Hydrophobicity
The histogram reports the different percentages of hydrophobicity in the Candida spp. It is reasonable to expect that the differences among the four species of Candida depend on the distribution and composition of the molecules involved in the link with other compounds. Indeed, CSH values obtained by a two-phase system showed that C. albicans and C. glabrata were less hydrophobic than other species of Candida, as reported in Fig. 1. The differences were approximately five times less hydrophobic with respect to C. krusei and seven times less hydrophobic with respect to C. tropicalis. These results are in accordance with other authors who demonstrated that C. albicans is less hydrophobic than C. tropicalis. (Silva-Dias et al 2015). Generally, hydrophobic cells are more adherent than hydrophilic cells to host tissue. The cell surface hydrophobicity of Candida species enhances virulence by promoting adhesion to host tissues (Goswami et al 2017).
Polysaccharide content
To confirm the differences observed in the hydrophobicity in Candida spp. The polysaccharide content in the cell wall was determined and is reported in Table 1. C. glabrata and C. krusei showed similar contents of total glucose, while it was decreased in C. albicans. It is very interesting to note that in C. tropicalis, the total glucose was halved compared to other species. Regarding the total cell wall polysaccharide content constituted by polysaccharide complexes with protein and b-glucan, all four strains showed similar behavior with small differences. However, there is a marked relative difference in the cell wall alkali-acid insoluble constituents, which is mostly due to mannoprotein, rigid β-1,3-glucan complexes with proteins and chitin. In particular, the content of the alkali-acid insoluble material per equal cell wall preparation amount of the C. glabrata and C. krusei strains was less than half that of the C. tropicalis strain (Table 1). C. albicans showed an intermediate value. It is interesting to note that the relative amounts of glucose and alkali-acid insoluble material (β 1,3-glucan) were inverted in all strains of Candida spp. However, the cell wall amount results were similar. In particular, the importance of the alkali-acid soluble component in the regulation of CSH has been demonstrated by other authors (Singleton et al 2005). Similar results were obtained in a previous study of resistant or sensitive strains of C. glabrata (Vitali et al 2016). From a functional point of view, it is possible to suggest that the increased amount of β 1,3-glucan in C. tropicalis can be responsible for the high hydrophobicity of this strain.
Table 1. Cell wall composition of different Candida species
|
Strains
|
% Glucose a
|
% Cell wall b
|
% Alkali-acid Insoluble c
|
C. albicans
|
30 ±1.05
|
13± 0.7
|
25±1.9
|
C. glabrata
|
44 ± 3.2
|
17±1.5
|
17±2.3
|
C. krusei
|
40± 1.7
|
15±1.4
|
13±2.1
|
C. tropicalis
|
20± 1.2
|
10±1.0
|
50±3.8
|
a Expressed as a percentage of cell dry weight and measured by the methods of Dubois et al.
b Expressed as a percentage of cell dry weight
c Expressed as percentage of alkali-acid insoluble cell wall material/ milligram of cell wall dry weight.
Antimicrobial activity of OVEO, carvacrol and thymol
In the last decade, there has been an increase in drug resistance in Candida spp. (Pappas et al 2017, H.J. Heusinkveld et al 2013). To overcome this problem, natural compounds can represent a valid alternative to decrease this phenomenon. In particular, the antimicrobial activity against Candida spp. was studied with the essential oil of Origanum vulgare and two phenol compounds, carvacrol and thymol. The antimicrobial activity was measured by the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC). Table 2 reports the MIC and MFC of C. albicans, C. glabrata, C. krusei and C. tropicalis. For all Candida spp., the MIC values of OVEO were between 780 and 1560 µg/mL, and those of MFC were between 780 and 3120 µg/mL for all strains tested. Additionally, other authors confirmed the antifungal activity of OVEO against C. albicans (Ebani et al 2018). These results indicated that C. albicans was less sensitive to OVEO than C. glabrata, C. tropicalis and C. krusei, in discordance with reports in the literature on Mentha suaveolens, Coridothymus capitatus, Origanum hirtum and Rosmarinus officinalis essential oils. (Spagnoletti et al 2016). C. albicans and C. tropicalis were more resistant to OVEO with high MFC values.
The MIC and MFC for carvacrol were between 97.5 and 195 mg/ml, while the MIC values of thymol were between 195 and 390 mg/ml and MFC were between 390 and 780 mg/ml. It is more evident that the antimicrobial activity of carvacrol is more efficacious than those of OVEO and thymol. In particular, in all Candida spp tested, the MIC values corresponded to MFC values for carvacrol. demonstrating the fungicidal activity of carvacrol at low doses. Similar antifungal activities of OVEO and carvacrol were also reported on Malassezia furfur (Vinciguerra et al 2019).
Table 2. MIC and MFC of Origanum vulgare essential oil (OVEO), carvacrol and thymol on Candida spp (mg/mL).
|
Strains
|
OVEO
|
Thymol
|
Carvacrol
|
OVEO
|
Thymol
|
Carvacrol
|
MIC
|
MFC
|
Candida albicans (CO23)
|
1560
|
195
|
195
|
3120
|
390
|
195
|
Candida glabrata(43976)
|
780
|
390
|
195
|
780
|
780
|
195
|
Candida krusei (45709)
|
780
|
390
|
97.5
|
780
|
390
|
97.5
|
Candida tropicalis (47829)
|
780
|
390
|
97.5
|
1560
|
390
|
97.5
|
MIC: Minimum inhibitory concentration mean expressed in µg/mL;
MFC: Minimum fungicidal concentration mean expressed in µg/mL.
Preclinical evaluation in G. mellonella larvae
Larvae of G. mellonella are broadly used as an invertebrate wax moth larva model to evaluate the virulence of microbial pathogens, measure the efficacy/safety of biologically active antimicrobial agents, and produce results similar to those that can be found using mammals (Cook and Mc Arthur). As a consequence, no ethical authorization was requisite for this study because there was no use of mammal or animal models. In this work, the viability of G. mellonella larvae was evaluated to measure the relative toxicity and antimicrobial activity of OVEO, carvacrol, and thymol at MIC concentrations. The viability of the larvae was evaluated on Candida spp. for a five-day study period. Figure 2 shows all graphs of G. mellonella infections with and without treatment with OVEO, carvacrol, and thymol. All substances used were nontoxic at the MIC concentration, and the survival rate was similar to that of the control. Only in C. albicans did OVEO (Fig. 2 A1) show slight toxicity, lowering survival by approximately 30% compared to the control. When larvae were treated after infection with an MIC concentration of OVEO, carvacrol or thymol, the survival rate increased in all cases compared with infection alone. Specifically, treatment with OVEO in C. albicans and C. glabrata (Fig. 2 B). A1, B1) increased the survival rate by approximately 30%, from 50% to 80% survival in both strains. in C. krusei by approximately 50% (Fig. 2C1), increasing from 40% to 90% survival. In C. tropicalis (Fig. 2 D1), there was only a 10% increase. After infection, the larvae were treated with carvacrol in C. albicans (Fig. 2 B). A2), there was an increase in survival of 100%; in C. glabrata, the survival rate increased only by 10% (Fig. 2 B2); in C. krusei (Fig. 2, C2), it increased by approximately 30%; and in C. tropicalis (Fig. 2, D2), there was no improvement in survival following carvacrol treatment. Finally, thymol treatment (Fig. 2 B). A3, B3, C3, and D3) in all strains of all Candida species showed a slight increase in survival rate of 20% in C. albicans, C. glabrata, and C. tropicalis, while it increased survival by 30% in C. krusei. It is evident that there are differences between the Candida spp., where the species less susceptible “in vivo” to the activity of natural compounds was C. tropicalis, confirming the differences reported in cell wall components (Table 1). These results can be compared with results reported by other authors with another essential oil on C. albicans (Kaskatepe et al 2022).
Release of cellular material that absorbs at 260 nm
The leakage of the cytoplasmic membrane was analyzed by determining the release of cellular materials, including nucleic acids, metabolites and ions, which were absorbed at 260 nm into the fungal suspensions as reported in figure 3. This approach for determining the mechanism of antifungal action of OVEO was applied against different strains of Candida spp. The results of the release of cellular material at an absorbance value of 260 nm from Candida spp. cells exposed to OVEO at the MIC concentrations for 1 h showed differences between C. glabrata cells and other Candida spp. C. glabrata and C. krusei were more sensitive to OVEO, relaxing high levels of cellular material with respect to C. albicans and C. tropicalis. In particular, C. glabrata and C. krusei reported OD values at 260 nm of 2.5 and 1.9, respectively.
C. tropicalis showed the same values of approximately 1. Nevertheless, no changes in the absorbance of untreated cells (control) of Candida spp. were observed during the study (data not shown). Marked leakage of cytoplasmic material is used as an indicator of gross and permanent injury to the cytoplasmic membrane and plasma membrane (Cox et al 1998). The same treatment with carvacrol or thymol did not induce the same release of material as reports for the essential oil, and the differences between the different species of Candida were not significant (Fig. 3). These results directly confirm the high leakage of the material absorbing at 260 nm from the fungal cells treated with OVEO compared to those treated with a single constituent, such as carvacrol and thymol, and confirm the different activity in “vivo” on G. mellonella larvae (Figure 2).
Furthermore, highlighting the differences observed between C. albicans and NAC, the release of cellular material was inversely proportional to insoluble material (see Table 1).
Confocal laser scanning microscopy
DAPI is a DNA-specific probe that forms a fluorescent complex by attaching to the minor groove of A-T rich sequences (Yun and Lee 2016); in this case, as reported in Figure 4, we used DAPI to stain the nucleus of yeast cells (DAPI, see blue in Fig. 4). DAPI can be used to stain mammalian cells as well as gram-positive and gram-negative bacteria. In yeast, the staining is weak and not nuclear. (Morales-Cruz et al 2016).
Propidium iodide (PI, see red in Fig. 4) is a membrane-impermeant nucleic acid intercalator. The dye is usually used to selectively color dead cells in a mixed live-dead population and is also used as a counterstain in multicolor fluorescent assays. PI is dead cell specific in all cell types, including mammalian cells, bacteria and yeasts. In this case, we used this fluorescent dye to reveal the damage to the yeast membranes because this probe is unable to penetrate the cytoplasm of healthy cells with integral plasma membranes.
CLSM observations of cells treated with OVEO, thymol and carvacrol for 1 h revealed red positivity to propidium iodide in Candida spp. cells (Fig. 4). This is evident in C. krusei cells treated with OVEO and especially with thymol.
This small monoterpene is small and can easily increase the fluidity of the membrane without inducing its disruption, allowing PI to stain the cytoplasm of fungal cells (Fig. 4, panels f and h, respectively). C. glabrata cells treated with OVEO appeared poorly stained with propidium iodide, revealing minor damage to fungal bio-membranes (Fig. 4, panel j). Fig. 4 (panels b, c, d, n, o and p, respectively) shows C. albicans and C. tropicalis cells treated with OVEO, carvacrol and thymol for 1 h. The observations demonstrated that the yeast cells of these two strains were less propidium iodide positive, demonstrating their minor susceptibility to different treatments (see the micrographs of C. krusei).
SEM observations
To study the effects of OVEO, carvacrol and thymol treatment on the surface morphology of all Candida spp. cells, SEM observations were performed in Figure 5. SEM micrographs of C. albicans treated with OVEO, carvacrol and thymol (Fig. 5, panels b, c and d, respectively) show the effects of OVEO and the two phenolic compounds on morphology and on the cellular surface. Fig. 5 (panel a) shows the untreated cells (control) with their typical shape, dimension, and surface morphology, while Fig. 5 panel b) C. albicans cells treated with OVEO at the MIC concentration show roughness on the cell walls (arrowheads) and the presence of amorphous material on their surfaces. Conversely, some fungal cells treated with carvacrol (Fig. 5 panel c) showed a slight roughness of the surface (arrow). On the other hand, treatment with thymol induced distinct ultrastructural changes, such as a reduction in cell volume. In fact, the yeast cells are deflated (Fig. 5 panel d, arrowheads). Fig. 5 (panels f, g and h, respectively) shows micrographs of
C. krusei, after treatment with OVEO, carvacrol and thymol. After incubation with carvacrol (panel g), the C. krusei cells have some evident morphological alteration, such as roughness and the presence of amorphous material (arrow) on their surface. In panels f and h, fungal cells treated with OVEO and thymol showed a significant reduction in cell volume (arrows).
Fig. 5 (panels j, k and l, respectively) shows C. glabrata cells treated with OVEO and two phenolic compounds at the MIC concentration. Fig. 5 (panel j) shows cells treated with OVEO where the surface morphology was completely destroyed and the cells appeared flat and empty of their contents. This is probably because OVEO damages the cytoplasmic membrane, leading to the leakage of electrolytes and inducing an increase in permeability (de Castro et al 2015). This was also confirmed by confocal microscopy images (Fig. 4j). Fig. 5 panel k shows cells treated with carvacrol at the MIC concentration in the presence of some bubbles on the cell walls (arrows). This material could be the set of biological macromolecules released by fungal cells damaged by treatment with carvacrol. In Fig. 5, panel l the treatment with thymol show cells without marked alterations of their morphology.
In Fig. 5 (panels n and o, respectively) yeast cells of C. tropicalis treated with both OVEO and the two phenolic compounds, no evident superficial modifications are observable. In fact, in Fig. 5 (panel o), fungal cells after incubation with carvacrol have amorphous materials on the surface (arrows), while fungal cells treated with OVEO (Fig. 5, panel n) do not induce any change, and furthermore, their surface morphology is similar to that of untreated control yeast cells (Fig. 5 panel m). Instead, in Fig. 5 (panel p), the scanning electron micrograph observations showed that thymol-treated fungal cells became flat with deep depressions, probably caused by damage to biomembranes, as revealed by CLSM observations. These data and the results of the present study, as has also been reported by D’Agostino et al. 2019, demonstrate that fungal strains can respond differently to treatment with phytocompless or single natural molecules depending on their sensitivity to natural compounds.