Introduction

Candida auris, initially identified in Japan in 2009, has become a widespread multidrug-resistant fungus known as a 'superbug' (Satoh et al. 2009). It has emerged as a global epidemic, leading to outbreaks of nosocomial infections with high mortality rates and rising levels of antifungal resistance (Ahmad et al. 2020). Over the past decade, C. auris infections have increased in over 40 countries and territories across six continents (Du et al. 2020), exhibiting genetic diversity across five distinct evolutionary clades (Chow et al. 2019). The COVID-19 pandemic has also facilitated the spread of C. auris (Ben Abid et al. 2023). In recent years, traditional antifungal drugs, including azoles, polyenes, and echinocandins, have been mainly used in clinical settings. The emergence of multidrug-resistant C. auris indicates its resistance to several standard antifungal drugs, including fluconazole, amphotericin B, and caspofungin. This situation also underscores the limitations of these drugs, including their high toxicity, significant drug interactions, and increased costs for specific formulations (Vélez et al. 2024). A significant challenge exists in treating fungal infectious diseases, emphasizing the need for new drugs and innovative approaches to treat notorious fungal infections.

Baicalein (BE) is a predominant constituent found in Scutellaria baicalensis, it exhibits various pharmacological effects, such as antioxidant, anti-inflammatory, antibacterial, antineoplastic, and antiviral properties (Yan et al. 2020). Additionally, it does not exhibit toxicity toward normal cells, including blood and liver cells (Wu et al. 2018). Its primary application is in treating acute and chronic inflammatory diseases (Li-Weber 2009). Candida albicans is a vital member of the Candida family. Previous research has shown that BE (0.023–370.1 μM) exhibits an inhibitory effect on C. albicans, resulting in reduced overall growth, biofilm formation, metabolic activity, and cell surface hydrophobicity. This effect is dosage‐dependent (Shirley et al. 2017). Other research has suggested the combination of BE and quercetin effectively reduces biofilm formation and mycelial growth of Candida albicans, while also increasing cell membrane permeability. RT-qPCR results indicate that the combination of BE and quercetin significantly enhances the expression of hypha-related genes (ALS3, HYR1, and CPH1) compared to BE or quercetin alone. Thus, the potential efficacy of combining BE and quercetin as anti-Candida agents has been proven. (Janeczko et al. 2022). BE treatment could also induce apoptosis in C. albicans cells by disrupting mitochondrial membrane potential. The study revealed a significant increase in intracellular ROS levels after processing with BE, as compared to untreated cells, accompanied by elevated expression of CAP1 (a key regulatory gene of oxidative stress response), SOD2 (manganese superoxide dismutase gene) and TRR1 (thioredoxin reductase gene). (Dai et al. 2009). In conclusion, these studies have shown that BE is effective as an antifungal agent and suggest its potential use as a solution for C. auris biofilm-related infections.

However, studies have shown the distinctive advantages of Traditional Chinese medicine in combating fungal infections, while the effect of baicalein (BE), an active ingredient extracted from the dried roots of Scutellaria baicalensis Georgi on C. auris, remains unknown. Therefore, this study aims to evaluate the potential of BE as an antifungal agent against the emerging multidrug-resistant C. auris by inhibiting adhesion, biofilm formation, and its detrimental effects on the cell membrane and cell wall. The findings from this study could significantly contribute to expanding therapeutic options for fungal infectious diseases, and it may hold substantial theoretical and research value.

Material and methods

Candida auris strains and cultivation

C. auris strains (C1, C2, C3, and C4) were provided by Prof. Huang Guanghua from FuDan University. C. auris was cultured on Yeast Peptone Dextrose (YPD) Agar, then activated in YPD Broth (Hopebio Biotechnology Co., Ltd, Shandong, China) and incubated at 37 °C for 12–14 h until the exponential phase. The fungal precipitate was obtained through centrifuging at 825 × g for 5 min, and the supernatant was discarded. Roswell Park Memorial Institute -1640 (RPMI-1640) medium (Thermo Fisher, New York, USA; pH 7.0) was then added to adjust the concentration to 2 × 103, 1 × 106, 2 × 106, 7 × 106, 1 × 107, 2 × 107, and 2 × 108 CFU/ml.

MIC

The minimum inhibitory concentration (MIC) of the drugs against the four C. auris strains was determined following the broth microdilution method in 96-well plates following the CLSI M27-M44S protocol guidelines of the American Committee for Standardization of Clinical Trials. The fungal suspension was adjusted to a concentration of 2 × 103 cells/ml. Drugs were then prepared in serial two-fold dilution, with BE (CAS 491–67-8, HPLC ≥ 98%, Shanghai Yuanye Biotechnology Co., Ltd) ranging from 0.23–1894.6 μM and FLZ (CAS 86,386–73–4, Shanghai Yuanye Biotechnology Co., Ltd) ranging from 209–3343.5 μM. Subsequently, 100 μl of the fungal suspension and 100 μl of the drug solution were added to each well. The well plates were incubated at 37 °C for 24 h. The MIC of the drug against Candida was determined by visually observing sterile growth (Zhang et al. 2024).

MFC

The minimum fungicidal concentration (MFC) of BE was carried out for four C. auris strains. The fungal suspension was adjusted to a concentration of 2 × 103 cells/ml. Drugs were then prepared in serial two-fold dilution, with BE ranging from 0.23–1894.6 μM and FLZ ranging from 209–3343.5 μM. Subsequently, 100 μl of the fungal suspension and 100 μl of the drug solution were added to each well. The well plates were incubated at 37 °C for 24 h. After incubation, the four wells were chosen before and after MIC values, and from all the wells, the 100 µL samples were transferred into YPD Agar medium. After 24 h incubation, the plates and existence of colonies were observed and the absence of colonies were noted as minimum fungicidal concentration and results were noted (Nandhagopal et al. 2024).

Spot assay

The C. auris cells were grown overnight in liquid YPD media at 37℃. They were exposed to varying concentrations of BE or FLZ at densities of 2 × 105, 2 × 104, 2 × 103 and 2 × 102 CFU/ml. After serially diluting cultures (5 μl), we spotted them on YPD agar plates. Growth differences were observed after 24 h of incubation at 37℃ (Zhang et al. 2024).

Time growth curve

During the logarithmic growth phase, the concentration of C. auris was adjusted to 2 × 106 cells/ml. Subsequently, 7.4, 14.8, 29.6, and 59.2 μM of BE and 209 μM of FLZ were added to the C. auris suspension. Additionally, a control group without drugs was established. After incubation for 0, 6, 12, 18, and 24 h, the cells were collected through centrifugation at 825 × g for 5 min. They were diluted with phosphate buffered saline (PBS) and spread onto the YPD agar medium. After 48 h of incubation at 37 °C, the colonies were counted as CFU/ml (Wei et al. 2024).

Transcriptome sequencing

Total RNA for transcriptome sequencing was extracted from C. auris in the control (n = 3) and BE (59.2 μM) (n = 3) groups. The concentration, purity, and integrity of the extracted RNA underwent assessment using a Nanodrop2000 system and agarose gel electrophoresis. After A-T base pairing with poly-A using magnetic beads with oligo (d-T), mRNA was isolated from the total RNA. This mRNA was then added to a fragmentation buffer to isolate a small fragment of approximately 300 bp, which was subsequently reverse-transcribed into cDNA. The adaptor was ligated after the formation of a stable double-stranded structure. Sequencing was conducted using the Illumina NovaSeq 6000 sequencing platform. Genes with a threshold of |log2FC|> 1 and a p-value < 0.05 were selected for further analysis. Gene set cluster analysis, sample relationship analysis, and expression difference analysis were employed to interpret the gene expression data (Wu et al. 2024).

Flocculation test

The concentration of C. auris during the logarithmic growth phase was adjusted to 2 × 106 cells/ml. Subsequently, 7.4, 14.8, 29.6, and 59.2 μM of BE and 209 μM of FLZ were added to the suspension of C. auris. Additionally, a control group that received no treatment was also included. The suspension was incubated at 37 °C in a constant temperature box for 24 h. After incubation, the tubes were vigorously oscillated for a few seconds, then 100 μl of the liquid from the bottom of the test tube was aspirated into a clean 96-well plate using a micropipette (Gelis et al. 2012). The liquid was examined under a DMi8 microscope (Leica, Wetzlar, Hesse, Germany) at 200 × magnification.

Cell surface hydrophobicity

The cell surface hydrophobicity (CSH) of C. auris was measured using the two‐phase water‐hydrocarbon. A suspension of C. auris (2 × 106 cells/ml) was added to a 6-well plate, together with concentrations of 7.4, 14.8, 29.6, and 59.2 μM of BE and 209 μM of FLZ. A control group without treatment was included. After biofilm formation, we removed the supernatant and resuspended the cells in sterile PBS. After centrifugation at 825 × g for 5 min, we removed the supernatant and resuspended the precipitate in PBS. The concentration was then adjusted to an OD600 of 1.0. We collected 1.2 mL of the fungal suspension from each group and added 0.3 mL of n-octane (Shanghai Aladdin Bio-Chem Technology Co., Ltd, China). After vertexing, we let the suspension stand for approximately 15 min to separate the two phases. The lower liquid phase was collected with a pipette and examined at a wavelength of 600 nm. After removing the aqueous phase, we measured the optical density (OD) value at 600 nm. The OD600 value of PBS without octane served as the blank control, and CSH was calculated as (OD600 control–OD600 experiment) /OD600 control (Zhang et al. 2024).

Adhesion

The slides, each with a 1 cm2 surface area, were soaked in a 75% ethanol solution for 24 h. Following that, they were rinsed with sterile PBS. A suspension of C. auris at a concentration of 2 × 106 cells/ml was added to a 6-well plate, along with different concentrations of BE (7.4, 14.8, 29.6, 59.2 μM) and FLZ (209 μM). The plates were subsequently incubated at 37 °C for 2 and 4 h (Zhang et al. 2015). Following this, the slides were stained in the dark for 30 min using a newly configured fluorescein diacetate (FDA, 10 μM, Sparkjade Biotechnology Co., Ltd., Shandong, China). The cells were observed and photographed using a DMi8 microscope (Leica, Wetzlar, Hesse, Germany) at 400 × magnification.

In each well of a six-well plate, 2 mL of Human keratinocyte cells (HaCaT) at a concentration of 2 × 105 cells/ml were added and allowed to culture for 24 h at 37 °C in an environment containing 5% CO2. Following this, a C. auris suspension at a concentration of 2 × 107 cells/ml was added to the six-well plate (Hu et al. 2023), along with varying concentrations of BE (7.4, 14.8, 29.6, 59.2 μM) and FLZ (209 μM). The plate was incubated in an incubator at 37 °C with 5% CO2 for 6 h. After that, the specimens were rinsed 2–3 times with sterile PBS and treated with 4% paraformaldehyde at room temperature for 15 min. Excess liquid was then shaken off, and the cells were washed again with sterile PBS before being stained using an enhanced Gram staining kit. The adherence of C. auris was observed under a DMi8 microscope (Leica, Wetzlar, Hesse, Germany) at 200 × magnification.

Proteinase activity

In a medium containing bovine serum albumin (BSA), 0.05% MgSO4, 2% glucose, 0.1% KH2PO4, 1% BSA, and 2% agar, 10 μl of a prepared fungal suspension (1 × 106 cells/ml) was mixed with equal quantities of drugs (7.4, 14.8, 29.6, 59.2 μM BE, and 209 μM FLZ). This mixture was then added dropwise onto the surface of the BSA medium. The mixture was then incubated at 37℃ for 3–4 days (Kumar et al. 2015). The control group did not receive any drugs. We analyzed the effect of the drugs on proteinase activity based on the PZ value, which was calculated as the colony diameter divided by the sum of the precipitation circle and the colony diameters. A PZ value of 1.0 indicated no proteinase activity, and no precipitation circle was observed around the colony. A PZ value less than 1.0 indicated a precipitation circle around the colony, with a larger circle indicating higher proteinase activity of C. auris.

Phospholipase activity

A fungal suspension (1 × 106 cells/ml) was mixed with equal drug quantities (7.4, 14.8, 29.6, 59.2 μM BE, and 209 μM FLZ) in egg yolk agar base containing 10% (v/v) sterile egg yolk emulsion (Hopebio Biotechnology Co., Ltd, Shandong, China). This mixture was then added dropwise onto the surface of the egg yolk medium and incubated at 37℃ for 2–4 days (Kumar et al. 2015; Larkin et al. 2017). The PZ value was determined using the method employed for proteinase activity.

Esterase activity

In the lipase medium (1% peptone, 0.055% CaCl2, 5% NaCl, 1.5% agar, and 0.5%Tween-80, 10 μl of a prepared fungal suspension (1 × 106 cells/ml) was mixed with equal drug quantities (7.4, 14.8, 29.6, 59.2 μM BE, and 209 μM FLZ) using a micropipette. The mixture was then added dropwise onto the surface of the BSA medium and incubated for 3–4 days at 37℃ (Oyardi et al. 2023). A control group without drugs was included. We evaluated the effect of BE on esterase secretion based on the PZ value, which was calculated using the same method as for proteinase activity analysis.

Hemolytic activity

On the Columbia CNA Blood Agar Plate (HuanKai Microbial Biotechnology Co., Ltd, Guangdong, China), we combined 10 μl of a prepared fungal suspension (1 × 106 cells/ml) with equal drug quantities (7.4, 14.8, 29.6, 59.2 μM BE, and 209 μM FLZ) using a micropipette. The mixture was then added dropwise to the surface of the Plate and incubated at 37 °C for 3–4 days (Kumar et al. 2015). A control group without drugs was included. We analyzed the effect of the drugs on hemolytic secretion using the HZ value, which was calculated as the colony diameter divided by the sum of the precipitation circle and the colony diameters. An HZ value of 1.0 indicated no hemolytic activity, and no precipitation circle was observed around the colony. An HZ value < 1.0 indicates the presence of a precipitation circle around the colony. A smaller HZ value correlates with a larger precipitation circle, indicating a more active secretion of hemolysin by C. auris.

XTT assay

The XTT assay was employed to assess the ability of the drug to inhibit C. auris biofilms. The fungal suspension was adjusted to 2 × 106 cells/ml, and the drug solution was prepared using a serial two-fold dilution method. The final concentrations ranged from 7.4–947.3 μM for BE and 209–3343.5 μM for FLZ. We combined 100 μl of fungal suspension with 100 μl of drug solution in each well, then incubated the plates at 37 °C for 24 h. Absorbance at 492 nm was measured for each well using an enzyme labeler. After incubation, we added 50 μl of XTT (740.2 μM) and menadione (10 μM) (MackLin Bio-Chem Technology Co., Ltd, Shanghai, China) to each well and incubated for 2 h in the absence of light. The OD measurements for each well were taken at 492 nm using a spectrophotometer (Larkin et al. 2017).

Violet crystal assay

The concentration of C. auris during the logarithmic growth phase was adjusted to 2 × 106 cells/ml. Subsequently, 7.4, 14.8, 29.6, and 59.2 μM of BE and 209 μM of FLZ were added to the C. auris suspension. A control group was also established without medication. After co-incubating the drug solution and fungal suspension for 24 h, we removed any non-adherent planktonic cells by rinsing the wells twice with sterile PBS. The wells were incubated with crystal violet staining solution (Yeasen Biotechnology Co., Ltd., Shanghai, China) for 45 min, washed twice with sterile PBS, and then incubated with 95% (v/v) ethanol for 45 min. Finally, the absorbance of each well was measured at 560 nm using a microplate reader (Zhang et al. 2024).

SEM

The coverslips were treated with 75% ethanol and placed in six-well plates. Cells of C. auris in the logarithmic growth phase were added at a concentration of 2 × 106 cells/ml. The C. auris suspensions were treated with 7.4, 14.8, 29.6, and 59.2 μM of BE and 209 μM of FLZ, while a control group without medication was established. After being cultured for 24 h at 37 °C, non‐adherent cells were rinsed with sterile PBS buffer and then immersed in pre-chilled 2.5% glutaraldehyde for a minimum of 2 h. The samples underwent dehydration in 30%, 70%, 95%, and 100% ethanol for 10 min. Following adequate drying at room temperature, the samples were gold‐coated, observed, and photographed using SEM (Larkin et al. 2017). Similar processing was applied to pig skin samples.

CLSM

The concentration of C. auris in the logarithmic growth phase was adjusted to 2 × 106 cells/ml. Subsequently, C. auris suspension was treated with 7.4, 14.8, 29.6, 59.2 μM of BE and 209 μM of FLZ. A control group without medication was also included. After incubating at 37 °C for 24 h, non‐adherent fungal cells were removed by rinsing with sterile PBS. Subsequently, a FunXite-1 dual-color fluorescent active probe (Lumiprobe Corporation, USA) was added, and the cells were stained for 1 h under light protection (Larkin et al. 2017). Fluorescent expression was observed using an FV3000 CLSM (Olympus, Shinjuku, Tokyo, Japan).

Flow cytometry

The concentration of C. auris cells was adjusted to 2 × 106 CFU/ml using PBS, followed by centrifugation at 825 × g for 5 min and resuspension in fresh PBS. Rhodamine 6G was added at a final concentration of 10 µM (Macklin Bio-Chem Technology Co., Ltd, Shanghai, China) to the C. auris cultures, which were then incubated at 35℃ for 50 min. Rhodamine 6G uptake was stopped by placing the C. auris on ice, followed by centrifugation at 825 × g for 5 min. The collected cells underwent three washes with PBS and then treated with different concentrations of BE (7.4, 14.8, 29.6, 59.2 μM) or FLZ (209 μM). The fluorescence intensity of R6G in cells (10,000 cells) was measured using flow cytometry (BD FACSCelesta, USA) with excitation at 488 nm and emission at 525 nm at time points:10, 30, 60, 90, and 120 min (Zhang et al. 2024).

Live/dead fluorescent staining

During the logarithmic growth phase, the concentration of C. auris was adjusted to 2 × 106 cells/ml. Subsequently, BE at concentrations of 7.4, 14.8, 29.6, and 59.2 μM, along with FLZ at 209 μM, were added to the C. auris suspensions. Additionally, a control group without any drug was established. Following a 24‐h incubation period, the biofilms underwent staining with FDA (10 µM) and PI (10 µM, Beyotime Biotechnology Co., Ltd., Shanghai, China) for approximately 5 min. The cells were subsequently rinsed with sterile PBS to remove excess staining (Sun et al. 2022). Fluorescent expression was examined using a DMi8 microscope (Leica, Wetzlar, Hesse, Germany) at a magnification of 200 × .

Reactive oxygen species

The fungal cells (2 × 106 cells/ml) were treated with 7.4, 14.8, 29.6, 59.2 μM of BE and 209 μM of FLZ for 24 h at 37 °C. No rats were allocated to the control group. Samples were stained using the ROS Assay Kit (Beyotime Biotechnology Co., Ltd, Shanghai, China) for 20 min at 37 °C. Excess 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) was removed with sterile PBS (Lei et al. 2023). Fluorescence was observed using a DMi8 microscope (Leica, Wetzlar, Germany) at × 630 magnification.

Cell wall assay

The fungal cells (2 × 106 cells/ml) were treated with 7.4, 14.8, 29.6, 59.2 μM of BE and 209 μM of FLZ for 24 h at 37 °C. Subsequently, the fungal cells were collected, washed with sterile PBS, and treated with a fungal suspension in calcofluor white (CFW, 10 μM, Sigma-Aldrich, Shanghai, China). Staining for chitin content was conducted in the dark at 37 °C for 30 min. As previously stated, samples were treated with aniline blue (Solarbio Science & Technology Co., Ltd, Beijing, China) and incubated in the dark at room temperature for 60 min. (Lei et al. 2023). Fluorescence was examined using a DMi8 microscope (Leica, Wetzlar, Germany) at × 630 magnification.

Aggregation assay

The concentration of C. auris during its logarithmic growth phase was adjusted to 2 × 108 cells/ml (Ramos et al. 2023). Subsequently, 7.4, 14.8, 29.6, and 59.2 μM, along with FLZ at 209 μM, were added to the C. auris suspension. A control group that received no treatment was also included. The fungal suspensions underwent incubation at 37 °C for 120 min. Subsequently, the liquid was examined with a DMi8 microscope (Leica, Wetzlar, Hesse, Germany) at a magnification of × 200.

Porcine skin model

The skin (the skin was obtained from a local abbatoir) was cleaned with PBS and 75% ethanol and then shaved. Using a biopsy punch, 12 mm full-thickness skin samples were collected and placed in 12-well plates containing 3 mL of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS, Hyclone, Logan, UT, USA). The samples were incubated for 6 h at 37 °C with 5% CO2. After rinsing in PBS and thorough drying, a 1 ml paraffin barrier was applied to the inner surface of the DMEM at the epidermal interface. C. auris, suspended in medium (107 cells/ml), was applied to the skin surface (10 μl). Following a 24-h incubation at 37 °C with 5% CO2 but without humidity, the skin samples were prepared for SEM and plated for viability assessment (Horton et al. 2020).

G. mellonella larvae infection model

The G. mellonella larvae (Jiacheng Insect Breeding, Guilin, China) were stored at 4 °C and revived at room temperature for 30 min before the experiments. Based on the survival study, a median lethal dose (LD50) of 7 × 106 cells/ml was determined for G. mellonella larvae. Larval-killing assays were conducted at 37 °C, with 10 randomly assigned larvae to each group inoculated with 10 µl of C. auris (7 × 106 cells/ml) through the last right proleg. Two hours later, the larvae were injected with sterile PBS, BE (7.4, 14.8, 29.6, 59.2 μM) and FLZ (209 μM) through the last left proleg. The treated larvae were then kept in darkness at 37 °C for 5 days, with daily recording of deceased larvae numbers (Pan et al. 2021).

RT-qPCR

Total cDNA extracted from C. auris was used to synthesize cDNA following the instructions of the manufacturer for the reverse transcription kit (Yeasen Biotechnology Co., Ltd., Shanghai, China). Table 1 depicts the primer sequences. The RT-qPCR amplification was conducted using a Light Cycler ® 96 for fluorescence quantification, with the following program: initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 50–60 °C for 20 s, and extension at 72 °C for 20 s. Gene expression levels were analyzed using the relative quantification method based on the cycle threshold values (2-ΔΔCt) (Wu et al. 2023).

Table 1 Primers sequences used for RT-qPCR
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Statistical analysis

The presented data are expressed as the mean ± standard deviation of at least three biological replicates. Statistical analysis utilized Graphpad Prism 9.5 and SPSS 23.0, employing one-way ANOVA followed by LSD or Welch's method. Statistical significance was determined at P < 0.05.

Results

BE demonstrates clear inhibitory effects against C. auris

We evaluated the inhibitory and fungicidal effect of BE on C. auris by determining the MIC and MFC values against four strains. The results showed MIC values ranging from 0.93–1.85 μM (Table 2) for all strains and MFC values were 7.4 μM (Table 2), indicating strong growth inhibition and fungicidal activity by BE across the tested strains. C. auris strains with the lowest MIC values (C. auris strains C1) were selected for further virulence experiments.

Table 2 MIC of BE against C. auris
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To investigate the effect of BE on the growth of C. auris, we used the spot assay to examine the effect of different concentrations of BE on the growth of C. auris. This method is crucial for detecting fungal activity by observing colony growth on a solid agar medium. Our study revealed that varying concentrations of C. auris treated with 7.4 and 14.8 μM of BE along with 209 μM of FLZ showed no significant change in colony growth compared to those in the control group. However, at 29.6 μM or 59.2 μM BE concentrations, C. auris growth was significantly inhibited, indicating the inhibitory effect of BE on C. auris growth (Fig. 1A).

Fig. 1
figure 1

Inhibitory effect of baicalein (BE) on C. auris growth. A After co-culturing for 24 h, the growth-inhibiting impact of BE on different concentrations of C. auris was detected using spot assay. B BE and C. auris were cultured together for different time periods (0, 6, 12, 18, and 24 h), and the impact of BE on inhibiting the growth of C. auris was assessed by counting the number of colonies. All data are presented as mean ± SD: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Control group

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Time-growth studies showed the time and concentration‐dependent antimicrobial activity of a drug, making it applicable as a microbiostatic drug. Here, we investigated the antifungal effects of BE on C. auris at different time intervals. These results showed that the antifungal effect of BE peaked at the 12th hour. However, over time, this effect diminished while still exhibiting a dependence on drug concentration (Fig. 1B). These findings suggest that BE may have a moderate antifungal effect on C. auris.

Effect of BE on the transcriptomics profile of C. auris

To gain further insight into the potential molecular mechanisms of BE against C. auris, we conducted transcriptome sequencing analysis on C. auris samples from the control and BE treatment groups using the Illumina high-throughput sequencing platform. Principal component analysis (Fig. 2B) revealed significant differences between these groups, indicating that pharmacological interventions can affect transcriptional patterns. Significantly, compared to the control group, genes in the treatment group exhibited a significant rightward shift.

Fig. 2
figure 2

Baicalein (BE) regulates the expression of genes associated with C. auris. A Heatmap of clustering analysis of all genes. B Principal components analysis (PCA) mapping of control and BE samples. C Volcano plots between BE and control groups. D–G Heatmap of clustering analysis of virulence-related genes

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This study focused on genes that exhibited differential expression in the BE treatment group (59.2 μM) compared to the control group. Overall, 4,062 differentially expressed genes, comprising 2,020 upregulated and 2,042 downregulated genes, were identified in the BE treatment group (Fig. 2A and C). This suggests that BE intervention significantly changed the gene expression of C. auris, highlighting the comprehensive effect of BE on C. auris and indicating its considerable potential against C. auris infections in the future. Additional clustering analysis of these genes, depicted in the heatmap, indicated that BE-regulated genes associated with adhesion, biofilm formation, efflux pumps, cell membranes, and cell walls in C. auris (Fig. 2D-G) (Jakab et al. 2024).

BE inhibits flocculating ability, cell surface hydrophobicity, and adherence of C. auris

Upon examining the flocculent under an inverted microscope, it was discovered that a significant number of C. auris adhered and accumulated into clusters in the control and 209 μM FLZ groups. Similarly, many C. auris adhered and formed clusters in the 7.4 and 14.8 μM BE concentration groups, while only a few clusters were observed in the 29.6 and 59.2 μM BE concentration groups (Fig. 3A).

Fig. 3
figure 3

Baicalein (BE) inhibits flocculation and cell surface hydrophobicity (CSH) of C. auris. A Images represent flocculation of C. auris. Scale bars 50 μm. B CSH of C. auris was measured by the water-hydrocarbon two-phase method. All data are presented as mean ± SD: ns = not significant, ***P < 0.001, ****P < 0.0001 vs. Control group

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The positive correlation between CSH and adhesion has established CSH as a reliable indicator of adhesion capability. Our results showed significantly lower relative hydrophobicity of the cell surface in the BE group (14.8, 29.6, and 59.2 μM BE) but not in the FLZ group (Fig. 3B).

Early adhesion plays a pivotal role as a virulence factor in Candida spp. Fluorescence microscopy examination revealed that a small quantity of C. auris adhered to the coverslips after a two-h incubation. Comparatively, the treatment group exhibited significantly fewer C. auris adhering to the coverslips than that of the treatment group (Fig. 4A). After 4 h of incubation, a significant increase was observed in the number of C. auris adhered to the coverslips under fluorescence microscopy. In the control and 209 μM FLZ groups, a significant number of adherent C. auris were observed, forming clusters. Increasing the BE concentration led to a decrease in the number of adherent C. auris cells, accompanied by a corresponding reduction in adherent clusters (Fig. 4B). Furthermore, BE reduced the adhesion of C. auris to HaCaT cells. In bright field observation, a significant number of C. auris adhered to the HaCaT cell surface in the control group and 209 μM FLZ group. However, as BE concentration increased, the number of C. auris cells and their adhesion to HaCaT cells decreased. At BE concentrations of 29.6 μM and 59.2 μM, respectively, a significant decrease was observed between the number of C. auris and their adhesion ability (Fig. 5A and B).

Fig. 4
figure 4

Baicalein (BE) inhibits the adhesive ability of C. auris. A Images represent the state of C. auris adhesion at 2 h. Scale bars 50 μm. Quantification of C. auris number by Image J. B Images represent the state of C. auris adhesion at 4 h. Scale bars 50 μm. Quantification of C. auris number by Image J. All data are presented as mean ± SD: ns = not significant, *P < 0.05, ***P < 0.001, ****P < 0.0001 vs. Control group

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Fig. 5
figure 5

Baicalein (BE) inhibits adhesion of C. auris to Human Keratinocytes Cells (HaCaT) cells. A Images represent C. auris adhering to the surface of HaCaT cells. B Quantification of C. auris number by Image J. All data are presented as mean ± SD: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Control group

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BE inhibits partial hydrolase secretion

This study examined the virulence characteristics of C. auris. The results revealed that the strain did not show proteinase or hemolytic activities, with only weak levels of phospholipase activities observed and strong levels of esterase activity detected. A significant inhibitory effect on phospholipase secretion in C. auris was only observed at BE concentrations of 29.6 μM and 59.2 μM. Similarly, esterase secretion in C. auris was inhibited only at a BE concentration of 59.2 μM (Fig 6A–D).

Fig. 6
figure 6

Baicalein (BE) inhibits partial hydrolase secretion in C. auris. A Images represent the growth of C. auris on a medium containing bovine serum albumin. Analyses the effect of BE on proteinase activity based on zone of precipitation (PZ) values. B Images represent the growth of C. auris on a lipase medium. Analyses the effect of BE on esterase activity based on PZ values. C Images represent the growth of C. auris on an egg yolk agar base. Analyses the effect of BE on phospholipase activity based on PZ values. D Images represent the growth of C. auris on a Columbia CNA Blood Agar Plate. Analyses the effect of BE on hemolytic activity based on zone of hemolytic activity (HZ) values. All data are presented as mean ± SD: ns = not significant, *P < 0.05, **P < 0.01 vs. Control group

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BE inhibits C. auris biofilm formation

Biofilms are created when a group of cells adhere together to produce a matrix of extracellular polymeric substances (EPS). We assessed the inhibitory effect of BE on C. auris biofilms by determining SMIC through the XTT method. Figure 7A depicts the inhibitory effect of BE on C. auris. The OD value at 492 nm for C. auris indicated that the SMIC ranged from 29.6–118.4 µM, indicating that BE can impede the formation of C. auris biofilm to some extent.

Fig. 7
figure 7

Baicalein (BE) inhibits C. auris biofilm formation. A Images represent the effect of BE on the metabolic viability of C. auris, measured at 492 nm. B Images represent the effect of BE on the biomass of C. auris biofilms, measured at 560 nm; The biomass of biofilms formed by C. auris was measured in 96-well plates using crystal violet staining. All data are presented as mean ± SD: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Control group

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Biofilms were detected using crystal violet staining, with C. auris staining levels assessed at the base of a 96-well plate. OD was measured at 560 nm using a microplate reader. The presence of crystal violet‐stained C. auris biofilms was indicated by a purple residue at the bottom of the 96-well plate. The control and 209 μM FLZ‐treated groups displayed strong crystal violet staining at the bottom of the plate, indicating robust C. auris biofilm growth. Conversely, BE‐treated wells exhibited reduced crystal violet staining in a concentration-dependent manner (Fig. 7B). The OD value at 560 nm confirmed that BE inhibited C. auris biofilm growth and biomass reduction.

The inhibitory effect of BE on C. auris biofilms was validated using SEM. The control and 209 μM FLZ groups showed distinct oval-shaped yeast morphology in C. auris. However, cells exposed to BE at a concentration of 29.6 µM and 59.2 µM exhibited significantly altered yeast cell topography, indicating an impaired ability to divide (Fig. 8A).

Fig. 8
figure 8

Baicalein (BE) disrupts the morphological structure of C. auris biofilm. A Images represent the biofilms of C. auris under scanning electron microscope (SEM) observation. B Images represent the morphological structure of C. auris biofilms under confocal laser scanning microscope (CLSM) observation

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The inhibitory effect of BE on C. auris biofilm formation was examined with CLSM. FUNXite-1, known for binding to the cell membrane and undergoing transformation within the cell, emitted green fluorescence. OLYMPUS FV3000 software and 3D reconstruction technology were utilized to generate a 3D view of the CLSM image. The Figure illustrates that the biofilm structure in control and 209 μM FLZ groups remained intact, with cells in good condition and exhibiting vigorous growth, resulting in thicker biofilm formation. However, the biofilm thickness of the 7.4 µM and 14.8 µM BE groups were relatively reduced. Additionally, a significant decrease was observed in the thickness of the C. auris biofilm, as well as a reduction in the quantities of cells in the 29.6 µM BE and 59.2 µM BE groups (Fig. 8B). These findings suggest that the absence of complete biofilm formation.

BE suppresses resistance in C. auris biofilm

Flow cytometry results showed that initial intracellular fluorescence levels were similar across all groups, gradually decreasing over time. However, the rate of fluorescence decline in the 29.6 µM BE and 59.2 µM BE groups was significantly slower than that of the control and 209 μM FLZ groups (Fig. 9), indicating that BE may inhibit the efflux pump activity of C. auris.

Fig. 9
figure 9

Effect of baicalein (BE) on efflux transporters in C. auris. A Flow cytometry images represent C. auris efflux pump protein expression after BE treatment at different times. B The mean fluorescence strength of the excretory transport protein in C. auris. All data are presented as mean ± SD: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Control group

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BE induces the apoptosis of C. auris

To determine the effect of BE on C. auris cell viability, we analyzed changes in differential expression between live and dead cells. The control group and 209 μM FLZ group exhibited intense green fluorescence, indicating mature biofilm formation. After incubation with 7.4 µM BE and 14.8 µM BE, C. auris showed a reduction in cell numbers, with most cells expressing green fluorescence, indicating an active state. However, incubation with 29.6 µM BE and 59.2 µM BE resulted in a significant increase in red fluorescence, indicative of an increase in dead cells (Fig. 10). Mitochondria generate ROS, which can affect cell membranes and walls integrity. Therefore, we quantified ROS production, as depicted in the Figure. Figure 11A and B depict a significant increase in fluorescence following BE treatment. Particularly, 74.32% and 78.44% of cells tested positive for ROS after treatment with 29.6 µM BE and 59.2 µM of BE, respectively. However, other groups exhibited considerably lower ROS levels. These findings indicate that BE significantly enhances ROS production.

Fig. 10
figure 10

Baicalein (BE) promotes the death of C. auris biofilm cells. Images represent the survival of C. auris in the biofilm state under fluorescence microscopic observation. Scale bars 50 μm

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Fig. 11
figure 11

The impact of baicalein (BE) on the generation of reactive oxygen species (ROS). A Images represent the accumulation of ROS, with green fluorescence observed. Scale bars 20 μm. B Histogram displays the proportion of cells that are positive for ROS. All data are presented as mean ± SD: ns = not significant, ****P < 0.0001 vs. Control group

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BE damages the cell wall integrity and inhibits C. auris aggregation

The fungal cell wall plays a crucial role in maintaining cell shape and controlling polarized growth. Candida yeasts can modify their cell wall composition and structure in response to environmental changes. Figure 12A illustrates that before BE treatment, CFW was uniformly distributed in yeast cells. However, after treatment with 29.6 µM BE, and 59.2 µM BE, a higher concentration of CFW was observed in the outer layer of C. auris cells (Malavia-Jones et al. 2023), particularly at the budding end (indicated by red arrows). CFW fluorescence analysis showed an increase in chitin content following BE treatment. Additionally, aniline blue staining assay results indicated a reduction in total glucan levels after BE treatment (Fig. 12B). These results indicated that BE altered the structure of the C. auris cell wall.

Fig. 12
figure 12

The effect of baicalein (BE) on the C. auris cell wall. A Images represent C. auris with or without BE treatment was stained with calcofluor white (CFW). The deposition of chitin is indicated by red arrows. Scale bars 20 μm. The fluorescence intensity of CFW-stained cells was used to measure the levels of chitin. B Images represent C. auris with or without BE treatment was stained with aniline blue. Scale bars 20 μm. The levels of glucan were quantified through the fluorescence intensity of cells stained with aniline blue. All data are presented as mean ± SD: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Control group

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Aggregation has recently been identified as an important virulence factor in C. auris and is closely associated with the cell wall. (Bing et al. 2024). The results showed that higher BE concentration led to significant inhibition of C. auris aggregation. Certain inhibition was also observed in the FLZ group (Fig. 13).

Fig. 13
figure 13

Baicalein (BE) inhibits the aggregation of C. auris. Images represent the aggregation of C. auris following treatment with BE. Scale bars 5 μm

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BE inhibits C. auris colonization on porcine skin and decreases its infection to G. mellonella larvae

A porcine skin model was created to simulate C. auris growth in the skin niche (Fig. 14A). The selection of pig skin was based on its similarity to human skin regarding skin layer thickness, skin cell types and distribution, and skin repair mechanisms.

Fig. 14
figure 14

Baicalein (BE) inhibit the adhesion of C. auris to porcine skin. A The porcine skin samples were immersed in Dulbecco's modified Eagle's medium (DMEM) with 10% Fetal Bovine Serum (FBS), and the paraffin method was used to separate the epidermal surface from the liquid medium. B C. auris biofilms were cultured on samples of porcine skin and observed using scanning electron microscope (SEM)

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SEM was employed to evaluate C. auris biomass dispersion and biofilm structure on the skin. Following C. auris introduction, a low‐magnification examination revealed fungal growth on the porcine skin surface. At higher magnification, multilayer biofilms containing yeast cells were observed in the control and the 209 μM FLZ group. A significant change was not observed in C. auris numbers on the porcine skin with 7.4 and 14.8 μM BE concentrations compared to the control group. This indicated that the inhibitory effect of C. auris on adherence to porcine skin was weak at these concentrations. However, at 29.6 and 59.2 μM BE concentrations, the number of C. auris on pig skin significantly decreased, showing only a single layer of adherence. The number of C. auris adhesions significantly decreased, resulting in a single layer (Fig. 14B). BE also enhanced larval survival. By the end of the 5th day, all larvae infected by C. auris had lost their vitality. At concentrations of 7.4, 14.8, and 29.6 μM BE and 209 μM FLZ, survival rates were 20%, 40%, and 20%, respectively, while 59.2 μM BE significantly increased survival to 70% in C. auris. (Fig. 15A and B) This indicates that BE enhances the viability of G. mellonella larvae.

Fig. 15
figure 15

Baicalein (BE) therapy influenced larval survival. A Images represent larvae (n = 10) infected by C. auris after treatment with BE. B The Kaplan–Meier method and log rank test were utilized to analyze survival curves and identify differences. All data are presented as mean ± SD: ns = not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Control group

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RT-qPCR results reveal the mechanisms of BE against C. auris

To confirm the transcriptome results, we selected the two most differentially expressed genes (DEGs) from each of the four groups of virulence-related genes for RT-qPCR. SCF1 and ALS7 encode adhesins that facilitate Candida adhesion. SNQ2 and YCF1 are classified as ATP-binding cassette (ABC) transporters, specifically belonging to the group of multiple drug resistance-associated protein-like transporters. ERG25 and ERG12 are closely associated with the biosynthesis of ergosterol within the cell membrane. This process is essential for maintaining the integrity and function of the cell membrane. RBT5 and PGA4 are essential components of the cell wall proteome. The results showed significant downregulation of all genes under BE treatment, consistent with the transcriptome data (Fig. 16). This indicated that BE might affect related mechanisms to inhibit C. auris.

Fig. 16
figure 16

Baicalein (BE) inhibits C. auris through potential mechanisms (A-H) RT-qPCR were used to detect expression of ALS7, SCF1, SNQ2, YCF1, ERG25, ERG12, RBT5, PGA4 mRNA. All data are presented as mean ± SD: *P < 0.05, **P < 0.01, ****P < 0.0001 vs. Control group

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Discussion

Recently, the proportion of non-C. albicans infections increase annually. All Candida pathogens, including C. albicans, cause superficial and systemic infections of varying severity. C. auris, also known as the “superbug,” emerges as a rapidly growing pathogen. Its rapid global spread over the last decade has triggered several hospital-acquired infection outbreaks (Ruiz-Gaitán et al. 2018), posing a significant threat to human health. C. auris infects patients of all ages, causing invasive infections (Du et al. 2020), including bloodstream infections in patients in intensive care units. It colonizes various human body parts, such as the skin, respiratory tract, and urinary tract. Its survival time in the environment is prolonged, making it difficult to remove. Consequently, using equipment such as implanted catheters and invasive medical instruments often results in cross-infection with C. auris (Biswal et al. 2017).

Additionally, the most prominent feature of C. auris is drug resistance or multidrug resistance, leading to treatment failure and challenges in epidemic control. In 2019, the Centers for Disease Control and Prevention (CDC) categorized pathogenic microorganisms into four threat levels in the antibiotic resistance threats report: urgent, serious, containing, and watch list threats. C. auris was classified as an urgent threat, emphasizing the importance of studying its drug resistance and mechanisms. The CDC has reported a series of drug patterns against C. auris by monitoring MIC data for commonly used antifungal drugs over the past decade. Specifically, 90% of C. auris strains showed high resistance to fluconazole, with a drug breakpoint of MIC ≥ 104.5 μM, 30% of the strains were resistant to the polyene drug amphotericin B, with a drug breakpoint of MIC ≥ 2.2 μM. A small minority of strains (< 5%) were resistant to the echinocandin drug caspofungin, with a drug breakpoint of MIC ≥ 1.8 μM (Chaabane et al. 2019). In our research, we discovered that the MIC of BE against C. auris ranged from 0.93–1.85 μM, indicating its potent growth inhibition activity against C. auris. Compared to the three common antifungal drugs mentioned above, BE demonstrated even greater effectiveness in inhibiting C. auris. Considering the toxicity and high cost associated with currently used antifungal agents, BE shows promising potential for the treatment of C. auris infection.

To understand the molecular interactions between BE and C. auris, we conducted a comparative analysis of the gene expression profiles of BE‐treated C. auris cells (59.2 µM) with untreated cells. Our findings reveal significant changes in the expression levels of 4,062 genes in the BE-treated group compared to those of the untreated control group. We specifically examined differentially expressed genes distinct to the BE-treated group and found downregulation of several genes associated with adhesion, biofilm formation, and other virulence factors in the presence of BE. This suggests that BE inhibits C. auris through multiple mechanisms.

C. auris enhances a significant virulence factor: its exceptional adhesion capability. This underscores the crucial need for early intervention during the early adhesion phase to control fungal infection effectively. Adhesion is facilitated by genes expressing adhesins, among which is the Surface Colonization Factor (SCF1), a distinct and prominent adhesion binding to various surfaces through cation-dependent interactions (Santana et al. 2023). The ALS (agglutinin-like sequence) gene family encodes proteins that facilitate the adherence of the yeast Candida albicans to endothelial and epithelial cells. ALS7 is a hypermutable contingency locus and plays a crucial role in the pathogenicity of Candida as an opportunistic human pathogen (Zhang et al. 2003). Flocculation is also closely associated with adhesin expression (Gelis et al. 2012). Our RT-qPCR findings revealed a significant reduction in the expression levels of SCF1 and ALS7 after BE treatment. Furthermore, BE significantly inhibited flocculation and CSH in C. auris, reducing its adherence to cells. The FLZ treatment, however, did not yield satisfactory results as it showed minimal inhibitory effect on the adhesion of C. auris. These findings suggest that targeting adhesion-related genes to disrupt the initial adhesion stages could be an effective treatment for treating C. auris infection.

Biofilm formation becomes a crucial factor in C. auris virulence as adhesion progresses. Biofilms exhibit higher resistance to antifungal agents than that of the planktonic cells. A previous study found that the MBEC of antifungals against biofilms significantly surpassed the MIC values against planktonic cells (Oyardi et al. 2023). Our study also revealed that SMIC values against biofilms were significantly higher than those of the MIC against planktonic cells. The XTT and crystal violet assays indicated that BE treatment suppressed the metabolic activity and biomass of C. auris biofilms, while FLZ treatment exhibited minimal inhibitory effect. CLSM and SEM images revealed densely packed/oval cells in C. auris biofilms. Furthermore, BE treatment resulted in a concentration‐dependent decrease in biomass and cell density of C. auris biofilms, while FLZ treatment did not exhibit inhibitory effects on biofilm formation.

Efflux pump expression is responsible for antimicrobial resistance within biofilm structures. In fungi, some pump proteins facilitate the transport of intracellular antifungal drugs to the extracellular milieu, leading to reduced drug concentrations within cells and subsequently reducing drug efficacy (Jangir et al. 2023). R6G was employed to measure intracellular fluorescence intensity after drug administration to C. auris, investigating whether the inhibitory effect of BE on Candida auriculata was related to drug efflux. Flow cytometry results showed that BE reduced biofilm resistance by affecting efflux pump expression. In contrast, the FLZ did not affect the expression of efflux pumps. SNQ2 and YCF1 are categorized as multiple drug resistance-associated protein-like transporters—ATP-binding cassette (ABC) transporters. Yeast survives in micro environments utilizing ABC transporters to circumvent toxins and a variety of drugs (Buechel and Pinkett 2020). Consistent with our RT-qPCR results, SNQ2 and YCF1 expression levels significantly decreased after BE intervention.

Candida spp. secrete hydrolytic enzymes, such as proteinases, phospholipases, esterase, and hemolysins, crucial for causing disease. These enzymes assist in binding to host tissues and damaging the host cell membrane. Phospholipases target cell membrane phospholipids, while esterase degrade ester bonds. Hemolysin enhances virulence by breaking down hemoglobin for iron extraction. These enzymes enable Candida spp. To infiltrate mucous membranes and blood vessels while evading host immune detection. A previous study found that various strains exhibited diverse virulence factors; however, all strains displayed proteinase activity. Additionally, 67.3% of these strains showed phospholipase activity, and 68.2% exhibited hemolysin activity (Larkin et al. 2017). Another study showed that 96% of strains exhibited proteinase activity, while none showed phospholipase activity (Shaban et al. 2020). Our study revealed no proteinase or hemolysin activity in any isolates. However, all isolates exhibited weak phospholipase and esterase activities. Among them, BE weakly inhibited the secretion of phospholipase and esterase, while FLZ did not exhibit any inhibitory effect. This suggests that C. auris may exhibit strain-dependent phenotypes that affect its pathogenicity. Various studies have observed variations in virulence traits among C. auris strains, which can be attributed to the distinct characteristics of isolates from different geographical clades (Oyardi et al. 2023).

The cell membrane plays a vital role in normal growth, reproduction, and metabolism of fungal cells. In our investigation into the effect of BE on C. auris structure, we evaluated cell membrane integrity using PI, a fluorescent dye that penetrates cells through damaged membranes and binds to nucleic acids, causing red fluorescence. These findings revealed a significant increase in red cells post‐BE treatment, while the FLZ treatment had no red cells. After BE intervention, our RT-qPCR results also showed reduced expression levels of ERG25 and ERG12, genes closely linked to ergosterol biosynthesis within the cell membrane. One of the essential membrane lipids is sterols, among which ergosterol is the most abundant in fungal cells and mainly resides in the plasma membrane. Meanwhile, ergosterol biosynthesis is one of the major targets of antifungal therapies (Jordá et al. 2023). Above findings have confirmed that BE has the capability to disrupt the cell membrane of C. auris and impact the formation of the cell membrane. ROS typically function as signaling molecules in normal physiological and fungal growth processes. However, excessive ROS can cause cell death or pathological alterations due to their damaging effects. Our study reveals that BE disrupts membrane integrity, leading to ROS accumulation, suggesting the potential of BE to induce apoptosis in C. auris. However, FLZ did not have any effect on C. auris apoptosis.

The fungal cell wall, comprising mannoprotein, β-glucan, and chitin, is essential for maintaining cell shape and protecting against external factors (Hasim and Coleman 2019). The inner chitin/glucan layer plays a crucial role in preserving cell morphology and providing structural stability. Our study reveals that BE induces cell wall destabilization, leading to increased chitin deposition and reduced glucan expression, while FLZ had little impact on the cell wall. After BE intervention, our RT-qPCR results also showed downregulated expression levels of RBT5 and PGA4, integral parts of the cell wall proteome. PGA4 is a 1,3-β-glucanosyltransferase responsible for elongating the 1,3-β-glucan chain in the cell wall and playing a crucial role in cell wall biosynthesis and morphogenesis (Plaine et al. 2008). RBT5 is an extracellular glycosylphosphatidylinositol (GPI)-anchored heme-binding protein, which plays a crucial role in facilitating heme–iron uptake and forming a cross-cell wall heme-transfer cascade (Kuznets et al. 2014). Additionally, cell wall proteins could potentially enhance the aggregative phenotype of C. auris and its adaptability, potentially affecting its persistence and virulence (Wang et al., 2024). Our findings suggest that BE inhibits the aggregation of C. auris, and FLZ also exhibits some inhibitory effects.

Candida colonization is a crucial step in the development of invasive candidiasis. Results from the porcine skin model showed that C. auris could grow and survive on skin surfaces and in environments with skin components, forming dense biofilms on porcine skin. This explains its tendency to colonize the skin, endure on hospital surfaces, and spread efficiently in healthcare settings. Similarly, we observed that BE effectively inhibited C. auris colonization of porcine skin, while FLZ did not show the same effectiveness. The G. mellonella larval model effectively exhibited the potential of BE to inhibit the invasion of organisms by C. auris, whereas FLZ also did not exhibit similar effects.

Conclusions

In conclusion, this study suggests that BE could serve as a novel antifungal and anti-biofilm agent. We showed its efficacy in inhibiting multidrug-resistant C. auris through in vitro phenotypic and genotypic analyses. However, further in vivo studies are necessary to elucidate the mechanisms of action of BE against C. auris and evaluate its potential clinical applications in the pharmaceutical industry.