Molecular Characterization and Sterol Profiles Identify Nonsynonymous Mutations in as a Major Mechanism Conferring Reduced Susceptibility to Amphotericin B in Candida kefyr.

The molecular basis of reduced susceptibility to amphotericin B (rs-AMB) among any yeasts is poorly defined. Genetic alterations in genes involved in ergosterol biosynthesis and total cell sterols were investigated among clinical Candida kefyr isolates. C. kefyr isolates ( = 81) obtained from 74 patients in Kuwait and identified by phenotypic and molecular methods were analyzed. An Etest was initially used to identify isolates with rs-AMB. Specific mutations in and involved in ergosterol biosynthesis were detected by PCR sequencing. Twelve selected isolates were also tested by the SensiTitre Yeast One (SYO), and total cell sterols were evaluated by gas chromatography-mass spectrometry and and sequencing. Eight isolates from 8 patients showed rs-AMB by Etest, including 2 isolates with additional resistance to fluconazole or to all three antifungals. SYO correctly identified 8 of 8 rs-AMB isolates. A nonsynonymous mutation in was detected in 6 of 8 rs-AMB isolates but also in 3 of 73 isolates with a wild-type AMB pattern. One rs-AMB isolate contained a deletion (frameshift) mutation in . One or more nonsynonymous mutations was detected in in 11 of 81 isolates with the rs-AMB or wild-type AMB pattern. Among 12 selected isolates, 2 and 2 isolates contained a nonsynonymous mutation(s) in and , respectively. Ergosterol was undetectable in 7 of 8 rs-AMB isolates, and the total cell sterol profiles were consistent with loss of function in 6 rs-AMB isolates and loss of activity in another rs-AMB isolate. Our data showed that is a major target conferring rs-AMB in clinical C. kefyr isolates. Some yeast species exhibit intrinsic resistance or rapidly acquire resistance to azole antifungals. Despite >50 years of clinical use, resistance to amphotericin B (AMB) among yeast species has been extremely rarely reported until recently. Reduced susceptibility to AMB (rs-AMB) among yeast species is, therefore, a matter of serious concern due to the availability of only four classes of antifungal drugs. Recent studies in Candida glabrata, Candida lusitaniae, and Candida auris have identified genes involved in ergosterol biosynthesis as the major targets conferring rs-AMB. The results of this study also show that nonsynonymous mutations in impair its function, abolish ergosterol from C. kefyr, and confer rs-AMB. Thus, rapid detection of rs-AMB among clinical isolates will help in proper management of invasive C. kefyr infections.