Evaluation of Mycobacterium tuberculosis associated with treatment failure for intrinsic and efflux pump mediated resistance: a comparative retro perspective cohort study

Abstract

Background: To treat tuberculosis is very complicated and difficult procedure that involves the administration of a panel of five antimicrobial drugs for the period of 6 months. The purpose of this study was to determine antimicrobial drug resistant features of Mycobacterium tuberculosis associated with treatment failure and to determine efficacy of the second line drugs and the efflux pump inhibitor verapamil against M. tuberculosis associated with treatment failure.Methods: The identity of isolates was confirmed by ZN staining and multiplex PCR through detection of Mycobacterium species specific loci rv0577, mtbk_20680, 16S rRNA, RD9, IS 1311, mass_3210 and mkan_rs12360. Drug susceptibly testing (DST) and efficacy of the efflux pump inhibitor verapamil were performed through MGIT 960. Mutations associated with drug resistance were determined through DNA sequencing of ropB, katG, pncA, rrs and eis loci. The transcription of efflux pump gene rv1258 was assessed by real time quantitative PCR. Results: Upon monitoring 1200 tuberculosis patients, 64 were found not-cured after six months of treatment course. From M. tuberculosis isolates recovered from sputum of these 64 patients, 3.1% isolates were detected resistant to four anti M. tuberculosis drugs (extreme drug resistant) 48.4% were resistant to three anti M. tuberculosis drugs (extensive drug resistant), 26.5% were resistant to two anti M. tuberculosis drugs (multi drug resistant). High frequency of resistance to the second line drug amikacin was detected in 26,5% isolates whereas moxifloxacin and linezolid resistance was detected in only 3.1% isolates. The Serine 315 in katG was the most frequent amino acid mutated in treatment failure group. Three novel mutations were detected at codons 99, 149 and 154 in pncA associated with pyrazinamide resistance. Rifampicin and isoniazid enhanced the transcription of the efflux pump gene rv1258 in drug susceptible isolates collected from the treatment failure patients whereas verapamil reduced minimum inhibitory concentrations of antimicrobial drugs in these isolates.Conclusion: The use of Amikacin as a second line drug is not appropriate as compare to moxifloxacin and linezolid. Verapamil enhanced anti-bacterial activity of rifampicin and isoniazid in drug susceptible M. tuberculosis isolates cured from treatment failure patients but not in drug resistant isolates.