Digital archive of theses discussed at the University of Pisa


Thesis etd-07032015-102207

Thesis type
Tesi di specializzazione (5 anni)
Thesis title
Molecular characterization of fluconazole resistance in Candida orthopsilosis clinical isolates
Course of study
relatore Prof.ssa Tavanti, Arianna
  • fluconazole
  • ERG11
  • Candida orhtopsilosis
  • qRT-PCR
  • resistance
Graduation session start date
Candida orthopsilosis is a human fungal pathogen belonging to the Candida parapsilosis species. This species is able to colonize virtually all body sites and is responsible for a wide spectrum of symptomatic infections in humans, ranging from mucosal to life-threatening mycoses. Although C. parapsilosis sensu lato species are not considered particularly prone to develop drug resistance, epidemiological investigations have highlighted a reduced susceptibility to azole and echinocandins, mainly for C. parapsilosis sensu stricto. So far, a very limited number of C. orthopsilosis fluconazole resistant clinical isolates have been reported. We recently isolated two sequential C. orthopsilosis strains isolated from a patient with candidemia, and two sequential isolates from a patient with onychomycosis characterized by a fluconazole resistant phenotype (MIC=64 and 32 μg/ml, respectively). These clinical isolates also showed a resistant phenotype to posaconazole and voriconazole and a dose dependent susceptibility to itraconazole. Identification of clinical isolates as C. orthopsilosis was obtained by MALDI-TOF mass spectrometry analysis and further confirmed with two independent DNA-based methods.
The aim of this study was to investigate the molecular mechanisms of fluconazole resistance in C. orthopsilosis. To this end, we evaluated the involvement of efflux multidrug transporters in the development of fluconazole resistance as well as the presence of amino acid substitutions in the cytochrome P-450 lanosterol 14 α -demethylase in C. orthopsilosis azole resistant and susceptible strains. Fluconazole target is codified in C. orthopsilosis by CORT0E05900, an ortholog of C. albicans ERG11. By comparing the obtained sequences, 38 different single nucleotide polymorphisms were identified, five of which induced non-synonymous amino acid substitution. All the other variations did not change the translated amino acid. Interestingly, only one of the non-synonymous amino acid substitution was found in the predicted protein of fluconazole resistant C. orthopsilosis isolates only.
SIFT, PROVEAN and PHYRE2 algorithms were used to predict the impact of the above mentioned non synonymous mutation on C. orthopsilosis Erg11 protein function. Analysis with SIFT and PROVEAN algorithms predicted the mutation Y132F to be damaging and deleterious, while PHYRE2 mapped the substituted amino acid right into the pocket of the protein. Previous identification and association with azole resistance of this polymorphism in different Candida species further strengthen the potential role played by Y132F substitution on enzymatic activity.
To evaluate the contribution of efflux pumps or target over-expression to the observed azole resistant phenotype, transcription levels of ERG11, and CDR1 and SNQ2 ABC type transporters were determined in azole sensitive and resistant C. orthopsilosis isolates by quantitative real-time RT-PCR. Results ruled out a major role of these genes in drug resistance, under the experimental condition tested.
The overall results indicate that a mutation in the coding sequence of C. orthopsilosis azole target gene is associated with azoles multi-resistance in a panel of C. orthospsilosis clinical isolates, thus providing the first insights on azole resistance in this opportunistic pathogen.