Defining the role of efflux pump inhibitors on anti-TB drugs in Rifampicin resistant clinical Mycobacterium Tuberculosis isolates

Pule, Caroline (2014-04)

Thesis (MScMedSc)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: Central dogma suggests that mutations in target genes is the primary cause of resistance to first and second-line anti-TB drugs in Mycobacterium tuberculosis. However, it was previously reported that approximately 5% of Rifampicin mono-resistant clinical M. tuberculosis did not harbor mutations in the rpoB gene. The present study hypothesized that active efflux plays a contributory role in the level of intrinsic resistance to different anti-TB drugs (Isoniazid, Ethionamide, Pyrazinamide, Ethambutol, Ofloxacin, Moxifloxacin, Ciprofloxacin, Streptomycin, Amikacin and Capreomycin in RIF mono-resistant clinical M. tuberculosis isolates with a rpoB531 (Ser-Leu) mutation. This study aimed to define the role of Efflux pump inhibitors (verapamil, carbonylcyanide m-chlorophenylhydrazone and reserpine) in enhancing the susceptibility to different anti-TB drugs in the RIF mono-resistant clinical isolates. The isolates were characterized by determining the level of intrinsic resistance to structurally related/unrelated anti-TB drugs; determining the effect of EPIs on the level of intrinsic resistance in the isolates and comparing the synergistic properties of the combination of EPIs and anti-TB drugs. To achieve this, genetic characterization was done by PCR and DNA sequencing. Phenotyping was done by the MGIT 960 system EpiCenter software to determine the MICs of the different anti-TB drugs and the effect of verapamil and carbonylcyanide m-chlorophenylhydrazone on determined MICs. Due to inability to test reserpine in a MGIT, a different technique (broth microdilution) was used for the reserpine experiment. Additionally; fractional inhibitory concentrations (FIC) indices were calculated for each of these drugs. The FIC assess the anti-TB drugs/inhibitor interactions. STATISTICA Software: version 11 was used for statistical analysis. Results revealed that the RIF mono-resistant isolates were sensitive at the critical concentrations of all 10 drugs tested, with the exception of Pyrazinamide. This could be explained by the technical challenges of phenotypic Pyrazinamide testing. A significant growth inhibitory effect was observed between the combination of EPI and anti-TB drug exposure in vitro. This suggests that verapamil, carbonylcyanide m-chlorophenylhydrazone and reserpine play a significant role in restoring the susceptibility (decrease in intrinsic resistance level) of the RIF mono-resistant isolates to all anti-TB drugs under investigation. Additionally, a synergistic effect was observed by the combination treatment of the anti-TB drugs with the different EPIs. Based on these findings, we proposed a model suggesting that efflux pumps are activated by the presence of anti-TB drugs. The activated pumps extrude multiple or specific anti-TB drugs out of the cell, this in turn decrease the intracellular drug concentration, thereby causing resistance to various anti-TB drugs. In contrast, the addition of EPIs inhibits efflux pump activity, leading to an increase in the intracellular drug concentration and ultimate cell death. This is the first study to investigate the effect of different efflux pumps inhibitors on the level of intrinsic resistance to a broad spectrum of anti-TB drugs in drug resistant M. tuberculosis clinical isolates from different genetic backgrounds. The findings are of clinical significance as the combination of treatment with EPI and anti-TB drugs or use of EPIs as adjunctives could improve MDR-TB therapy outcome.

AFRIKAANSE OPSOMMING: Sentrale dogma beweer dat mutasies in teiken gene die primêre oorsaak van die weerstandheid teen anti-TB-middels in Mycobacterium tuberculosis is. Vorige studies het getoon dat ongeveer 5% van Rifampisien enkelweerstandige kliniese M. tuberculosis isolate nie ‘n mutasie in die rpoB geen het nie. Die hipotese van die huidige studie was dat aktiewe pompe 'n bydraende rol speel in die vlak van intrinsieke weerstandheid teen 10 verskillende anti-TB-middels (Isoniasied, Ethionamied, Pyrazinamied, Ethambutol, Ofloxacin, Moxifloxacin, Siprofloksasien, Streptomisien, Amikasien and Capreomycin) in RIF enkelweerstandige kliniese M . tuberculosis isolate met 'n rpoB531 (Ser-Leu) mutasie. Die doel van hierdie studie was om die rol van uitpomp inhibeerders (verapamil, carbonylcyanide m-chlorophenylhydrazone en reserpien) te definieer in die verbetering van die werking vir verskillende anti-TB-middels in die RIF enkelweerstandige kliniese isolate. Die doelstellings van die studie was om die vlak van intrinsieke weerstandigheid teen struktureel verwante/onverwante anti-tuberkulose middels asook die effek van die EPIs op die vlak van intrinsieke weerstand in die isolate is bepaal. Verder is sinergistiese eienskappe van die kombinasie van EPIs en anti-TB-middels ondersoek. Hierdie doelstellings is bereik deur genetiese karakterisering deur PKR en DNS volgorde bepaling. Fenotipering is gedoen deur gebruik te maak van MGIT 960 EpiCenter sagteware om die Minimum Inhibisie Konsentrasie (MIC) van die verskillende anti-TB-middels en die effek van verapamil en carbonylcyanide m-chlorophenylhydrazone op die MIC te bepaal. Reserpien kan nie in die MGIT sisteem getoets word nie, and daarom is 'n ander tegniek (mikro-verdunning) is gebruik om die effek van reserpien te toets. Fraksionele inhiberende konsentrasies (FIC) is bereken vir elk van hierdie middels die anti-TB-middels / inhibeerder interaksies te bepaal. STATISTICA v11 sagteware is gebruik vir alle statistiese analises. Resultate van hierdie studie toon dat die RIF enkelweerstandige isolate sensitief is teen kritieke konsentrasies van al die middels, met die uitsondering van Pyrazinamied. Weerstandigheid van Pyrazinamied kan wees as gevolg van welbekende tegniese probleme met die standaard fenotipiese pyrazinamied toets. ‘n Beduidende groei inhiberende effek is waargeneem tussen die kombinasie van EPI en anti-TB middel blootstelling in vitro. Dit dui daarop dat verapamil, CCCP en reserpine 'n belangrike rol speel in die herstel van die sensitiwiteit (afname in intrinsieke weerstand vlak) van die RIF enkelweerstandige isolate aan alle anti-TB-middels wat ondersoek is. Daarbenewens is 'n sinergistiese effek waargeneem deur die kombinasie van die verskillende anti-TB-middels en die verskillende EPIs. Op grond van hierdie bevindinge het ons ‘n model voorgestel wat toon dat uitvloei pompe geaktiveer word deur die teenwoordigheid van anti-TB-middels en die geaktiveerde pompe dan verskeie of spesifieke anti-TB-middels uit die sel pomp. Dus verminder die intrasellulêre konsentrasie van die middel en veroorsaak daardeur weerstandigheid teen verskeie anti-TB-middels. Die byvoeging van EPIs inhibeer uitvloei pompe se werking en lei tot 'n toename in die intrasellulêre konsentrasie van die middels en uiteindelik die dood van die selle. Hierdie is die eerste studie wat die effek van verskillende uitvloei pompe inhibeerders op die vlak van intrinsieke weerstand teen 'n breë spektrum van anti-TB-middels in die middel-weerstandige kliniese isolate ondersoek. Die bevindinge kan van belangrike kliniese belang wees aangesien die kombinasie van behandeling met EPI en anti-TB-middels die uitkoms MDR-TB terapie kan verbeter.

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