title: Stepwise emergence of azole, echinocandin and amphotericin B multidrug resistance in vivo in Candida albicans orchestrated by multiple genetic alterations.
abstract: OBJECTIVE
  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.
authors.affiliation: Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Rasmus Hare
authors.initials: RH
authors.lastname: Jensen
authors.affiliation: Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Karen Marie Thyssen
authors.initials: KM
authors.lastname: Astvad
authors.affiliation: Institute of Microbiology, University of Lausanne and University Hospital Center (CHUV), Lausanne, Switzerland.
authors.forename: Luis Vale
authors.initials: LV
authors.lastname: Silva
authors.affiliation: Institute of Microbiology, University of Lausanne and University Hospital Center (CHUV), Lausanne, Switzerland.
authors.forename: Dominique
authors.initials: D
authors.lastname: Sanglard
authors.affiliation: Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Rene
authors.initials: R
authors.lastname: Jørgensen
authors.affiliation: Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark.
authors.forename: Kristian Fog
authors.initials: KF
authors.lastname: Nielsen
authors.affiliation: Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmark Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark.
authors.forename: Estella Glintborg
authors.initials: EG
authors.lastname: Mathiasen
authors.affiliation: Clinical Microbiology Laboratory, Herlev Hospital, Herlev, Denmark.
authors.forename: Ghazalel
authors.initials: G
authors.lastname: Doroudian
authors.affiliation: Public Health and Research Institute, New Jersey Medical School-Rutgers Biomedical and Health Sciences, Newark, NJ, USA.
authors.forename: David Scott
authors.initials: DS
authors.lastname: Perlin
authors.affiliation: Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmark maca@ssi.dk.
authors.forename: Maiken Cavling
authors.initials: MC
authors.lastname: Arendrup
mesh_terms: D000368:Aged; D000666:Amphotericin B / Q000494:pharmacology*; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D001393:Azoles / Q000494:pharmacology*; D002176:Candida albicans / Q000145:classification / Q000187:drug effects* / Q000235:genetics; D002177:Candidiasis / Q000188:drug therapy* / Q000382:microbiology; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D026141:Drug Resistance, Multiple, Fungal; D054714:Echinocandins / Q000494:pharmacology*; D005838:Genotype; D006801:Humans; D007915:Lepidoptera / Q000382:microbiology; D008297:Male; D008826:Microbial Sensitivity Tests; D058885:Multilocus Sequence Typing; D009154:Mutation; D017422:Sequence Analysis, DNA; D016019:Survival Analysis; D014774:Virulence
chemical_list: D000935:Antifungal Agents; D001393:Azoles; D004271:DNA, Fungal; D054714:Echinocandins; D000666:Amphotericin B
keywords: antifungal resistance; molecular typing; mycology; resistance mechanisms