ERG6 and ERG2 are major targets conferring reduced susceptibility to amphotericin B in clinical Candida glabrata isolates in Kuwait. Molecular basis of resistance to amphotericin B in Candida glabrata

Objectives: Candida glabrata is intrinsically less susceptible to azoles and resistance to echinocandins and amphotericin B has also been detected. Molecular mechanisms of reduced susceptibility (RS) to amphotericin B (AMB) were investigated in C. glabrata strains in Kuwait by sequence analyses of genes involved in ergosterol biosynthesis. Methods: Eight RS-AMB and 5 AMB-susceptible C. glabrata isolates were used. Antifungal susceptibility testing was done by Etest and by reference broth microdilution. PCR-sequencing of three (ERG2, ERG6 and ERG11) genes was performed by using gene-specific primers. Total cell sterol content was analyzed by gas chromatography-mass spectrometry. Phylogenetic relationship among the isolates was investigated by multilocus sequence typing. Results: AMB-susceptible isolates contained only synonymous mutations in ERG2, ERG6 or ERG11 and total sterol content of 1 isolate was similar to reference strain. A nonsynonymous (AGA48AAA, R48K) ERG6 mutation was found in both RS-AMB and AMB-susceptible isolates. Four RS-AMB isolates contained novel nonsense mutations at Trp286/Tyr192/Leu341 and two isolates contained nonsynonymous (V126F or C198F) mutation in ERG6 and their sterol content were consistent with ERG6 deficiency. Two other RS-AMB isolates contained novel nonsynonymous (G119S or G122S) ERG2 mutation and their sterol content were consistent with ERG2 deficiency. Isolate Kw861/13 also contained Y141H + L381M mutations while 7 RS-AMB isolates contained only synonymous mutations in ERG11. All isolates with ERG6/ERG2/ERG11 mutations were genotypically distinct strains. Conclusions: Our data show that ERG6 and ERG2 are major targets conferring RS-AMB in clinical C. glabrata isolates in Kuwait.