Antifungal Effects of Pterostilbene on Candida albicans, Candida dubliniensis, and Microcosm Biofilms of Denture Stomatitis

Abstract

Natural compound-based strategies have gained attention as alternatives to conventional antifungal therapies, particularly in the management of Candida infections affecting the oral cavity, such as denture stomatitis. Our aim was to investigate the antifungal activity of the polyphenol pterostilbene (PTE) on clinical Candida isolates and microcosm biofilms from denture stomatitis, as well as to evaluate its toxicity and therapeutic efficacy in Galleria mellonella. PTE exhibited fungicidal effects against Candida albicans and Candida dubliniensis at 32 µg/mL. Time-kill assays demonstrated complete inhibition of viability for both strains within 8 h of exposure. In addition, PTE exhibited broad-spectrum antimicrobial activity, significantly reducing the counts of streptococci, mutans streptococci, staphylococci, and yeasts within microcosm biofilms. In vivo, PTE showed no signs of toxicity in G. mellonella at concentrations up to 20× MIC. Prophylactic treatment with PTE enhanced larval survival in experimental candidiasis caused by both C. albicans and C. dubliniensis. Moreover, prophylactic treatment decreased the fungal burden of C. albicans in the G. mellonella hemolymph, while the recruitment of hemocytes involved in host defense mechanisms remained unaltered. In summary, PTE demonstrated antimicrobial activity against Candida planktonic cells and complex biofilms associated with denture stomatitis, exhibiting favorable biocompatibility and in vivo antifungal efficacy in G. mellonella model.

1. Introduction

The Candida genus comprises yeast species that are part of the normal human microbiota and are commonly found on the skin, in the gastrointestinal and genitourinary tracts, and in the oral cavity [1]. Under healthy conditions, Candida spp. behaves as commensal. However, disruptions in host homeostasis—such as compromised immune defenses or factors like broad-spectrum antibiotic use, diabetes mellitus, chemotherapy, or the use of medical and dental devices (e.g., catheters and prostheses)—can promote yeast overgrowth [2,3]. These conditions may shift Candida from a commensal to a pathogenic state, leading to infections of the skin and mucous membranes, and potentially to hematogenous dissemination [4,5].

Candida colonization and infection are driven by several fungal virulence factors, including morphological transitions, adhesion and filamentation capabilities, secretion of hydrolytic enzymes, and biofilm formation [6,7]. Among these, biofilm formation plays a critical role. Biofilms are complex, structured communities of microorganisms embedded in a self-produced extracellular matrix. In the oral cavity, Candida infections are closely linked to the yeast’s ability to adhere and form biofilms on both biotic (oral mucosa) and abiotic (dentures) surfaces [8,9]. This process is a major contributor to Candida-associated denture stomatitis—a condition affecting from 20 to 75% of denture wearers [10,11].

Conventional treatment of Candida spp. infections primarily rely on antifungal agents from the polyene, azole, and echinocandin classes [12]. However, prolonged use of these drugs presents several limitations, including hepatotoxicity and nephrotoxicity, limited bioavailability, and the emergence of resistant strains [13,14,15]. For example, resistance to fluconazole, a widely used antifungal for managing Candida infections, has been estimated to range between 0.5% and 13% among Candida species [16]. To overcome these challenges, the search for alternative therapies—particularly those based on natural compounds such as polyphenols—has gained increasing attention [17].

Polyphenols are low molecular weight organic compounds characterized by one or more phenolic groups and are widely distributed in various plant species. In humans, they exhibit a range of biological activities, including anticancer, anti-inflammatory, antioxidant, and antimicrobial effects. These therapeutic properties are largely attributed to their antioxidant properties, which include the ability to reduce reactive oxygen and nitrogen species, enhance the enzymatic activity of the antioxidant system, and mitigate inflammation [18,19].

Among these, pterostilbene has emerged as a promising polyphenol with notable antimicrobial properties. It belongs to the stilbene class and is characterized by high lipophilicity and a broad spectrum of pharmacological activities. Naturally occurring in fruits such as blueberries, grapes, and plums, pterostilbene is synthesized by plants as a secondary metabolite in response to environmental stressors, including pathogen invasion and excessive ultraviolet radiation exposure [20]. The antibacterial activity of pterostilbene has been demonstrated against Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus [21,22]. In addition, pterostilbene has shown antifungal effects against filamentous fungi such as Aspergillus flavus [23] and yeasts including Candida albicans [24]. However, the effects of PTE on complex heterotypic biofilms and its therapeutic efficacy in vivo remain unexplored.

In this study, we investigated the antifungal activity of pterostilbene against clinical isolates of Candida spp. and microcosm biofilms associated with denture stomatitis. Furthermore, we evaluated its antifungal and immunomodulatory effects in an in vivo experimental candidiasis model using the invertebrate host Galleria mellonella.

2. Materials and Methods

2.1. Clinical Samples

In this study, clinical samples were collected from the lesions of denture stomatitis of two patients (P1 and P2). From these samples, Candida strains were isolated and identified by mass spectrometry (MALDI-TOF). The isolates of patient 1 and patient 2 were identified as Candida dubliniensis (strain P1) and Candida albicans (strain P2), respectively. In addition, both clinical samples of patient 1 (sample 1) and patient 2 (sample 2) were used to form microcosm biofilms.

The collections were approved by the Human Research Ethics Committee of the Institute of Science and Technology of São Paulo State University (ICT/UNESP) under number 5.827755. These strains and samples are kept frozen in a freezer at −80 °C in the Oral Microbiology and Immunology Laboratory (ICT/UNESP).

2.2. Activation of Candida Strains and Pterostilbene Preparation

Candida dubliniensis P1 and C. albicans P2 were cultured on Sabouraud dextrose agar (SDA; Kasvi, Pinhais, PR, Brazil) at 37 °C for 24 h prior to each assay. The polyphenol pterostilbene (PTE; Sigma-Aldrich, St. Louis, MI, USA) and the antifungal fluconazole (FCZ; Sigma-Aldrich) were used. Stock solutions of PTE and FCZ at 10,000 µg/mL were prepared in dimethyl sulfoxide (DMSO; Sigma-Aldrich).

2.3. Susceptibility Assay

The susceptibility of Candida spp. to PTE and FCZ was determined using the broth microdilution method according to manual E.Def 7.3.2 of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Stock solutions of PTE and FCZ were diluted in RPMI 1640 (Sigma-Aldrich) culture medium with L-glutamine, pH 7.0, without sodium bicarbonate, supplemented with 2% glucose, and buffered with 0.165 M 3-(N-morpholino) propanesulfonic acid (MOPS; Sigma-Aldrich). We used concentrations ranging from 0.5 to 512 µg/mL for PTE, and from 0.12 to 64 µg/mL for FCZ.

Fungal inoculum was prepared from cultures of C. dubliniensis P1 and C. albicans P2 on SDA for 24 h at 37 °C. The cells were suspended in distilled water, homogenized, and standardized to a final concentration of 5.0 × 10⁵ cells/mL. Microplates containing 100 µL of 2-fold serial dilutions of PTE and FCZ were inoculated with 100 µL of the adjusted inoculum. Growth (100 µL of RPMI + 100 µL of fungal inoculum) and sterility (100 µL of RPMI + 100 µL of distilled water) controls were included. The reference strain Pichia kudriavzevii (formerly Candida krusei) ATCC 6258 was used as quality control. After incubation at 37 °C for 24 h, the absorbance of microplates was measured in a spectrophotometer (Epoch, Biotek Instruments, Winooski, VT, USA) at 530 nm. The minimum inhibitory concentration (MIC) was considered as the lowest concentration capable of inhibiting at least 90% of fungal growth for PTE, and 50% for FCZ, compared to the growth control.

For the minimum fungicidal concentration (MFC) assay, 10 μL was removed from each well without visible growth, cultured on SDA plates, and incubated for 24 h at 37 °C. The minimum fungicidal concentration (MFC) was determined as the lowest concentration of PTE capable of preventing 100% fungal growth after plating. Then, the MFC/MIC ratio was performed. PTE was considered fungicidal if the MFC/MIC ratio was ≤4 and fungistatic if the ratio was >4 [25].

2.4. Time-Kill Curves

Time-kill assays were performed using the RPMI medium supplemented with 2% glucose as the growth medium. Fungal inoculum was prepared from C. dubliniensis P1 and C. albicans P2 cultures diluted in distilled water, quantified, and adjusted to a final concentration of 2.5 × 10⁵ cells/mL. Then, both strains were treated with PTE at MIC, followed by incubation at 37 °C. Non-treated cells were used as negative controls. Aliquots were taken at 0, 2, 8, 24, and 48 h after exposure and plated on SDA to determine the colony-forming units (CFU/mL).

2.5. Effects of PTE on Microcosm Biofilms of Denture Stomatitis

Microcosm biofilms were formed from clinical samples P1 and P2, collected from two volunteers clinically diagnosed with oral candidiasis associated with denture use. Cervical brushes were used to collect specimens from both the palatal lesion and the surface of the dentures, which were then stored in brain heart infusion (BHI, Kasvi, Paraná, Brazil) broth. The presence of yeast cells suggestive of Candida spp. was confirmed through microscopic analysis and by growth on SDA supplemented with 0.5% chloramphenicol, as well as on a chromogenic selective medium (Chromagar Candida Plus; Difco, Franklin Lakes, NJ, USA) for presumptive identification of Candida species.

After collection, both samples were thawed and homogenized. Subsequently, 400 μL of each sample was added to 20 mL of fresh BHI broth with 5% sucrose and incubated at 37 °C with 5% CO₂ for 24 h. After this period, the suspension was centrifuged (5000 rpm) for 10 min, the supernatant was discarded, and the cells were resuspended in 10 mL of fresh BHI broth with 5% sucrose. Then, 225 µL of the previously grown sample was added to each well, and the microplates were incubated at 37 °C for 96 h with 5% CO₂. The wells were washed, and 225 µL of fresh BHI broth with 5% sucrose was added every 24 h.

After biofilm formation, the microcosm biofilms were treated for 24 h with PTE at 1×, 5×, 10× and 20× MIC. Non-treated groups were used as negative controls. Then, the biofilms were disrupted using an ultrasonic homogenizer (Sonoplus HD2200, Bandelin Electronic, Berlin, Germany) with a power of 7 W for 30 s. From the solution obtained, serial dilutions were performed and plated in five different culture media: BHI for non-selective counting, Mitis Salivarius agar (MS; Difco) for counting streptococci, Mitis Salivarius Bacitracin Sucrose agar (MSBS; Difco) for counting mutans streptococci, Mannitol agar (MAN; Difco) for counting staphylococci, and SDA with chloramphenicol for counting yeasts. The plates were then incubated at 37 °C for 24 h under 5% CO₂ to determine microbial viability (CFU/mL) [26].

2.6. Standardization of Candida Inoculation Conditions and PTE Treatment in G. mellonella

To establish a model of experimental infection with C. dubliniensis P1 and C. albicans P2, as well as the PTE treatment protocol in G. mellonella, we evaluated the toxicity of PTE and the effects of infection at varying Candida concentrations.

To assess toxicity, PTE was injected at different concentrations (1×, 10× and 20× MIC) into the last proleg of G. mellonella larvae (n = 10) using a 10 µL 26G Hamilton syringe (Hamilton Company, Reno, NV, USA). A group inoculated with PBS was included as a control. Larvae were monitored for 5 days to generate survival curves. Larvae were considered dead if they did not respond to touch [27].

To determine the appropriate inoculum for Candida strains, G. mellonella larvae (n = 10) were inoculated with three different concentrations of each strain (5 × 10⁷, 1 × 10⁸ and 5 × 10⁸) using a Hamilton syringe. A group inoculated with PBS was included as control. Larvae were then monitored for 5 days, and those that did not respond to touch were considered dead [27].

2.7. Study of the Prophylactic and Therapeutic Effects of PTE in Experimental Candidiasis in G. mellonella: Analysis of Survival Curves

Since the toxicity of PTE on G. mellonella larvae and the inoculum concentration were established, two different treatment approaches were evaluated. In the first approach (prophylactic), larvae were pre-treated with PTE at 20× MIC prior (24 h before) to infection with C. dubliniensis P1 or C. albicans P2 (1 × 10⁸ cells/mL). In the second approach (therapeutic), larvae were treated with PTE at 20× MIC after (1 h) infection with C. dubliniensis P1 or C. albicans P2 (1 × 10⁸ cells/mL). Treatment with PTE was inoculated in the last right proleg, while Candida spp. was injected in the last left proleg using a 10 µL 26G Hamilton syringe. Then, larvae were incubated at 37 °C and monitored for 5 days. Larvae were considered dead if they did not respond to touch [27].

2.8. Study of Prophylactic Effects of PTE in Experimental Candidiasis in G. mellonella: Analysis of Fungal Burden and Hemocyte Recruitment

Since the prophylactic approach exhibited promising results compared to the therapeutic, we assessed the effects of PTE treatment in fungal burden and hemocyte recruitment. Thus, larvae (n = 12) were inoculated with PTE at 20× MIC prior (24 h before) inoculation with C. dubliniensis P1 or C. albicans P2, both at 1 × 10⁸ cells/mL. Control groups with larvae inoculated with C. dubliniensis P1 or C. albicans P2 and not treated were included. After 1 h of infection with Candida spp., larvae were cut ventrally in the cephalocaudal direction, and the hemolymph was diluted in saline solution for insects (IPS; 2% NaCl; glucose 0.1 M; sodium citrate 30 mM; citric acid 26 mM and EDTA 10 mM).

To study the antifungal effects of PTE, the hemolymph of the larvae (n = 6) was diluted and plated in ASD. After incubation for 24 h at 37 °C, the fungal burden in the hemolymph (CFU/mL) was determined [26]. To investigate the immunomodulatory effects of PTE, 10 μL of the hemolymph from each larva (n = 6) was dispensed into a hemocytometer and the number of hemocytes for each experimental group was quantified [27].

2.9. Statistical Analysis

Statistical analysis was performed using GraphPad Prism, version 6.0, for Windows (GraphPad Software, San Diego, CA, USA), with p < 0.05 considered significant. The results of CFU/mL counting (time-kill curves, microcosm biofilm assays and fungal burden in G. mellonella) and hemocyte counting were analyzed by ANOVA, followed by Tukey’s test to compare different groups. The survival curve of G. mellonella larvae was analyzed using the Kaplan–Meier method and the Log-rank test (Mantel–Cox).

3. Results

3.1. PTE Exhibited Fungicidal Effects on Candida Planktonic Cells

In the broth microdilution tests, the MIC of PTE was 32 μg/mL for both Candida strains. The MIC of FCZ was 0.25 μg/mL for C. dubliniensis P1 and C. albicans P2, and 16 μg/mL for the quality control strain (P. kudriavzevii ATCC 6258). The MFC was 32 μg/mL for C. dubliniensis P1 and C. albicans P2, with an MFC/MIC ratio of 1, confirming the fungicidal activity of PTE on both Candida strains (Table 1).

Table 1.

Minimum inhibitory concentration (MIC; µg/mL), minimum fungicidal concentration (MFC; µg/mL), and MFC/MIC ratio values of pterostilbene (PTE) and fluconazole (FCZ) on Candida spp.

Strains	MIC (µg/mL)		MFC	MFC/MIC
	PTE	FCZ	PTE	PTE
Candida dubliniensis P1	32	0.25	32	1
Candida albicans P2	32	0.25	32	1
Pichia kudriavzevii ATCC 6258	32	16	ND	ND

ND: not determined.

Time-kill curve analysis demonstrated the inhibitory effects of PTE against Candida strains. PTE reduced the growth of C. dubliniensis P1 and C. albicans P2 within 2 h (Figure 1). Furthermore, complete inhibition of both strains was observed after 8 h of treatment with PTE. Importantly, the inhibitory effects persisted throughout the 48 h analysis, confirming its fungicidal effects on both Candida strains (Figure 1).

Figure 1.

Treatment with pterostilbene (PTE) inhibited Candida growth. Viability (log₁₀ CFU/mL) of (A) Candida dubliniensis P1 and (B) C. albicans P2 non-treated (NT) and treated with PTE for 48 h. NG: no growth. * Significant difference (p < 0.05) compared to the NT group.

3.2. PTE Demonstrated Broad Antimicrobial Activity Against Microcosm Biofilms Associated with Denture Stomatitis

Since PTE exhibited effects on Candida planktonic cells, we investigated its activity on denture stomatitis microcosm biofilms formed from patient 1 (sample P1) and patient 2 (sample P2). Treatment of biofilms from sample P1 with PTE reduced the microbial count in BHI, MS, MSBS, MAN, and ASD. In the non-selective microorganism count (BHI), a reduction of 1.15 log₁₀ CFU/mL was observed following treatment with PTE at 1× MIC. At concentrations of 5×, 10×, and 20× MIC, the reductions were 1.71, 2.00, and 1.98 log₁₀ CFU/mL, respectively (Figure 2A). Regarding the streptococci count (MS), microbial viability was decreased in a dose-dependent manner, with reductions observed at 5× (0.77 log₁₀ CFU/mL), 10× (1.80 log₁₀ CFU/mL), and 20× MIC (2.88 log₁₀ CFU/mL) (Figure 2B). For the mutans streptococci group (MSBS), treatment with PTE at 10× MIC resulted in a reduction of 1.04 log₁₀ CFU/mL, while at 20× MIC, the decrease was 3.04 log₁₀ CFU/mL (Figure 2C). Staphylococcal growth (MAN) was reduced at all tested concentrations of PTE, ranging from 0.37 log₁₀ CFU/mL at 1× MIC to 1.80 log₁₀ CFU/mL at 20× MIC (Figure 2D). The yeast count (SDA) was significantly reduced after treatment with PTE at 5×, 10× and 20× MIC, with reductions of 0.16, 0.49, and 2.21 log₁₀ CFU/mL, respectively (Figure 2E).

Figure 2.

Pterostilbene (PTE) reduced the microbial viability of the microcosm biofilm formed from clinical sample P1. Microbial viability (log₁₀ CFU/mL) of microcosm biofilms from the sample P1, either non-treated (NT) and treated with pterostilbene (PTE) at 1×, 5×, 10×, and 20× the minimum inhibitory concentration (MIC), plated on (A) Brain Heart Infusion (BHI), (B) Mitis Salivarius (MS), (C) Mitis Salivarius Bacitracin Sucrose (MSBS), (D) Mannitol (MAN), and (E) Sabouraud dextrose (SDA) media. Different letters represent a statistically significant difference (p < 0.05).

PTE also decreased microbial viability of the microcosm biofilm formed from clinical sample P2 across all tested media, with significant reductions observed at 10× and 20× MIC. Treatment with PTE significantly decreased the microbial count in non-selective media (BHI), with reductions of 0.85 and 1.02 log₁₀ CFU/mL at 10× and 20× MIC, respectively (Figure 3A). In the streptococci count (MS), reductions of 0.19 log₁₀ CFU/mL at 5× MIC, 0.97 log₁₀ CFU/mL at 10× MIC, and 2.18 log₁₀ CFU/mL at 20× MIC were observed (Figure 3B). While the mean CFU count for mutans group streptococci (MSBS) in the non-treated (NT) group was 7.33 log₁₀ CFU/mL, the count in the group treated with PTE at 20× MIC decreased to 5.12 log₁₀ CFU/mL (Figure 3C). For staphylococci counts (MAN), viability was reduced at PTE concentrations of 1×, 5×, 10×, and 20× MIC, with mean reductions of 0.42, 1.23, 1.57, and 2.33 log₁₀ CFU/mL, respectively. (Figure 3D). The yeast count (SDA) was significantly reduced following treatment with PTE at 20× MIC, resulting in a decrease of 2.30 log₁₀ CFU/mL (Figure 3E).

Figure 3.

Pterostilbene (PTE) reduced the microbial viability of the microcosm biofilm formed from clinical sample P2. Microbial viability (log₁₀ CFU/mL) of microcosm biofilms from the sample P2, either non-treated (NT) and treated with pterostilbene (PTE) at 1×, 5×, 10×, and 20× the minimum inhibitory concentration (MIC), plated on (A) Brain Heart Infusion (BHI), (B) Mitis Salivarius (MS), (C) Mitis Salivarius Bacitracin Sucrose (MSBS), (D) Mannitol (MAN), and (E) Sabouraud dextrose (SDA) media. Different letters represent a statistically significant difference (p < 0.05).

3.3. G. mellonella Larvae Did Not Have Toxic Reactions After PTE Administration, and Were Susceptible to Infections Caused by Clinical Candida Isolates

Toxicity analysis revealed that G. mellonella larvae maintained full viability following inoculation with PTE at concentrations up to 20× MIC. Based on these findings, we selected the 20× MIC for subsequent assays using G. mellonella larvae.

In the experimental infection of G. mellonella with C. dubliniensis P1, 100% of larvae inoculated with 5 × 10⁸ cells/mL succumbed within one day. In the group inoculated with 1 × 10⁸ cells/mL, 10% of larvae died on day 1, while mortality reached 100% by day 2. In addition, 60% of larvae inoculated with 5 × 10⁷ cells/mL died by day 2, and the remaining 40% succumbed on day 3 (Figure 4A).

Figure 4.

Candida strains caused experimental infection in G. mellonella larvae. Survival curves of G. mellonella larvae inoculated with PBS, (A) C. dubliniensis P1 and (B) C. albicans P2 at 5 × 10⁷, 1 × 10⁸ and 5 × 10⁸ cells/mL.

For C. albicans P2, 100% mortality occurred within one day for larvae inoculated with 5 × 10⁸ cells/mL. In the group inoculated with C. albicans P2 at 1 × 10⁸ cells/mL, 100% mortality was observed by day 5, while 60% of larvae inoculated at 5 × 10⁷ cells/mL remained viable throughout the five-day monitoring period (Figure 4B). Based on these results, the standardized inoculum for experimental infection in G. mellonella larvae was established at 1 × 10⁸ cells/mL for both Candida strains.

3.4. Prophylactic Treatment with PTE Increased the Survival Rate of G. mellonella Larvae Infected by Candida Strains

To investigate the in vivo antifungal and immunomodulatory effects of PTE, we evaluated both prophylactic and therapeutic strategies in an experimental model of candidiasis using G. mellonella. In infections with C. dubliniensis P1, no mortality was observed in the prophylactic group on day 1, while the therapeutic group showed 10% mortality. By day 5, mortality rates reached 20% in the prophylactic group and 10% in the therapeutic group. Both treatment strategies significantly improved larvae survival (Figure 5A).

Figure 5.

Prophylactic treatment with pterostilbene (PTE) improved the survival of G. mellonella larvae following experimental infection with Candida spp. (A) Survival curves of G. mellonella larvae pre-treated PTE (prophylactic PTE) at 20× minimum inhibitory concentration (MIC), followed by inoculation with Candida dubliniensis P1 (1 × 10⁸ cells/mL). Additional groups included larvae inoculated with C. dubliniensis P1 and either non-treated (NT), and post-treated with PTE (therapeutic PTE). (B) Survival curves of G. mellonella larvae pre-treated with PTE (prophylactic PTE) at 20× MIC, followed by inoculation with Candida albicans P2 (1 × 10⁸ cells/mL). Additional groups included larvae inoculated with C. albicans P2 and either non-treated (NT), and post-treated with PTE (therapeutic PTE). * Significant difference (p < 0.05) compared to the NT group.

In infections with C. albicans P2, prophylactic treatment resulted in 10% larval mortality on day 1, whereas therapeutic treatment led to 20% mortality. By day 5, mortality increased to 40% in the prophylactic group and reached 90% in the therapeutic group. Prophylactic administration of PTE significantly enhanced larval survival, while the therapeutic approach showed no significant protective effect (Figure 5B).

3.5. Prophylactic Treatment with PTE Reduced Fungal Burden in G. mellonella Infected with C. albicans Without Alter the Hemocyte Count

Given the superior outcomes observed with the prophylactic approach compared to the therapeutic strategy, we assessed its effects on fungal burden in the hemolymph of G. mellonella larvae and on hemocyte recruitment. Prophylactic treatment with PTE had no effect on fungal burden in the hemolymph of G. mellonella larvae infected with C. dubliniensis P1 (Figure 6A). However, in larvae pre-treated with PTE and infected with C. albicans P2, a significant reduction in fungal burden was observed (Figure 6B).

Figure 6.

Prophylactic treatment with pterostilbene (PTE) reduced fungal burden in G. mellonella infected with Candida albicans. (A) Fungal burden (CFU/mL) in the hemolymph of larvae infected with C. dubliniensis P1 and either non-treated (NT) or pre-treated with PTE (PTE). (B) Fungal burden (log₁₀ CFU/mL) in the hemolymph of larvae infected with C. albicans P2 and either non-treated (NT) or pre-treated with PTE (PTE). * Significant difference (p < 0.05) compared to the NT group.

Subsequently, the immunomodulatory effects of PTE on G. mellonella were analyzed. Prophylactic treatment with PTE did not affect hemocyte counting compared to the control and non-treated groups for both Candida strains (Figure 7).

Figure 7.

Prophylactic treatment with pterostilbene (PTE) did not impact the hemocyte counting in G. mellonella larvae. (A) Hemocyte counting (hemocytes/mL) in the hemolymph of larvae inoculated with PBS (control), infected with C. dubliniensis P1 and either non-treated (NT) or pre-treated with PTE (PTE). (B) Hemocyte counting (hemocytes/mL) in the hemolymph of larvae inoculated with PBS (control), infected with C. albicans P2 and either non-treated (NT) or pre-treated with PTE (PTE).

4. Discussion

The treatment of oral Candida infections has been hindered by limitations in the current antifungal arsenal, stimulating the search for new antifungal agents [13,15,28]. In this study, we investigated the antifungal effects of PTE against C. albicans and C. dubliniensis associated with denture stomatitis using both in vitro and in vivo approaches.

The effects of PTE were initially investigated on planktonic cells of Candida. Microdilution assays confirmed its fungicidal effects against C. dubliniensis and C. albicans at 32 μg/mL. Previous studies have reported similar inhibitory effects of PTE at concentrations ranging from 16 to 32 μg/mL against C. albicans and non-albicans Candida species, including Candida tropicalis and Nakaseomyces glabratus (formerly Candida glabrata) [29,30]. In addition, Simonetti et al. [29] observed that the treatment with PTE at 32 μg/mL significantly decreased fungal growth after 4 h. In the present study, PTE completely inhibited fungal growth at 8 h of treatment, confirming its fungicidal mode of action.

PTE is a dimethyl ether derivative of the polyphenol resveratrol, distinguished by the methoxyl groups. These structural features confer increased lipophilicity to PTE and enhanced cellular absorption [31]. Due to this chemical similarity, comparisons between the biological activities of PTE and resveratrol have garnered significant scientific interest. However, a previous study by our research group [32] demonstrated that resveratrol failed to inhibit C. albicans strains at concentrations up to 256 μg/mL. Moreover, a high fungal burden (10⁶ cells/mL) was still observed at 48 h of resveratrol treatment. Although resveratrol is known for its broad biological activity, our findings indicate that PTE is significantly more effective on planktonic cells, inhibiting C. albicans growth at concentrations at least 8× lower. However, further studies are needed to elucidate the mode of action of PTE on fungal cells and to strengthen its potential as a therapeutic agent to overcome drug resistance. Hu et al. [23] reported that PTE treatment led to decreased conidial germination and, consequently, reduced growth of A. flavus. Nonetheless, the mode of action of PTE on Candida and other fungal species has not yet been investigated.

We subsequently studied the antimicrobial activity of PTE in microcosm biofilms associated with denture stomatitis. Denture stomatitis is a chronic, multifactorial inflammatory condition frequently observed in denture wearers [33]. Clinically, this condition presents as petechiae, diffuse erythema, or granular hyperplasia, typically restricted to the mucosal areas in contact with the prosthesis. These lesions can cause discomfort and burning sensations, significantly impairing the quality of life of affected individuals [11,34]. Denture stomatitis is commonly associated with infections caused by Candida spp. and various bacterial pathogens. These microorganisms possess the capacity to form biofilms—complex structures that pose a significant challenge to antifungal therapy due to their heightened resistance [35,36,37]. In our study, PTE exhibited broad-spectrum antimicrobial activity, demonstrated by its ability to reduce the viability of streptococci, staphylococci, mutans streptococci, and yeasts. Beyond its relevance to denture stomatitis, these microorganisms are also implicated in other oral infections such as periodontitis and dental caries [38,39].

Previous studies have established the antimicrobial activity of PTE against biofilms of C. albicans [24] and staphylococci [21]. PTE was shown to reduce the viability and biomass of C. albicans monotypic biofilms at concentrations up to 20 µg/mL [24]. These antibiofilm effects were associated with decreased cellular surface hydrophobicity and inhibition of hyphal formation. Moreover, gene expression analysis confirmed downregulation of key pathways related to morphological transition, ergosterol biosynthesis, antioxidant defense, cell surface structure, and protein folding [30,40]. In a separate study, Vanková et al. [21] reported antibacterial effects of PTE against Staphylococcus aureus and Staphylococcus epidermidis monotypic biofilms, with minimum biofilm eradication concentrations ranging from 50 to 75 µg/mL. Wang et al. [41] demonstrated that PTE impaired S. aureus pathogenesis by targeting critical virulence factors, including β-lactamase biofilms and α-hemolysin. In addition, treatment with PTE elevated reactive oxygen species (ROS) levels against S. aureus, leading to cellular damage characterized by irregular morphology, membrane disruption, leakage of nucleic acids and proteins, reduced DNA content, and activation of apoptosis pathways [42].

In the present study, PTE reduced the microbial viability of C. albicans and staphylococci at concentrations of 640 and 32 µg/mL, respectively. The discrepancy observed in biofilm inhibitory activity values is likely attributable to the nature of the biofilms examined. While most studies have assessed the effects of PTE on monotypic biofilms, our investigation evaluated its activity against complex heterotypic microcosm biofilms. Microcosm biofilms are in vitro models that simulate the conditions of biofilm formation and development in a controlled setting [43,44,45]. Unlike monotypic models, microcosm systems incorporate biological material from the target environment, capturing the complexity of inoculum dynamics seen in natural biofilm formation. This added sophistication enables them to better mimic in vivo conditions [46,47]. Such methodological distinctions probably account for the notable differences in PTE concentrations observed between our study and previous reports. Importantly, our study demonstrated that PTE effectively reduced the viability of streptococci, mutans streptococci, and C. dubliniensis within biofilm microcosms at concentrations of 160, 320, and 640 µg/mL, respectively. To our knowledge, this is the first report describing the antimicrobial activity of PTE against microcosm biofilms and specifically targeting streptococci, mutans streptococci, and C. dubliniensis. PTE fully inhibited both C. albicans and C. dubliniensis planktonic cells at 32 µg/mL. However, as expected, higher concentrations of PTE were required to significantly reduce the viability of biofilms formed by both Candida strains. This finding highlights the challenges associated with biofilm treatment, as these structures are more resistant to antifungal therapies [9,45]. Even in this context, treatment with PTE at 640 µg/mL decreased the viability of mature biofilms formed by C. albicans and C. dubliniensis by approximately 50%.

Given the in vitro antifungal effects of PTE against Candida spp. planktonic cells and their antimicrobial activity in microcosm biofilms associated with denture stomatitis, we extended our investigation to an in vivo experimental candidiasis model using G. mellonella larvae. The G. mellonella model has been widely recognized as a suitable system for evaluating drug toxicity and the antimicrobial efficacy of novel compounds, yielding consistent results with mammalian models [48,49]. Moreover, its low cost and ease of handling allow for high-throughput testing, making it an efficient alternative to conventional animal models [50]. In our study, PTE exhibited no toxicity toward G. mellonella larvae at concentrations up to 640 μg/mL. Supporting our findings, Yang et al. [51] demonstrated the biocompatibility of PTE with mammalian cells, showing no cytotoxic effects on keratinocytes (HaCaT) and human neutrophils at concentrations up to 256 μg/mL. However, mild toxicity was observed at higher concentrations of PTE. In addition, Li et al. [30] reported that PTE was non-toxic to mice at 64 μg/mL, reinforcing its safety profile across biological systems.

Since PTE was not toxic for G. mellonella larvae, we assessed its effectiveness using both prophylactic and therapeutic approaches. Larvae pre-treated with PTE exhibited increased survival and reduced early mortality. In contrast, therapeutic administration of PTE did not influence larval survival outcomes. Notably, Rossoni et al. [52] previously demonstrated that the pre-treatment of G. mellonella with extracts from Lacticaseibacillus paracasei 28.4 (formerly Lactobacillus paracasei) prolonged larval survival in larvae experimentally infected with Candidozyma auris (formerly Candida auris), through mechanisms involving direct antifungal activity and enhanced immune cell recruitment.

Based on these observations, we hypothesized that PTE might exert both antifungal and immunomodulatory effects in vivo. Prophylactic treatment with PTE significantly reduced the fungal burden of C. albicans in the hemolymph—an effect not observed in larvae infected with C. dubliniensis. This finding underscores the importance of investigating candidiasis caused by non-albicans Candida species, given their rising prevalence in clinical settings [53]. Regarding immunomodulation, pre-treatment with PTE did not influence the recruitment of hemocytes—key immune cells in the hemolymph involved in phagocytosis, nodulation, pathogen encapsulation, coagulation, and melanization [54]. These findings suggest that the enhanced survival observed during C. albicans infection may be primarily attributed to the antifungal properties of PTE rather than its influence on innate immune responses. However, further studies are warranted to investigate differences in virulence factors between the strains used, including filamentation capacity, adhesion, invasion and enzymatic activity, which may help explain the in vivo outcomes.

In summary, PTE demonstrated antifungal activity against planktonic cells of C. albicans and C. dubliniensis, as well as broad-spectrum antimicrobial effects on microcosm biofilms associated with denture stomatitis, effectively reducing the viability of staphylococci, streptococci, and yeasts. In vivo assays confirmed the biocompatibility of PTE in G. mellonella larvae. Furthermore, prophylactic treatment with PTE enhanced larval survival, likely due to a reduction in fungal burden within the hemolymph during C. albicans infection. However, further studies on PTE are needed to elucidate its broad-spectrum mechanism against non-albicans Candida species using a large number of clinical strains, particularly C. dubliniensis, which is a clinically relevant species in oral candidiasis.

Author Contributions

Conceptualization: P.H.F.d.C. and J.C.J. Methodology, formal analysis, investigation and data curation: P.H.F.d.C., M.d.F.d.S., A.S.F., J.C.G., P.M.N.d.L., G.M.d.C., K.K.K., F.d.C.R., M.T.G. and J.C.J. Funding acquisition, project administration and supervision: J.C.J. Writing—original draft: P.H.F.d.C., M.d.F.d.S., G.M.d.C. and J.C.J. All authors contributed to revision of the manuscript. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

This research was approved by the Human Research Ethics Committee of the Institute of Science and Technology of São Paulo State University (ICT/UNESP) under number 5.827755 on 19 December 2022.

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq–310265/2022-3 and 407032/2023-1).