isoniazid and amikacin) for TB patients is 6-9 months for normal patients but if Mycobacterium tuberculosis (Mtb) becomes multidrug resistant, it takes 20-30 months to treat. According to the World Health Organization in 2018, there were about half a million new cases among which 78% were multidrug resistant TB. This antibiotic resistance is due in part to its ability to survive in the macrophage in our body by entering a non-replicating persistent state. Mtb also contains efflux pumps that increase antibiotic tolerance by pumping out the drugs. Therefore, if the efflux pump activity can be blocked by using efflux pump inhibitors, then it might increase antibiotic susceptibility of the pathogen. In our study, we used Mycobacterium smegmatis (Msm) as a model organism for Mtb and subjected it to a combination of three stresses (low oxygen, low pH and low nutrients) that mimic the physiological stresses in the human body and report that these conditions produced a non-replicating state in Msm. This is the first report of the use of this combination of stresses to produce a non-replicating state in Msm. Our results show that non-replicating Msm became completely tolerant to isoniazid and displayed increased tolerance to rifampin and clarithromycin by nearly 2-fold when compared to log-phase cells. Moreover, the efflux pump inhibitor verapamil decreased the antibiotic tolerance of the nonreplicating Msm to the antibiotics by 6-10 fold and the efflux pump inhibitor piperine decreased tolerance to the antibiotics by 2-4 fold. Also, in this study we attempted to construct a gene knockout mutant lacking two potential ATP-binding cassette transporters to study their functions as drug exporters. However, we were unable to obtain homologous recombination mutants. Further studies on efflux pump inhibitors could potentially enable greater understanding of antibiotic tolerance mechanisms in non-replicating, drug tolerant Mtb and enable the development of novel therapies that shorten treatment time for tuberculosis.