Potent anti-biofilm properties of plumbagin against fluconazole-resistant Candida auris

Abstract

Background and aim: The escalation of fungal infections is driving an increase in disease and mortality rates. In particular, the emergence of Candida auris (C. auris), which shows powerful resistance to the antifungal drug fluconazole, is becoming a global concern. Furthermore, several biological hurdles need to be overcome by candidate therapeutics because C. auris has the ability to form biofilm. Therefore, this study aimed to investigate the antifungal and anti-biofilm effects of plumbagin, a natural extract, against fluconazole-resistant C. auris (FRCA).

Experimental procedure: The minimum inhibitory concentrations (MICs) of fluconazole and plumbagin were determined against clinically isolated C. auris. Inhibition of biofilm formation and eradication effects of plumbagin against FRCA were confirmed through minimum biofilm inhibition concentration (MBIC) and minimum biofilm eradication concentration (MBEC) assays. Additionally, the inhibition of metabolic activity in biofilm cells was verified through quantification by XTT reduction assay and visualization by confocal laser scanning microscopy (CLSM). The relative expression levels of the azole resistant gene ERG11, the efflux pump gene CDR1, and the extracellular matrix gene KRE6, were measured.

Results and conclusion: Plumbagin exhibits antifungal efficacy against C. auris and has been shown to effectively inhibit both the formation and eradication of biofilms produced by FRCA. Furthermore, the metabolic activity inhibition in biofilm cells was both quantified and visually observed. The downregulation of all genes (ERG11, CDR1, and KRE6) by plumbagin was confirmed. Taken together, this study demonstrates that plumbagin has antifungal and anti-biofilm efficacy against FRCA, indicating its potential as an alternative to antifungal agents and a valuable resource in combating FRCA infections.

Keywords: Antifungal; Efflux pump; Extracellular matrix; Fluconazole-resistant Candida auris; Plumbagin; anti-Biofilm.

Graphical abstract

Fig. 1

The inhibitory effect of plumbagin on C. auris NCCP32683 biofilm. (A) Biofilm stained with 0.5 % crystal violet. (B) The stained biofilm was quantified at a wavelength of 595 nm and converted to a percentage. The error bars represent the means ± standard deviation (SD). Statistical significance is indicated by ***p < 0.001.

Fig. 2

The eradication effect of plumbagin on preformed biofilm of C. auris NCCP32683. (A) Anti-biofilm effects were assessed through biofilm eradication concentration (BEC) assay using 0.5 % crystal violet staining. (B) The stained biofilm was quantified at a wavelength of 595 nm and expressed as a percentage. The error bars represent the means ± standard deviation (SD). Statistical significance is indicated by ***p < 0.001.

Fig. 3

The inhibitory effect of plumbagin on the metabolic activity of C. auris NCCP32683 in biofilm. The preformed biofilm was treated with plumbagin and assayed by XTT reagent. After measuring the absorbance at 475 nm and 650 nm, values were calculated according to the A_475nm (Test) - A_475nm (Blank) - A_650nm (Test) formula. The error bars represent the means ± standard deviation (SD). Statistical significance is indicated by *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 4

CLSM image visualizing metabolic activity of C. auris NCCP32683 in biofilm. Fungal cultures (1 × 10⁶ CFU/mL) exposed to plumbagin were incubated at 37 °C for 24 h, followed by staining with SYTO9 (green) and PI (red). SYTO9 fluorescence, representing live cells, was detected at wavelength of 525 nm, while PI fluorescence, indicating dead cells was detected at 640 nm. All images taken at × 40 magnification and acquired using ZEN software.

Fig. 5

Expression levels of efflux pump and extracellular matrix genes in C. auris NCCP32683. (A) Azole resistance gene (ERG11), (B) efflux pump gene (CDR1), and (C) extracellular matrix gene (KRE6) were analyzed. The target gene was normalized to ACT1. The error bars represent the means ± standard deviation (SD). Statistical significance is indicated by *p < 0.05, **p < 0.01, and ***p < 0.001 compared to control.

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