Two promising Bacillus-derived antifungal lipopeptide leads AF4 and AF5 and their combined effect with fluconazole on the in vitro Candida glabrata biofilms

Abstract

Introduction: Candida species are endowed with the ability to produce biofilms, which is one of the causes of pathogenicity, as biofilms protect yeasts from antifungal drugs. Candida glabrata (Nakaseomyces glabrata) is one of the most prevalent pathogenic yeasts in humans and a biofilm producer. Methods: The study was aimed at evaluating the combined effects of two highly promising antifungal biomolecules (AF₄ and AF₅) lipopeptide in nature, chromatographically purified to homogeneity from Bacillus subtilis (B. subtilis) and the standard antifungal fluconazole (at different concentrations) to demonstrate C. glabrata biofilm formation inhibition. Biofilm production and inhibition were evaluated by quantification of the biofilm biomass and metabolic activity using crystal violet (CV) staining and XTT reduction assays, respectively. Microscopic techniques such as confocal scanning laser microscopy (CSLM) and scanning electron microscopy (SEM) were employed to visualize biofilm formation and inhibition. Results and Discussion: Compared to untreated and fluconazole-treated biofilms, an enhanced in vitro anti-biofilm effect of the antifungal lipopeptides AF₄/AF₅ alone and their combinations with fluconazole was established. The lipopeptides AF₄/AF₅ alone at 8 and 16 μg/mL exhibited significant biomass and metabolic activity reductions. SEM and CSLM images provided evidence that the lipopeptide exposure results in architectural alterations and a significant reduction of C. glabrata biofilms, whereas (2', 7'-dichlorofluorescin diacetate (DCFDA) and propidium iodide (PI) analyses showed reactive oxygen species (ROS) generation along with membrane permeabilization. The estimation of exopolysaccharides (EPS) in AF₄/AF₅-treated biofilms indicated EPS reduction. The combinations of fluconazole (64/128 μg/mL) and AF₄/AF₅ lipopeptide (16 μg/mL) were found to significantly disrupt the mature (24 h) biofilms as revealed by CSLM and SEM studies. The CSLM images of biofilms were validated using COMSTAT. The FTIR-analyses indicate the antibiofilm effects of both lipopeptides on 24 h biofilms to support CSLM and SEM observations. The combinations of fluconazole (64/128 μg/mL) and AF₄/AF₅ lipopeptide were found to disrupt the mature biofilms; the study also showed that the lipopeptides alone have the potentials to combat C. glabrata biofilms. Taken together, it may be suggested that these lipopeptide leads can be optimized to potentially apply on various surfaces to either reduce or nearly eradicate yeast biofilms.

Keywords: Antifungal lipopeptide; Bacillus sp.; CV assay; Candida glabrata; XTT assay; biofilm inhibition; confocal microscopy.

FIGURE 1

(A) Mean absorbance values at 590 nm obtained from the total biomass quantification by CV assay of CG 2001 biofilm. The solid rectangular black and gray bars indicate 6 and 24 h biofilms respectively. (B) Effect of FLC and FLC plus (AF₄/AF₅) on CG ATCC 2001 biofilm formation by XTT assay. Graph shows (black bars) 6 h, (white bars) 24 h biofilm. Results were normalized to control (untreated), which was taken as (100%). Data represent the means ± SD of three individually performed experiments with the error bars.

FIGURE 2

Scanning electron micrographs of CG 2001 24 h preformed biofilm. (A) control (untreated) and (B,C) AF₄ 8 μg/mL and 16 μg/mL treatments alone respectively, (D) treated with FLC 32 μg/mL and (E,F) AF₅ 8 μg/mL and 16 µg/mL-treatments alone respectively. (G,J) treated with FLC 64 μg/mL and FLC 128 μg/mL alone respectively, (H) treated with FLC 64 μg/mL/AF₄ (16 μg/mL), (K) treated with FLC 128 μg/mL/AF₄ (16 μg/mL) and (I) treated FLC 64 μg/mL/AF₅ (16 μg/mL), (L) treated with FLC 128 μg/mL/AF₅ (16 μg/mL). Images are shown at 4 µm scale bars. Arrows indicate the cell surface damages and deformities.

FIGURE 3

CSLM images of biofilm-associated CG cells. Concanavalin A-Alexa 488 (green), and FUN-1 (red) staining at 60 × 2 oil immersion objective and 2× magnification. Con-A stains the extracellular polysaccharides and FUN-1 stains the metabolically active cells. Each image (A–D) is control (untreated), and (E–H) is treated with FLC 32 μg/mL, and (I–P) are treated with AF₄ 8 and 16 μg/mL respectively. (Q–X) AF₅ 8 and 16 μg/mL respectively. 3D reconstruction images were obtained from Z-stack. Scale bar: 10 µm.

FIGURE 4

Confocal images of 24 h biofilm of CG ATCC 2001 treated with varying concentrations of FLC alone and in combination with AF₄/AF₅. Images were acquired using the confocal scanning laser microscope, con-A, Alexa Flour 488 conjugate (green), and FUN-1 (red) staining at ×60 oil immersion objective and ×2 magnification. Con-A stains the extracellular polysaccharides, and FUN-1 stains the metabolically active cells. The images (A–D) show biofilms treated with 64 μg/mL, while the images (M–P) show biofilms treated with FLC 128 μg/mL. Images (E–H) depict biofilms treated with FLC 64 μg/mL and AF₄ (16 μg/mL), while images (I–L) show biofilms treated with FLC 64 μg/mL and AF5 (16 μg/mL). Images (Q–T) show biofilms treated with FLC 128 μg/mL and AF₄ (16 μg/mL), and (U–X) show biofilms treated with FLC 128 μg/mL and AF₅ (16 μg/mL). Z-stack 3D reconstruction images were obtained, and the scale bar is 10 µm.

FIGURE 5

CSLM-COMSTAT analysis of various parameters of the CG 2001 biofilms untreated and treated at 24 h. (A) Biomass (µm³/µm²), (B) Mean thickness (µm), (C) Surface to bio-volume ration (µm³/µm²), and (D) Roughness coefficient.

FIGURE 6

(A) Intracellular ROS generation in C. glabrata pre-formed biofilm. The CSLM images of C. glabrata biofilm cells stained with DCFDA and PI after antifungal lipopeptide treatment. The units of the values in brackets and FLC are µg/mL. Scale bar 50 μm. (B) The ROS generation in AF₄ and AF₅ treated biofilm were measured using DCFDA dye in terms of fluorescence intensity at excitation of 485 nm and emission of 520 nm. Data represent the means ± SD of two individually performed experiments.

FIGURE 7

Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra analysis of untreated C. glabrata 24 h biofilms (A,B), AF₄ and AF₅ (8 μg/mL and 16 μg/mL) treated biofilms respectively, (C) shows spectra of merged AF₄ and AF₅ along with FLC (32 and 64 μg/mL)-treated biofilms, (D) Main absorption bands and assignments for ATR-FTIR spectra of C. glabrata biofilms.

FIGURE 8

Effects of FLC, AF₄/AF₅ alone and various combinations of AF₄/AF₅ and FLC at different concentrations on the EPS layer of CG biofilms. AF₄ at 16 μg/mL, and AF₅ at 8 μg/mL and 16 μg/mL showed reduction in exopolysaccharide.