Biofilm formation in clinical isolates of Candida Albicans and Candida Dubliniensis modulation of erggene expression by fluconazole

Bujdakova1, H., Borecka-Melkusova1, S., Kolecka1, A., Kucharikova1, S., Chorvat2, D., Gasperik3, J.

Author address: 

1 Comenius University, BRATISLAVA, Slovak Republic 2International Laser Centre, BRATISLAVA, Slovak Republic 3Institute of Molecular Biology SAS, BRATISLAVA, Slovak Republic

Abstract: 

Biofilm-associated candidiasis presents a new class of diseases connected with using medical devices which serve as an optimal substrate for adherence of Candida. Formation of biofilm can be influenced by different conditions (for example pH) and biofilm is characterized by decreased susceptibility to antifungal agents, including fluconazole (FLC). This work was focused on the study of biofilm formation by C. albicans and C. dubliniensis clinical isolates from different clinical materials and the expression of ERG genes involved in ergosterol biosynthesis pathway with main focus on ERG11 gene encoding a key enzyme 14-alpha lanosterol demethylase- the target for FLC. Additionally, the effect of pH to biofilm formation and to the expression of ERG11 gene was determined. C. albicans and C. dubliniensis strains identified by growth on CHROMagar Candida was submitted for determination of genotype using primer specific albicans/dubliniensis PCR. Susceptibility/resistance to FLC was determined by microdilution method according to NCCLS M27-A2. Biofilm was prepared in microtitre plates and quantified by XTT reduction assay supplemented by the dry weight measurement. Additionally, biofilm formed on coverslips in Petri dishes at the same condition like biofilm in microtitre plates, was observed by confocal scanning laser microscopy (CSLM). The expression of ERG genes was determined in both planktonic as well as in biofilm forming culture using the reverse transcription- PCR . The four time points (1.5h, 6h, 24h, 48h) of biofilm formation were selected for collection of RNA. All experiments were prepared in the presence or absence of 0.5 x MIC95 of FLC. It is of interest that while the expression of ERG genes in C. albicans proved to be strain dependent and generally decreased in the presence of FLC, the formation of biofilm by C. dubliniensis in the FLC presence was characteristic by decreased expression of ERG1 and ERG11 genes expression of ERG3 and ERG25 genes was markedly increased. Expression of the ERG11 gene was maximal in mature biofilm and proved to be pH dependent. While the presence of FLC in YNB+0.9% of D-glucose with non-adjusted pH (about 5.6) did not significantly affect the ERG11 expression, upregulation of this gene was proved in the same medium with FLC when pH=7.0. The changes in biofilm formed at the presence of FLC correlated with observation of 48h-biofilm using CSLM confirming reduction not only in thickness of biofilm, but also in partial inhibition of yeast-mycelial transformation during biofilm formation. Presented results confirmed effect of FLC on biofilm formation, but its efficiency was limited by pH of environment, susceptibility/resistance of isolates, clinical material and Candida species.
2007

abstract No: 

o.15

Full conference title: 

3rd Trends in Medical Mycology
    • TIMM 3rd (2011)