Overcoming Candida albicans biofilm drug resistance via azole-sophorolipid synergy

Abstract

Antimicrobial resistance is a momentous global threat, demanding innovative approaches to combat drug-resistant pathogens. As a prevalent fungal pathogen, Candida albicans exhibits increasing resistance to conventional antifungals, especially the azoles. This study explores a novel approach combining sophorolipids (SLs), a glycolipid biosurfactant, with clinical azoles, including fluconazole (FLZ), itraconazole (ITZ), and ketoconazole (KCZ), against C. albicans biofilms. SLs from the yeast Starmerella riodocensis exhibited promising metabolic reduction and antibiofilm activity against Candida biofilms, with a biofilm inhibitory concentration (BIC₅₀) of 512 mg/L. Among the tested azoles, ITZ exhibited the highest antibiofilm efficacy, prompting further investigation of SLs combinations. The ITZ–SLs combination markedly enhanced antibiofilm activity against preformed biofilms, with ITZ and SLs concentrations reduced by 16-fold and 4-fold, respectively, compared with their individual treatments (achieving a BIC₅₀ of 1 mg/L ITZ and 128 mg/L SLs). Quantitative real-time polymerase chain reaction analysis revealed significant downregulation of essential biofilm-associated genes such as BCR1, EFG1, and CDC28, demonstrating SLs’s ability to inhibit various stages of biofilm development and stability. Thus, the synergy observed with azole drugs, particularly ITZ and SLs, was highly effective in biofilm removal, highlighting the compatibility of anti-biofilm biosurfactant SLs with some antifungal agents.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-36278-8.

Introduction

Antimicrobial resistance (AMR) is a growing global health threat, largely driven by the overuse of antibiotics and antifungals¹. Infections caused by resistant pathogens contribute substantially to morbidity and mortality worldwide, and fungal resistance has become a critical concern in clinical settings². Among fungal pathogens, Candida albicans is a major opportunistic yeast that forms resilient biofilms on host tissues and medical devices³. These biofilms, composed of adherent cells embedded in a protective extracellular matrix, significantly reduce the efficacy of conventional antifungal agents. Biofilm-associated resistance arises from restricted drug penetration, altered metabolic activity, and the upregulation of efflux pumps and expression of antimicrobial resistance encoding genes⁴. Consequently, infections caused by C. albicans biofilms remain difficult to eradicate and are often associated with recurrent or persistent disease⁵.

Current antifungals, including azoles, polyenes, and echinocandins, are widely used to treat fungal infections⁶. Many antifungal agents are less effective against Candida biofilms due to the physiological barrier that reduces the permeability of drugs through the extracellular matrix⁷. Triazoles, a subclass of azoles, are widely prescribed due to their oral availability and broad spectrum of activity against various pathogenic fungi, including C. albicans⁸. Since their introduction in the 1980s⁷, azoles have become the most widely used class of antifungals in clinical settings. These synthetic heterocyclic compounds function by disrupting ergosterol biosynthesis, which is crucial for maintaining fungal cell membrane integrity⁹. Their clinical utility is further constrained by drug–drug interactions via cytochrome P450 inhibition and by rising resistance in non-albicans species¹⁰. Candida auris, in particular, shows alarming resistance to fluconazole, underscoring the urgent need for alternative or complementary strategies¹⁰.

Combination therapy has emerged as one promising approach to overcome these limitations¹¹. By pairing antifungal agents from different classes, combination regimens can broaden the spectrum of activity, lower the required doses, and reduce the risk of resistance¹². Several studies have evaluated the effectiveness of antifungal drug combinations in combating Candida biofilms. Synergistic effects have been reported for azoles with amphotericin B¹² while combining echinocandins has shown synergistic or additive effects against Candida biofilms, supporting the potential of drug combinations to improve outcomes in biofilm-associated infections¹³.

Beyond classical antifungal combinations, glycolipid biosurfactants such as sophorolipids (SLs) have attracted attention for their wide range of applications. SLs, produced by non-pathogenic yeasts such as Starmerella bombicola and Starmerella riodocensis^14,15, are amphiphilic molecules¹⁶ valued for their biodegradability, low toxicity, and surface activity¹⁷. They have been reported to exhibit antimicrobial¹⁷, antiviral¹⁸, anticancer¹⁹, and immunomodulatory activities²⁰, as well as synergy with certain antifungal drugs²⁰.

Most studies so far have focused on SLs from S. bombicola, typically lactonic forms, which have shown promising activity in planktonic cultures or in silico models but limited validation under biofilm conditions²¹. In contrast, S. riodocensis produces structurally distinct acidic, dimeric, and trimeric SLs not commonly found in other yeasts²². These unique features suggest potentially different biological activities and raise the possibility that S. riodocensis SLs could provide enhanced or complementary anti-biofilm effects.

To date, little is known about the effects of S. riodocensis SLs on Candida biofilms or their potential in combination with azole antifungals. Addressing this gap, the present study evaluates the activity of S. riodocensis SLs, alone and in combination with azoles, against C. albicans biofilms. The findings aim to provide insights into the potential of sophorolipid–azole combinations as alternative strategies for managing biofilm-associated candidiasis.

Results

SLs Production, Purification, and characterization

The yeast strain S. riodocensis strain GT-2564R¹⁵ was employed for the production and extraction of crude SLs. Cultures grew in a production medium containing palm oil, glucose, yeast extract, and urea as primary carbon and nitrogen sources for 7 days. Following the extraction, crude SLs were purified via silica column chromatography to enrich the active compound. During the purification, 1 g of crude SLs extract was loaded onto the column, yielding approximately 0.3–0.5 g of purified SL. The purified SLs were subsequently characterized using HPLC to evaluate their composition. HPLC analysis produced chromatograms that enabled the identification of characteristic SL peaks, confirming the successful production, extraction, and purification of SLs. Peaks corresponding to acidic SLs were identified at retention times (RTs) of 32.944, 33.807, 34.307, 34.905, 35.301, and 37.020 min, while lactonic SLs were detected at RTs of 43.968, 47.906, 48.390, 53.700, and 58.497 min (Fig. 1). These findings confirmed the presence of both acidic and lactonic SLs in the purified samples, indicating the production of a diverse range of SLs forms.

Fig. 1.

HPLC analysis chromatograms of purified SLs extracted from Starmerella riodocensis grown in yeast extract-containing medium. The chromatogram highlights the separation profile, showing differences in the composition of purified SLs.

Antifungal activity of SLs against planktonic Candida albicans

To confirm that the observed antibiofilm effects were not due to direct antifungal activity, the planktonic susceptibility of C. albicans to crude and purified sophorolipids (SLs) was evaluated using the EUCAST broth microdilution protocol²³. SLs showed no inhibition of planktonic growth at concentrations up to 1,024 mg/L. In contrast, reference azole antifungals inhibited growth within expected ranges: fluconazole (MIC₅₀ = 0.5 mg/L), itraconazole (MIC₅₀ = 0.03 mg/L), and ketoconazole (MIC₅₀ = 0.06 mg/L)²⁴. These results confirm that the reductions in biofilm formation observed in subsequent assays are independent of the fungicidal or fungistatic activity of SLs (Table 1).

Table 1.

Antifungal and antibiofilm activities of sophorolipids and antifungal drugs against Candida albicans.

Antifungal Agent	MIC₅₀ (mg L⁻¹)	Metabolic activity (IC₅₀) (mg L⁻¹)		Biofilm Inhibition (BIC₅₀) (mg L⁻¹)
	Planktonic cells	Young biofilms	Preformed biofilms	Young biofilms	Preformed biofilms
Sophorolipid (crude)	> 1024	512	1024	512	2048
Sophorolipid (purified)	> 1024	256	512	256	512
FLZ	0.5	32	128	> 32	> 128
ITZ	0.03	4	16	8	16
KCZ	0.06	8	32	> 8	> 32

Biological effect of SLs on metabolic activity and biofilm formation of C. albicans

Initial experiments were conducted to assess the inhibitory effects of crude SLs extracted from S. riodocensis grown in specific media against C. albicans, focusing on both young biofilms (90 min incubation, corresponding to the early adhesion phase) and preformed biofilms (24 h incubation, representing established biofilm structures)²⁵. Metabolic activity was assessed using the XTT assay and expressed as IC₅₀, the lowest concentration of an antimicrobial agent that reduces the metabolic activity of biofilm-embedded cells by 50%. Biofilm biomass was quantified using the crystal violet staining assay and expressed as BIC_50, the lowest concentration of an antimicrobial substance required to reduce total biofilm biomass by 50%²⁶ as described in the methodology section (Table 1).

Crude SLs achieved IC₅₀ at 512 mg/L for young biofilms (90 min) and 1,024 mg/L for preformed biofilms (24 h). In fact, crude SLs demonstrated significant antibiofilm activity, achieving a BIC₅₀ of 512 mg/L against young biofilms and 2,048 mg/L against preformed biofilms (Fig. 2). The notable difference in IC₅₀ and BIC₅₀ values suggests that young biofilms are more susceptible to crude SLs treatment than preformed biofilms, which typically exhibit increased tolerance due to their structural complexity and extracellular matrix accumulation. These preliminary results suggest that crude SLs possess the ability to inhibit biofilm formation in Candida species.

Fig. 2.

Effects of SLs crude treatments on young biofilms (90 min) and preformed biofilms (24 h) of C. albicans. (a) Percentage of metabolic activity reduction of C. albicans biofilms. (b) Percentage of biofilm biomass of C. albicans, treated with crude SLs at different concentrations. The percentage inhibition of biofilms was compared with the untreated biofilms. Data are presented as mean ± standard deviation/standard error of the mean. Asterisks indicate significant differences between the two groups, where p < 0.05.

In the next phase of the experiment, purified SLs significantly reduced the metabolic activity and biofilm formation of young C. albicans biofilms, achieving IC₅₀ and BIC₅₀ at concentrations below 256 mg/L, which is half the concentration required for crude SLs (Fig. 3). A similar pattern of metabolic activity and biofilm biomass inhibition was observed against preformed biofilms, at 512 mg/L. The IC₅₀ values indicated that metabolic inhibition required half the crude concentration, while BIC₅₀ values were 4-fold lower (Table 1). Thus, purification significantly enhanced SL activity against both young and preformed Candida biofilms.

Fig. 3.

Effects of purified SLs treatments on young biofilms (90 min) and preformed biofilms (24-h) of Candida albicans (a) Percentage of metabolic activity reduction of C. albicans biofilms (b) Percentage of biofilm biomass of C. albicans, treated with purified SLs at different concentrations. The percentage inhibition of biofilms was compared with the untreated biofilms. Data are presented as mean ± standard deviation/standard error of the mean. Asterisks indicate significant differences between the two groups, where * p < 0.05.

Inhibitory effect of clinical antifungal Azoles on C. albicans metabolic activity and biofilm formation

In this study, three azole drugs (FLZ, ITZ, and KCZ) were employed to evaluate their effects on C. albicans metabolic activity and biofilm formation. Various concentrations of FLZ were used to treat C. albicans biofilms, with up to 32 mg/L applied to young biofilms and 128 mg/L to preformed biofilms. IC₅₀ was observed at 32 mg/L against young biofilms, while biofilm biomass was reduced by approximately 40% at this concentration (Fig. 4a). For preformed biofilms, FLZ concentrations up to 128 mg/L achieved an IC₅₀ but failed to reach a BIC₅₀, indicating limited efficacy against preformed biofilms (Fig. 4b).

Fig. 4.

Effects of clinical drug treatments on young biofilms (90 min) and preformed biofilms (24 h) of C. albicans. (a) FLZ against C. albicans young biofilms (b) FLZ against C. albicans preformed biofilms (c) ITZ against C. albicans young biofilms (d) ITZ against C. albicans preformed biofilms (e) KCZ against C. albicans young biofilms (f) KCZ against C. albicans preformed biofilms. The percentage inhibition of biofilms was compared with the untreated biofilms. Data are presented as mean ± standard deviation/standard error of the mean. Asterisks indicate significant differences between the two groups, where * p < 0.05.

ITZ, a widely used antifungal, was administered at a concentration of 8 mg/L against young biofilms and 16 mg/L against preformed biofilms. The results demonstrated IC₅₀ at a concentration of 4 mg/L, with a BIC₅₀ of 8 mg/L against young biofilms (Table 1). For preformed biofilms, a higher concentration of 16 mg/L was used. ITZ demonstrated its BIC₅₀ at the highest concentration applied, 16 mg/L, while IC₅₀ was achieved at 8 mg/L (Fig. 4d).

KCZ (imidazole class) exhibited a similar inhibition pattern. At 8 mg/L, KCZ achieved IC_50, while BIC₅₀ failed to reach at this concentration against young biofilms (Fig. 4e). Against preformed biofilms, IC₅₀ was achieved at 32 mg/L, while KCZ reached approximately 45% inhibition of biofilm biomass at 32 mg/L, without exceeding the 50% threshold (Fig. 4f). These data demonstrate that all tested azoles reduce biofilm viability, though preformed biofilms remain largely tolerant. These active concentrations were subsequently employed in combination with SLs treatments to explore potential synergistic effects between azoles and crude SLs in inhibiting C. albicans biofilms.

Combination of crude SLs with Azoles against Candida biofilms

The effects of crude SLs in combination with azoles (FLZ, ITZ, and KCZ) were evaluated against both young (90 min) and preformed (24 h) C. albicans biofilms. Combination efficacy was assessed using IC₅₀ and BIC₅₀ values derived from XTT reduction and crystal violet staining assays, respectively. Multiple SLs-azole combinations across a range of tested concentration pairs exhibited inhibition of metabolic activity and reduction of biofilm biomass, demonstrating a concentration-dependent combined effect (Tables 2 and 3).

Table 2.

Summary of FICI values obtained using the metabolic Inhibition assay against Candida albicans young and preformed biofilms using the combination of sophorolipids (SLs) and antifungal drugs fluconazole (FLZ), Itraconazole (ITZ), and ketoconazole (KCZ).

Combination	Biofilm type	SLs Concentration Range (mg L⁻¹)	Azole concentration range (mg L⁻¹)	FICI Range	Interaction type
Crude SLs + FLZ	Young	128	8	0.5	Synergistic
		64–128 256	16 8–16	0.6–0.8 0.8–1.0	Additive
	Preformed	256	32	0.5	Synergistic
		64–256 512	64 16–64	0.6–0.8 0.6–1.0	Additive
Crude SLs + ITZ	Young	16 32–128	0.5–2 0.5–1	0.2–0.5 0.2–0.5	Synergistic
		32–128 256	2 0.5–2	0.6–0.8 0.6–1.0	Additive
	Preformed	16–32 64 128–256	4–8 4 2–4	0.3–0.5 0.3 0.3–0.5	Synergistic
		64–256 512	8 2–8	0.6–0.8 0.6–1.0	Additive
Crude SLs + KCZ	Young	16 128	4 1–2	0.5 0.4–0.5	Synergistic
		32–128 256	4 1–4	0.6–0.8 0.6–1.0	Additive
	Preformed	256 512	16 2–16	0.8 0.6–1.0	Additive
Purified SLs + ITZ	Young	8 16–64	1–2 0.5–1	0.3–0.5 0.3–0.5	Synergistic
		16–64 128	2 0.5–2	0.6–0.8 0.6–1.0	Additive
	Preformed	8 16 32 64	4–8 4 2 1–4	0.3–0.5 0.3 0.4 0.3–0.5	Synergistic
		16–64 128	8 1–8	0.6-0.8 0.6–1.0	Additive

Table 3.

Summary of FICI values obtained using the biofilm Inhibition assay against Candida albicans young and preformed biofilms using the combination of sophorolipids (SLs) and antifungal drugs fluconazole (FLZ), Itraconazole (ITZ) and ketoconazole (KCZ).

Combination	Biofilm type	SLs concentration range (mg L⁻¹)	Azole concentration range (mg L⁻¹)	FICI Range	Interaction type
Crude SLs + FLZ	Young	128	8	0.5	Synergistic
		64–128 256	16 8–16	0.6–0.8 0.8–1.0	Additive
	Preformed	256–1024	32–64	0.6–1.0	Additive
Crude SLs + ITZ	Young	16 64–128	4 2	0.5 0.4–0.5	Synergistic
		32–128 256	4 2–4	0.6–0.8 0.8–1.0	Additive
	Preformed	512	4	0.5	Synergistic
		256–512 1024	8 4	0.6–0.8 0.8–1.0	Additive
Crude SLs + KCZ	Young	64 128	2 1–2	0.4 0.4–0.5	Synergistic
		64–128 256	4 1–4	0.6–1.0	Additive
	Preformed	512	8	0.5	Synergistic
		256–512 1024	16 8–16	0.6–0.8 0.8–1.0	Additive
Purified SLs + ITZ	Young	16–32 64	1–2 0.5–2	0.2–0.4 0.3–0.5	Synergistic
		16–64 128	4 0.5–4	0.6–0.8 0.6–1.0	Additive
	Preformed	8–16 32 64 128	8 4 2–4 1–4	0.5 0.3 0.3 0.3–0.5	Synergistic
		32–128 256	8 1–8	0.6–0.8 0.6–1.0	Additive

Against young biofilms, the SLs-FLZ combination yielded IC₅₀ values at 8–16 mg/L FLZ in combination with 64–256 mg/L SLs (Table 2), while corresponding BIC₅₀ values were observed at comparable concentration ranges, indicating the potent anti-biofilm activity (Table 3). For preformed biofilms, IC₅₀ values were obtained with 32–64 mg/L FLZ combined with 64–512 mg/L SLs, whereas BIC₅₀ values required a higher SLs concentration (≥ 256 mg/L). These findings suggest that SLs enhance azole accessibility but do not completely overcome fluconazole resistance in preformed biofilms (Supplementary Fig. S1).

The combination, SLs-ITZ, exhibited a stronger synergistic trend. Against young biofilms, IC₅₀ values were observed at 0.5 mg/L ITZ, while BIC₅₀ values were obtained at 2 mg/L ITZ in combination with 64 mg/L SLs, defining synergistic effects in combination. For preformed biofilms, this combination resulted in markedly reduced effective concentrations by 8-fold, yielding IC₅₀ values at 2 mg/L ITZ and 128 mg/L SLs. Similarly, a 4-fold enhancement in biofilm inhibition was observed, yielding BIC₅₀ values of 4 mg/L and 512 mg/L of ITZ and SLs, respectively (Tables 2 and 3).

For the SLs–KCZ combination, young biofilms treated with 1 mg/L KCZ and 128 mg/L SLs exhibited both IC₅₀ and BIC₅₀ values, indicating synergistic interaction. In preformed biofilms, combinations of 2–16 mg/L KCZ with 512 mg/L SLs yielded IC₅₀ values, corresponding to an additive effect, while BIC₅₀ values were observed at 8 mg/L KCZ combined with 512 mg/L SLs (Tables 2 and 3).

Combination of purified SLs with ITZ against Candida biofilms

Given the superior performance of ITZ in combination with crude SLs, further experiments were conducted using purified SLs to determine whether increased purity enhanced synergistic activity. The combination of purified SLs and ITZ demonstrated potent synergistic effects against both young and preformed biofilms.

For young biofilms, the purified SLs–ITZ combination yielded IC₅₀ values at 0.5 mg/L ITZ in combination with 16 mg/L SLs, while BIC₅₀ values were observed at 0.5 mg/L ITZ combined with 64 mg/L SLs, indicating strong synergistic interaction. This synergy likely resulted from enhanced drug penetration and disruption of the less-developed extracellular matrix, suggesting that the combination can lower antifungal doses and reduce potential side effects and resistance. For preformed biofilms (24 h), which are characterized by a more robust extracellular matrix, purified SLs further enhanced ITZ efficacy. The purified SLs–ITZ combination yielded an IC₅₀ at 1 mg/L ITZ in combination with 64 mg/L SLs, representing a two-fold improvement compared with the crude SLs–ITZ combination. Biofilm inhibition was also enhanced, with a BIC₅₀ of 1 mg/L ITZ combined with 128 mg/L SLs (Tables 2 and 3). These results demonstrate that even preformed biofilms, which typically exhibit increased tolerance to antifungal treatment, can be effectively targeted using purified SLs–ITZ combinations. The enhanced efficacy of this combination against both metabolic activity and biofilm biomass suggests a complementary mode of action and highlights its potential for future antifungal applications.

Cytotoxicity activity of sophorolipid

The cytotoxicity of purified SLs was evaluated on RAW 264.7 macrophages using the MTT assay. Cells treated with SLs at concentrations ranging from 6.25 to 100 mg/L maintained high viability, with values exceeding 86% at the highest tested concentration (100 mg/L) (Supplementary Table S9). Although these tested concentrations are lower than the BIC₅₀ values for antibiofilm activity (256–512 mg/L), previous studies have shown that sophorolipids exhibit low or negligible cytotoxicity even at concentrations up to 500 mg/L in various mammalian cell lines, including human keratinocytes²⁷. Additional studies are underway to examine the toxicity of different forms of sophorolipids at various concentrations for further applications.

Expression analysis of hyphal and biofilm associated genes

qRT-PCR analysis was performed to investigate the molecular impact of SLs treatment on C. albicans, focusing on hyphal and biofilm-associated genes under hyphal-inducing and biofilm conditions. A concentration of 256 mg/L purified SLs was selected, corresponding to the BIC₅₀ determined against young biofilms in our inhibition assays (Table 1).

Hyphal-Inducing conditions

Under hyphal-inducing conditions, non-synchronized cells were treated with SLs for 2 h. Overall, SLs exposure led to significant downregulation of multiple genes spanning different stages of biofilm development (Fig. 5a). Adhesion-related genes: Strong suppression was observed for adhesins ALS1 (7.69-fold downregulation) and ALS3 (5.55-fold), as well as HWP1 (6.25-fold), a major hyphal wall protein required for host adhesion and biofilm maturation. These findings indicate that SLs strongly inhibit early adhesion processes. In contrast, YWP1 was only modestly downregulated (1.69-fold), consistent with its reported role as a negative regulator of biofilm formation²⁸.

Fig. 5.

Effect of sophorolipid (SLs) treatment on C. albicans biofilm-associated gene expression. (a) Relative expression levels of biofilm-associated genes in non-synchronized C. albicans cells grown at 37 °C, 200 rpm (OD₆₀₀ = 0.5) in RPMI-1640 medium supplemented with 10% fetal bovine serum and treated with 256 mg/L SLs for 2 h under hyphal-inducing conditions. (b) Relative expression levels of biofilm-associated genes in non-synchronized C. albicans cells treated with 256 mg/L SLs for 6 h under biofilm-inducing conditions. (c) Relative expression levels of biofilm-associated genes in G2/M-synchronized C. albicans cells (nocodazole-treated) exposed to 256 mg/L SLs for 6 h under biofilm-inducing conditions. (d) Graphical summary illustrated the functional roles and regulatory interactions of key genes affected by SLs treatment during Candida albicans biofilm formation. The scheme was constructed based on the experimental findings of this study. (Bio render program was used to create the graphical summary for this study).

Cell wall integrity and surface proteins, genes encoding glycosylphosphatidylinositol (GPI)-anchored proteins such as PGA10²⁹ (7.69-fold), PGA55 (3.70-fold), and PGA59 (2.70-fold) were also downregulated, suggesting that SLs affect cell wall remodeling and reduce cell-surface stability during biofilm establishment. The cyclin-dependent kinase CDC28 (3.85-fold downregulation) and its hyphal-specific regulator HGC1 (5.26-fold) were suppressed, pointing to inhibition of hyphal elongation and cell cycle progression under SLs treatment. The fatty acid synthase gene FAS2 was reduced by 5.26-fold, indicating possible impairment of lipid biosynthesis, which is essential for membrane formation and hyphal growth. The transcription factors BCR1 (4.55-fold downregulation) and ACE2 (3.57-fold) were also suppressed, both of which regulate adhesion and biofilm-related genes, suggesting that SLs interfere with upstream regulatory circuits controlling biofilm development. Genes associated with biofilm matrix and late-stage development, including EFG1 (2.78-fold) and RLM1 (1.78-fold), were moderately affected, suggesting a weaker impact of SLs on maturation processes compared to early adhesion. Finally, dispersal-associated genes NRG1 and PES1 showed only modest reductions (1.72-fold and 2.08-fold, respectively), indicating that SLs primarily target the initial and proliferation phases rather than dispersal. Collectively, these results demonstrate that SLs broadly suppress key adhesion-, hyphal-, and regulatory genes, with the most pronounced effects observed during the early adhesion and filamentation stages of C. albicans biofilm development.

Expression analysis under biofilm conditions

qRT-PCR analysis was conducted to investigate the impact of SLs on Candida albicans biofilm formation, focusing on key genes involved in adhesion, proliferation, and maturation. In non-synchronized biofilm-forming populations analyzed after 6 h, SLs treatment reduced ACE2 (2.9-fold), consistent with a moderate effect on the adhesion stage (Fig. 5b). The expression of other adhesion and regulatory genes, including ALS1, ALS3, HWP1, BCR1, EFG1, PGA55, and NRG1, showed only mild changes. Since cells are heterogeneous in terms of which stage of the cell cycle they are in, it is important to consider whether the differences in gene expression are related to the number of cells at each stage.

While adhesion genes like ALS1 (2.25-fold), ALS3 (1.90-fold), and HWP1 (2.13-fold) were still suppressed, the fold changes indicated milder inhibition. Similarly, BCR1 (1.92-fold), EFG1 (1.49-fold), RLM1 (1.20-fold), and HGC1 (1.52-fold) transcription factors and hyphal regulators maintained higher expression levels, suggesting ongoing biofilm regulatory activity. The cell cycle gene CDC28 (1.27-fold) remained less affected, reflecting a heterogeneous cell cycle distribution.

Previous studies have demonstrated a connection between biofilm development and cell cycle progression, showing enhanced biofilm formation when yeast cells accumulate in the G₂/M phase and reduced biofilm capability during the G₁/G₀ phase³⁰. To analyze this relationship, nocodazole, a microtubule polymerization inhibitor commonly used in cell cycle studies, was applied to synchronize C. albicans cells at the G₂/M checkpoint before initiating biofilm formation³⁰. This synchronization approach allowed for targeted assessment of SLs’ effects during a key cell cycle stage involved in biofilm regulation.

In G₂/M-synchronized cells treated with SLs, significant downregulation was observed in key biofilm-related genes. Adhesion genes such as ALS1 (5.13-fold), ALS3 (4.44-fold), HWP1 (4.4-fold), and ACE2 (4.49-fold) showed marked suppression. Transcription regulators involved in biofilm maturation, like BCR1 (2.99-fold), EFG1 (2.82-fold), and RLM1 (2.37-fold), were also notably repressed. Furthermore, the hyphal regulator HGC1 (3.30-fold) and adhesin PGA55 (2.74-fold) had reduced expression, indicating interference with multiple biofilm stages. The cell cycle kinase CDC28 was moderately downregulated, consistent with arrested proliferation (Fig. 5c).

Together, these results indicate that SLs primarily disrupt biofilm formation when C. albicans cells are synchronized in G₂/M, with the most pronounced effects on adhesion and maturation stages. In non-synchronized populations, the effect is largely confined to ACE2, highlighting that susceptibility to sophorolipid mediated biofilm inhibition is strongly influenced by the cell cycle stage.

Discussion

The present study provides new insights into the antibiofilm potential of sophorolipids (SLs) derived from Starmerella riodocensis strain GT-2564R, focusing on their activity against Candida albicans and their synergistic interaction with azole antifungal agents. The findings highlight efficacy of SLs and the remarkable enhancement of azole activity when used in combination with these glycolipid biosurfactants. Together with gene expression analyses supporting a biofilm-specific mechanism, the results suggest that SLs represent promising natural adjuvants for combating drug-resistant Candida biofilms.

HPLC analysis confirmed the presence of both acidic and lactonic SLs, eluting between 30 and 60 min of RT (Fig. 1). The earlier elution of acidic SLs reflects their lower hydrophobicity, while lactonic SLs typically more hydrophobic are recognized for stronger antimicrobial properties³¹. To be consistent with previous reports, another study identified a composition of acidic and lactonic forms³² confirming a consistent elution pattern where acidic forms appeared earlier and lactonic forms later in chromatogram. These results demonstrate the ability of S. riodocensis to produce a balanced mixture of functionally diverse SLs.

In this study, both crude and purified SLs inhibited C. albicans biofilm formation in a dose-dependent manner, a pathogen known for forming resilient biofilms on medical devices and host tissues³³, while exhibiting no direct fungicidal or fungistatic activity against planktonic cells. This is consistent with the observations that SLs generally lack strong antifungal activity in free-living cells but interfere with biofilm formation (Table 1). The absence of growth inhibition at concentrations up to 1024 mg/L confirmed that SLs act primarily through antibiofilm pathways rather than conventional antifungal toxicity, unlike reference azoles, which displayed activity at reported MICs (fluconazole 0.5 mg/L, itraconazole 0.03 mg/L, ketoconazole 0.06 mg/L)²⁴. This distinction is crucial, as biofilm formation represents a key virulence factor in C. albicans pathogenicity and a major contributor to antifungal resistance.

A clear distinction was observed between the activities of crude and purified SLs. Purified SLs exhibited stronger inhibition of both young and preformed biofilms, requiring half the concentration of crude SLs to achieve comparable IC₅₀ values for metabolic inhibition. The BIC₅₀ values for purified SLs were 256 mg/L for young biofilms and 512 mg/L for preformed biofilms, whereas crude SLs required 512 mg/L and 2048 mg/L, respectively. The higher purity of this fraction likely explains its stronger antibiofilm activity compared with the crude extract, which may contain residual medium components or other metabolites. The higher BIC₅₀ values of crude SLs, therefore, reflect lower effective bioactive content. The higher susceptibility of young biofilms compared with preformed biofilms underscores the progressive resistance that develops as the extracellular matrix becomes more complex and denser³⁴. The biofilm maturation process involves the accumulation of polysaccharides, proteins, and extracellular DNA, which collectively reduce the penetration of antifungal agents³⁵. The observation that purified SLs retained activity against preformed biofilms, albeit at higher concentrations, suggests that they can disrupt matrix integrity and cellular adhesion forces, possibly through amphiphilic interactions that alter surface hydrophobicity and permeability³⁶.

Although azoles effectively inhibit planktonic growth by targeting ergosterol biosynthesis³⁷. The IC₅₀ and BIC₅₀ values of FLZ, ITZ, and KCZ confirmed their reduced activity against biofilm-associated cells, particularly in preformed biofilms. These results agree with previous reports that azole efficacy declines sharply once biofilm formation has occurred, primarily due to reduced drug penetration, efflux pump activation, and metabolic dormancy of biofilm-embedded cells³⁸. While increasing azole concentrations improves biofilm-associated Candida inhibition, high dosage is neither clinically desirable nor sustainable due to potential host toxicity and the risk of resistance development. Instead, these findings emphasize combination approaches such as SLs-azoles treatments, which enhance drug efficacy without requiring elevated drug concentrations.

Building on this rationale, the combination assays of SLs with azoles provided strong evidence for synergistic and additive interactions, particularly with itraconazole. The combination of crude SLs with FLZ yielded predominantly additive inhibition patterns against both young and preformed biofilms, suggesting that SLs enhance drug accessibility to the biofilm but do not fully overcome intrinsic azole resistance. In contrast, the combination with ITZ exhibited clear synergistic behavior, achieving substantial reductions in both metabolic activity and biomass at concentrations several-fold lower than monotherapy. Specifically, the crude SLs-ITZ combination achieved BIC₅₀ at 4 mg/L ITZ with 512 mg/L SLs against preformed biofilms, representing a 4-fold improvement over ITZ alone. This synergistic effect became more pronounced when purified SLs were used, with BIC₅₀ values reduced to 1 mg/L ITZ and 128 mg/L SLs. Such enhancement likely results from the increased membrane-permeabilizing properties of purified SLs, facilitating greater intracellular accumulation of ITZ (Tables 1 and 3).

Mechanistically, the synergistic effect between SLs and azoles may arise from multiple, complementary effects. SLs, being amphiphilic glycolipids, can increase cell membrane permeability and disrupt lipid microdomains³⁶, thereby facilitating azole entry and access to their intracellular target, lanosterol 14α-demethylase³⁷. Moreover, by interfering with the biofilm matrix, SLs may reduce the local concentration of extracellular polysaccharides that impede drug diffusion³⁹. These physicochemical interactions could account for the substantial reduction in drug dosage required to achieve comparable or enhanced inhibition. Such combination effects are clinically relevant, as lowering azole concentrations may mitigate cytotoxicity and slow the emergence of resistance.

Among the azole antifungals tested, ITZ exhibited superior inhibitory activity against C. albicans biofilms compared with FLZ and KCZ. This enhanced efficacy can be partly attributed to ITZ’s higher lipophilicity and favorable pharmacokinetics, which promote its accumulation in lipid-rich membranes and penetration through the biofilm extracellular matrix⁴⁰. ITZ is often considered superior to FLZ and KCZ for treating C. albicans biofilms due to these distinct mechanisms of action, while this enhanced penetration allows ITZ to reach effective concentrations within biofilms, where drug resistance mechanisms often limit the efficacy of other antifungals⁴¹. Studies have shown that ITZ possesses favourable pharmacokinetic properties, including good penetration into tissues and a relatively long half-life⁴². In contrast, FLZ, being more polar and highly water soluble, and with minimal protein binding, exhibits limited biofilm penetration⁴². While lipophilicity facilitates drug distribution within the biofilm, the extracellular matrix remains a significant physical barrier, highlighting the importance of SLs-mediated matrix disruption in enhancing antifungal efficacy.

The enhanced efficacy of purified SLs in synergy with ITZ against preformed biofilms is particularly significant. Preformed biofilms represent one of the most challenging Candida infection states due to their protective architecture and reduced metabolic activity, which limit the effectiveness of antifungals⁴³. The ability of SLs–ITZ combinations to achieve IC₅₀ and BIC₅₀ values at markedly lower concentrations underscores a promising therapeutic strategy. The dual mode of action, membrane permeability, and biofilm matrix disruption may explain this improvement. Importantly, such synergistic interactions also highlight the potential for integrating natural biosurfactants as antifungal adjuvants in current treatment regimens.

Cytotoxicity assays further supported the potential biocompatibility of purified SLs for biomedical applications. The compounds exhibited minimal toxicity toward RAW 264.7 macrophages, maintaining over 85% cell viability even at concentrations up to 100 mg/L. Although the BIC₅₀ values observed for biofilm inhibition were higher than the cytotoxicity test range, the high cell viability suggests that SLs remain well-tolerated at sub-cytotoxic levels relevant to antifungal activity. This observation is consistent with previous studies reporting that SLs are biodegradable, non-immunogenic, and well-tolerated in mammalian systems^44,45.

qRT-PCR analysis provided mechanistic insight into how SLs interfere with Candida albicans biofilm development. Under hyphal-inducing conditions, SL treatment caused strong suppression of adhesion- and hypha-associated genes such as ALS1, ALS3, HWP1, BCR1, and ACE2, which are essential for biofilm initiation and morphological transition⁴⁶. Previous studies have shown that mutants lacking ALS3 or HWP1 form defective biofilms with reduced metabolic activity, supporting our findings⁴⁶. In contrast, YWP1 showed only mild downregulation, consistent with its role as a negative regulator of biofilm formation its deletion enhances rather than suppresses biofilm growth⁴⁶.

SLs also downregulated genes encoding GPI-anchored cell wall proteins (PGA10, PGA55, PGA59), suggesting weakened adhesion and reduced cell wall integrity²⁸. Suppression of FAS2, a fatty acid biosynthesis gene, further implies interference with lipid metabolism, potentially affecting membrane fluidity and biofilm resilience⁴⁷. The reduced expression of CDC28 and HGC1, key regulators of cell cycle progression and hyphal elongation, indicates inhibition of the yeast-to-hypha transition, a critical virulence factor. Moderate repression of EFG1 and RLM1 supports the idea that SLs also affect biofilm maturation and matrix organization^48,49, while limited changes in dispersal-associated genes (NRG1, PES1) suggest that SLs act mainly during the early biofilm stages^50,51.

Under biofilm-forming conditions, transcriptional profiles revealed stage-dependent responses to SLs. In non-synchronized biofilms, gene expression was heterogeneous, reflecting the mixed population of cells at different developmental stages⁵². ACE2 was the only gene significantly downregulated (approximately 2.9-fold), suggesting that SLs primarily target the adhesion phase of biofilm formation under these conditions. As ACE2 regulates cell wall remodeling and daughter-cell separation, its repression may lead to impaired adhesion and delayed biofilm initiation. Other key genes (ALS1, ALS3, HWP1, BCR1, EFG1) showed moderate suppression, indicating partial inhibition of early biofilm signaling pathways⁵².

Nocodazole-mediated G₂/M arrest amplified glycolipid-induced downregulation of biofilm genes associated with adhesion, proliferation, and maturation. Adhesion appears to be the biofilm stage most affected by SLs, with significant repression of ACE2 and BCR1. The latter is a master regulator of biofilm formation, controlling matrix production and genes involved in cell surface properties, thereby enhancing adherence. ALS3, an adhesin critical for biofilm stability and regulated by BCR1, is also a prominent target of SLs activity. Additionally, PGA55, encoding a GPI-anchored adhesin-like protein localized at budding sites and hyphal tips, was downregulated in synchronized cells, further indicating disruption at the proliferation stage⁵³. RLM1, a transcription factor essential for cell wall stress response and biofilm maturation, indicates that RLM1 may act as a novel cell cycle transcription factor regulating a cell controlling a cell wall–associated “START checkpoint” that governs the G₁/S phase transition. This checkpoint appears to coordinate biofilm development with cell size and cycle progression⁵⁴. Candida albicans biofilm development occurs through four distinct stages: initial attachment to surfaces (0–2 h), formation of microcolonies (2–4 h), hyphal growth and proliferation (4–6 h), and eventual maturation into a complex, multilayered biofilm with extracellular matrix and polymorphic cells over 24–48 h⁵⁵. Our findings show that SLs reduce the expression of genes involved in adhesion, proliferation, and maturation, supporting existing models of biofilm development. Future studies employing detailed time-course analyses will be important to delineate the precise effects of SLs at each stage of biofilm formation.

Taken together, these findings indicate that SLs target multiple transcriptional programs governing adhesion, hyphal growth, lipid metabolism, and biofilm matrix stability. While, previous work from our laboratory demonstrated SLs treatments induce hyphal deformation and cell surface alterations in C. albicans, as observed by scanning electron microscopy¹⁴. These findings support the interpretation that the transcriptional downregulation of biofilm-associated genes observed here correlates with functional effects at the cellular level.

The structural characteristics of SLs from S. riodocensis may contribute to their distinctive activity profile. Unlike S. bombicola, which mainly produces lactonic SLs⁵⁶, S. riodocensis generates a higher proportion of acidic forms along with unique dimeric and trimeric SLs, which have not been reported in S.bombicola²². Variations in acetylation pattern, polymerization, and fatty acid branching likely alter hydrophobicity and molecular interactions, potentially enhancing permeability and biofilm disruption capacity²². Such structural diversity may account for the observed differences in biological activity between the two species.

In conclusion, this study highlights the promising antibiofilm potential of combining SLs with existing clinical azole drugs to address the growing resistance of C. albicans. The combination of SLs with ITZ demonstrated significant enhancements in antibiofilm activity while reducing the metabolic impact on the fungal pathogen. The SLs-ITZ synergy provides a valuable strategy for modifying pathogen–drug interactions for developing novel approaches to antifungal therapies. Downregulation of key genes involved in biofilm formation further supports the efficacy of SLs in disrupting C. albicans virulence. Exploration of the mechanistic action and development of SLs-drug combination may further result in effective strategies to combat the escalating global challenge of drug-resistant fungal infections.

Materials and methods

Production of sophorolipids

A yeast strain, Starmerella. riodocensis strain GT-2564R, obtained from the Excellent Research Laboratory of Yeast Innovation, KMUTT, and employed for the production of SLs¹⁵. The inoculum was cultivated in YPD media at 30 °C overnight. Subsequently, the cultured cells were transferred to the production medium, and the cultivation was continued in a glucose-rich yeast extract medium⁵⁷ composed of 100 g/L glucose, 10 g/L yeast extract (Thermo Fisher Scientific, Waltham, MA, USA), 1 g/L urea, and 100 g/L palm oil. Fermentation was carried out at 30 °C for 7 days under constant agitation (150 rpm) to support efficient cell growth and metabolite production. The initial pH was maintained at 4.5 before adding the inoculum to the medium. After fermentation, the cells were separated from the broth by centrifugation. The supernatant, containing the crude SLs, was subjected to liquid-liquid extraction. In this step, ethyl acetate (Quality Reagent Chemical, QREC, New Zealand) was used in a 1:2 (v/v) ratio to extract SLs from the aqueous layer. The organic phase was collected and concentrated by evaporation under reduced pressure using a rotary evaporator (Büchi Rotavapor; Büchi Labortechnik AG, Flawil, Switzerland), resulting in crude SLs.

The C. albicans strain ATCC90028 was used in this study to conduct the antibiofilm assay. Morpholinepropanesulfonic acid (MOPS)-buffered RPMI 1640 (R6504; Sigma-Aldrich, St. Louis, MO, USA) was used as the growth medium, with incubation at 37 °C.

SLs purification and characterization

Crude sophorolipid extract was purified by silica gel column chromatography (200–300 mesh, Acros Organic) using a glass column (15 × 300 mm, packed with 2.5 g silica gel). The column was pre-equilibrated with chloroform containing 1% methanol (v/v), and 1 mL of crude extract solution (0.5 mL SLs in 0.5 mL chloroform) was applied. Elution with 80 mL of chloroform: methanol (99:1, v/v) yielded 1 mL fractions, which were analyzed by TLC (silica gel 60 F254 plates, Merck). Spots were visualized with p-anisaldehyde spray, heating (110 °C, 5 min), and iodine vapor. Fractions with identical TLC profiles were pooled.

Following purification, fractions were characterized by gradient elution HPLC (Shimadzu, Japan) equipped with a UV detector (207 nm) and a VertiSep™ C18 column (5 μm, 4.6 × 250 mm). Commercial standards of acidic SLs (non-acetylated, 0.500 g, lot 0000037112) and lactonic SLs (di-acetylated, 0.265 g, lot 714831903) from Biosynth Carbosynth (Compton, UK; product codes DL71484, DL71483) were used for comparison. The mobile phase consisted of a linear acetonitrile gradient in water: 0–20% (0–15 min), 20–80% (15–40 min), and 80–100% (40–50 min), followed by isocratic elution at 100% acetonitrile for 20 min, at a flow rate of 0.5 mL/min. The gradient was adapted from Kim et al.⁵⁸ and used to separate acidic and lactonic sophorolipid isoforms^57,59. Retention times and UV spectra matched the reference standards, confirming the identity of acidic and lactonic SLs.

Antifungal susceptibility assay (MIC₅₀ determination)

The antifungal activity of crude and purified sophorolipids (SLs) against Candida albicans was evaluated using broth microdilution according to EUCAST (E.DEF 7.3.1) protocols²³. Inoculum was adjusted to 1 × 10⁵ CFU/mL as specified, and two-fold serial dilutions of SLs (2–1,024 mg/L) were prepared in 96-well plates with RPMI 1640 (MOPS-buffered). Fluconazole, itraconazole, and ketoconazole were included as reference antifungals within guideline breakpoint ranges. Controls included medium-only (blank) and growth-only wells. Plates were incubated at 37 °C for 24 h, and optical density at 600 nm was measured using Multiskan Sky microplate reader (Thermo Fisher Scientific). MIC₅₀ was defined as the lowest concentration reducing growth by ≥ 50% relative to the untreated control.

Biofilm formation assay

Candida albicans was cultured overnight in RPMI 1640 medium, supplemented with 2% glucose, at 37 °C with shaking at 150 rpm to ensure robust growth. For biofilm assays, cells were washed and resuspended in standard RPMI 1640 (without additional glucose) to avoid high glucose-induced alterations in biofilm morphology and matrix composition. Biofilms were established in 96-well plates at 1 × 10⁶ CFU/mL, with 200 µL of cell suspension per well. To assess SL’s and azole effect on different biofilm stages, cells were allowed to adhere for 90 min (young biofilms at the early stage of the adhesion phase²⁵ or 24 h (preformed biofilms). Wells were then washed three times with 0.15 M sterile PBS to remove any non-adherent cells. Treatments were applied after the washing, ensuring planktonic cells were not exposed to treatments.

Each well was loaded with 200 µL of RPMI 1640 (without additional glucose) containing the desired concentration of SLs or azoles and was incubated at 37 °C for 24 h. Treatments were applied separately (SLs or Azole) for young and preformed biofilms. Untreated wells served as negative controls, and DMSO was used as the vehicle control at concentrations below 5%. Crude SLs were tested at 1–2,048 mg/L, purified SLs at 1–1,024 mg/L, and azoles across relevant ranges (fluconazole 0.5–128 mg/L; itraconazole 0.125–16 mg/L; ketoconazole 0.125–32 mg/L) against both young and preformed Candida albicans biofilms. SLs and azole combinations were applied based on their individual BIC₅₀ values, using fixed-ratio concentrations to evaluate potential synergistic, additive, or antagonistic effects on both young and preformed Candida albicans biofilms.

Quantification of metabolic activity of C. albicans (XTT Assay)

The metabolic activity of C. albicans biofilms was quantified using the XTT reduction assay, following a slightly modified version of the protocol outlined by Watchaputi et al.⁶⁰. To prepare the working solution, XTT was dissolved in PBS at a concentration of 0.5 g/mL, and a final concentration of 1 µM menadione was added to facilitate electron transfer. After the SLs treatment incubation, wells were washed twice with PBS, and 200 µL of the XTT-menadione solution was added to each well. The plates were incubated in the dark at 37 °C for 2 h to allow the reaction to proceed. The assay relies on the reduction of XTT, which is converted into a formazan product by mitochondrial dehydrogenases, indicating the level of metabolic activity. The resulting colour change, due to XTT reduction, was measured at an absorbance of 490 nm using a Multiskan Sky microplate reader (Thermo Fisher Scientific).

Crystal Violet-Based biofilm analysis

To assess biofilm formation, the wells were first rinsed three times with PBS to remove non-adherent cells and then left to air-dry for 30 min. After drying, 110 µL of a 1% (w/v) crystal violet solution was added to each well to stain the biofilm matrix, allowing for visualization and quantification of the biofilm structures. Following a 45-minute incubation period, any excess stain was carefully removed by washing the wells three times with PBS. To solubilize the bound crystal violet, 200 µL of absolute ethanol was added to each well. A 100 µL aliquot of the ethanol–crystal violet solution was then transferred to a new 96-well plate. Finally, the absorbance of the solubilized dye was measured at 590 nm using a microplate reader to quantify the extent of biofilm formation.

SLs - Drug combination

The interaction between SLs and potent clinical antifungal drugs was tested using the checkerboard assay on both young and preformed C. albicans biofilms. The primary goal was to evaluate the potential synergistic effects of SLs in combination with commercial antifungal drugs, including triazoles such as FLZ, ITZ, and the imidazole ketoconazole (KCZ), against C. albicans biofilm formation. In addition to testing SLs alone, drug combination assays were conducted using crude SLs with azoles to investigate possible synergism. After evaluating the efficacy of these combinations, the most effective crude SLs-azole combinations were selected for further testing with purified SLs, allowing for a direct comparison of the effects of crude versus purified SLs in combination with azoles in inhibiting C. albicans biofilms.

Candida cell suspension was prepared at 1 × 10⁶ CFU/mL in RPMI-1640 medium (R6504; Sigma-Aldrich). The cells were treated with various concentrations of SLs and clinical antifungals. The concentrations of SLs and azoles were determined based on prior experiments that had demonstrated significant biofilm disruption at BIC₅₀. Both the XTT assay and antibiofilm assay (CV assay) were performed to determine the metabolic and antibiofilm activity of SLs in combination with the two triazoles (FLZ and ITZ) and the imidazole (KCZ).

The Fractional Inhibitory Concentration Index (FICI) was calculated based on BIC₅₀ values using the formula,

Where the BIC₅₀ of drug A alone and the BIC₅₀ of drug B alone are the biofilm inhibitory concentrations at 50% inhibition for drugs A and B when used alone, and the numerators are the BIC₅₀ values of the drugs when used in combination. FICI was calculated to evaluate the nature of the interactions and classified as synergistic (FICI ≤ 0.5), additive (0.5 < FICI ≤ 1.0), indifferent (1.0 < FICI < 4.0), and antagonistic (FICI ≥ 4.0)⁶¹. Three independent biological replicates were performed, each with two independent replicates per condition. Checkerboard testing with dual (XTT/CV) endpoints was used as an accepted first-line method to screen for synergy across biofilm conditions⁶².

Cytotoxicity assay (MTT assay)

Cytotoxic effects of the compounds on RAW 264.7 cells were assessed using the MTT assay. Cells were treated with various concentrations of the test compound for 24 h. After incubation, 0.5 mg/mL of MTT solution was added to each well and incubated for 2 h to allow formation of formazan crystals. The medium was then removed, and 100 µL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan. Absorbance was measured at 570 nm using a microplate reader. Cell viability (% cell survival) was calculated relative to untreated control cells.

Gene expression analysis by qRT-PCR

Quantitative real-time PCR (qRT-PCR) was performed to examine transcriptional responses of Candida albicans under both hyphal-inducing and biofilm-forming conditions.

Hyphal conditions

C. albicans cells were adjusted to an optical density (OD₆₀₀) of 0.5 in RPMI-1640 medium supplemented with 10% fetal bovine serum to induce hyphal growth and early biofilm formation. Cells were treated with 256 mg/L of SLs at 37 °C with agitation at 200 rpm for 2 h. This concentration, based on the BIC₅₀ determined for young biofilms, represents a sub-inhibitory dose sufficient to elicit transcriptional responses without eradicating biofilms. Following treatment, cells were washed twice with DEPC-treated water, mechanically disrupted by vortexing with sterile glass beads, and total RNA was extracted using the method of Schmitt et al.⁶³, and DNase I (Qiagen, Hilden, Germany) treatment to remove genomic DNA. The RNA was then purified using the Qiagen RNeasy Mini Kit, and its quality was verified using agarose gel electrophoresis to visualize clear RNA bands. The RNA concentration and purity were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific) to ensure adequate RNA quality for downstream applications.

Biofilm conditions with cell synchronization

For biofilm-specific analysis, C. albicans ATCC 90,028 cells were first synchronized into the G₂/M phase of the cell cycle using nocodazole, as described by Juanes (2016) with modifications⁶⁴. Cells were grown in YPD medium supplemented with 15 µg/mL nocodazole, adjusted to OD₆₀₀ = 0.5, and incubated at 30 °C for 3 h with shaking (150 rpm). After incubation, the cell cultures were washed twice with PBS. According to the biofilm formation procedure described by Kuloyo et al.⁶⁵, cells were adjusted to 1 × 10⁶ cells/mL in RPMI-1640 medium (without glucose). Biofilm initiation was achieved by allowing the cells to adhere to polystyrene plates for 90 min at 37 °C without agitation. Non-adherent cells were removed by PBS washing, after which the medium was replaced with RPMI-1640 (glucose-free) containing 256 µg/mL of SLs. The plates were incubated for a further 6 h at 37 °C to allow biofilm development under treatment. Biofilms were then collected by scraping the plate surface, washed with DEPC-treated water, and processed for RNA extraction and purification as outlined above.

cDNA synthesis and qRT-PCR

For both hyphal and biofilm samples, cDNA was synthesized using the qPCRBIO cDNA synthesis kit (PCRBIOSYSTEMS, UK). qRT-PCR was performed using Luna^® Universal qPCR Master Mix (NEB, USA). Thermal cycling conditions followed the protocol described by Haque et al.²⁰. The housekeeping gene ACT1 was used for normalization. Relative gene expression levels were calculated using the 2^–ΔΔCt method, with untreated C. albicans cells serving as the reference control. Each biological replicate (n = 3) was analyzed in triplicate technical replicates. Table 3 lists the gene-specific primer sequences used in this study, including primers for the housekeeping control gene ACT1.

Data acquisition

At least two independent studies, each performed in triplicate, were conducted for each experiment. The findings are presented as mean ± standard deviation. The statistical analysis was performed using GraphPad Prism version 8.0.0 for Windows (GraphPad Software, San Diego, CA, USA; www.graphpad.com), employing multiple t-test comparisons to assess the differences between groups. A significance threshold of p ≤ 0.05 was used. All two-dimensional graphs (Figs. 1, 2, 3, 4 and 5) were generated using GraphPad Prism, Version 8.0.0 (GraphPad Software). Three-dimensional surface plots (supplementary figures S1-S4) were generated using XYZ Mesh, Version 9.0 (Gray Technical, LLC, Flemingsburg, KY, USA) with default mesh interpolation settings.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization and Validation: L.A.C.B.J., & N.S.: Investigation; Formal Analysis; Data Curation; Visualization: L.A.C.B.J. & N.S, Methodology and Software, L.A.C.B.J., K.W., & N.S.: Writing-Original Draft Preparation, L.A.C.B.J.; Writing-review and editing, K.W., C.B., & N.S.: Supervision and Project Administration, C.B., & N.S; Resource and Funding acquisition L.A.C.B.J., K.W., & N.S. All authors have read, reviewed, and agreed to the published version of the manuscript.

Funding

This research project is supported by King Mongkut’s University of Technology Thonburi (KMUTT), Thailand Science Research and Innovation (TSRI), and National Science, Research and Innovation Fund (NSRF) Fiscal year 2025, National Research Council of Thailand (NRCT) and King Mongkut’s University of Technology Thonburi for Mid-Career Researcher grant (No. N42A650315) to N.S., Excellent Research Laboratory for Yeast Innovation of Fiscal Year 2023 (No. 6620864) to N.S. L.A.C.B.J. received the Scholarship for the Development of High-Quality Research Graduates in Science and Technology Petchra Pra Jom Klao Ph.D. Research Scholarship (KMUTT-NSTDA) from King Mongkut’s University of Technology Thonburi. It is also partially supported by King Mongkut’s University of Technology Thonburi’s Postdoctoral Fellowship in fiscal year 2024 under KIRIM Research Project ID 28074 to K.W.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article and its Supplementary materials.

Declarations

Competing interests

The authors declare no competing interests.