The effect of antifungal combination on transcripts of a subset of drug-resistance genes in clinical isolates of Candida species induced biofilms

Biofilm formation is often associated with increased Candida resistance toward antifungal agents. Therefore, the current study aimed to assess the incidence of biofilm formation among Candida isolates and to investigate the effect of high doses of fluconazole {FLC}, voriconazole {VOC} and amphotericin B {AMB}, singly and in combination on mature biofilms. Moreover, it aimed to assess the expression of selected genes (CDR1, KRE1 and SKN1) responsible for Candida biofilm resistance. The study included 49 patients; samples were collected from the King Khalid Hospital, Riyadh, Saudi Arabia. Isolates were prepared for biofilm formation and quantification using 0.4% (w/v) crystal violet. Minimum Inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) were conducted by the broth microdilution method. Biofilm eradication was evaluated using counting, XTT stain intensity and observed under the inverted microscope. Selected genes were evaluated in Candida biofilms under the effect of antifungal exposure using QPCR. The major isolates were Candida albicans (65.3%) followed by Candida tropicalis and Candida glabrata. 77.6% of the strains were biofilm formers. AMB showed susceptibility in 87.8% of isolates, followed by VOC (77.6%) and FLC (67.3%). MIC50 and MIC90 were (0.03, 0.125), (0.5, 8), (2, >128) μg/ml for AMB, VOC and FLC, respectively. 34.7% and 18.4% of the isolates were antagonistic to AMB/FLC and AMB/VOC, respectively. Mature biofilms of ten selected isolates were found resistant to FLC (1000 μg/ml). VOR and AMB concentration required to inhibit biofilm formation was 16-250 fold higher than the MIC for planktonic cells. Isolates showed significant reduction with antifungal combination when compared with the untreated controls (p value ⩽ 0.01), or using fluconazole alone (p value ⩽ 0.05). High doses of the antifungals were employed to assess the effect on the persisters' selected gene expression. Marked over expression of SKN1 and to a lesser extent KRE1 was noticed among the mature biofilms treated with AMB alone or in combination after 1 h of exposure, and SKN1 expression was even more sharply induced after 24 h. No statistically significant over expression of CDR1 was observed in biofilms after exposure to high doses of FLC, VOC or any of the combinations used.