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

[摘要] 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.

[发布机构] Stellenbosch University