ERG6 and ERG2 are major targets conferring reduced susceptibility to amphotericin B in clinical Candida glabrata isolates in Kuwait [Mechanisms of Resistance]

Candida glabrata is intrinsically less susceptible to azoles and resistance to echinocandins and reduced susceptibility to 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. A total of 1646 C. glabrata isolates were tested by Etest and results for 12 selected isolates were confirmed by reference broth microdilution. PCR-sequencing of three (ERG2, ERG6 and ERG11) genes was performed for all RS-AMB and 5 selected wild-type C. glabrata isolates 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. Wild-type isolates contained only synonymous mutations in ERG2, ERG6 or ERG11 and total sterol content was similar to reference strains. A nonsynonymous (AGA48AAA, R48K) ERG6 mutation was found in both RS-AMB and wild-type isolates. Four RS-AMB isolates contained novel nonsense mutations at Trp286/Tyr192/Leu341 and 2 isolates contained nonsynonymous (V126F or C198F) mutation in ERG6 and their sterol content were consistent with ERG6 deficiency. Two other RS-AMB isolates contained a novel nonsynonymous (G119S or G122S) ERG2 mutation and their sterol content were consistent with ERG2 deficiency. Of 8 RS-AMB isolates, 1 fluconazole-resistant isolate also contained nonsynonymous Y141H+L381M mutations while 7 isolates contained only synonymous mutations in ERG11. All isolates with ERG6/ERG2/ERG11 mutations were genotypically distinct strains. Our data show that ERG6 and ERG2 are major targets conferring RS-AMB in clinical C. glabrata isolates.

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