Stepwise emergence of azole, echinocandin and amphotericin B multidrug resistance in vivo in Candida albicans orchestrated by multiple genetic alterations

Received 2 April 2015; returned 14 April 2015; revised 22 April 2015; accepted 24 April 2015Objectives:The objective of this study was to characterize the underlying molecular mechanisms in consecutiveclinical Candida albicans isolates from a single patient displaying stepwise-acquired multidrug resistance.Methods: Nine clinical isolates (P-1 to P-9) were susceptibility tested by EUCAST EDef 7.2 and Etest. P-4, P-5, P-7,P-8 and P-9 were available for further studies. Relatedness was evaluated by MLST. Additional genes wereanalysed by sequencing (includingFKS1, ERG11, ERG2 and TAC1) and gene expression by quantitative PCR(CDR1, CDR2 and ERG11). UV-spectrophotometry and GC-MS were used for sterol analyses. In vivo virulencewas determined in the insect model Galleria mellonella and evaluated by log-rank Mantel–Cox tests.Results: P-1+P-2 were susceptible, P-3+P-4 fluconazole resistant, P-5 pan-azole resistant, P-6+P-7 pan-azoleand echinocandin resistant and P-8+P-9 MDR. MLST supported genetic relatedness among clinical isolates. P-4harboured fourchangesinErg11(E266D, G307S, G450EandV488I), increasedexpression ofERG11andCDR2andachange in Tac1 (R688Q).P-5, P-7, P-8 and P-9 had an additional change in Erg11 (A61E), increased expression ofCDR1,CDR2andERG11(except forP-7) and a different aminoacid changein Tac1(R673L). Echinocandin-resistantisolates harboured the Fks1 S645P alteration. Polyene-resistant P-8+P-9 lacked ergosterol and harboured aframeshift mutation in ERG2 (F105SfsX23). Virulence was attenuated (but equivalent) in the clinical isolates,but higher than in the azole- and echinocandin-resistant unrelated control strain.Conclusions: C. albicans demonstrates a diverse capacity to adapt to antifungal exposure. Potentially novelresistance-inducing mutations in TAC1 , ERG11 and ERG2 require independent validation.Keywords: mycology, molecular typing, antifungal resistance, resistance mechanisms