The S862C amino acid change in CpMrr1 confers fluconazole resistance in Candida parapsilosis

Background: Candida parapsilosis is an opportunistic pathogen with increasing rates of resistance to fluconazole and voriconazole. Recently, in an outbreak at the Azienda Ospedaliero-Universitaria Pisana, a new amino acid substitution, S862C in the CpMrr1 protein, was found only in azole-resistant strains. The contribution of this mutation to the acquisition of an azole-resistant phenotype was investigated in this study. Methods: Antifungal resistance in C. parapsilosis clinical strains isolated from the outbreak (n= 16) was tested by the broth microdilution method and Etest strip. WGS and Sanger sequencing analyses were used for the detection of SNPs. A CRISPR-Cas9–based genome editing strategy was used to induce the C2585G substitution in the CpMRR1 gene of susceptible C. parapsilosis isolates to investigate its role in the acquisition of azole resistance. Results: The A395T and the newly found C2585G substitution in the CpMRR1 gene were present in all resistant isolates, but not in the susceptible ones. Such mutations were later induced in the C. parapsilosis reference strain ATCC 22019 and in two azole-susceptible clinical isolates in homozygosis, and in heterozygosis only for ATCC 22019 and one azole-susceptible clinical isolate. Both heterozygous and homozygous mutants carrying the C2585G mutation were fluconazole resistant, with some clones also presenting intermediate susceptibility or resistance to voriconazole. Conclusions: To the best of our knowledge, this is the first study to report the effect on azole resistance of a novel C2585G nucleotide substitution in the CpMRR1 gene found in clinical isolates recovered during an outbreak of azole-resistant C. parapsilosis in a healthcare setting.