Objectives: The aim of this study was to investigate the molecular mechanisms involved in a decreased susceptibility to azole antifungals in unmatched C. glabrata clinical isolates recovered from different patients treated in two university hospitals in Slovakia. The attention was also paid to cell surface hydrophobicity and in vitro biofilm formation associated with virulence of fungal pathogens. Methods: Drug susceptibilities were determined by microbroth dilution method in 96-well plates according to the proposed CLSI (formerly NCCLS), M27-A standard guidelines and by E-test assay. PCR was carried out with a high-fidelity KOD Hot Star DNA polymerase (Sigma-Aldrich, St. Louis, USA). The DNA sequence was determined with the ABI Prism 3100 DNA sequencer. The quantitative real time PCR was carried out in the 7900 HT Fast Real-Time PCR System (Applied Biosystems, Foster City, USA). Cell surface hydrophobicity was measured by the water-octane two phases assay. Biofilm formation was quantified biochemically as XTT reduction. Results: Molecular mechanisms of resistance to azole antifungals were investigated in ten unmatched C. glabrata clinical isolates. Overexpression of the CgPDR1 gene encoding the main multidrug resistance transcription factor and its target genes CgCDR1 and CgCDR2 coding for drug efflux transporters was observed in six fluconazole resistant isolates. Sequence analysis of the PCR amplified DNA fragments of their CgPDR1 gene identified the L347F and H576Y amino acid substitutions in CgPdr1p in one and five clinical isolates, respectively. Sequencing of the CgERG11 gene encoding 14C-lanosterol demethylase and analysis of its expression identified the E502V amino acid substitution in five of ten isolates and showed no upregulation of this gene in any clinical isolates. No significant association was observed among the drug susceptibility profiles, mechanisms of azole resistance and the cell surface hydrophobicity correlating with the ability of isolates to form biofilm. Conclusion: The results demonstrate that decreased susceptibilities of C. glabrata clinical isolates to azole antifungals are associated with upregulation of drug efflux transporter genes due to novel gain-of-function mutations in gene encoding CgPdr1p transcription factor.
Full conference title:
19th European Congress of Clinical Microbiology and Infectious Diseases
- ECCMID 19th (2009)