Rhizopus oryzae intrinsic reduced voriconazole susceptibility is due to Cyp51Ap.

MACEDO, D.; LEONARDELLI, F.; GAMARRA, S.; GARCIA, G.

Congreso; XVIII Congreso Argentino de la Sociedad Argentina de Infectología/XVIII Congress of the international society of Infectious Diseases. 2018; 2018

SADI

Background:Rhizopus oryzae shows intrinsic reduced voriconazole (VRC) susceptibility. The molecular mechanism for VRC intrinsic resistance has not been described yet; however, azole resistance in filamentous fungi has mostly been linked to the CYP51 gene. The in vitro susceptibility patterns of Mucormycetes to azoles resemble those observed in Aspergillus fumigatus isolates with amino acid substitutions at the C-terminal portion of the Cyp51Ap protein. Cyp51Ap enzymes of A. fumigatus, C. elegans and R. oryzae share a homology higher than 50%. The aim of this study was to verify that the reduced VRC susceptibility of R. oryzae is due to the Cyp51Ap. Methods & Materials:An A. fumigatus strain (LMDM-1229) with the R. oryzae CYP51A was generated. A. fumigatus LMDM-32 (ΔKU80 and ΔCYP51A) was used as parental strain and R. oryzae LMDM-597 was used to obtain the CYP51A. The transformation plasmid LMDM-p134 used to A. fumigatus electroporation was generated by fusion PCR. Transformants were selected on fluconazole (FLC) plates since LMDM-32 strain is 8 times more sensitive to FLC than its wild type counterpart. A FLC resistance phenotype was expected by the incorporation of a functional CYP51A (from R. oryzae LMDM-597) in A. fumigatus LMDM-32 genome. Transformants genomic DNA were extracted and the homologous recombination was checked by PCR. VRC and FLC susceptibility testing were performed following the CLSI documents M38-A2 and M51-A.Results:Fluconazole resistant transformants obtained. The mutant strain LMDM-1229 showed the genomic integration and the homologous recombination of the R. oryzae CYP51A. Parental strain showed low VRC CIM value (0.06 µg/ml), while LMDM-1229 strain showed 256-fold higher MICs (16 µg/ml). This value mimicked the R. oryzae reduced VRC susceptibility phenotype (MICs of 16 µg/ml). The FLC and VRC susceptibility differences between the parental and mutant strains were confirmed by disk diffusion susceptibility testing.Conclusion There are few tools for selecting transformant colonies when the genotype-phenotype relationship in filamentous fungi is studied. Here, we propose FLC as a new selective agent and we demonstrated that is very effective tool. The 256-fold increases in VRC MICs observed in the transformant strain support the assumption that CYP51A is the responsible for intrinsic voriconazole resistance in this Mucormycete.