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

Abstract

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.

In vitro antifungal activity of organic compounds derived from amino alcohols against onychomycosis

“…The 2014 WHO report on global surveillance of antimicrobial resistance revealed that antimicrobial resistance, including antifungal resistance, is no longer a prediction for the future, as it is occurring worldwide now and is increasing the risk of being unable to properly treat common infections in the community and hospitals. For this reason, fostering innovative research and developing new vaccines, diagnostics, infection treatment options, and other tools is urgently needed 22, 58, 59, 60…”

Synergistic Interaction of Fluconazole/Amphotericin B on Inhibition of Enzymes Contributes to the Pathogenesis of Candida Tropicalis

“…Our results are in partial agreement with Khodavandi et al 23 revealed that fluconazole in combination with terbinafine significantly down-regulated the expression of ERG1, 3, and 11 genes in C. albicans. Ibrahim et al 37 investigated the effect of high doses of fluconazole alone and in combination with voriconazole and amphotericin B on the expression levels of CDR1, KRE1 and SKN1 genes responsible for Candida biofilm resistance. Significant up-regulation of SKN1 expression and to a lesser extent KRE1 was observed in Candida biofilms treated with amphotericin B alone or in combination.…”

Differential effects of antifungal agents on expression of genes related to formation of Candida albicans biofilms

“…It is well-known that C. albicans biofilms once formed exhibit an intrinsic resistance to several antifungal agents. Although several studies [6][7][8]17 have used transcript profiling to explore whether biofilm transcriptional responses contribute to antifungal resistance, there is still much information to be learned to exclude or adopt the hypotheses put forth on the mechanisms of biofilm drug resistance. To our knowledge, up to A > 2-fold change in gene expression between drug-treated vs. untreated cells was considered significant.…”

“…The basis of recalcitrance of biofilms to antifungal agents is not fully understood. Among the hypotheses formulated for this resistance, the possibility that differential gene expression may reduce the action of antifungals against C. albicans biofilms has been considered . On the other hand, antifungal‐exposed biofilms showing no significant changes in certain gene expression have led to the conclusion that recalcitrance to antifungals could be due to the intrinsic properties of the biofilm extracellular matrix, which prevents antifungals from reaching biofilm cells …”

“…Among the hypotheses formulated for this resistance, the possibility that differential gene expression may reduce the action of antifungals against C. albicans biofilms has been considered. [5][6][7] On the other hand, antifungal-exposed biofilms showing no significant changes in certain gene expression have led to the conclusion that recalcitrance to antifungals could be due to the intrinsic properties of the biofilm extracellular matrix, which prevents antifungals from reaching biofilm cells. 8 Three critical genes were selected for analysis in this study: BGL2 (927 bp) involved in cell wall metabolism and especially in (1?3)-b-D-glucan synthesis, ECE1 (1470 bp) related to cell elongation 9 and SUN41 (1127 bp) involved in hyphal formation, cell wall integrity and virulence in systemic candidiasis.…”