title: Candida parapsilosis Resistance to Fluconazole: Molecular Mechanisms and In Vivo Impact in Infected Galleria mellonella Larvae.
abstract: Candida parapsilosis is the main non-albicans Candida species isolated from patients in Latin America. Mutations in the ERG11 gene and overexpression of membrane transporter proteins have been linked to fluconazole resistance. The aim of this study was to evaluate the molecular mechanisms in fluconazole-resistant strains of C. parapsilosis isolated from critically ill patients. The identities of the nine collected C. parapsilosis isolates at the species level were confirmed through molecular identification with a TaqMan qPCR assay. The clonal origin of the strains was checked by microsatellite typing. The Galleria mellonella infection model was used to confirm in vitro resistance. We assessed the presence of ERG11 mutations, as well as the expression of ERG11 and two additional genes that contribute to antifungal resistance (CDR1 and MDR1), by using real-time quantitative PCR. All of the C. parapsilosis (sensu stricto) isolates tested exhibited fluconazole MICs between 8 and 16 μg/ml. The in vitro data were confirmed by the failure of fluconazole in the treatment of G. mellonella infected with fluconazole-resistant strains of C. parapsilosis. Sequencing of the ERG11 gene revealed a common mutation leading to a Y132F amino acid substitution in all of the isolates, a finding consistent with their clonal origin. After fluconazole exposure, overexpression was noted for ERG11, CDR1, and MDR1 in 9/9, 9/9, and 2/9 strains, respectively. We demonstrated that a combination of molecular mechanisms, including the presence of point mutations in the ERG11 gene, overexpression of ERG11, and genes encoding efflux pumps, are involved in fluconazole resistance in C. parapsilosis.
authors.affiliation: Laboratório Especial de Micologia, Disciplina de Infectologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors.forename: Ana Carolina R
authors.initials: AC
authors.lastname: Souza
authors.affiliation: Division of Infectious Diseases, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, Rhode Island, USA.
authors.forename: Beth Burgwyn
authors.initials: BB
authors.lastname: Fuchs
authors.affiliation: Universidade de Brasília, Brasília, DF, Brazil Hospital Santa Luzia, Brasília, DF, Brazil.
authors.forename: Henrique M S
authors.initials: HM
authors.lastname: Pinhati
authors.affiliation: Laboratório Especial de Micologia, Disciplina de Infectologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors.forename: Ricardo A
authors.initials: RA
authors.lastname: Siqueira
authors.affiliation: Department of Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina Hospital, Nijmegen, Netherlands.
authors.forename: Ferry
authors.initials: F
authors.lastname: Hagen
authors.affiliation: Department of Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina Hospital, Nijmegen, Netherlands Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, Netherlands.
authors.forename: Jacques F
authors.initials: JF
authors.lastname: Meis
authors.affiliation: Division of Infectious Diseases, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, Rhode Island, USA.
authors.forename: Eleftherios
authors.identifier: http://orcid.org/0000-0002-4624-0777
authors.initials: E
authors.lastname: Mylonakis
authors.affiliation: Laboratório Especial de Micologia, Disciplina de Infectologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil arnaldolcolombo@gmail.com.
authors.forename: Arnaldo L
authors.initials: AL
authors.lastname: Colombo
mesh_terms: D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D002175:Candida / Q000187:drug effects* / Q000472:pathogenicity*; D025141:Drug Resistance, Fungal; D015725:Fluconazole / Q000494:pharmacology*; D007814:Larva / Q000382:microbiology*; D008826:Microbial Sensitivity Tests; D009036:Moths / Q000382:microbiology*
chemical_list: D000935:Antifungal Agents; D015725:Fluconazole