Fluorene derivatives as potent antifungal and antibiofilm agents against fluconazole-resistant Candida albicans

Abstract

Candida albicans ranks as one of the most common causes of fungal sepsis in hospitalized patients around the world with an increasing mortality rate. The current antifungal drugs in use face several limitations including fungal resistance due to biofilm formation. This has complicated the treatment landscape, necessitating the need for continued search for effective therapeutic options against drug-resistant C. albicans threats. Therefore, this study investigated eighteen fluorene derivatives for their antifungal and antibiofilm potential against C. albicans. Two fluorene derivatives namely: 9,9-bis(4-hydroxyphenyl) fluorene (BHPF) and fluorene-9-acetic acid (FAA) were identified as potential inhibitors of Candida biofilms, achieving 97 % and 89 % inhibition at 10 μg/mL. Microscopic studies also confirmed their antibiofilm efficacy, with BHPF demonstrating activities comparable to amphotericin B. Furthermore, BHPF inhibited planktonic cell growth at concentration as low as 5 μg/mL. Both BHPF and FAA exhibited fungicidal activity and also inhibited C. albicans virulence factors such as cell aggregation and hyphal formation. Notably, neither compound showed propensity for resistance development over 15 passages. Additionally, toxicity evaluations in both plant and Caenorhabditis elegans model revealed non-to mild toxicity, and the ADMET prediction also satisfied the criteria for drug-likeliness. The results of this multifaceted investigation highlight the potential of BHPF and FAA as novel antifungal agents targeting C. albicans infections and biofilm-related challenges.

Highlights

• •Antibiofilm activity of fluorene derivatives against C. albicans.
• •Fluorene derivatives inhibit cell aggregation and hyphal formation.
• •Fluorene derivatives induce ROS production and demonstrates low propensity for resistance development.
• •Fluorene derivatives demonstrates low toxicity and good bioavailability.

1. Introduction

Over the years, fungal infections have posed increasing threats to the global health, specifically with respect to opportunistic pathogens, including Candida albicans. As a commensal and ubiquitous fungus, C. albicans remains one of the foremost causes of opportunistic infections in immunocompromised individuals with the ability to invade nearly every part of the human host, from superficial to deep tissues. Infections caused by the pathogen range from superficial mucosal, vaginitis, oral thrush to life-threatening systemic infections such as candidemia [1,2]. Candidemia ranks as the fourth most common fungal sepsis in hospitalized patients around the world, accounting for a 30–40 % mortality rate [3,4]. The rise in prevalence is attributed to its adaptability, coupled with virulence mechanisms such as hyphal transformation, protease secretion, and quorum sensing, which enhance its ability to cause disease [5].

Another hallmark of C. albicans pathogenicity is its capacity to form biofilms. Biofilms are intricate, three-dimensional networks of microbial cells adhering to host tissues or abiotic surfaces, and protected by the extracellular polysaccharide matrix (EPS). The EPS provides structural support and shields the embedded microorganisms from environmental stressors, antifungal agents, and host immune defense [6]. The ability of C. albicans to form biofilms on medical devices is facilitated by its dimorphic nature, which allows the transition between yeast and hyphal forms. This morphological versatility plays a critical role in biofilm initiation, development and maturation [7,8]. C. albicans biofilms are associated with chronic infections associated with medical devices and tissues. Also, cases of recurrent candidemia have been strongly linked to biofilm, with an alarming mortality rate of ∼50 % [9]. The current armamentarium against fungi is mainly azoles and polyenes, which are now limited due to problems ranging from poor oral bioavailability, toxicity, resistance to reduced spectrum [10,11]. Biofilms have further complicated resistance to these antifungal treatments posing challenges for clinical management. Hence, necessitating novel therapeutic strategies that aim at inhibiting biofilm formation and enhancing drug penetration into biofilm-embedded cells.

In the search of novel antifungal agents, heterocyclic compounds have emerged as a promising scaffold due to their structural diversity and tunable pharmacological properties. In particular, fluorene, a tricyclic aromatic hydrocarbon, serves as a crucial functional component in numerous medications such as, lumefantrine (antimalarial), imirestat (aldose reductase inhibitor), cicloprofen (analgesic and anti-inflammatory), pavatrin (antispasmodic), indecainide (antiarrhythmic), and hexafluronium bromide (muscle relaxant) [[12], [13], [14], [15]]. Its aromatic rigid structures provide a versatile framework for the design of bioactive compounds, enabling the incorporation of diverse functional groups that are appropriate for targeting cellular processes and microbial enzymes [13]. The hydrophobic nature of fluorene derivatives enables them to interact with microbial membranes, potentially disrupting membrane integrity and permeability [16]. They are widely employed as essential building blocks for pharmaceuticals, drugs, and industrial fine chemicals [17]. With rising issues of conventional antibiotics resistance, fluorene derivatives have garnered attention as promising agents capable of overcoming conventional resistance mechanisms, such as efflux pump activity and enzyme inactivation. Their structural adaptability and unique mechanisms of action underscore their potential in the development of novel antimicrobials to address this global health challenge [18]. Recent studies have revealed the antimicrobial activity of fluorene derivatives, particularly against some fungi and bacteria [15,19,20], yet their activity against C. albicans and its biofilm formation remains underexplored.

Therefore, we hypothesized that fluorenes could serve as effective control agents against C. albicans infections and biofilm formation. To achieve this, eighteen fluorene derivatives were screened for their antifungal and antibiofilm potential against C. albicans. The effect of the active derivatives was further assessed on planktonic cell viability while the antibiofilm effect was visualized with phase-contrast and scanning electron microscopes. Also, they were evaluated for their impact on critical virulence factors such as filamentous protrusion, hyphal formation and cell aggregation, all of which are associated with Candida pathogenesis. To understand the risk of resistance, the propensity of C. albicans to develop resistance against these derivatives were examined using passage assay in addition to unveiling their possible antifungal mechanism of action. Lastly, the toxicity profile of the active fluorenes was preliminarily examined in C. elegans and seed germination models and also simulated with ADME-Tox analysis.

2. Materials and methods

2.1. Candida strain, growth conditions, and reagents

Candida albicans DAY185, an azole-resistant strain, was sourced from the Korean Culture Centre for Microorganisms (KCCM). The organism was streaked from glycerol stock at −80 °C onto solid potato dextrose agar (PDA) to obtain pure colonies and was incubated at 37 °C. A single colony was then inoculated into 25 mL of potato dextrose broth (PDB) and incubated for 48 h at 37 °C with shaking at 250 rpm.

Eighteen fluorene derivatives (Table S1) used in this study were procured from Combi-blocks Inc. (San Diego, USA). Amphotericin B (AMB) was sourced from Sigma-Aldrich (St. Louis, MO, USA), and fluconazole and itraconazole were purchased from Tokyo Chemical Industry (Tokyo, Japan) and were used as positive controls. Dimethyl sulfoxide (DMSO) was used as the solvent for dissolving all chemical compounds maintaining a final concentration below 0.1 % (v/v) which did not interfere with cell growth or biofilm formation of C. albicans.

2.2. Crystal violet biofilm inhibition assay

Biofilms inhibition assay for C. albicans was investigated as previously described [21] with slight modification. Briefly, C. albicans culture was diluted in PDB to ∼10⁶ CFU/mL and treated with or without fluorene derivatives at 10 and 50 μg/mL. Dose-dependent treatment with 9,9-Bis(4-hydroxyphenyl) fluorene and fluorene-9-acetic acid were performed at 0.5, 1, 2, 5 and 10 μg/mL and the antifungal fluconazole and itraconazole were tested at 10, 50, 200, 500 and 1000 μg/mL. Subsequently, 300 μL of the samples was transferred to 96-well polystyrene plates (SPL Life Sciences, Korea) and incubated for 24 h at 37 °C without agitation. After incubation, planktonic cells growth was measured by recording the optical density at 620 nm using a Multiskan EX microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The minimum inhibitory concentration (MIC), which is the lowest concentration at which no visible growth was observed in the wells was determined based on the broth dilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [22]. For biofilm quantification, the medium containing planktonic cells was carefully discarded, and the wells were gently washed three times with distilled water (dH₂O) to remove unattached cells. Biofilm cells were subsequently stained with 0.1 % crystal violet (CV) for 20 min. Excess CV was removed by rinsing with dH₂O and solubilized with 95 % ethanol. The absorbance of the dissolved CV was measured at 570 nm using a Multiskan EX microplate reader. All experiments were obtained from two independent cultures, tested in triplicate wells.

2.3. Dispersal assay for preformed biofilm

The ability of fluorene derivatives to disperse preformed C. albicans biofilms was further evaluated as previously described [23] with slight modification. C. albicans DAY185 was diluted in PDB to a final cell density of ∼10⁶ CFU/mL and inoculated into 96-well microtiter plates without fluorene derivatives. Plates were incubated at 37 °C for 4, 8 and 24 h under static conditions to allow biofilm formation. After incubation, planktonic cells were removed by gentle pipetting, and the wells were washed with phosphate-buffered saline PBS (pH 7.4) to remove non-adherent cells. Subsequently, fresh 300 μL PDB with or without fluorene derivatives at 10, 20, 50, 100 and 200 μg/mL was added to the wells followed by an additional 24 h incubation under the same condition. Biofilm was quantified using crystal violet staining as described in section 2.2 and absorbance was measured at 570 nm to quantify the extent of biofilm retention or dispersal.

2.4. Cell growth assessment

The cell growth assessment of C. albicans treated with fluorene derivatives was performed as previously described [24]. Briefly, C. albicans culture (∼10⁶ CFU/mL) was diluted in PDB and treated with or without fluorene derivatives (0, 1, 2, 5, 10 or 20 μg/mL). Thereafter, 300 μL aliquots of treated cultures and negative control were dispensed into 96-well plates and incubated at 37 °C under static conditions. C. albicans cell growth was assessed by measuring the absorbance at 620 nm at 2 h intervals for 24 h.

2.5. Time -kill kinetics assay

The fungicidal or fungistatic effects of fluorene derivatives were investigated and compared to a known antifungal agent (Amphotericin B) [25]. C. albicans culture (∼10⁶ CFU/mL) was inoculated in 2 mL tubes with or without the compounds at concentrations corresponding to MIC or 4 × MIC and were incubated at 37 °C with shaking at 250 rpm. Culture samples (100 μL) were collected at 0, 4, 8, 12 and 24 h, serially diluted, plated on PDB agar plates, and subsequently incubated at 37 °C for 48 h. After incubation, colonies were counted, and the results were expressed as CFU/mL using the formula

$$\text{CFU}/\text{mL}=\frac{\text{Number}\text{of}\text{colony}\text{units}\times \text{dilution}\text{factor}}{\text{Volume}\text{plated}\left(\text{mL}\right)}$$

2.6. Biofilm visualization by live imaging and SEM

To visualize the antibiofilm activities of fluorene derivatives against C. albicans, 300 μL of C. albicans culture (∼10⁶ CFU/mL) containing different concentrations of fluorene derivatives (0, 5, or 10 μg/mL) were dispensed into 96-well plates and incubated at 37 °C. Afterward, planktonic cells were washed three times with PBS (pH 7.4), and the biofilms were visualized with the iRiS™Digital Cell Imaging System (Logos BioSystems, Anyang, Korea). The resulting biofilm images were reconstructed as 2D and 3D color-coded representations using ImageJ (https://imagej.nih.gov/ij/index.html).

The SEM analysis was carried out following the described procedure [26]. Briefly, C. albicans cells were diluted to ∼10⁶ CFU/mL and treated with or without fluorene derivatives (0, 5, or 10 μg/mL) and amphotericin B (1 μg/mL). 300 μL of the treated cells were dispensed into 96-well plates containing sterile nylon filter membranes (0.4 × 0.4 mm²). After incubating the plates for 24 h at 37 °C without agitation, biofilms attached to the membranes were fixed using a 2.5 % glutaraldehyde and 2 % formaldehyde solution for 24 h. The biofilms were then dehydrated in a series of ethanol concentrations (30, 50, 70, 80, 95, and 99 %), followed by critical-point drying using an HCP-2 apparatus (Hitachi, Tokyo, Japan). After gold sputter-coating, the samples were examined under a Hitachi S-4800 scanning electron microscope at an accelerating voltage of 15 kV.

2.7. Hyphal development, cell aggregation and colony morphology assay

The hyphae formation and cell aggregation were observed as previously described [27]. Briefly, PDB medium was inoculated at a dilution of ∼10⁶ CFU/mL with or without fluorene derivatives at concentrations (0, 5, or 10 μg/mL), cultures were incubated under static condition at 37 °C for 24 h. Post incubation, hyphal formation and cell aggregation were observed under bright field using the iRiS™ Digital Cell Imaging System.

Colony morphology analysis was investigated as previously reported [27]. Briefly, C. albicans was streaked onto solid PDA plates with or without fluorene derivatives at varying concentrations (0, 5, or 10 μg/mL), incubated at 37 °C under static conditions and the colony morphology was observed under an iRiS™ Digital Cell Imaging System over a period of 7 days.

2.8. Reactive oxygen species (ROS) assay and resistance development

The potential of fluorene derivatives to induce ROS production in C. albicans was investigated as earlier reported with slight modification [28]. C. albicans cells were cultured to the logarithm growth phase, harvested, resuspended in PBS to a concentration of 10⁵ CFU/mL, and treated with or without H₂O₂ (positive control), AMB or fluorene derivatives at ½ × MIC, 1 X MIC and 4 × MIC for 3 h at 37 °C and 250 rpm. Following treatment, 5(6)-carboxy- 2′7′-dichlorofluorescein (10 μM) was added to cell suspensions and incubated in the dark for 30 min at 37 °C. Fluorescence intensity (FI) was measured using a multimode microplate reader. The excitation wavelength was at 506 nm, and emission intensities were recorded at 524 nm. FI was normalized with growth (OD₆₀₀). Untreated cells were processed similarly and used as controls.

The potential of C. albicans to develop resistance against fluorene derivatives and amphotericin B was studied by monitoring changes in MIC values. The initial MIC was determined using the broth dilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines in 14 mL tubes. C. albicans culture (∼10⁶ CFU/mL) was exposed to sub-inhibitory concentrations (sub-MICs) and incubated at 37 °C and 250 rpm for 24 h. For each passage, visibly grown cultures were diluted to ∼10⁶ CFU/mL and reinoculated. The process was repeated for 15 passages (days) thereafter, the graph of MIC values against the passages were generated to determine resistance development over time. A four-fold increase in MIC was used as the threshold to indicate resistance development [29].

$$Foldchange=\frac{{MIC}_{afterpassage}}{{MIC}_{initial}}$$

2.9. Toxicity studies of fluorene derivatives using plant and nematode models

The seed germination assay was observed as previously described [30]. Briefly, the effect of fluorene derivatives (0, 2, 5, 10, 20 and 50 μg/mL) on the germination of radish seed (Raphanus sativus) was investigated for their toxicity. Seeds were washed and dried, thereafter seeds (n = 9/plate per test concentration) were placed on the soft agar plates (0.7 % agar; 0.86 g/L Murashige and Skoog medium) with or without fluorene derivatives. The set up was incubated at room temperature for 7 days while the total length of plant was recorded daily.

The cytotoxicity of fluorene derivatives was evaluated using nematode model (C. elegans) as previously described [31,32]. Briefly, synchronized young adult nematodes were washed twice with M9 buffer and approximately 30 strains fer-15(b26); fem-1(hc17) worms were added to 96-well plates. Each well contained 200 μL of M9 buffer supplemented with fluorene derivatives at concentrations of 0, 5, 10, 20 and 50 μg/mL. The plates were incubated at 25 °C in the dark without shaking for 7 days. Worm viability was assessed based on their responses to plate tapping and exposure to LED lights for 20–30 seconds, using the iRiS™ Digital Cell Imaging System. Results were expressed as the percentage of live worms. Two independent experiments were performed in triplicate.

2.10. Predictions of absorption, distribution, metabolism, and excretion (ADME) properties

ADME (Absorption, Distribution, Metabolism, and Excretion) analysis was performed to assess the drug-likeness properties of the most active fluorene derivatives in this study. This in silico assessment provided preliminary evidence of their suitability for further clinical development prior to in vitro and in vivo confirmation. The toxicity and drug-likeness characteristics of 9,9-bis(4-hydroxyphenyl) fluorene and fluorene-9-acetic acid were predicted using several online platforms such as preADMET (https://preadmet.qsarhub.com/), molinspiration (https://www.molinspiration.com), and GUSAR (http://www.way2drug.com/gusar/) on the 7th June 2024. These platforms evaluated the bioavailability and other pharmacokinetic properties, including but not limited to Lipinski's rule of five [33].

2.11. Statistical analysis

Two independent cultures and three replicates were used for all experiments, and results are presented as means ± SDs. The Student's t-test was employed to analyze the significance of differences, and statistical significance was accepted for p < 0.05.

3. Results

3.1. Antibiofilm activities of fluorene derivatives on biofilm and planktonic cell of C. albicans

Among the eighteen fluorene derivatives initially screened at 10 and 50 μg/mL 9,9-bis(4-hydroxyphenyl) fluorene (BHPF) (#4) demonstrated the highest antibiofilm activity, achieving 97 % biofilm inhibition at 10 μg/mL followed by fluorene-9-acetic acid (FAA) (#16) and 9,9-dimethyl-2-nitro-9h-fluorene (#14) with biofilm inhibition of 89 and 87 % respectively (Fig. 1A). Interestingly, compounds #3, #5, #6, and #11 showed a concentration-dependent increase in biofilm biomass, with higher concentrations resulting in enhanced biofilm formation compared to lower concentrations, suggesting a paradoxical effect (Fig. 1A). Furthermore, BHPF and FAA displayed minimum inhibitory concentrations (MICs) of 5 and 100 μg/mL while, 9,9-dimethyl-2-nitro-9h-fluorene (#14) exceeded 300 μg/mL. For comparison, conventional azole antifungal drugs used as positive controls (fluconazole and itraconazole) showed MIC values of >1000 μg/mL, confirming the resistant phenotype of the C. albicans DAY185 strain used in this study. Therefore, due to their notable antibiofilm activities and relatively low MIC values, BHPF and FAA were selected for further investigation.

Fig. 1.

Initial antibiofilm screening of fluorene derivatives. Compounds (#4: BHPF) and (#16: FAA), which showed the most significant antibiofilm activity, are highlighted in red and blue, respectively, and were selected for further analysis (A). Dotted horizontal line indicates the mean OD₅₇₀nm value of the untreated control (∼3). Dose-dependent antibiofilm activities of selected compounds (B, C) and antifungal fluconazole and itraconazole used as positive controls (D, E). Live microscope and 3D-color coded images of biofilm inhibitory effects of BHPF and FAA (F). ∗Denotes a significant difference at p < 0.05 and error bars represent the standard deviation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Further antibiofilm evaluation at various concentrations revealed that both BHPF and FAA significantly inhibited Candida biofilm formation. Specifically, at 5 and 10 μg/mL, BHPF displayed superior activity with 91 and 97 % inhibition respectively, whereas FAA showed 42 and 89 % biofilm biomass reduction at the same concentration. The antibiofilm effect of BHPF is largely attributable to growth inhibition, while FAA acted more selectively, preventing biofilm formation without suppressing cell growth, a property peculiar to antibiofilm agents (Fig. 1B and C). As expected, fluconazole showed no inhibitory activity even at concentrations up to 1000 μg/mL. In contrast, itraconazole demonstrated substantial biofilm inhibition, achieving 76 and 90 % inhibition at 500 and 1000 μg/mL, respectively, concentrations that are 50 to 200-fold higher than those observed for FAA and BHPF (Fig. 1D and E). The live imaging microscope evaluation confirmed the antibiofilm potentials of both fluorene derivatives with BHPF being more remarkable at 5 and 10 μg/mL (Fig. 1F). Furthermore, the potential of BHPF and FAA to disperse preformed C. albicans DAY185 biofilm was assessed at varying concentrations of 10, 20, 50, 100, and 200 μg/mL after biofilm formation for 4, 8, and 24 h. Notably, BHPF displayed a dose-dependent ability to disperse preformed biofilms. In early biofilms (4 h), treatment with 10–200 μg/mL of BHPF and FAA reduced biomass by 88–91 % and 32–93 %, respectively. In contrast, mature biofilms (8 and 24 h) were far less susceptible, showing only slight reductions even at higher doses of 100 and 200 μg/mL (Fig. S1A–F). Taken together, BHPF and FAA showed strong antibiofilm activity against C. albicans, with BHPF being more potent at low doses and superior to two conventional azoles (Fig. 1). However, while both dispersed early biofilms effectively, mature biofilms remained largely resistant, suggesting that timing of administration may be critical for optimal therapeutic benefit.

3.2. Effects of fluorene derivatives on Candida planktonic cell growth

The effects of the fluorene derivatives were investigated on the planktonic cells of C. albicans over a period of 24 h. The result showed that BHPF inhibited cell growth at a concentration as low as 5 μg/mL whereas FAA displayed no inhibitory effect even at 20 μg/mL (Fig. 2A and B), suggesting an antibiofilm activity independent of planktonic cell growth inhibition. Similarly, the time-kill kinetics studies of BHPF and FAA, alongside amphotericin B highlighted significant fungicidal effects against C. albicans cells. At MIC, all treatments exhibited fungistatic effects compared to the untreated control after 24 h. However, at 4 × MIC, no viable cells were detected in the BHPF (20 μg/mL) and AMB (4 μg/mL) treated samples after 12 h, indicating complete eradication of the fungal cells. (Fig. 2C).

Fig. 2.

Effects of BHPF and FAA on C. albicans planktonic cell growth (A, B), and time-kill kinetics studies of fluorene derivatives (C). MICs values of BHPF, FAA and AMB are 5, 100 and 1 μg/mL respectively.

3.3. Fluorene derivatives inhibited cell aggregation and hyphal formation

In C. albicans, cell aggregation and the yeast-to-hyphal transition are essential steps for biofilm formation [34]. Also, it is closely associated with increased virulence and resistance to antifungal agents. Therefore, we investigated the effects of fluorene derivatives on colony morphology, cell aggregation, and hyphal formation (Fig. 3). When viewed under the light microscopy, colony morphology analysis on PDA plates revealed that compared to untreated control, smaller, less filamentous colonies (hyphal and pseudohyphae) were observed in samples treated with BHPF at 5 μg/mL and total inhibition at 10 μg/mL. At both concentrations examined, BHPF displayed superior inhibitory activities compared with FAA (Fig. 3A). Also, BHPF significantly reduced cell aggregation at 5 μg/mL and was better than FAA with significant effects at 10 μg/mL in the liquid medium (Fig. 3B). Furthermore, untreated samples showed significant hyphal elongation while drastic reduction was observed in groups treated with AMB and the fluorene derivatives (Fig. 3C, Fig. S2A). In addition, SEM observation corroborated the anti-hyphal and antibiofilm formation effects of fluorene derivatives and AMB. Specifically, prolific hyphal formation with a combination of few yeast cells and hyphae were clearly observed in untreated samples while fewer yeast cells without hyphae were observed in BHPF, FAA and AMB treated samples (Fig. 3D, Fig. S2B).

Fig. 3.

Effects of the fluorene derivatives on the colony morphology of C. albicans(A), cell aggregation (B), dimorphic transition of C. albicans(C), and SEM images of anti-hyphal formation activities of fluorene derivatives (D). Blue, red and yellow scale bars represent 200, 100 and 5 μm, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.4. Fluorene derivatives induced ROS production and displayed no propensity for resistance development

To further unveil the possible antibiofilm mechanism, we evaluated the ROS inducing potential of fluorene derivatives and amphotericin B while hydrogen peroxide (H₂O₂, 700 μg/mL) served as a positive control. Interestingly, BHPF and AMB induced ROS level comparable to H₂O₂ at ½ × MIC, 1 X MIC and 4 × MIC. On the other hand, FAA induced lower ROS levels but significantly higher than the untreated control sample (Fig. 4A).

Fig. 4.

Production of ROS in C albicans when treated with BHPF, FAA and AMB (A), and resistance development potential of C. albicans to the fluorene derivatives (B). MIC values of BHPF, FAA and AMB are 5, 100 and 1 μg/mL, respectively while H₂O₂ (700 μg/mL) served as positive control in the ROS assay. ∗Denotes a significant difference at p < 0.05 and error bars represent the standard deviation.

Furthermore, due to the growing resistance of fungi to conventional antifungal treatments, it is pertinent to investigate the potentials of new antifungal entities to address this challenge. Hence, we investigated the propensity of C. albicans to develop resistance to fluorene derivatives over 15 passages (days) by tracking changes in the MIC. The results showed that BHPF exhibited a 3-fold change in MIC over the 15 days’ period while FAA and AMB increased by 1.5-fold, indicating no propensity for resistance development as a four-fold increase from the original MIC was considered as resistance [29] (Fig. 4B).

3.5. Effects of fluorene derivatives in plant and nematode models

The environmental effects and potential toxicities of the active fluorenes were assessed using radish seeds (Raphanus sativus) and a nematode (C. elegans) model, respectively. The results revealed that both BHPF and FAA slightly reduced the plant lengths at MIC while further reduction was observed with increasing concentrations (Fig. 5A–D). Also, the toxicity of the fluorenes was determined in C. elegans. Interestingly, the nematodes survived BHPF treatment after 7 days, indicating its non-toxic potential. On the other hand, FAA was slightly toxic at higher concentration of 50 μg/mL with 22 % death after 4 days (Fig. 5E and F). Therefore, our results show that fluorene derivatives, in their active antibiofilm concentrations (5–10 μg/mL), exhibited slight effects on nematode and plant growth survival.

Fig. 5.

The effects of BHPF and FAA on total plant (Raphanus sativus) length (A–D), and their cytotoxic effects in C. elegans model (E–F). Panels A, B show representative images from n = 6 independent samples, while C, D and E, F depict the mean ± SD of 6 replicates. ∗Denotes a significant difference at p < 0.05.

3.6. In-silico absorption, distribution, metabolism, excretion (ADME) profiling of fluorene derivatives

ADME analysis of the fluorene derivatives was carried out to predict their physicochemical, toxicological, and bioavailability profiles (Table S2). Lipophilicity, measured by MLOGP (Moriguchi Log P) is a key determinant of drug-likeness since it reflects a compound's tendency to partition into fats rather than water, thereby influencing membrane permeability and solubility. FAA complied fully with Lipinski's Rule of Five, suggesting favorable oral drug-like properties, while BHPF had one violation due to an MLOGP above 4.15, indicating greater lipophilicity. Both compounds nevertheless met the Veber and Muegge criteria, which further assess molecular flexibility and hydrogen bonding capacity to predict oral bioavailability. High plasma protein binding was observed for both derivatives (>96 %), which may prolong their circulation time but may reduce the fraction of free drug available for immediate activity.

Predictions of intestinal uptake using the Caco-2 cell permeability model showed moderate absorption potential, with FAA displaying higher values than BHPF (Table S2), suggesting more efficient intestinal transport. In addition, blood-brain barrier (BBB) penetration was examined, as central nervous system access can have implications for both efficacy and toxicity. BHPF exhibited higher BBB permeability and lipophilicity, pointing to a stronger ability to cross biological membranes. Both compounds also demonstrated high gastrointestinal absorption, further supporting their oral suitability. Toxicity predictions indicated that neither FAA nor BHPF is likely to be carcinogenic in mice, and BHPF additionally showed lower predicted fish toxicity, suggesting a favorable drug-like profile. Taken together, these results show that FAA and BHPF possess drug-like properties with promising oral absorption potential and low predicted toxicity, supporting their further exploration as antifungal candidates.

4. Discussion

Biofilm mediated resistance in C. albicans remains a major challenge contributing to its recalcitrant pathogenicity. This has resulted in prolonged hospitalization, increased rate of morbidity and mortality, specifically among patients with compromised immune systems, causing tremendous economic burdens on healthcare systems [6,35,36]. Clinical azole antifungal drugs fluconazole and itraconazole exhibited high MICs (>1000 μg/mL), confirming the resistant phenotype of C. albicans DAY185 (Fig. 1D and E). This observation underscores the urgent need for alternative therapeutic strategies that target fungal virulence traits.

In this study, fluorene derivatives, distinctively 9,9-bis(4-hydroxyphenyl) fluorene (BHPF with an MIC of 5 μg/mL) and fluorene-9-acetic acid (FAA) demonstrated strong antibiofilm and antifungal properties against C. albicans, with 97 and 89 % biofilm inhibition respectively, at 10 μg/mL (Table S1, Fig. 1A). Microscopic observations further substantiated these results with significant reductions in biofilm density and hyphal integrity upon treatment with BHPF and FAA (Fig. 1F). Our results are consistent with previous reports that o-aryl-carbamoyl-oxymino-fluorene derivatives effectively inhibited biofilm formation and growth of E. faecalis, S. aureus, P. aeruginosa, E. coli, at 0.009–5 mg/mL and 0.156–10 mg/mL respectively [20]. Similarly, 9,9-bis(4-hydroxyphenyl)-fluorene exhibited antimicrobial activity against S. aureus and B. subtilis [37]. The significant antibiofilm activity displayed by the fluorene derivatives underscores their potential to combat biofilm mediated Candida infection. Of note, the increased biofilm biomass observed at higher concentrations of compounds #3, #5, #6, and #11 may reflect a paradoxical effect (Fig. 1A), previously reported with antifungals such as echinocandins. In such cases, microbial biofilms activate compensatory stress responses, including chitin upregulation, efflux pump expression, or cell wall remodeling that reduce susceptibility and promote persistence under drug pressure [[38], [39], [40]]. These adaptive mechanisms, more prominent in biofilm than planktonic states, may explain the unexpected biomass increase despite elevated compound exposure.

Also, the ability of BHPF and FAA to disperse preformed C. albicans biofilms (Fig. S1A–F) highlights their potential clinical relevance. Although dispersal of mature biofilms was limited, both compounds effectively disrupted early biofilms, which are critical stages for infection establishment. Partial biofilm disruption as observed for mature biofilms can enhance susceptibility to antifungal therapy and host immune defenses, reducing the risk of persistent or relapsing infections [41,42]. This stage-specific activity suggests that BHPF and FAA could be particularly valuable as adjunctive agents or preventive strategies for device-associated infections, helping to limit biofilm formation and improve treatment outcomes.

Furthermore, BHPF's superior efficacy compared to FAA is notable, particularly in inhibiting C. albicans planktonic cells at concentrations as low as 5 μg/mL. Conversely, FAA displayed no inhibitory effect on planktonic cells at higher doses (20 μg/mL) (Fig. 2A and B), suggesting that its activity is mediated through a mechanism independent of planktonic cell inhibition. Speculatively, FAA's mechanism of action may involve interference with biofilm specific processes such as adhesion, extracellular matrix biosynthesis, yeast-to-hyphal transition, or quorum sensing, rather than directly targeting cell growth. Similar observations were reported for other antibiofilm compounds that selectively inhibited biofilm without affecting the planktonic cell viability [[43], [44], [45], [46]]. Importantly, such selective antibiofilm activity is advantageous, as it minimizes the selective pressure for resistance development thereby making them valuable drug candidates. Antibiofilm compounds such as BHPF and FAA are particularly promising given that biofilms confer intrinsic tolerance to antifungal drugs and contribute to persistent infections in vivo. Additionally, at 4 × MIC, both compounds along with AMB displayed fungicidal activities (Fig. 2C). This observation does not only suggest the potential of the active compounds to disrupt biofilm matrix but also inactivate the Candida community that might recolonize the surface upon dispersal.

Notably, both compounds inhibited cell aggregation and hyphal formation of C. albicans (Fig. 3A–D) as further validated by SEM images showing the absence of microcolonies and hyphal formation in groups treated with BHPF and FAA compared with AMB (Fig. 3D,S2A, B). Our results corroborate other findings where antifungal agents inhibited cell aggregation and hyphal formation in C. albicans [[47], [48], [49], [50]]. Moreover, cell aggregation and hyphal formation remain the key virulence factors in the pathogenesis of C. albicans where they facilitate biofilm development, tissue penetration, phagocytosis resistance and secrete immune-suppressing proteins such as candidalysin that enhance fungal resistance [36,[51], [52], [53]]. Therefore, the suppression of these virulence attributes by the fluorene derivatives depict their potential to break fungal resilience to antifungal agents for enhanced treatment of biofilm-mediated Candida infection. In addition, the reactive oxygen species (ROS) production is a well-known antifungal mechanism, contributing to oxidative stress and membrane damage in fungal cells [54]. The present study revealed FAA as a moderate inducer of ROS while BHPF induced ROS levels comparable to amphotericin B (AMB) and H₂O₂, suggesting oxidative damage as a mode of action (Fig. 4A). Our result aligns with previous studies on AMB and other antifungal agents such as echinocandins and azole that rely on ROS-mediated fungal cell death [28,54]. Furthermore, resistance development remains a pressing challenge in antifungal therapy, particularly with azoles and echinocandins [55,56]. The low propensity for resistance observed with BHPF and FAA in this study is notable (Fig. 4B). These findings corroborate previous reports that biofilm-active compounds may target pathways less prone to genetic mutations [29]. Hence, the capacity to evade resistance suggests fluorene derivatives as viable alternatives or adjuncts to existing treatments, especially in mitigating resistance associated with biofilm-forming fungal pathogens.

Overall, both BHPF and FAA showed superior activities than the fluorene backbone. We speculate that the substitutions at the 9th position are important and responsible for the observed activities. In particular, the addition of carboxylic acid (-COOH) and two hydroxylated phenyl groups at the 9th positions compared to fluorene core without any substitution potentiated the antifungal and antibiofilm efficacies of BHPF and FAA respectively. Generally, the –COOH group adds polarity and increases the potential for hydrogen bonding or ionic interactions with key biomolecules [57,58]. This was also reported for hydroxyl groups (-OH) on phenyl rings, increasing their interaction with biofilm matrices and cell membranes. Usually, the phenyl groups introduce steric bulk, improving interaction with hydrophobic pockets in bacterial targets [59,60]. Therefore, the dual –OH groups and extended aromatic surface which provide strong interactions with microbial targets, significantly improved the efficacy of BHPF compared to FAA and fluorene. Additionally, –OH groups contribute to increased oxidative stress as confirmed in this study, causing cellular damage. These characteristics make hydroxyl groups valuable in designing effective antimicrobial agents [61,62]. Moreover, based on our initial screening, substitutions on the fluorene scaffold including Cl, Br, F and NH₂ did not enhance the antimicrobial activities of the derivatives (Table S1).

The ADME analysis to support the therapeutic and safety potentials of BHPF and FAA was assessed (Table S2). Specifically, both compounds exhibited high gastrointestinal absorption and plasma protein binding (>96 %), essential for systemic bioavailability. Also, the superior blood-brain barrier permeability (8.86) and lipophilicity (2.79) of BHPF suggest its potential in treating central nervous system fungal infections, a challenging area in antifungal therapy as reported [63,64]. Additionally, the higher Caco-2 permeability displayed by FAA indicates efficient intestinal absorption and oral bioavailability (Table S2). Although mammalian cytotoxicity assays were not conducted, the Raphanus sativus and C. elegans models, alongside in silico predictions, revealed mild toxicity at active antibiofilm concentrations of BHPF and FAA (Fig. 5). These models are widely used for preliminary safety evaluation, and prior studies demonstrated that C. elegans toxicity rankings correlate with mammalian LD₅₀ outcomes [65,66]. While no prior mammalian toxicity data exist for these active fluorene derivatives, our findings suggest a low risk of host damage at effective doses. Nonetheless, comprehensive cytotoxicity assessment in relevant mammalian cell lines is necessary and should be prioritized in future studies.

This study demonstrates that BHPF and FAA are promising antifungal candidates, capable of addressing key challenges such as biofilm, hyphal formation and resistance development. Their multifaceted mechanisms of action, including ROS induction and hyphae inhibition position them as strong alternatives to conventional antifungals like azoles and AMB. However, future research should focus on elucidating the molecular pathways targeted by these compounds in biofilm disruption and investigating their potential synergy with existing antifungal agents to enhance therapeutic outcomes.

5. Conclusion

This study emphasized the significant potential of BHPF and FAA as effective antifungal agents against C. albicans. Their strong antibiofilm and antivirulence activities, low propensity to resistance development, ROS production as a possible mechanism of action, favorable ADME profiles, and low toxicity make them promising candidates for further development. By addressing critical gaps in antifungal therapy, these fluorene derivatives pave the way for novel solutions to combat resistant fungal infections.

CRediT authorship contribution statement

Oluwatosin Oluwaseun Faleye: Writing – original draft, Visualization, Software, Methodology, Investigation, Formal analysis, Data curation. Amra Yunus: Writing – original draft, Visualization, Software, Methodology, Investigation, Formal analysis, Data curation. Jin-Hyung Lee: Writing – review & editing, Resources, Project administration, Methodology, Funding acquisition. Jintae Lee: Writing – review & editing, Resources, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Data availability

Data will be made available on request.