Synergistic Anticandidal Effects of Six Essential Oils in Combination with Fluconazole or Amphotericin B against Four Clinically Isolated Candida Strains

The development of opportunistic pathogenic Candida strains insensitive to several classes of antifungals has emerged as a major health care problem during the last years. Combinational therapy of natural products (e.g., essential oils, EOs) with conventional antifungals has been suggested as a promising alternative to overcome this medical problem. The present study investigates the potential antifungal activity of EOs extracted from some selected medicinal plants, alone and in combination with two common conventional antifungals (fluconazole and amphotericin B) against four clinical Candida isolates. MIC assays indicated that EOs induced strong anticandidal activities with MIC values ranging from 0.162 to 4.950 mg/mL. The combination of amphotericin B with Thymus leptobotrys, Origanum compactum and Artemisia herba alba EOs provided a synergistic effect against C. krusei only, with MIC gain of four-fold, and additive effect against remaining strains (MIC gain = two-fold). Interesting synergistic interactions were observed by combining all studied EOs with fluconazole, with reduction rates of their MICs ranging from 16 to 512-fold. This synergistic effect was very pronounced with the combination of T. leptobotrys EO and fluconazole. These findings indicate that studied EOs can be used as anti-candidals in combination with antifungals, particularly fluconazole, to counteract the emergence of resistant Candida spp.


Introduction
The incidence of systemic fungal infections has been increasing significantly and currently affects millions of people worldwide [1][2][3]. Yeasts of Candida genus are reported to be responsible for 80% of fungal infections, which are recognized as one of the most common nosocomial contaminations, producing important morbidity and mortality rates, particularly in immunocompromised patients [4,5]. Within 200 yeast species, Candida albicans has been described as the most common pathogen found in severe candidiasis infections, but other non-albicans Candida spp. such as C. glabrata, C. tropicalis, C. krusei, and C. parapsilosis are becoming increasingly insidious [6][7][8]. The current treatment of these candidiasis infections remains essentially based on the use of common polyenes (e.g., amphotericin B) and azole antifungals (e.g., fluconazole), mainly targeting ergosterol in the fungal cell membrane or its biosynthetic pathway [9][10][11]. However, the fungistatic property of many of these antifungals, in addition to the increased therapy cost and the emergence of clinical drug resistance, limit their success in clinical practice [9,10,12,13]. The development of novel antifungal agents is becoming difficult and challenging due to the eukaryotic nature of Candida cells, which results in a limited number of drug targets. The required antifungals should be specific against the pathogen's targets, which are not shared with human hosts; otherwise, these antifungals should display a selective toxicity to the fungal cell, while being safe towards the human host cell [14]. Alternatively, the synergy between conventional drugs and natural antimicrobial products has been described as an emerging strategy to minimize the effective doses of standard antifungals, minimizing their side effects and their related toxicity, while enhancing their biological efficacies [15,16]. Among natural products, essential oils (EOs) from medicinal plants constitute rich sources of bioactive compounds with strong antimicrobial activities and low cytotoxicity against the host [17]. Several EOs have been reported to present high synergistic interactions with conventional antimicrobials against several pathogenic micro-organisms, including polyene and azole-resistant Candida isolates [15,18,19]. In fact, many EO components target multiple metabolic pathways in Candida cells, which can overcome or delay the emergence of drug resistance [19][20][21]. Additionally, some terpenoids, when used in combination, are able to transform the fungistatic nature of fluconazole into a fungicidal drug, and to inhibit the antifungal efflux by blocking drug transporter pumps [15,18]. (Asteraceae) and Ammodaucus leucotrichus (Coss. &Durieu) (Apiaceae) are extensively used in Moroccan folk medicine in different forms to treat many fungal diseases [22]. Moreover, their antibacterial, antifungal, antiparasitic and antiviral activities have been reported in many previous studies [23][24][25][26][27][28]. To the best of our knowledge, no study has yet been published about synergistic interactions of their EOs with conventional antifungals. Therefore, the aim of the present work was to evaluate the capacity of these EOs to enhance the anticandidal effects of amphotericin B and fluconazole, when used in combination at significantly low concentrations, against four clinically isolated fungal strains, namely C. albicans, C. glabrata, C. krusei and C. parapsilosis.

Anticandidal Activity
The anticandidal activities of studied EOs towards Candida strains were evaluated using broth microdilution assays. The results given in Table 3 demonstrated that all EOs expressed strong anticandidal effects with MIC values ranging from 0.162 mg/mL to 4.950 mg/mL (Table 3). A. leucotrichus, O. compactum and T. leptobotrys EOs displayed the strongest inhibitory activity against all tested Candida strains, with comparative MIC values ranging from 0.162 mg/mL to 0.596 mg/mL. These values were, interest-ingly, lower than those of fluconazole (MIC = 1 mg/mL for all Candida strains). EOs of T. saturioiedes and T. pallidus expressed high anticandidal activities at concentrations ranging from 0.373 mg/mL to 2.598 mg/mL, while A. herba alba EO showed poor effect towards all tested strains, with MIC values between 2.475 mg/mL and 4.950 mg/mL. The relatively strong activity of A. leucotrichus observed in our study is in line with those works reporting the effect of this oil on some Candida strains [28,[44][45][46]. This strong activity can be mainly explained by the presence of high content of perillaldehyde, an oxygenated monoterpene previously tested for its potent antimicrobial activity [47][48][49]. O. compactum and Thymus species have long been known for their large spectrum of activity against numerous pathogenic strains [23,25,50]. Their antifungal effect is attributed to the presence of bioactive antimicrobial compounds, especially carvacrol, thymol, borneol, p-cymene and γ-terpinene. The anticandidal effect of these monoterpenoids has been well demonstrated, with carvacrol being more active than the other thyme oil constituents [15,[51][52][53], and can explain the relative high activities of T. leptobotrys and O. compactum which contain higher percentages of this phenolic monoterpene compared to the remaining studied thymes. The moderate anticandidal effect of A. herba alba obtained in our study is consistent with that previously reported [41].

Synergistic Effect of EOs with Conventional Antifungals
The results of the synergistic interactions (FICI values and MIC gain of the antifungals) between studied EOs and conventional antifungals fluconazole and amphotericin B are reported in Tables Tables 4 and 5, respectively. The combinations of studied EOs and fluconazole gave very pronounced synergistic effects regarding all tested Candida strains, with FICI values between 0.25 and 0.31 (Table 4). Among these combinations, the one prepared by T. leptobotrys EO showed the greater synergism, with promising reduction in fluconazole MICs of Candida strains up to 512 fold. The addition of T. satureioides or T. pallidus EOs reduced the fluconazole MICs for C. albicans, C. glabrata and C. krusei by 456 fold and by 64 fold for C. parapsilosis. EOs of O. compactum and A. leucotrichus decreased the MIC values of fluconazole 128 fold for C. albicans and C. glabrata, 64 fold for C. krusei and 16 fold for C. parapsilosis, while A. herba alba EO reduced the fluconazole MICs for C. albicans, C. glabrata and C. krusei by 64 fold and C. parapsilosis by 32 fold. From these results, it can be observed that the synergistic effect of EOs with fluconazole was stronger than that obtained with amphotericin B (Table 5). Indeed, the combination of amphotericin B with sub-MICs of T. leptobotrys, A. herba alba or O. compactum EOs gave synergistic effects against C. krusei only, with FICI = 0.5 and four-fold gains, while additive effects (FICI = 0.75) and gains of two-fold were obtained against the other tested Candida strains. Remaining EOs gave MIC gains of 1 towards all tested Candida and FICI = 1.25, indicating indifferent outcome. Interestingly, for all combinations and for the two conventional antifungals studied, none was found to be antagonistic against the tested strains.
To the best of our knowledge, the combinations of the EOs and antifungals (fluconazole and amphotericin B) have not been investigated previously. However, the studied EOs are characterized by the presence of some major compounds known to possess strong antimicrobial effects and synergism interactions with many antimicrobials [20,54,55]. In fact, carvacrol and thymol (principal components of studied Origanum and Thymus EOs) were reported to interact with the cytoplasmic membrane by its entry between the acyl chains of phospholipids, leading to the disruption of its fluidity and permeability [20,56,57]. This mechanism may facilitate the permeability of fluconazole through the fungal membrane to the intracellular target, acting together on the ergosterol biosynthesis pathway. Carvacrol and thymol were reported to increase the effectiveness of fluconazole by chemo-sensitizing the Candida cells to the antifungal and decreasing its extrusion by efflux pumps [15].

Plant Material and EOs Extraction
Aerial parts of T. leptobotrys, T. pallidus, T. satureioides, O. compactum, A. herba alba and fruits of A. leucotrichus were collected from different wild locations in Morocco (Table 1) and identified by one of the authors (Abbad. A). Plants collected were dried in the shade at room temperature (≈25 • C) and voucher specimens were deposited at the Laboratory of Microbial Biotechnologies, Agrosciences and Environment of the Faculty of Science Semlalia, University Cadi Ayyad, Marrakech. Each dried plant material was submitted to four successive steam-distillations (4 × 200 g) for about 3 h using a Clevenger-type apparatus, and recovered EOs were dried with anhydrous sodium sulfate and stored in hermetically sealed vials at 4 • C until use.

GC/MS Analysis
Qualitative and quantitative analysis of the EO chemical compounds was carried out using a gas chromatograph equipped with a TG-5MS column (length: 30 m; internal diameter: 0.25 mm, thickness film: 0.25 mm) and coupled to a mass selective detector ISQ (Single Quadrupole Mass spectrometer). The carrier gas was helium with flow rate of 1.0 mL/min. EO samples (20 µL) were diluted in 2 mL of hexane and 2.0 µL of the dilution was injected, using split mode. The injection temperature was 260 • C and the column temperature was programmed from 100 to 260 • C at a rate of 4 • C/min with a hold of 10 min at 246 • C. The transfer line and ion source temperatures were held at 230 • C with EI ionization (70 eV) and the m/z scan range was of 41-500. Identification of the individual components was carried out by matching their mass spectra with WILEY275, NBS75K, and Adams terpene library [58], and standards of the main components where possible. For semi-quantification purposes, the normalized peak area of each compound was used without any correction factors to establish abundances.

Candida Strains
The yeast strains used in this study were provided by the Moroccan coordinated collection of microorganisms. C. albicans (CCMM L4), C. glabrata (CCMM L7), C. krusei (CCMM L10) and C. parapsilosis (CCMM L18) are clinically isolated fungal strains originating from patients suffering from acute candidiasis. They were cultured in Sabouraud Dextrose Agar at 28 • C for 48 h [59].

Determination of the Minimum Inhibitory Concentration (MIC)
The anticandidal activities of EOs were evaluated using broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines M27-A3 [60]. EO dilutions were performed in Sabouraud Dextrose Broth supplemented with dimethylsulfoxide (DMSO), at a final concentration of 1%, to enhance oil solubility. A negative control was prepared using the same concentration of DMSO. Then, 100 µL of each dilution was mixed with the same volume of cell suspension at 1-2 × 10 3 cells/mL. The microplates were incubated at 28 • C for 48 h and the MIC value was defined as the lowest EO concentration that inhibits macroscopic growth of the tested strains. Fluconazole and amphotericin B were used as positive controls.

Synergistic Effect of EOs with Conventional Antifungals
Synergistic interactions between conventional antifungals (fluconazole and amphotericin B) and EOs, were determined using microdilution assay [16]. Briefly, 50 µL of a serial dilution of antifungals (from MIC to 1/512 MIC) were added to microwells containing 50 µL of the EO at sub-inhibitory concentrations (1/4 MIC), and previously seeded by 100 µL of cell suspensions. The microplates were incubated at 28 • C for 48 h. The MIC values of antifungals in combination with sub-MICs of EOs were determined and fractional inhibitory concentration index (FICI) was calculated using the following formula: FICI = FIC of EO + FIC of antifungal FIC of EO = MIC of EO in combination with antifungal/MIC of EO alone, and FIC of antifungal = MIC of antifungal in combination with EO/MIC of antifungal alone.
The MIC gain of the antifungal was determined as MIC of antifungal alone/MIC of antifungal in combination with EO.

Conclusions
The present work provides new information regarding the anticandidal potential of some selected Moroccan EOs and their synergistic effects with two common antifungals (fluconazole and amphotericin B). The results indicate that all EOs studied possess anticandidal activity, with those extracted from A. leucotrichus, O. compactum and T. leptobotrys being more effective. The addition of the different EOs at sub-inhibitory concentration reduces the fluconazole and amphotericin B MICs of the tested Candida strains by 16 to 512-fold and one to four-fold, respectively. Among the EOs examined, T. leptobotrys EO combined high anticandidal effect and high synergistic interaction with two conventional antifungals, mainly fluconazole. These findings showed that the studied EOs, in particular T. leptobotrys EO, may be used as effective anticandidal agents to restore the efficacy of these common antifungal drugs for combating resistant-Candida strains. Additional studies are necessary to determine the mechanism of these synergistic antifungal associations.