Objectives: Candida glabrata is among the second or third most commonly isolated yeast species causing candidemia and other invasive infections in critically ill patients. The species is intrinsically less susceptible to azole antifungal drugs and increasing reports of breakthrough infections in patients receiving echinocandins have been described in recent years limiting the choice of antifungal drugs to treat such infections. This study investigated the molecular mechanisms responsible for reduced susceptibility to amphotericin B (RS-AMB) in a collection of amphotericin B-resistant C. glabrata strains isolated from different patients in Kuwait.
Methods: Twelve C. glabrata strains with RS-AMB were used. Six amphotericin B susceptible isolates were also analyzed for comparison purposes. Antifungal susceptibility testing was done by Etest and/or by Vitek 2 system. The sequences of three (ERG2, ERG6 and ERG11) genes involved in ergosterol biosynthesis were determined. Sterol content of the isolates was analyzed by gas chromatography-mass spectrometry (GC-MS). Phylogenetic relationship among the isolates was investigated by multilocus sequence typing.
Results: None of the six amphotericin B-susceptible isolates contained a resistance conferring mutation in ERG2, ERG6 or ERG11 and the sterol content of two representative isolates was similar to that of the wild-type reference strain (ATCC90030). Among RS-AMB isolates, four isolates contained a nonsynonymous (AGA48AAA, R48K) mutation in ERG6. however, this mutation was also present in three amphotericin B-susceptible isolates. Three isolates contained a nonsense mutation (two isolates at codon Trp286 and one isolate at codon Tyr192), one isolate (Kw105-3/13) contained deletion of a nucleotide (nucleotide 1021) resulting in premature termination due to frame shift at codon Leu341 and two other isolates contained a nonsynonymous (V126F or C198F) mutation in ERG6. The sterol content of these six isolates were consistent with ERG6 deficiency. Two other isolates contained a nonsynonymous (G119S or G122S) mutation and the sterol content of these two isolates were also consistent with ERG2 deficiency. Isolate Kw105-3/13 also contained Y141H + L381M mutations while another isolate contained a nonsense mutation at codon 495 in ERG11. The remaining 10 RS-AMB isolates did not contain any nonsynonymous mutation in ERG11. Multilocus sequence typing data showed that nearly all individual patient isolates and all isolates with ERG6 or ERG2 or ERG11 mutations were genotypically distinct strains.
Conclusions: We have identified the molecular basis of decreased susceptibility to amphotericin B among clinical C. glabrata isolates. Our data show that ERG6 and ERG2 are major targets conferring resistance to amphotericin B in eight of 12 isolates. Four isolates involved novel nonsense/deletion mutations in ERG6 that resulted in truncation of the ERG6 protein and consequent changes in the sterol content of the cells. Two other isolates contained a novel nonsynonymous mutation in ERG6 including one isolate with C198F mutation that has been described previously. Interestingly, two other isolates contained nonsynonymous (G119S or G122S) mutations in the same region (Thr121) of ERG2 protein that has previously been shown to be involved in causing reduced susceptibility to amphotericin B and changes in the sterol composition of C. glabrata.

ERG6 and ERG2 are major targets conferring reduced susceptibility to amphotericin B in clinical Candida glabrata isolates