Determination of the mechanism of synergy of SQ109 with rifampicin and isoniazid in Mycobacterium smegmatis

Manunu, Bayanika (2015-03)

Thesis (MSc)--Stellenbosch University, 2015

Thesis

ENGLISH ABSTRACT : Multidrug resistance tuberculosis (MDR-TB) is a serious concern in the public health environment globally and the understanding of its mechanisms may help to prevent the emergence and spread of resistant strains of Mycobacterium tuberculosis (Mtb). Several compounds are being tested in clinical trials and SQ109 was identified as a promising new anti-TB drug because of its bactericidal activity against Mtb and demonstrated synergistic activity with the fist-line TB drugs. This study focussed on the mechanism of synergy of SQ109 with rifampicin (RIF) and isoniazid (INH) in Mycobacterium smegmatis (Msmeg). The influence of SQ109 on efflux in Msmeg was evaluated using two approaches. Firstly, accumulation and efflux of ethidium bromide (EtBr) was monitored using a semi-automated fluorometric assay and secondly efflux and accumulation of RIF in Msmeg was assessed using tandem mass spectrometry. Although SQ109 resulted in a slight decrease in EtBr efflux by Msmeg in some of the assays performed, this decrease was not consistently seen. SQ109 appeared to have no significant influence on the efflux or accumulation of RIF in Msmeg, suggesting that it does not act to inhibit efflux in this organism. Six spontaneous SQ109-resistant mutants were generated in Msmeg and bactericidal activity of SQ109, RIF and INH against wild-type and mutant strains of Msmeg was assessed. The minimum inhibitory concentrations (MICs) for all three drugs increased in the mutant strains compared to the wild-type. Drug-drug interaction studies performed on one of the SQ109-resistant mutants revealed a change from synergy to additivity for both SQ109/RIF and SQ109/INH combinations, suggesting that identification of the genes harbouring mutations in these strains would shed light on the mechanism of synergy of SQ109 with RIF and INH. Sanger sequencing revealed that none of the SQ109-resistant mutants harboured mutations in MSMEG_0250 (mmpL3 homologue), a gene previously implicated in SQ109 resistance in M. tuberculosis. Preliminary whole genome sequencing data for six SQ109-resistant mutants identified SNPs in 10 genes, however the role of these genes in SQ109 resistance and synergy with RIF and INH in Msmeg remains to be verified.

AFRIKAANSE OPSOMMING : Multi-middel weerstandige tuberkulose (MDR-TB) is ‘n ernstige probleem in globale publieke gesondheid. Kennis van die meganisme van middelweerstandigheid kan help om die ontwikkeling en versprei van weerstandige Mycobacterium tuberculosis (Mtb) te voorkom. Verskeie middele word tans in kliniese proewe getoets. SQ109 is identifiseer as ‘n belowende nuwe anti-TB middel as gevolg van die kiemdodende aktiwiteit wat dit teen Mtb toon en die sinergistiese aktiwiteit wat dit met die eerstelyn TB middele toon. Hierdie studie fokus op die meganisme van sinergie van SQ109 met rifampisien (RIF) en isoniasied (INH) in Mycobacterium smegmatis (Msmeg). Twee benaderings is gebruik om die invloed van SQ109 op effluks in Msmeg te evalueer. Eerstens is opbou en effluks van ethidium bromied (EtBr) gemonitor deur van ‘n semi-outomatiese fluorometriese toets gebruik te maak. Tweedens is effluks en opbou van rifampicin (RIF) in Msmeg ondersoek deur van tandem massaspektrometrie gebruik te maak. Alhoewel SQ109 ‘n effense afname in EtBr effluks in Msmeg in sommige van die eksperimente veroorsaak het, is die afname nie herhaaldelik deur al die eksperimente gesien nie. Dit het geblyk dat SQ109 geen beduidende invloed op effluks of opbou van RIF in Msmeg gehad het nie, wat daarop dui dat dit nie as ‘n effluks inhibeerder in die organisme optree nie. Ses spontane SQ109 weerstandige mutante is in Msmeg gegenereer en die kiemdodende aktiwiteit van SQ109, RIF en INH teen die wilde-tipe en mutante is ondersoek. Die minimum inhiberende konsentrasie (MIC) vir al drie middels is verhoog in die mutante in vergelyking met die wilde-tipe. Middel-middel interaksie studies uitgevoer vir een van die SQ109 weerstandige mutante het getoon dat daar ‘n verandering van sinergie to additiwiteit vir beide SQ109/RIF en SQ109/INH kombinasies was. Dit het voorgestel dat die identifisering van gene waarin mutasies voorkom in die SQ109 mutante kan lei tot die identifisering van die meganisme van sinergie van SQ109 met RIF en INH. Sanger DNA volgordebepaling het getoon dat geen van die SQ109 mutante mutasies in die MSMEG_0250 (mmpL3 homoloog), ‘n geen wat voorheen geassosieer is met SQ109 weerstandigheid in M. tuberculosis, gehad het nie. Met voorlopige heel genoom volgorde bepaling vir die ses SQ109 mutante is SNPs in 10 gene identifiseer, maar die rol van die gene in SQ109 weerstandigheid en sinergie met RIF en INH in Msmeg moet verifieer word.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/96616
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