The standard C. albicans strain DAY185 used in this study was obtained from the Korean Culture Center of Microorganisms (http://www.kccm.or.kr/). Streaking and subculturing of C. albicans strain was performed using potato dextrose agar (PDA) or potato dextrose broth (PDB), unless otherwise specified. C. albicans strain was preserved at −80°C in 1 ml of PDB supplemented with 30% glycerol stock and, when needed, streaked on PDA plates. Plates were incubated for 48 h at 37°C, and a fresh single colony was then inoculated into 25 ml of PDB and cultured overnight at 37°C. CLEO and cedar wood oil were obtained from Jin Aromatics (Anyang, Gyeonggi Province, Korea) dissolved in ethanol and the final ethanol concentration in the experiments was always kept below 0.1% (vol/vol). All compounds tested (camphor, camphene, camphorquinone, camphorsulfonic acid, fenchone, fenchyl alcohol, α-thujone, borneol, and thymol) were purchased from Sigma-Aldrich (St. Louis, USA) and dissolved at 0.01% (vol/vol) in ethanol. To determine cell growths, turbidities were measured at 620 nm using a spectrophotometer (UV-160, Shimadzu, Japan). Minimum inhibitory concentrations (MICs) were determined using Clinical Laboratory Standards Institute (CLSI) broth dilution method with slight modification (Alastruey-Izquierdo et al., 2015), using 96-well polystyrene plates (SPL Life Sciences, Korea). C. albicans cells were inoculated and cultured overnight in PDB medium (dilution 1:100) at varying concentrations (0.1–1%, v/v, or w/v) of tested compounds for 24 h at 37°C. MIC was defined as the lowest concentration that inhibited at least 80% of microbial growth, as assessed by spectrophotometry (620 nm) and confirmed by colony counting. MICs of essential oils and tested compounds are expressed as percentages (v/v or w/v).

Static biofilm formation assays were performed in 96-well polystyrene plates, as previously reported (Lee et al., 2011). Briefly, overnight cultures of C. albicans strains were inoculated into PDB (total volume 200 μl) at an initial turbidity of 0.05 at 600 nm and cultured with or without CLEO or its main components at varying concentrations (0.0005 to 0.05%, v/v or w/v) for 24 h without shaking at 37°C. Biofilm formation was quantified after washing three times with H₂O to remove all non-adherent cells, staining with crystal violet for 20 min, rinsing three times with H₂O, and extracting the crystal violet with 95% ethanol. Absorbance was measured at 570 nm, and results are presented as the averages of at least six replicates.

A colorimetric XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt] reduction assay was performed using established procedures (Ramage et al., 2001; Nett et al., 2011). Briefly, overnight cultures of C. albicans strains were inoculated into PDB (total volume 300 μl) at an initial turbidity of 0.1 at 600 nm and cultured with or without camphor or fenchyl alcohol at different concentrations for 24 h without shaking at 37°C. A XTT reduction kit (Sigma-Aldrich, St. Louis, USA) was used to measure the metabolic activity of biofilm cells. XTT and menadione solutions were mixed at 20:1 (v/v) immediately prior to the assay. PBS was then added to XTT-menadione solution (3.76:1 v/v) and 200 μl of this mix was added to each well containing pre-washed biofilms, and incubated in the dark for 3 h at 37°C. The colored supernatant (100 μl) so obtained was transferred to new microtiter plates, and optical densities were measured at 450 nm. Similarly, the metabolic activity of planktonic cells was measured by using culture supernatants.

To determine the planktonic and biofilm cell viability, colony-forming units were examined (Martinez et al., 2010). A 100 μl of planktonic cells were aspirated from culture supernatants and transferred to new microcentrifuge tubes containing 900 μl of PBS. Similarly, biofilms were removed from the bottom of the plates with a sterile tip to dissociate biofilm cells. The samples transferred to tubes containing PBS were mixed gently and serially diluted onto PDA plates, and incubated at 37°C for 24 h, and colony forming units were calculated.

The chemical constituents of CLEO were determined by GC-MS (Jeol JMS 700 mass spectrometer) equipped with an Agilent 6890N GC DB-5 MS fused silica capillary column (30 × 0.25 m i.d., film thickness 0.25 μm). The conditions used have been previously described (Kim et al., 2015). Briefly, electron ionization was performed at 70 eV, helium was used as carrier at 1 ml/min, and temperatures of the GC injector and MS transfer line were 280 and 250°C, respectively. After injections, the GC oven temperature was maintained at 50°C for 2 min, increased to 250°C at 10°C per min, and held at 250°C for 10 min. Diluted samples (1/100, v/v, in methanol) of volume 1.0 ml were injected manually in split-less mode. The relative percentages of selected essential oil components are expressed as percentages. Components were identified using GC retention times on a DB-5 capillary column and by computer matching mass spectra using the Wiley and NIST libraries.

C. albicans cells were cultured in 96-well polystyrene plates (SPL Life Sciences, Korea) without shaking in the absence or presence of CLEO or its components. Planktonic cells were then removed by washing with PBS buffer three times. C. albicans cells were stained with carboxyfluorescein diacetate succinimidyl ester (Catalog #: C34554, Invitrogen, Molecular Probes, Inc, Eugene, USA; Weston and Parish, 1990), which is a minimally fluorescent lipophile, which becomes highly fluorescent when its loses its acetyl groups due to the action of esterases in cells. Biofilms were visualized by excitation using an Ar laser at 488 nm (emission wavelength 500–550 nm) under a confocal laser microscope (Nikon eclipse Ti, Tokyo) equipped with a 20 × objective (Kim et al., 2012). Color confocal images were constructed using NIS-Elements C version 3.2 (Nikon eclipse). For each experiment, at least 10 random positions in two independent cultures were examined.

SEM was used to observe hyphal formation, as previously described (Lee et al., 2014). Briefly, a nylon filter was cut into 0.5 × 0.5 cm pieces and placed in 96-well plates containing 200 μL cells of turbidity 0.05 at 600 nm. Cells were incubated in the presence or absence (untreated control) of CLEO, camphor, or fenchyl alcohol at 37°C for 24 h without shaking. They were then fixed with glutaraldehyde (concentration 2.5%) and formaldehyde (concentration 2%) for 24 h, and post-fixed by treating them with sodium phosphate buffer, osmium, an ethanol series (50, 70, 80, 90, 95, and 100%), and isoamyl acetate. After critical-point drying, cells were examined under a S-4100 scanning electron microscope (Hitachi, Japan) at a voltage of 15 kV and magnifications ranging from x 2,000 to x 10,000.

For transcriptional analysis, C. albicans was inoculated into 25 ml of PDB broth in 250 ml shake flasks at a starting OD₆₀₀ of 0.1, and then cultured at 37°C for 4 h with agitation (250 rpm) in the presence or absence of camphor or fenchyl alcohol (0.01%). RNase inhibitor (RNAlater, Ambion, TX, USA) was added to prevent RNA degradation. Total RNA was isolated using a hot acidic phenol method (Amin-ul Mannan et al., 2009) and further proceed to clean up this RNA with Qiagen RNeasy mini Kit (Valencia, CA, USA).

qRT-PCR was used to determine the transcription levels of hypha- and biofilm-related genes (HGC1, HYR1, RAS1, SAP1, TEC1, UME6, ECE1, ECE2, RBT1, and EED1) in C. albicans treated with or without camphor or fenchyl alcohol. Gene specific primers were used and ACT1 was used as housekeeping controls (Supplementary Table 1). The qRT-PCR method used has been previously described (Lee et al., 2011). qRT-PCR was performed using a SYBR Green master mix (Applied Biosystems, Foster City, USA) and an ABI StepOne Real-Time PCR System (Applied Biosystems) on two independent cultures.

To investigate the effects of camphor and fenchyl alcohol on the virulence of C. albicans, the nematode C. elegans was infected with C. albicans as previously described (Manoharan et al., 2017). Briefly, a freshly prepared overnight C. albicans culture (100 μl) was spread onto a lawn on PDA plates and incubated for 48 h at 37°C. Synchronized adult C. elegans fer-15; fem-1 nematodes were then allowed to feed on the C. albicans yeast lawn for 4 h at 25°C. Worms were collected and washed three times with sterile M9 buffer. Approximately 10 worms were then pipetted into single wells of 96-well plates containing PDB medium and treated with the solutions (300 μl) of investigated compounds at final concentrations of 0.01 or 0.001%. For control experiments, M9 buffer was added to medium. For toxicity assays, 10 non-infected worms were pipetted into single wells of 96-well dish containing M9 buffer and solutions of the compounds (300 μl) were added to final concentrations of 0.05, 0.1, or 0.5%. Plates were then incubated at 25°C for 4 days with gentle shaking. Three independent experiments were conducted in triplicate. Results are expressed as percentages of alive or dead worms after 4 days of incubation, and photographs were taken of worms treated at 0.01% using an iRiS™ Digital Cell Imaging System (Logos Bio Systems, Korea).

At least two independent experiments were conducted and results are expressed as means ± standard deviations. The student's t-test was used to determine the significances of differences between treated and non-treated samples. Statistical significance was accepted for p < 0.05, and significant changes are indicated using asterisks in figures.

Previously, we investigated the antibiofilm activities of 83 essential oils against C. albicans DAY185 (Manoharan et al., 2017). Of these, CLEO and cedar wood oil at 0.01% inhibited C. albicans biofilm formation by more than 85% without affecting planktonic cell growth. Previously, cedar wood oil has been reported to inhibit fungal spores and vegetative cells (Yooussef et al., 2016), and thus, CLEO was examined to determine its antibiofilm activity. CLEO was found to dose-dependently inhibit C. albicans biofilm formation (Figure 1), to reduce biofilm formation by 87% at a concentration of 0.01% and completed inhibited biofilm formation at 0.1% (Figure 1). Hence, 0.01% would be considered as reasonable concentration for further study.

GC-MS analysis identified 19 compounds in CLEO (Table 1). The major components of CLEO were; fenchone (14.2%), β-thujone (28.0%), α-thujone (20.3%), camphor (8.7%), 1-borneol (4.7%), and endobornyl acetate (4%) (Table 1), which are volatile compounds, mainly monoterpenes. However, a previous study reported different compounds in the ethanol extract of cedar wood and cedar leaf (Loizzo et al., 2008). In particular, himachalene and germacrene were found to be the most characteristics compounds in cedar wood and cedar leaf, respectively. These findings show that the chemical compositions of essential oils vary, and are probably dependent on plant age, soil, and climatic conditions (Polovka and Suhaj, 2010; Kamatou et al., 2012).

Antibiofilm efficacies of major components of CLEO and some of their derivatives were examined using C. albicans. Nine compounds, namely, camphor, camphene, camphorquinone, camphorsulfonic acid, fenchone, fenchyl alcohol, α-thujone, borneol, and thymol at concentrations of 0.01% significantly inhibited C. albicans biofilm formation (Figure 2). In addition, α, β-thujone (mixture of α-thujone and β-thujone) had showed similar activity as α-thujone (data not shown). Consequently, β-thujone could have similar activity against C. albicans biofilms. In particular, camphor, fenchone, fenchyl alcohol, borneol, and thymol at 0.005% inhibited biofilm formation by >80% (Figure 2). It appears that combination of camphor and fenchone-related compounds was probably responsible for the observed antibiofilm activity of CLEO. Borneol and thymol have well-known antimicrobial and antibiofilm effects. These compounds have also been found in thymus oils and their activities have been previously evaluated on Candida spp. (Khan et al., 2014; Bona et al., 2016). Although, camphor and fenchyl alcohol are components of many essential oils, their antibiofilm activities have not been described, and thus, they were included in the present study.

The cell growth of C. albicans was investigated to confirm antibiofilm activities of CLEO (Figure 3A), camphor (Figure 3B) and fenchyl alcohol (Figure 3C). Notably, planktonic cell growth was not affected by CLEO, camphor, or fenchyl alcohol even at concentrations of ≤ 0.05%. The antimicrobial activities of CLEO and its major components were initially evaluated using the microdilution method using C. albicans (Figure 3D). The MIC of CLEO was found to be 0.5% (v/v), whereas camphor (w/v) and fenchone (v/v) had MICs of 0.5%. Camphorquinone, fenchyl alcohol, and thymol were more active than CLEO with MICs of 0.1–0.3% (w/v). Interestingly, MIC values were 50-times higher than the concentrations (~0.01%) required for antibiofilm activity. These results confirm that biofilm formation by C. albicans was effectively reduced by the antibiofilm activities of these compounds and not by their antimicrobial activities.

The effects of camphor and fenchyl alcohol on C. albicans biofilms and planktonic cells were quantified using XTT reduction assay and cell viabilities. The metabolic activity of C. albicans biofilm cells was significantly reduced by 40 and 76% by camphor at 0.005 and 0.01%, respectively. Similarly, fenchyl alcohol reduced the metabolic activity of C. albicans biofilms by 66% at the concentration of 0.005% (Figure 4A), and 0.01% reduced biofilms 81%, but no significant effect were seen on planktonic cells. To confirm XTT results, colony forming units were enumerated and expressed as percentage of survival cells (Figure 4B). As expected, biofilm cells were significantly susceptible to 0.005 and 0.01% (>90%) of camphor and fenchyl alcohol (Figure 4B), whereas no significant effect was observed on planktonic cells. These results were consistent with XTT assay and MIC values of camphor and fenchyl alcohol treated cells.

To investigate the effects of CLEO, camphor and fenchyl alcohol on cellular morphology and hyphal growth, we used confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). CLSM images showed that C. albicans formed dense biofilms and hyphal cells in non-treated control samples, whereas in the presence of CLEO, camphor, or fenchyl alcohol at 0.01% biofilm adherence and thicknesses were reduced (Figure 5A). SEM revealed that in the non-treated controls, C. albicans biofilms were a mixture of yeast cells and hyphae (Figure 5B). However, CLEO, camphor, or fenchyl alcohol at 0.01% inhibited hyphal growth (Figure 5B). Taken together, these results showed CLEO, camphor, and fenchyl alcohol potently inhibited hyphal formation and biofilm formation by C. albicans.

To investigate the anti-biofilm mechanism of camphor and fenchyl alcohol, qRT-PCR was performed to examine the expression changes of hypha specific genes and biofilm related genes in treatment and non-treated C. albicans cells. Camphor and fenchyl alcohol significantly downregulated the expression of some hypha-specific genes and adhesive related genes. Notably, the expression of hypha-specific gene ECE1 was downregulated by 14.2-fold after camphor (0.01%) treatment, while adhesive related genes such as ECE2 (HWP1), RBT1 and EED1 were also downregulated by 4.7-, 3.5-, 1.7-fold, respectively when compared to their respective controls (Figure 6). Similarly, the expression of ECE1, ECE2, and RBT1 were significantly downregulated by 2.3-, 2-, and 6.2-fold, respectively after fenchyl alcohol (0.01%) treatment. Nevertheless, HGC1, HYR1, RAS1, SAP1, TEC1, and UME6 were not affected after camphor or fenchyl alcohol treatment. Taken together, qRT-PCR results showed that both camphor and fenchyl alcohol affected the expression of some hypha-specific genes and adhesive related genes, supporting the reduction of biofilm formation and hypha production (Figures 2, 5).

Antihyphal effects of active compounds such as camphor and fenchyl alcohol were examined in C. elegans infected with C. albicans. Microscopic observations of infected nematodes revealed that C. albicans hyphae pierced worms' cuticle and caused death. However, no hyphal formation was observed in worms treated with camphor, fenchyl alcohol, or fluconazole (the positive control) at 0.01% (w/v) (Figure 7A). In C. elegans, C. albicans caused a significant decrease in survival (5%) in non-treated controls, whereas >70% of nematodes survived 4 days in the presence of camphor at 0.01%, >50% survived 4 days in the presence of 0.01% fenchyl alcohol (Figure 7B), and >50% survived 4 days in the presence of fluconazole (a commercial antifungal agent).

We also examined the toxicities of CLEO, camphor, and fenchyl alcohol by treating worms with different concentrations for 4 days in the absence of Candida (Figure 7C). Camphor and fenchyl alcohol at concentrations of 0.05 and 0.1% did not affect nematode viability or survival (Figure 7C), whereas nematode survival decreased substantially after exposure at >0.5%. These results suggest camphor and fenchyl alcohol at the concentrations administered (< 0.05%) do not have toxic effects on the nematode.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.