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

Objectives: The objective of this study was to characterize the underlying molecular mechanisms in consecutive clinical 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 were analysed by sequencing (including FKS1, 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 virulence was 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-azole and echinocandin resistant and P-8+P-9 MDR. MLST supported genetic relatedness among clinical isolates. P-4 harboured four changes in Erg11 (E266D, G307S, G450E and V488I), increased expression of ERG11 and CDR2 and a change in Tac1 (R688Q). P-5, P-7, P-8 and P-9 had an additional change in Erg11 (A61E), increased expression of CDR1, CDR2 and ERG11 (except for P-7) and a different amino acid change in Tac1 (R673L). Echinocandin-resistant isolates harboured the Fks1 S645P alteration. Polyene-resistant P-8+P-9 lacked ergosterol and harboured a frameshift 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 novel resistance-inducing mutations in TAC1, ERG11 and ERG2 require independent validation.