The Inhibition of Non-albicans Candida Species and Uncommon Yeast Pathogens by Selected Essential Oils and Their Major Compounds

The epidemiology of yeast infections and resistance to available antifungal drugs are rapidly increasing, and non-albicans Candida species and rare yeast species are increasingly emerging as major opportunistic pathogens. In order to identify new strategies to counter the threat of antimicrobial resistant microorganisms, essential oils (EOs) have become an important potential in the treatment of fungal infections. EOs and their bioactive pure compounds have been found to exhibit a wide range of remarkable biological activities. We investigated the in vitro antifungal activity of nine commercial EOs such as Thymus vulgaris (thyme red), Origanum vulgare (oregano), Lavandula vera (lavender), Pinus sylvestris (pine), Foeniculum vulgare (fennel), Melissa officinalis (lemon balm), Salvia officinalis (sage), Eugenia caryophyllata (clove) and Pelargonium asperum (geranium), and some of their main components (α-pinene, carvacrol, citronellal, eugenol, γ-terpinene, linalool, linalylacetate, terpinen-4-ol, thymol) against non-albicans Candida strains and uncommon yeasts. The EOs were analyzed by GC-MS, and their antifungal properties were evaluated by minimum inhibitory concentration and minimum fungicidal concentration parameters, in accordance with CLSI guidelines, with some modifications for EOs. Pine exhibited strong antifungal activity against the selected non-albicans Candida isolates and uncommon yeasts. In addition, lemon balm EOs and α-pinene exhibited strong antifungal activity against the selected non-albicans Candida yeasts. Thymol inhibited the growth of all uncommon yeasts. These data showed a promising potential application of EOs as natural adjuvant for management of infections by emerging non-albicans Candida species and uncommon pathogenic yeasts.


Introduction
To date, the majority of fungal infections in humans are still due to Candida albicans (63-70%). However, other Candida species, such as C. krusei and C. parapsilosis, Cryptococcus neoformans and other uncommon yeasts, usually found in the environment and as skin or mucosal colonizers in humans, are emerging as opportunistic pathogens [1][2][3]. Particularly, C. krusei is responsible for invasive fungal infections in patients with severely compromised immunity undergoing bone marrow or stem-cell transplantation and in patients with malignant hematological disease [4,5]. C. parapsilosis is currently one of the leading species of catheter-related candidemia and fungal infections in neonates and patients in intensive care units. C. neoformans generally affects immunocompromised individuals, but patients with no apparent underlying immunodeficiency has been reported in some cases [1,3,6]. Finally, other rare yeast species such as Sporobolomyces spp. have been reported as a cause of sporadic invasive bloodstream infections in patients in intensive care units and with central venous catheters, as well as in AIDS patients [6]. Moreover, some common species, such as Saccharomyces cerevisiae can be responsible for uncommon clinical cases. This yeast is generally considered a safe organism very useful in agro-alimentary and pharmaceutical fields. However, several isolates have shown virulence features, being able to produce opportunistic infections, mainly in immunodeficient patients [6].
Available antifungal drugs, such as polyenes, triazoles and echinocandins have challenges to cope with the evolving nature of drug-resistant fungal pathogens. Recent trends in acquired antifungal resistance include increased azole resistance among non-albicans Candida species isolates, and echinocandin resistance in C. glabrata. In addition, some fungal species are intrinsically resistant to certain drugs (e.g., C. krusei and fluconazole, C. neoformans and echinocandins, or C. lusitaniae and amphotericin B). It should be noted that emerging pathogenic yeasts could show resistance to multiple classes of available agents (for example, C. auris) [7][8][9]. In order to identify new strategies to counter the threat from antimicrobial-resistant infections (AMRs), including those caused by fungal pathogens, plant-derived compounds have become an important potential for counteracting the therapeutic failures in the treatment of fungal infections.
Amongst phytochemicals, essential oils (EOs) produced by aromatic plants as secondary metabolites and their bioactive pure compounds have been found to exhibit a wide range of remarkable biological activities. Particularly EOs produced by botanical species belonging to Lamiaceae (e.g., Thymus vulgaris, Origanum vulgare, Lavandula vera) and Pinaceae (e.g., Pinus sylvestris) are of great pharmaceutical interest. Nevertheless, literature data on the action of the above mentioned EOs against emerging yeast pathogens, except for C. neoformans, is still limited, being an open field of new research [3,10].

Results
In this study, the chemical compositions of F. vulgare, L. vera, M. officinalis, P. sylvestris, S. officinalis, T. vulgaris, E. caryophyllata, O. vulgare, and P. asperum EOs are presented in Table 1, in according to data gathered from GC-MS and GC-FID phytochemical analyses.
The MIC and MFC values of EOs, and comparator azole drugs (FLC and VRC) against non-albicans Candida species are reported in Table 2. Based on susceptibility testing results, many tested clinical isolates were found to be resistant to FLC (C. krusei MIC range = 4-256 mg/mL and C. norvegensis MIC range = 32-128 mg/mL), and to VRC (C. valida MIC range = 0.06-2 mg/mL and C. norvegensis MIC range = 0.25-1 mg/mL).
As can be seen, anticandidal activity of the EOs components against non-albicans Candida species was observed only for α-pinene (MIC range 0.002-0.016%, v/v) while γ-terpinene and linalylacetate did not show any effect on the non-albicans Candida species isolates. Carvacrol, eugenol, linalool, and thymol (MIC range 0.06-0.25%, v/v) were also effective against C. parapsilosis and other non-albicans Candida species (Table 2). Anethole  [3,11]. b Relative percentages of individual components expressed as the % peak area relative to the total composition of the EO obtained by the GC-MS analysis.    The results in Table 3 indicate that all EOs and components were considerably potent against all non-Candida yeasts. Pine EO exhibited inhibitory effect towards all the tested microorganisms (range 0.004-0.12 %, v/v). In particularly, this EO displayed the highest inhibitory activity (MIC range 0.015-0.06% v/v) on C. neoformans isolates susceptible to azole drugs. The major component, α-pinene (content 55.7%), was effective against C. neoformans and other all non-Candida spp., whereas the major component of thyme red, thymol (content 26.5%), effective pure compound, inhibited the growth of all C. neoformans isolates at a concentration of 0.002-0.008% (v/v) than thyme EO alone (MIC range 0.06-0.12% v/v). Thymol was also more effective on P. etchellsii/carsonii, K. japonica, S. cerevisiae and S. salmonicolor (MIC range 0.03-0.12% v/v). Oregano and thyme red EOs and α-pinene, carvacrol components exhibited slightly lower antimicrobial activity than pine EO (MIC range 0.03-0.12 versus 0.004-0.12% v/v, respectively) ( Table 3).

Discussion
Candida spp. has been reported as the most common cause of fungal infections worldwide [5]. By studying the growing problem of antimicrobial drug resistance, the antimicrobial properties of EOs could be a valuable resource. EOs, as they are known from the literature, are complex mixtures, and the synergistic mechanisms that occur between the components of EOs are important to determine their effects. The biological properties of EOs strictly depend upon their complex phytochemical compositions, which mainly include terpenes (monoterpenes and sesquiterpenes), oxygenated terpenoids (e.g., alcohols and phenols), and other aromatic and aliphatic compounds, at quite different concentrations. As each EO composition varies depending on the plant species and EO chemotype considered, overall bioactivity could be attributed either to the major components of each EO or to combinations of multiple bioactive pure constituents.
In this study, by GC-MS analysis, the main components constitute for some oils over 50% of the EO and the monoterpenes were the most representative molecules. The terpene profiles of lavender, lemon balm, pine, oregano, clove and thyme EOs in this study were found to be similar to those present in the literature data [4,12,13]. In fact, the phytochemical composition proved to be highly rich in phenolic monoterpenes and only minor differences were observed.
In our study, pine EO exhibited a high inhibitory activity on the growth of all nonalbicans Candida isolates with low MIC values ranging from 0.03% to 0.12% (v/v) and 0.004-0.12% (v/v) against non-Candida yeasts ( Table 2). α-pinene, the main component of pine EO, is fairly effective in inhibiting growth stronger than pine EO alone against all tested Candida strains, displaying MIC ranging from 0.002% to 0.016% (v/v) ( Table 2) and confirming the good pine EO antimicrobial activity. These findings on α-pinene and pine EO are consistent with previous studies in which many authors reported the good activity of these compounds [3,14]. Moreover, in our previous study on C. neoformans, pine EO exhibited a high inhibitory activity on the growth of all azole susceptible isolates, with low MICs [3]. However, our data are in contrast with studies of Motiejūnaite et al. [15] that in 2004 reported that pine EO was very active only against bacteria but not fungi. Although it is known that the compounds making up a larger proportion of the EOs are not necessarily responsible for the majority of the total activity, in this case pine EO activity could be attributed to the presence of major components, such as α-pinene (55.7%). The mechanism by which α-pinene exerts its activity may be due to its action on cell integrity, inhibiting respiration and ion transport processes, and increasing membrane permeability in C. albicans [16]. This monoterpene showed different fungistatic or fungicidal activities when tested against non-albicans Candida isolates. In particularly, α-pinene exhibited important fungicidal activity against C. lusitaniae ( Table 2), demonstrating that it would always be advisable to evaluate the activity of oils and components on the single yeast. This finding is in accordance with the report of Salehi et al. [17] who demonstrated that α-pinene has a strong effect against a number of different bacteria and fungi.
The activity of pine EO is particularly high and interesting against non-Candida yeasts, i.e., C. neoformans, in agreement with Scalas et al. [3], though a number of the 'rare yeasts' are encountered as frequent colonizers of human skin and mucosal surfaces [6]. However, in the immunosuppressed host, invasive infections may occur, some being related to non-Candida yeasts low pathogenicity.
In our study all non-Candida yeasts were sensitive to the EOs studied (Table 3). In particular, the EO of lemon balm is reported for its high antibacterial and anticandidal activity against the human pathogenic C. albicans [18]. Our data showed that this EO exhibited the highest antifungal activity on the non-albicans Candida species too. The composition of the oil from M. officinalis purchased from Azienda Agricola Aboca includes monoterpene aldehydes and is dominated by citronellal (25.2%) and limonene (12.4%). This composition shows differences with oil composition from different countries in the world [18]. Citronellal displayed antifungal properties against C. valida (MIC/MFC = 0.06%, v/v), probably as a result of make membranes porous [19]. Other studies have shown that the lipophilicity of EOs allows them to separate from the aqueous phase in the membrane structures of fungi, resulting in membrane expansion, and increased membrane fluidity and permeability, alteration of intra-membrane proteins, inhibition of respiration, and alteration of ionic transport processes in fungi and leakage of cellular content [20].
Against non-albicans Candida isolates, oregano, thyme red (chemotype thymol 26.5%), geranium and clove EOs and carvacrol (62.6% in O. vulgare EO), eugenol (82.7% in E. caryophyllata EO), linalool (41.9% in L. vera EO) and thymol (26.5% in T. vulgaris EO) components exhibited a little lower antimicrobial activity than pine and lemon balm EO (range 0.03-0.12%, v/v) so that the antifungal activity may be attributed to the presence of some elements like carvacrol, eugenol, linalool and thymol which are already well known to exhibit antimicrobial activity ( Table 2). The antifungal effects of thyme red EO and thymol were widely reported in literature, and it is one of the ten most commercial oils worldwide [13,[21][22][23], but our results showed that is less efficacy than pine.
Sakkas et al. [13] reported that the antifungal activity of EOs high in carvacrol and thymol depends on their phenolic alcohol content and the antimicrobial potency is determined by their chemical composition. The EO of oregano, rich in carvacrol, inhibited C. albicans while the effect of other chemotypes poorer in phenolic components was lower. Our data demonstrated that oregano and carvacrol have interesting antifungal activity against non-Candida yeasts. EOs rich in phenols alter the permeability and function of cell membrane proteins. Due to the variety of molecules present in the plant extract, their antimicrobial activity cannot be attributed to a single mechanism but to several mechanisms in the various external and internal cell sites of the microorganism [10,13,24].
Additionally, in our study, the EOs have demonstrated potential as antifungal agents when used against non-albicans Candida species and non-Candida yeasts especially in the presence of yeast resistant to FLC and/or VRC. When the susceptibility profiles among Candida spp. were compared, it was observed that C. norvegensis isolates were the least susceptible species to FLC and VRC but on the contrary, this yeast was significantly more susceptible to more EOs and components tested.
EO main components (α-pinene, carvacrol, citronellal, eugenol, G-terpinene, linalool, linalylacetate, terpinen-4-ol, and thymol) were purchased from Sigma-Aldrich (Milan, Italy; ≥98% purity) and used as received without any further purification. All samples were protected from light and humidity and stored at 4 • C until use. EO analyses were performed both by gas chromatography with flame-ionization (Agilent Technologies, Waldbronn, Germany) (GC-FID) and by gas chromatography-mass spectrometry (Agilent Technologies) (GC-MS) as previously reported [3,25]. The compounds were identified by the comparison of their linear retention indices (LRIs) relative to C8-C40 n-alkanes (Sigma-Aldrich, Milan, Italy) [26]. Identification of several constituents was carried out by the comparison of their mass spectra with those recorded in the National Institute of Standards and Technology (NIST version 2.0d, 2005) and, when necessary, identification was carried out by co-injection of available reference compounds. The relative percentage amounts of individual components were expressed as the percentage peak area relative to the total composition of the EO obtained by the GC-FID analysis. Quantitative data were acquired as the mean of triplicate analyses for each sample [3].

Antifungal Drugs
FLC and VRC powders (≥98% purity by HPLC) were purchased from Sigma-Aldrich, Milan, Italy) (n • F8929 and PZ0005, respectively). FLC stock solutions were made up in sterile distilled water, while VRC stock solutions were made up in 100% dimethyl sulfoxide (Sigma-Aldrich, Milan, Italy) and stored at −20 • C until use.

Yeast Isolates
Forty-seven clinical isolates were collected from various specimens (blood, normally sterile body fluids, deep tissue, genital tract, gastrointestinal tract, respiratory tract) from hospitalized patients in Turin (Italy) during 2019. Yeast isolates were sub-cultured onto CHROMagar Candida (Becton Dickinson, Milan, Italy) to ensure purity. Then, they were identified by the API ID32C identification systems (BioMérieux, Rome, Italy), stored at −80 • C in MicrobanksTM (Pro-Lab Diagnostics, Neston, UK), and sub-cultured at least twice on Sabouraud dextrose agar (SDA, Oxoid, Milan, Italy) at 35 • C for 24 h (Candida and non-Candida spp.). Slowly growing isolates, such as S. cerevisiae and C. neoformans were incubated for 48-72 h.

Yeast Inocula
Inocula were prepared in sterile saline to reach a 0.5 McFarland turbidity standard and diluted in RPMI 1640 broth medium to a final concentration of 0.5-2.5 × 10 3 colony forming unit/mL(CFU/mL) [3,25].
The microtiter plates were incubated at 35 • C for 24 h (Candida and non-Candida spp.) or 48-72 h (S. cerevisiae and C. neoformans). EO and EO component MICs were determined visually as the lowest concentration at which no yeast growth was observed. MICs of azoles were determined as the lowest drug concentration that produced ≥50% inhibition of growth relative to growth control.
CLSI susceptible and resistant breakpoints for FLC were ≤2 and ≥8 mg/mL, respectively, while for VRC were ≤0.12 and ≥1 mg/mL, respectively. Minimum fungicidal concentration (MFC) was determined by subculturing 10 µL of broth taken from all the wells containing either azoles or EO/EO component concentrations without visible growth of yeast onto SDA plates. MFC was defined as a ≥99.9% reduction in the number of CFUs from the starting inoculum count [25,29].

Statistical Analysis
All tests were performed in triplicate in separate experiments. Descriptive statistics were used for MIC, MFC, and modal results were calculated.

Conclusions
The epidemiology of yeast infections and resistance to available antifungal drugs are rapidly increasing and non-albicans Candida species and rare yeast species are increasingly emerging as major opportunistic pathogens. In order to identify new strategies to counter the threat of antimicrobial resistant microorganisms, essential oils (EOs) have become an important potential in the treatment of fungal infections.
We investigated the in vitro antifungal activity of nine commercial EOs (clove, fennel, geranium, lavender, lemon balm, oregano, pine, thyme red, sage), and some of their main components against non-albicans Candida strains and uncommon yeasts.
Together with our previous data about this topic [3,29], these results show a promising potential application of EOs as natural adjuvants for the management of infections by emerging non-albicans Candida species and uncommon pathogenic yeasts, encouraging adequately controlled and randomized clinical investigations.

Data Availability Statement:
The data presented in this study are available on request from the first author Narcisa Mandras. The data are not publicly available due to privacy.