<i>ERG6</i> and <i>ERG2</i> Are Major Targets Conferring Reduced Susceptibility to Amphotericin B in Clinical <i>Candida glabrata</i> Isolates in Kuwait

Abstract

<i>Candida glabrata</i> is intrinsically less susceptible to azoles, and resistance to echinocandins and reduced susceptibility (RS) to amphotericin B (AMB) have also been detected. The molecular mechanisms of RS to AMB were investigated in <i>C. glabrata</i> strains in Kuwait by sequence analyses of genes involved in ergosterol biosynthesis. A total of 1,646 <i>C. glabrata</i> isolates were tested by Etest, and results for 12 selected isolates were confirmed by reference broth microdilution. PCR sequencing of three genes (<i>ERG2</i>, <i>ERG6</i>, and <i>ERG11</i>) was performed for all isolates with RS to AMB (RS-AMB isolates) and 5 selected wild-type <i>C. glabrata</i> isolates by using gene-specific primers. The total cell sterol content was analyzed by gas chromatography-mass spectrometry. The phylogenetic relationship among the isolates was investigated by multilocus sequence typing. Wild-type isolates contained only synonymous mutations in <i>ERG2</i>, <i>ERG6</i>, or <i>ERG11</i>, and the total sterol content was similar to that of the reference strains. A nonsynonymous <i>ERG6</i> mutation (AGA48AAA, R48K) was found in both RS-AMB and wild-type isolates. Four RS-AMB isolates contained novel nonsense mutations at Trp286, Tyr192, and Leu341, and 2 isolates contained a nonsynonymous mutation in <i>ERG6</i> (V126F or C198F); and the sterol content of these isolates was consistent with <i>ERG6</i> deficiency. Two other RS-AMB isolates contained a novel nonsynonymous <i>ERG2</i> mutation (G119S or G122S), and their sterol content was consistent with <i>ERG2</i> deficiency. Of 8 RS-AMB isolates, 1 fluconazole-resistant isolate also contained nonsynonymous Y141H plus L381M mutations, while 7 isolates contained only synonymous mutations in <i>ERG11</i> All isolates with <i>ERG6</i>, <i>ERG2</i>, and <i>ERG11</i> mutations were genotypically distinct strains. Our data show that <i>ERG6</i> and <i>ERG2</i> are major targets conferring RS-AMB in clinical <i>C. glabrata</i> isolates.