**1. Introduction**

The genus *Candida* refers to a yeast that is part of the microbiota of healthy individuals living in commensalism with the human. However, in some cases, few *Candida* species tend to be opportunistic fungal pathogens, causing candidiasis. Candidiasis is among the common human infections; its symptoms vary according to the location of the infection in the body. Most of the infections may lead to minor symptoms such as slight localized rashes, redness, itching, and discomfort, though symptoms can be severe or even fatal if left without treatment in immunocompromised individuals [1–3]. Mostly, candidiasis is attributed to *Candida albicans*, however; non-albicans *Candida* species, including *C. parapsilosis*, *C. tropicalis*, and *C. glabrata*, have been reported to cause 30% to 54% of *Candida* infections [4–6]. Furthermore, the ability of these species to exhibit multidrug resistance, which may cause failure of the antifungal therapy, has been reported in earlier studies [6].

The ability of *Candida* spp to shift the commensal to pathogenic lifestyle is attributed to the presence of several virulence factors. Predominantly, the capability of switching morphology between yeast and hyphal forms, and the ability to form biofilms are the major properties crucial to *Candida* spp pathogenesis. The *Candida* infections are accompanied

**Citation:** Alfarrayeh, I.; Pollák, E.; Czéh, Á.; Vida, A.; Das, S.; Papp, G. Antifungal and Anti-Biofilm Effects of Caffeic Acid Phenethyl Ester on Different *Candida* Species. *Antibiotics* **2021**, *10*, 1359. https://doi.org/ 10.3390/antibiotics10111359

Academic Editors: Luís Melo and Andreia S. Azevedo

Received: 30 September 2021 Accepted: 3 November 2021 Published: 7 November 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

by the formation of biofilms on host tissues, organs, or abiotic surfaces such as urinary catheters, which result in high morbidity and mortality [7–9]. As with all microbial biofilms, *Candida* biofilms are highly resistant to antimicrobial treatment. Several factors might contribute to this resistance, including the physiological state of *Candida* cells in the biofilm, extracellular polymeric substances, overexpression of membrane-localized drug efflux pumps, variations in sterol content in the fungal membrane, and different developmental phases of cells through the biofilm [10]. Thus, the effectiveness of the current therapeutic agents against *Candida* biofilms is considered low, with a few exceptions [7–9]. The biofilms of *Candida* were 30 to 2000 times more resistant than planktonic cells to many antifungal agents, including amphotericin B, fluconazole, itraconazole, and ketoconazole [11]. As a result, the need to provide natural alternatives to these synthetic antifungal agents has arisen. The antifungal mechanisms of action of these natural alternatives can include the inhibition of germination and biofilm formation, the disruption of cell wall integrity, the alteration of cell membrane permeability, or the induction of apoptosis [9].

Caffeic acid phenethyl ester (CAPE), also called phenylethyl caffeate or phenethyl caffeate, is one of the promising natural alternatives of synthetic antimicrobial drugs. This polyphenolic ester compound is a major component of temperate propolis (poplartype) and can be produced in the laboratory by reacting caffeic acid with phenethyl alcohols. It consists of hydroxyl groups within the catechol ring, which is crucial for many biological activities [12,13]. No information was available in the literature about the LD<sup>50</sup> of CAPE in animal models or on normal human cells. However, Koru and coworkers investigated cytotoxicity in the human multiple myeloma cell line with LD<sup>50</sup> at 24, 48, and 72 h, found at 49.1, 30.6, and 22.5 µg/mL, respectively [14]. The CAPE has a wide range of biological activities, such as inhibition of nuclear factor κ-B, cell division restriction, termination of the cell cycle, and apoptotic induction [15]. It has been studied extensively as the most important individual component of propolis. Existing studies focused on its potential therapeutic properties such as its antibiotic, antioxidant, anti-inflammatory, anti-oxidative stress, antitumor, antidiabetic, anti-neurodegeneration, and anti-anxiety properties [13,16,17]. Numerous studies demonstrated the antibacterial activity of CAPE against different bacterial species [18–22]. However, few studies that have investigated the antifungal activity of CAPE as a single molecule or in combination with some antibiotics were found in the literature. De Barros and coworkers recently reported the ability of CAPE to inhibit the growth of both fluconazole-sensitive and fluconazoleresistant strains of *C. albicans* [23]. Moreover, Sun and coworkers found the synergistic effects of CAPE with caspofungin and fluconazole against *C. albicans* and fluconazoleresistant *C. albicans*, respectively [24,25]. The synergism with caspofungin was associated with a loss in iron homeostasis induced by CAPE, leading to functional defects in the mitochondrial respiratory chain and energy depletion, which increases the susceptibility of *C. albicans* to caspofungin [25].

Presently, CAPE has been given close attention for its important therapeutic effects in many diseases, including carcinomas, internal organ damage, metabolic diseases, inflammatory diseases, and microbial infections. CAPE may be a promising natural product for clinical application in the future [21]. One of the major advantages is that CAPE is devoid of some negative aspects of crude extracts of propolis, which includes, the inability to be standardized, which is a keystone of implementing it as therapy in the field of medicine [16]. This study aimed to investigate how CAPE, as a single molecule, affects planktonic growth, biofilm-forming abilities, mature biofilms, and cell death of some *Candida albicans* and non-albicans *Candida* species and strains.

#### **2. Results**

#### *2.1. Susceptibility of Candida Planktonic Cells to CAPE*

The antifungal effect of CAPE on nine *Candida* strains has been studied. The results of the minimal inhibitory concentration (MIC80) values for CAPE against different *Candida* species and strains are given in Table 1. It has been found that CAPE has a strain- and

dose-dependent effect. The MIC<sup>80</sup> values ranged from 12.5 to 100 µg/mL. The highest inhibitory effect was seen against *C. glabrata* SZMC 1378, *C. glabrata* SZMC 1374, and *C. parapsilosis* SZMC 8008 compared with the other strains. However, the most resistant strain was *C. albicans* SZMC 1423. **Table 1.** MIC<sup>80</sup> values of CAPE against the different *Candida* strains. **Strain MIC<sup>80</sup> (µg/mL)** *C. albicans* ATCC 44829 50

The antifungal effect of CAPE on nine *Candida* strains has been studied. The results

of the minimal inhibitory concentration (MIC80) values for CAPE against different *Candida* species and strains are given in Table 1. It has been found that CAPE has a strain- and dose-dependent effect. The MIC<sup>80</sup> values ranged from 12.5 to 100 µg/mL. The highest inhibitory effect was seen against *C. glabrata* SZMC 1378, *C. glabrata* SZMC 1374, and *C. parapsilosis* SZMC 8008 compared with the other strains. However, the most resistant strain


**Table 1.** MIC<sup>80</sup> values of CAPE against the different *Candida* strains. *C. albicans* SZMC 1423 100

was *C. albicans* SZMC 1423.

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#### *2.2. Effect of CAPE on Candida Biofilm-Forming Ability* The biofilm-forming ability is a crucial property related to the pathogenicity of *Can-*

The biofilm-forming ability is a crucial property related to the pathogenicity of *Candida*. In this experiment, the effect of CAPE was tested on the biofilms of four different *Candida* strains with high biofilm-forming abilities. The results demonstrate that CAPE has a dose-dependent inhibitory effect on the biofilm formation in four strains (Figure 1). The minimal biofilm inhibitory concentration (MBIC) values were 50, 50, 50, and 100 µg/mL for *C. albicans* SZMC 1424, *C. glabrata* SZMC 1374, *C. parapsilosis* SZMC 8007, and *C. tropicalis* SZMC 1366, respectively. *dida*. In this experiment, the effect of CAPE was tested on the biofilms of four different *Candida* strains with high biofilm-forming abilities. The results demonstrate that CAPE has a dose-dependent inhibitory effect on the biofilm formation in four strains (Figure 1). The minimal biofilm inhibitory concentration (MBIC) values were 50, 50, 50, and 100 µg/mL for *C. albicans* SZMC 1424, *C. glabrata* SZMC 1374, *C. parapsilosis* SZMC 8007, and *C. tropicalis* SZMC 1366, respectively.

**Figure 1.** Effect of CAPE on the biofilm-forming ability of four *Candida* species. The dashed line **Figure 1.** Effect of CAPE on the biofilm-forming ability of four *Candida* species. The dashed line represents the MBIC. Data are shown as mean ± SD from three independent experiments.

represents the MBIC. Data are shown as mean ± SD from three independent experiments.

*2.3. Effect of CAPE on Candida Biofilm Eradication*

#### *2.3. Effect of CAPE on Candida Biofilm Eradication* was investigated. Treatment of the mature biofilms of *C. albicans* SZMC 1424, *C. glabrata*

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The effect of CAPE on the mature biofilms of four biofilm-forming *Candida* species was investigated. Treatment of the mature biofilms of *C. albicans* SZMC 1424, *C. glabrata* SZMC 1374, *C. tropicalis* SZMC 1366, and *C. parapsilosis* SZMC 8007 with different concentrations of CAPE caused a partial eradication. The maximum eradications (19–49%) for the mature biofilms of *C. glabrata* SZMC 1374, *C. albicans* SZMC 1424, and *C. parapsilosis* SZMC 8007 were achieved at 25, 50, and 50 µg/mL, respectively. Moreover, the eradication process was found to be dose-independent above 50 µg/mL in the case of these strains. On the other hand, the mature biofilms of *C. tropicalis* SZMC 1366 were the most resistant to CAPE, and the maximum eradication was achieved at 100 µg/mL (Figure 2). SZMC 1374, *C. tropicalis* SZMC 1366, and *C. parapsilosis* SZMC 8007 with different concentrations of CAPE caused a partial eradication. The maximum eradications (19–49%) for the mature biofilms of *C. glabrata* SZMC 1374, *C. albicans* SZMC 1424, and *C. parapsilosis* SZMC 8007 were achieved at 25, 50, and 50 µg/mL, respectively. Moreover, the eradication process was found to be dose-independent above 50 µg/mL in the case of these strains. On the other hand, the mature biofilms of *C. tropicalis* SZMC 1366 were the most resistant to CAPE, and the maximum eradication was achieved at 100 µg/mL (Figure 2).

The effect of CAPE on the mature biofilms of four biofilm-forming *Candida* species

**Figure 2.** Effect of CAPE on mature biofilms of four *Candida* species. Data are shown as mean ± SD from three independent experiments. **Figure 2.** Effect of CAPE on mature biofilms of four *Candida* species. Data are shown as mean ± SD from three independent experiments.

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

*2.4. Biosorption of CAPE by Candida Cells* Biosorption may be defined as "the removal/binding of desired substances from aqueous solution by biological material" [26]. The results revealed that the biosorption of CAPE by different *Candida* strains occurs rapidly, followed by a maximum biosorption observed within the first 30 to 90 min (Figure 3). According to the amount of CAPE biosorbed, two groups can be recognized: the first group was able to biosorb 53–63 µg/mL of CAPE, and it includes *C. albicans* SZMC 1424, *C. parapsilosis* SZMC 8007, and *C. parapsilosis* Biosorption may be defined as "the removal/binding of desired substances from aqueous solution by biological material" [26]. The results revealed that the biosorption of CAPE by different *Candida* strains occurs rapidly, followed by a maximum biosorption observed within the first 30 to 90 min (Figure 3). According to the amount of CAPE biosorbed, two groups can be recognized: the first group was able to biosorb 53–63 µg/mL of CAPE, and it includes *C. albicans* SZMC 1424, *C. parapsilosis* SZMC 8007, and *C. parapsilosis* SZMC 8008; in contrast, the second group was able to biosorb 74–86 µg/mL, and it includes *C. albicans* ATCC 44829, *C. albicans* SZMC 1423, *C. tropicalis* SZMC 1366, *C. tropicalis* SZMC 1512, *C. glabrata* SZMC 1374, and *C. glabrata* SZMC 1378.

SZMC 1512, *C. glabrata* SZMC 1374, and *C. glabrata* SZMC 1378.

SZMC 8008; in contrast, the second group was able to biosorb 74–86 µg/mL, and it includes *C. albicans* ATCC 44829, *C. albicans* SZMC 1423, *C. tropicalis* SZMC 1366, *C. tropicalis*

**Figure 3.** Cellular uptake of CAPE by nine *Candida* strains. Data are shown as mean ± SD from three independent experiments. **Figure 3.** Cellular uptake of CAPE by nine *Candida* strains. Data are shown as mean ± SD from three independent experiments. **Figure 3.** Cellular uptake of CAPE by nine *Candida* strains. Data are shown as mean ± SD from

#### *2.5. Induction of Apoptotic Cell Death in Candida spp. by CAPE* three independent experiments.

*2.5. Induction of Apoptotic Cell Death in Candida spp by CAPE* Cells of the nine *Candida* strains treated with sub-lethal concentrations of CAPE were analyzed by double staining with CF*®*488A Annexin V and propidium iodide (PI). The apoptotic cells with externalized phosphatidylserine were detected by CF*®*488A Annexin V, while necrotic cells were detected by PI staining. The results shown in Figure 4 demonstrate CAPE-induced apoptosis in six of the tested strains at different levels. Among these strains, *C. albicans* ATCC 44829 and *C. albicans* SZMC 1423 revealed the highest percentage of early apoptotic cells (69.8 and 70.2%, respectively), whereas almost no apoptosis was seen in *C. glabrata* SZMC 1374, *C. parapsilosis* SZMC 8008, and *C. glabrata* SZMC 1378 Cells of the nine *Candida* strains treated with sub-lethal concentrations of CAPE were analyzed by double staining with CF®488A Annexin V and propidium iodide (PI). The apoptotic cells with externalized phosphatidylserine were detected by CF®488A Annexin V, while necrotic cells were detected by PI staining. The results shown in Figure 4 demonstrate CAPE-induced apoptosis in six of the tested strains at different levels. Among these strains, *C. albicans* ATCC 44829 and *C. albicans* SZMC 1423 revealed the highest percentage of early apoptotic cells (69.8 and 70.2%, respectively), whereas almost no apoptosis was seen in *C. glabrata* SZMC 1374, *C. parapsilosis* SZMC 8008, and *C. glabrata* SZMC 1378 (apoptotic cells ≤ 2%). On the other hand, no necrosis was observed in any of the tested strains (necrotic cells ≤ 1%). Examples of the scatter plots can be found in the supplementary material (Figures S1–S7). *2.5. Induction of Apoptotic Cell Death in Candida spp by CAPE* Cells of the nine *Candida* strains treated with sub-lethal concentrations of CAPE were analyzed by double staining with CF*®*488A Annexin V and propidium iodide (PI). The apoptotic cells with externalized phosphatidylserine were detected by CF*®*488A Annexin V, while necrotic cells were detected by PI staining. The results shown in Figure 4 demonstrate CAPE-induced apoptosis in six of the tested strains at different levels. Among these strains, *C. albicans* ATCC 44829 and *C. albicans* SZMC 1423 revealed the highest percentage of early apoptotic cells (69.8 and 70.2%, respectively), whereas almost no apoptosis was seen in *C. glabrata* SZMC 1374, *C. parapsilosis* SZMC 8008, and *C. glabrata* SZMC 1378 (apoptotic cells ≤ 2%). On the other hand, no necrosis was observed in any of the tested strains (necrotic cells ≤ 1%). Examples of the scatter plots can be found in the supplementary material (Figures S1–S7).

**Figure 4.** Cell death induced by CAPE treatment in nine *Candida* strains as determined by annexin V and PI staining. Data are shown as mean ± SD from three independent experiments. **Figure 4.** Cell death induced by CAPE treatment in nine *Candida* strains as determined by annexin V and PI staining. Data are shown as mean ± SD from three independent experiments.

**Figure 4.** Cell death induced by CAPE treatment in nine *Candida* strains as determined by annexin

V and PI staining. Data are shown as mean ± SD from three independent experiments.

#### *2.6. Effect of Caspase Inhibitor on the Growth of CAPE-Treated Candida Cells 2.6. Effect of Caspase Inhibitor on the Growth of CAPE-Treated Candida Cells*

To investigate whether yeast caspase Yca1p is involved in CAPE-induced apoptotic cell death, pre-incubation with the pan-caspase inhibitor Z-VAD-FMK was applied for 1 h. The growth of the sub-lethal CAPE concentration-treated *Candida* strains that had apoptosis was analyzed with and without pre-incubation with Z-VAD-FMK. As shown in Figure 5, a significant increase in the viability was observed in *C. albicans* ATCC 44829, *C. albicans* SZMC 1424, *C. tropicalis* SZMC 1366, and *C. tropicalis* SZMC 1512 that are pre-incubated with the pan-caspase inhibitor Z-VAD-FMK. However, the viability of CAPE-treated *C. albicans* SZMC 1423 and *C. parapsilosis* SZMC 8007 was not affected by the pre-incubation with the pan-caspase inhibitor Z-VAD-FMK. To investigate whether yeast caspase Yca1p is involved in CAPE-induced apoptotic cell death, pre-incubation with the pan-caspase inhibitor Z-VAD-FMK was applied for 1 h. The growth of the sub-lethal CAPE concentration-treated *Candida* strains that had apoptosis was analyzed with and without pre-incubation with Z-VAD-FMK. As shown in Figure 5, a significant increase in the viability was observed in *C. albicans* ATCC 44829, *C. albicans* SZMC 1424, *C. tropicalis* SZMC 1366, and *C. tropicalis* SZMC 1512 that are preincubated with the pan-caspase inhibitor Z-VAD-FMK. However, the viability of CAPEtreated *C. albicans* SZMC 1423 and *C. parapsilosis* SZMC 8007 was not affected by the preincubation with the pan-caspase inhibitor Z-VAD-FMK.

**Figure 5.** Effect of the pan-caspase inhibitor Z-VAD-FMK on the viability of six *Candida* strains treated with sub-lethal concentrations of CAPE. Data are shown as mean ± SD from three independent experiments. \*\*\* *p* <0.001 indicates a significant increment of the viability compared with the viability without pre-incubation with the pan-caspase inhibitor Z-VAD-FMK. **Figure 5.** Effect of the pan-caspase inhibitor Z-VAD-FMK on the viability of six *Candida* strains treated with sub-lethal concentrations of CAPE. Data are shown as mean ± SD from three independent experiments. \*\*\* *p* <0.001 indicates a significant increment of the viability compared with the viability without pre-incubation with the pan-caspase inhibitor Z-VAD-FMK.

#### *2.7. CAPE-Induced Subcellular Cell Death Markers Determined by TEM 2.7. CAPE-Induced Subcellular Cell Death Markers Determined by TEM*

To visualize the changes in intracellular morphology of the cells after CAPE treatment, transmission electron microscopy imaging was performed on *Candida* cells exposed to sub-lethal concentrations of CAPE. The TEM micrographs of *C. tropicalis* SZMC 1366, *C. albicans* SZMC 1423, and *C. parapsilosis* SZMC 8007 (Figures 6–8, respectively) mainly revealed typical hallmarks of apoptosis, including nuclear chromatin margination, nuclear blebs, condensation in the nucleus, vacuolization, plasma membrane detachment, enlarged lysosomes, cytoplasm fragmentation, cell wall distortion, and whole-cell shrinkage. However, very few cells displayed signs of necrosis, such as membrane disintegration and loss of cytoplasm density, whereas the TEM micrographs of *C. glabrata* SZMC 1374 (Figure 9) mainly revealed smaller necrotic signs. To visualize the changes in intracellular morphology of the cells after CAPE treatment, transmission electron microscopy imaging was performed on *Candida* cells exposed to sub-lethal concentrations of CAPE. The TEM micrographs of *C. tropicalis* SZMC 1366, *C. albicans* SZMC 1423, and *C. parapsilosis* SZMC 8007 (Figures 6–8, respectively) mainly revealed typical hallmarks of apoptosis, including nuclear chromatin margination, nuclear blebs, condensation in the nucleus, vacuolization, plasma membrane detachment, enlarged lysosomes, cytoplasm fragmentation, cell wall distortion, and whole-cell shrinkage. However, very few cells displayed signs of necrosis, such as membrane disintegration and loss of cytoplasm density, whereas the TEM micrographs of *C. glabrata* SZMC 1374 (Figure 9) mainly revealed smaller necrotic signs.

**Figure 6.** (**A**) TEM micrographs of the control *Candida tropicalis* SZMC 1366 cell structure demonstrates intact membranes, small unevenly scattered condensed chromatin grains, and homogenous cytoplasm structure. (**B**–**D**) TEM micrographs of *C. tropicalis* treated with a sub-lethal concentration of CAPE: (**B**) Late-stage disintegration with membrane fingerprints, vacuolization, plasma membrane detachment (arrowheads), and vacuole formation (double arrowheads). Fine granular homogenous cytoplasm organization disappeared, and a dense, compact cytoplasm with signs of fragmentation, rounded cell shape, and whole-cell shrinkage was seen. (**C**) Necrotic cell with membrane ruptures (arrowheads) and loss of cytoplasm density. (**D**) Several peripheral vacuoles show plasma membrane involvement (arrowheads). Nuclear bleb formation (double arrowheads). **Figure 6.** (**A**) TEM micrographs of the control *Candida tropicalis* SZMC 1366 cell structure demonstrates intact membranes, small unevenly scattered condensed chromatin grains, and homogenous cytoplasm structure. (**B**–**D**) TEM micrographs of *C. tropicalis* treated with a sub-lethal concentration of CAPE: (**B**) Late-stage disintegration with membrane fingerprints, vacuolization, plasma membrane detachment (arrowheads), and vacuole formation (double arrowheads). Fine granular homogenous cytoplasm organization disappeared, and a dense, compact cytoplasm with signs of fragmentation, rounded cell shape, and whole-cell shrinkage was seen. (**C**) Necrotic cell with membrane ruptures (arrowheads) and loss of cytoplasm density. (**D**) Several peripheral vacuoles show plasma membrane involvement (arrowheads). Nuclear bleb formation (double arrowheads).

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**Figure 7.** TEM micrographs of *C. albicans* SZMC 1423 treated with a sub-lethal concentration of CAPE exhibit different markers of cellular deterioration. (**A**) Severe cell wall distortion. (**B, C**) Appearance of enlarged lysosomes was rather frequently detected (arrowheads). (**D**) Isolation membranes precondition of cytoplasm fragmentation (arrowheads). (**E, F**) Nucleus fragmentation and marginal condensation (arrowheads). **Figure 7.** TEM micrographs of *C. albicans* SZMC 1423 treated with a sub-lethal concentration of CAPE exhibit different markers of cellular deterioration. (**A**) Severe cell wall distortion. (**B**,**C**) Appearance of enlarged lysosomes was rather frequently detected (arrowheads). (**D**) Isolation membranes precondition of cytoplasm fragmentation (arrowheads). (**E**,**F**) Nucleus fragmentation and marginal condensation (arrowheads). *Antibiotics* **2021**, *10*, x FOR PEER REVIEW 9 of 17

**Figure 8.** TEM micrographs of *C. parapsilosis* SZMC 8007 treated with a sub-lethal concentration of CAPE. Both apoptotic and necrotic cell structural changes were observed in samples. (**A**) Nuclear chromatin margination and condensation (arrowhead) and blebs (double arrowhead) detached from the nucleus are typical apoptotic hallmarks. (**B**) Few necrotic cells were also present. Note membrane disintegration, obvious vacuolization, and loss of cytoplasm density. (**C**) Peripheral vacuole formation refers to Golgi fragmentation and cell membrane separation from the cell wall (arrowheads). Nuclear blebs (double arrowhead). (**D**) Nuclear condensation (arrow) and extremely large lysosomal bodies (arrowhead). Note the swollen mitochondria (double arrowheads). **Figure 8.** TEM micrographs of *C. parapsilosis* SZMC 8007 treated with a sub-lethal concentration of CAPE. Both apoptotic and necrotic cell structural changes were observed in samples. (**A**) Nuclear chromatin margination and condensation (arrowhead) and blebs (double arrowhead) detached from the nucleus are typical apoptotic hallmarks. (**B**) Few necrotic cells were also present. Note membrane disintegration, obvious vacuolization, and loss of cytoplasm density. (**C**) Peripheral vacuole formation refers to Golgi fragmentation and cell membrane separation from the cell wall (arrowheads). Nuclear blebs (double arrowhead). (**D**) Nuclear condensation (arrow) and extremely large lysosomal bodies (arrowhead). Note the swollen mitochondria (double arrowheads).

**Figure 9.** TEM micrographs of *C. glabrata* SZMC 1374 treated with a sub-lethal concentration of CAPE. (**A, B, and C**) Mainly smaller necrotic signs were detected. Arrowheads denote small, mainly peripheral vacuoles typical of all samples. (**B**) Cell wall disintegration was also observed **Figure 9.** TEM micrographs of *C. glabrata* SZMC 1374 treated with a sub-lethal concentration of CAPE. (**A**–**C**) Mainly smaller necrotic signs were detected. Arrowheads denote small, mainly peripheral vacuoles typical of all samples. (**B**) Cell wall disintegration was also observed (double arrowhead).
