**3. Discussion**

(double arrowhead).

**3. Discussion**

This study focused on the antifungal and anti-biofilm effects of CAPE, which is one of the main biologically active components of propolis, on different *Candida* species in-This study focused on the antifungal and anti-biofilm effects of CAPE, which is one of the main biologically active components of propolis, on different *Candida* species including *C. albicans* and non-albicans *Candida* species. Moreover, we also investigated some of the mechanisms that might be involved in CAPE-induced cell death.

cluding *C. albicans* and non-albicans *Candida* species. Moreover, we also investigated some of the mechanisms that might be involved in CAPE-induced cell death. The *Candida* spp are still considered the most important opportunistic fungal pathogens that cause fungal infections worldwide. They are among the fourth to sixth most common nosocomial bloodstream isolates, according to estimates. Although *C. albicans* was the most frequent species isolated during candidemia, a greater role of non-albicans *Candida* spp has been observed in recent years [6,27]. Moreover, the extensive use of azole antifungals has resulted in the development of multidrug resistance in many *Candida* strains [28,29]. This resistance could be attributed to a change in drug intracellular accu-The *Candida* spp are still considered the most important opportunistic fungal pathogens that cause fungal infections worldwide. They are among the fourth to sixth most common nosocomial bloodstream isolates, according to estimates. Although *C. albicans* was the most frequent species isolated during candidemia, a greater role of non-albicans *Candida* spp has been observed in recent years [6,27]. Moreover, the extensive use of azole antifungals has resulted in the development of multidrug resistance in many *Candida* strains [28,29]. This resistance could be attributed to a change in drug intracellular accumulation, a change in membrane sterol composition, a change in efflux pump performance, or a change in ERG11 (the gene that is responsible for the production of the lanosterol-14-demethylase enzyme, the target of these medications) [6].

mulation, a change in membrane sterol composition, a change in efflux pump perfor-

The biological activities of CAPE have been widely studied. However, limited number of studies were found in the literature concerning the antifungal activity. This study aimed to add some information about the activity of CAPE against *C. albicans* and nonalbicans *Candida* species. Our findings showed that CAPE has high abilities to inhibit planktonic growth and biofilm formation as well as an ability to partially eradicate the mature biofilms of the different strains of *Candida*. Those results are in agreement with the results obtained by De Barros and coworkers, where the ability of CAPE to inhibit the

The biological activities of CAPE have been widely studied. However, limited number of studies were found in the literature concerning the antifungal activity. This study aimed to add some information about the activity of CAPE against *C. albicans* and non-albicans *Candida* species. Our findings showed that CAPE has high abilities to inhibit planktonic growth and biofilm formation as well as an ability to partially eradicate the mature biofilms of the different strains of *Candida*. Those results are in agreement with the results obtained by De Barros and coworkers, where the ability of CAPE to inhibit the growth of both fluconazole-sensitive and fluconazole-resistant strains of *C. albicans* has been reported [23]. Another study performed by Possamai Rossatto and coworkers showed similar results, where CAPE was also able to inhibit the growth of *C. auris* with MIC values ranging from 1 to 64 µg/mL. Furthermore, CAPE was able to inhibit the biofilm formation and phospholipase production of *C. auris* [30]. Our findings also showed the ability of CAPE to enter the cells of *Candida* spp rapidly. Such results were also observed by Cigut and coworkers in the yeast *Saccharomyces cerevisiae*. They found that, out of the four examined compounds (caffeic acid, p-coumaric acid, ferulic acid, and CAPE), CAPE was the only one that was able to enter the *S. cerevisiae* cells [31].

Phenolic compounds, including CAPE, are effective inhibitors of iron absorption [25]. According to Sun and coworkers, the antifungal mechanism of CAPE may include intracellular iron starvation due to its ability to form insoluble complexes with iron ions, which leads to the prevention of iron absorption by cells [25]. Using *C. albicans*-infected nematodes, Breger and coworkers showed that CAPE was able to inhibit the in vivo filamentation of *C. albicans*, leading to prolonged survival of infected nematodes [32]. Other studies attributed the antifungal activity of CAPE to its action on RNA, DNA, and cellular proteins, which are probable targets of this compound [13]. On the other hand, Su and coworkers suggested that the cytotoxicity of CAPE could be related to its apoptotic effect on the cells [33]. Marin and coworkers found that CAPE was able to induce the genes that are responsible for apoptosis and oxidative stress response. They reported that some polyphenolic compounds may have pro-oxidant activity, which can induce oxidative stress in the cells through the production of high levels of reactive oxygen species or inhibition of the system antioxidants [34]. In our study, we found that CAPE can induce apoptosis in five *Candida* strains, which are *C. albicans* ATCC 44829, *C. albicans* SZMC 1423, *C. albicans* SZMC 1424, *C. parapsilosis* SZMC 8007, *C. tropicalis* SZMC 1366, and *C. tropicalis* SZMC 1512. Surprisingly, *C. parapsilosis* SZMC 8007, *C. glabrata* SZMC 1374, and *C. glabrata* SZMC 1378 did not exhibit apoptotic cell death, which indicates that different species of *Candida* and, in one case, different strains of the same species have different cell death responses to CAPE. Moreover, the TEM images of CAPE-treated *Candida* cells showed the typical hallmarks of apoptosis in most *Candida* species. Notably, the apoptotic hallmarks were almost the same in different *Candida* species. Similar apoptotic hallmarks have been reported by several previous studies on *C. albicans*. De Nollin and Borgers (1975) reported the alterations of the surface micromorphology in *C. albicans* after treatment with miconazole. Shrinkage of protoplasm, abnormal cell and nuclear morphology, and vacuolization were also observed in plantaricin peptide-treated cells of *C. albicans* [35]. Distortion of the cell walls and membranes, which caused alterations of the surface micromorphology, could be explained due to a change in the permeability of the cell membrane, which could cause an osmotic imbalance, leading to alterations and indentations of the cell wall in collapsed cells [36].

Furthermore, we investigated the mechanisms involved in CAPE-induced apoptosis in *Candida* spp. Application of the broad-range pan-caspase inhibitor Z-VAD-FMK significantly reduced CAPE-induced apoptosis in *C. albicans* ATCC 44829, *C. albicans* SZMC 1424, *C. tropicalis* SZMC 1366, and *C. tropicalis* SZMC 1512. Such results suggest that this compound induced yeast caspase (Yca1p)-dependent apoptosis in these strains. Since CAPE can increase the permeability of the plasma membrane to ions [20], it can cause depolarization in mitochondria. This could lead to the release of cytochrome c and other proapoptotic factors into the cytosol, which in turn leads to the activation of yeast metacaspase Yca1p, resulting in the activation of caspase cascade inducing apoptosis [37]. However,

the pre-incubation of CAPE-treated *C. albicans* SZMC 1423 and *C. parapsilosis* SZMC 8007 with the pan-caspase inhibitor Z-VAD-FMK did not affect their viability, which means that the CAPE-induced apoptosis in these strains was Yca1p-independent. This suggests that it could be due to the release of the apoptosis-inducing factor Aif1p from the mitochondria triggered by CAPE. These results support the fact that CAPE not only has species- and strain-dependent cell death responses in *Candida* but also could induce apoptotic cell death through different mechanisms.

#### **4. Materials and Methods**

#### *4.1. Materials*

For our experiments, CAPE (Sigma-Aldrich, Buchs, Switzerland); sodium dodecyl sulfate; crystal violet; peptone; yeast extract (Merck, Germany); agar-agar (Fluka, Buchs, Switzerland); a modified version of RPMI 1640 medium (containing dextrose 1.8% (*w*/*v*), MOPS 3.4% (*w*/*v*), and adenine 0.002% (*w*/*v*)) (Sigma-Aldrich, St. Louis, MI, USA); potassium dihydrogen phosphate; disodium hydrogen phosphate (Reanal, Budapest, Hungary); dimethyl sulfoxide; ethanol (VWR Chemicals, Paris, France); sodium chloride (VWR Chemicals, Debrecen, Hungary); glucose (VWR Chemicals, Leuven, Belgium); adenine; calcium chloride; magnesium chloride; potassium chloride (Scharlau Chemie S.A, Sentmenat, Spain); Z-VAD-FMK (Biovision, Milpitas, CA, USA); glutardialdehyde solution; osmium tetroxide; propylene oxide; Durcupan (R) ACM components A/M, B, C, and D (Sigma-Aldrich, Darmstadt, Germany); 0.22 µm vacuum filters (Merck Millipore, Guyancourt, France); sterile 96-well microtiter plates for susceptibility testing (Costar®, Phoenix, AZ, USA) and for biofilm assays (Sarstedt AG & Co. KG, Nümbrecht, Germany, Catalog number: 83.3924.500); CF®488A Annexin V and PI apoptosis Kit (Biotium, Fremont, CA, USA); and methanol (Chemolab Ltd., Budapest, Hungary) were used. All of the chemicals used in the experiments were of analytical or spectroscopic grade.

#### *4.2. Instruments Used in the Experiments*

A Thermo Scientific Heraeus B12 incubator (Thermo Fisher Scientific, Waltham, MA, USA), Sanyo orbital incubator (Sanyo, Japan), Sanyo autoclave (Sanyo, Japan), Hitachi U-2910 UV/Vis spectrophotometer (Hitachi, Japan), WTW pH meter (inoLab, Germany), Multiskan EX plate reader (Thermo Fisher Scientific Inc., Vantaa, Finland), benchtop centrifuge (Hettich, Buford, GA, USA), Ultramicrotome Reichert Jung Ultracut E (LabX, Midland, ON, Canada), JEOL-1200EX Transmission electron microscope (TEM), and Attune NxT flow cytometer (Thermo Fisher Scientific Inc., Massachusetts, USA) were used throughout the experiments.

A Thermo Scientific Heraeus B12 incubator (Thermo Fisher Scientific, Waltham, MA, USA), Sanyo orbital incubator (Sanyo, Japan), Sanyo autoclave (Sanyo, Japan), Hitachi U-2910 UV/Vis spectrophotometer (Hitachi, Japan), WTW pH meter (inoLab, Germany), Multiskan EX plate reader (Thermo Fisher Scientific Inc., Vantaa, Finland), benchtop centrifuge (Hettich, USA), Ultramicrotome Reichert Jung Ultracut E (LabX, Canada), JEOL-1200EX Transmission electron microscope (TEM), and Attune NxT flow cytometer (Thermo Fisher Scientific Inc., Massachusetts, USA) were used throughout the experiments.

#### *4.3. Test Microorganisms, Culture Media, and Growth Conditions*

Four species of *Candida* were used: *C. albicans*, *C. tropicalis*, *C. glabrata*, and *C. parapsilosis*. Nine strains were included, one of which was the American Type Culture Collection (ATCC) strain, and the others were *Candida* isolates obtained from Szeged Microbial Collection (SZMC), University of Szeged, Hungary (Table 2). All strains were maintained at the Department of General and Environmental Microbiology, Institute of Biology, University of Pécs, Hungary. All strains were grown in yeast extract peptone dextrose (YPD) broth (yeast extract 1%, peptone 2%, and glucose 2% in distilled water, pH 6.8) or on YPD plates.


**Table 2.** *Candida* species and strains used in the study.

#### *4.4. Preparation of Stock Solution of CAPE*

The stock solution was freshly prepared by dissolving CAPE in ethanol at a concentration of 10 mg/mL. The stock solution was kept in the freezer at −20 ◦C.

#### *4.5. Antifungal Susceptibility Testing*

The broth microdilution method was performed to determine the minimal inhibitory concentration (MIC80) according to the protocol of the National Committee for Clinical Laboratory Standards Institute with some modifications [38]. Briefly, a standardized initial inoculum (10<sup>6</sup> cells/mL) was applied in all experiments. The experiments were performed in sterile, flat-bottom 96-well microplates. To obtain final CAPE concentrations ranging from 400 to 3.125 µg/mL, equal volumes (100 µL) of cell suspension and CAPE-containing YPD medium were added into the wells. Negative controls (media and cell suspension without CAPE) and blanks (media with CAPE) were included in each experiment. The concentration of the solvent was constantly fixed at 1%. The plates were incubated at 35 ◦C, and the absorbance was measured after 48 h at 600 nm using a Multiskan EX plate reader. The MIC<sup>80</sup> of CAPE was determined as the lowest concentration that causes an 80% reduction in the growth when compared with that of the negative control.

#### *4.6. Biofilm-Forming Ability Assay*

Biofilm formation assay was performed using the crystal violet staining method as described previously [39]. To inoculate the test microplates, a stationary-phase yeast culture was prepared using an inoculum size equivalent to 0.5 McFarland standard. The culture was shaken thoroughly and then diluted at 1:100 using RPMI-1640 medium. A series of two-fold dilutions were prepared from the stock solution of CAPE. In the test microplates, equal volumes (100 µL) of each dilution were added to identical volumes (100 µL) of the diluted cell suspensions to obtain final concentrations from 100 to 1.562 µg/mL CAPE in the wells. In each experiment, the negative controls and blanks were included. The concentration of the solvent was constantly fixed at 1%. The microplates were kept in an incubator at 35 ◦C for 48 h; afterward, the liquid part was discarded and the remaining biofilms were repeatedly washed with phosphate-buffered saline (PBS) (pH 7.4). Formalin in PBS 2% (*v*/*v*) was used to fix the biofilms; then, the crystal violet 0.13% (*w*/*v*) staining was applied for 20 min at room temperature. The excess crystal violet stain was discarded, and the wells were washed thoroughly and repeatedly with PBS buffer. The estimation of biofilm mass was conducted by adding sodium dodecyl sulfate (SDS) in ethanol (1% *w*/*v*) solution to each well to extract the stain overnight, and the absorbance of the solution was measured at 600 nm using a Multiskan EX plate reader. The minimum biofilm inhibitory concentration (MBIC) was defined as the lowest concentration of CAPE that was able to inhibit 90% of the biofilm-forming ability.

#### *4.7. Biofilm Eradication Assay*

The effect of CAPE on mature biofilms was verified as described by Nostro and coworkers [40]. Briefly, for the inoculation of the assay microplates, stationary-phase yeast cultures were prepared using an inoculum size equivalent to 0.5 McFarland standard and thereafter diluted 1:100 using RPMI-1640 medium. Microplates containing diluted cell suspension were kept in an incubator at 35 ◦C for 48 h. After the biofilm maturation, CAPE treatment was applied. Accordingly, the original RPMI culture was discarded and replaced with a CAPE-containing RPMI medium with concentrations ranging from 100 to 1.562 µg/mL. Negative controls and blanks were included in each experiment. The concentration of the solvent was constantly fixed at 1%. After 48 h of incubation at 35 ◦C, the liquid part of the media was discarded, and the remaining biofilms were washed, fixed, stained, and estimated as mentioned in the previous section (Section 4.6).

#### *4.8. Biosorption of CAPE by Candida Cells*

To determine the cellular biosorption of CAPE, YPD broth cultures of *Candida* strains were grown overnight at 35 ◦C and 150 rpm in an orbital shaker. The number of yeast cells was adjusted to 10<sup>7</sup> cells/mL in each case, and the cultures were treated with 100 µg/mL CAPE and incubated at 35◦C with shaking at 150 rpm. The concentration of the solvent was constantly fixed at 1%. Samples were taken at the time points 0, 5, 10, 15, 20, 30, 60, and 120 min after admission and centrifuged (5000 rpm, 5 min), and the absorbance of the cell-free supernatants was measured at 330 nm (absorption maximum of CAPE) using a Hitachi U-2910 UV/Vis spectrophotometer. A calibration curve of two-fold serial dilutions of CAPE from 100 to 0.781 µg/mL was constructed and used to evaluate the biosorption levels of *Candida* cells [41].

## *4.9. Cell Death Examination Assay*

The YPD broth media were inoculated with *Candida* cells (10<sup>6</sup> cell/mL) from fresh YPD plate cultures and incubated at 35 ◦C with shaking at 150 rpm until reaching the mid-exponential phase depending on their growth curves. Media containing sub-lethal concentrations (MIC80) of CAPE were inoculated with 2.5 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/mL of the midexponential phase cultures of different *Candida* species and strains and incubated at 35 ◦C with shaking for 3 h. Untreated cell samples were included as negative controls in each experiment. The concentration of the solvent was constantly fixed at 1% in all experiments. After the incubation period, cells were harvested and washed with PBS. The CF®488A Annexin V and PI apoptosis Kit was used according to the manufacturer's instructions to identify apoptosis and necrosis. In brief, *Candida* cells were re-suspended in 1X annexin V binding buffer at a concentration of 5 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/mL. To 100 µL of this solution, 5 µL of CF®488A Annexin V and 2 µL of PI working solution were added. The tubes were gently vortexed and incubated for 20 minutes at room temperature in the dark. After incubation, 400 µL of 1X annexin V binding buffer was added to each tube and analyzed using an Attune NxT flow cytometer. Annexin V is responsible for the detection of phosphatidylserine translocation from the inner to outer leaflets of the plasma membrane, whereas PI is a membrane-impermeant DNA-binding dye that is usually used to selectively stain dead cells in a cell population. PI is excluded by living cells and early apoptotic cells but stains necrotic and late apoptotic cells with compromised membrane integrity.

#### *4.10. Caspase Inhibitor Assay*

Caspase inhibitor assay was performed as described by Yue and coworkers [42] with some modifications. Briefly, cells were divided into two groups. The first group was pretreated for 1 h at 35 ◦C with the broad-spectrum caspase inhibitor Z-VAD-FMK (final concentration 77 µM) before incubation with CAPE. The second group was used as a control (not treated with the caspase inhibitor). For microplate assays, the cells were harvested by centrifugation, washed twice with PBS, and then re-suspended in YPD broth. The cell density was adjusted to 2 <sup>×</sup> <sup>10</sup><sup>6</sup> cell/mL. Equal volumes (100 µL) of cell suspension

and CAPE-containing YPD medium were dispensed into the wells to obtain the final CAPE concentration equal to the MIC<sup>80</sup> of each strain. Negative controls (media and cell suspension without CAPE) and blanks (media with CAPE) were included in each experiment. The concentration of the solvent was constantly fixed at 1%. The plates were incubated at 35 ◦C, and the absorbance was measured after 48 h at 600 nm using a Multiskan EX plate reader.

#### *4.11. Ultrastructural Examination of Candida Species by TEM*

Media containing sub-lethal concentrations of CAPE were inoculated with 2.5 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/mL of the mid-exponential phase cultures of different species of *Candida* and incubated at 35 ◦C with shaking for 3 h to induce apoptosis. After the incubation period, the cells were harvested by centrifugation (5000 rpm, 5 min). The pellets were immediately washed and re-suspended with modified PBS (a mixture of 50 mM K2HPO<sup>4</sup> and KH2PO<sup>4</sup> (pH 7.0), supplemented with 0.5 mM MgCl2) and incubated at room temperature for 15 min to achieve equilibrium. Then, the samples were fixed overnight in 2.5% glutaraldehyde fixative buffered with modified PBS. The samples were then washed 4 times with modified PBS, and after short centrifugal sedimentation (1000 rpm, 2 min) preparation continued with a 2 % osmium tetroxide post-fixation on ice for 2 h. The cells were then washed twice with distilled water for 15 min and stained 'en bloc' in 1% aqueous uranyl acetate for 30 min. After two further washing steps with distilled water and short sedimentation, the cells were dehydrated in 70, 96, and 100% ethanol for 15 min each, subsequently. The cells were treated with propylene oxide twice for 10 min each time and then infiltrated for 1 h in a propylene oxide/Durcupan epoxy resin mixture (1:1) at room temperature. After 1 h, the cells were transferred to fresh epoxy resin drops for another 1 h. The resin was then changed, and the samples were left overnight in the fresh resin drops at room temperature. On the next day, the resin was changed twice while incubating at 40 ◦C for 2 h, subsequently. Finally, the samples were encapsulated in fresh epoxy resin and left at 56 ◦C for a two-day-long polymerization. Serial ultrathin sections were cut with Reichert Ultramicrotome, collected onto 300 mesh Nickel grids, counterstained on drops of uranyl acetate and Reynolds solution of lead citrate, washed thoroughly in sterile distilled water, and examined with a JEOL-1200 EX TEM at 80 KeV [43].

#### *4.12. Statistical Analysis*

All assays were carried out in triplicate, and data were expressed as mean ± standard deviation (SD). For data processing and visualization of the results, Microsoft Office Excel 2016 was used. Data were statistically analyzed through two-sample t-tests using Past3.21 software (University in Oslo, Oslo, Norway).

#### **5. Conclusions**

CAPE could be considered a promising natural antifungal agent. It has a concentrationand strain-dependent effect on the viability and biofilm-forming ability of the different *Candida* species. Moreover, it has a partial ability to eradicate the mature biofilms of biofilmforming strains of *Candida*. In most *Candida* species and strains, the antifungal mechanism involves the induction of apoptotic cell death in treated cells. However, in other *Candida* species and strains, no apoptotic cell death was observed. This information suggests that CAPE may have a species- and strain-dependent cell death response in *Candida*.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/antibiotics10111359/s1, Figure S1: Representative flow cytometry scatter plot showing apoptosis of *C. albicans* ATCC 44829 treated with 50 µg/mL of CAPE, Figure S2: Representative flow cytometry scatter plot showing apoptosis of *C. albicans* SZMC 1423 treated with 100 µg/mL of CAPE, Figure S3: Representative flow cytometry scatter plot showing apoptosis of *C. albicans* SZMC 1424 treated with 50 µg/mL of CAPE, Figure S4: Representative flow cytometry scatter plot showing apoptosis of *C. parapsilosis* SZMC 8007 treated with 25 µg/mL of CAPE, Figure S5: Representative flow cytometry scatter plot showing apoptosis of *C. parapsilosis* SZMC 8008 treated with 12.5 µg/mL

of CAPE, Figure S6: Representative flow cytometry scatter plot showing apoptosis of *C. glabrata* SZMC 1374 treated with 12.5 µg/mL of CAPE, Figure S7: Representative flow cytometry scatter plot showing apoptosis of C. glabrata SZMC 1378 treated with 12.5 µg/mL of CAPE.

**Author Contributions:** Conceptualization, I.A. and G.P.; methodology, I.A., A.V. and E.P.; validation, I.A, E.P. and G.P.; formal analysis, all authors; investigation, I.A. and E.P.; resources, I.A., E.P., A.V. and G.P.; data curation, I.A.; writing—original draft preparation, I.A. and G.P.; writing—review and editing, I.A., E.P., Á.C., A.V., S.D. and G.P.; visualization, I.A., E.P., Á.C., A.V., S.D. and G.P.; supervision, G.P. and I.A.; project administration, G.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This research was also connected to the project GINOP-2.3.3-15-2016-00006 (Széchenyi 2020 Programme).

**Conflicts of Interest:** The authors declare no conflict of interest.

## **Abbreviations**

CAPE: Caffeic Acid Phenethyl Ester; MIC, Minimal Inhibitory Concentration; MBIC, Minimal Biofilm Inhibitory Concentration; PBS, Phosphate-Buffered Saline; PI, Propidium Iodide; YPD, Yeast extract Peptone Dextrose.

#### **References**

