Objectives: Compared with C. albicans there are relatively few studies examining the virulence factors of C. tropicalis, and its mechanisms of drug-resistance. C. tropicalis accounts for a significant proportion of Candida bloodstream infection, and traditionally is considered as second to C. albicans in terms of virulence and clinical importance. Here we investigated the molecular mechanism responsible for cross-resistance to fluconazole and voriconazole in C. tropicalis clinical strains isolated from bloodstream infections. Materials and methods: During a 10-year retrospective surveillance of candidemia we collected ten C. tropicalis isolates, showing resistance to fluconazole and voriconazole. For comparative purpose an equal number of azole-susceptible strains were studied. Isogenicity of the isolates was investigated by RAPD (primers OPE03, GC70 and UBC703) and Maldi-TOF analysis. Antifungal susceptibility testing was performed by Sensititre procedure and broth microdilution method. Quantification of the expression of the CtMDR1, CtCDR1 and CtERG11 genes was performed by real-time PCR, using SYBR Green chemistry. To stimulate the espression of efflux pumps genes, all the isolates were cultured in presence and absence of various fluconazole concentrations. The primers have been designed with the primer express 3.0 software (Applied Biosystems). For ERG11 sequencing, five pairs of oligonucleotide primers (Bouchara et al., 2005) were used. Results: The resistant isolates, coming from four different hospitals, clustered in three groups. No significant differences were found in the expression levels of the resistant isolates compared to the susceptible ones, even if cultured in the presence of sub-MIC concentration of fluconazole. Comparison of the CtERG11 gene sequences of the ten C. tropicalis resistant isolates with the available corresponding sequence in the GenBank database (accession number M23673) revealed the point mutation Y132F in the coding region. This mutation is located in the region between the B’ and C helices that have been postulated to be involved in inhibitor- or substrate-induced structural changes. Conclusions: In contrast to what observed in C. albicans, where azole resistance is usually the result of a combination of different mechanisms, in C. tropicalis we found a unique single point mutation sufficient to induce a decreased affinity of fluconazole and its derivative voriconazole.
Full conference title:
22nd European Congress of Clinical Microbiology and Infectious Diseases
- ECCMID 22nd (2012)