Fungicidal versus Fungistatic activity of five Iranian essences against fluconazole resistant Candida species

Introduction Incidence of invasive fungal infections (IFIs) has been increased in recent years (1). Candida species are the most frequent fungal pathogens isolated from IFIs. Although infections with Candida albicans have been the most prevalent, infections due to non-albicans Candida have been increased significantly in the last decades (2,3). The increase in prevalence of these species might be as a result of higher resistance to conventional antifungals (4). Development of new antifungals is challenging; because there are only few targets for antifungal actions and toxicity level of many antifungals is relatively high (5). In recent years, some researchers have focused on natural compounds including essential oils (EOs) and herbal extracts derived from medicinal plants and other biomaterials with antifungal activities (6). EOs are aromatic compounds derived from plant materials (7). EOs have shown to possess antimicrobial, insecticidal and antioxidant properties (8). It is believed that these properties are related to the functions of their components in plants (9). Iran is a country rich in medicinal plants and EOs from these plants could be potent antifungals (10). Conventional antifungals are divided into 2 groups of fungicidal and fungistatic compounds (11). Fungicidal http://www.herbmedpharmacol.com doi: 10.15171/jhp.2018.43


Introduction
Incidence of invasive fungal infections (IFIs) has been increased in recent years (1).Candida species are the most frequent fungal pathogens isolated from IFIs.Although infections with Candida albicans have been the most prevalent, infections due to non-albicans Candida have been increased significantly in the last decades (2,3).The increase in prevalence of these species might be as a result of higher resistance to conventional antifungals (4).Development of new antifungals is challenging; because there are only few targets for antifungal actions and toxicity level of many antifungals is relatively high (5).In recent years, some researchers have focused on natural compounds including essential oils (EOs) and herbal extracts derived from medicinal plants and other biomaterials with antifungal activities (6).EOs are aromatic compounds derived from plant materials (7).EOs have shown to possess antimicrobial, insecticidal and antioxidant properties (8).It is believed that these properties are related to the functions of their components in plants (9).Iran is a country rich in medicinal plants and EOs from these plants could be potent antifungals (10).Conventional antifungals are divided into 2 groups of fungicidal and fungistatic compounds (11).Fungicidal activity of antifungals is a benefit in most IFIs, especially in early stages of the disease, including systemic candidiasis, cryptococcal meningitis in HIV patients and aspergillosis in immunocompromised individuals (12,13).This study was designed to evaluate the fungicidal and/or fungistatic activities of 5 Iranian EOs against fluconazole (FLC) resistant Candida species and to study the effect of EOs' chemical compositions on their antifungal properties.

Materials and Methods
Isolation of Candida species Candida species were isolated from patients having candidiasis, referred to Mycology Research Center, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran, during 2016-2018.A total of 50 isolates from different organs were cultured on Sabouraud Dextrose agar (Merck, Germany) at 30°C for 7 days.Candida isolates were identified by germ tube test and RAPID yeast plus system (Remel Inc., Lenexa, KS, USA).Isolates were kept at 4°C before use (14).

Preparation of essential oils
In the present study, leaves of Artemisia aromatica A. Nelson, Andropogon citratus and Origanum majorana var.majoranoides, bark of Cinnamomum cayennense and flower buds of Syzygium aromaticum were purchased from Pakan Bazr Company, Isfahan, Iran.The plants were authenticated taxonomically at Pharmacognosy Department, Faculty of Pharmacy, University of Tehran, Iran.EOs were extracted by hydro-distillation of plant parts in a Clevenger-type apparatus at 100°C for 5 hours.The EOs were collected and dried over anhydrous sodium sulfate.The chemical composition of each EO was analyzed using a 6890 N Agilent Gas chromatograph coupled to a 5975 C Agilent Mass-selective detector (Agilent Technologies, Avondale, PA, USA) (15).EOs were stored in a dark glass bottle at 4°C until used.
Preparation of antifungal agents FLC, itraconazole (ITC) and voriconazole (VRC) standards were prepared from Sigma Company (Sigma, St. Louis, MO, USA).Antifungal stocks were prepared by dissolving 64 mg antifungal in 50 mL DMSO.Stocks were kept at -20°C before use.

Determination of minimum inhibitory concentration
Broth microdilution method was used to study antifungal susceptibility of Candida isolates to mentioned antifungal agents and studied EOs according to Clinical and Laboratory Standards Institute (CLSI) M27-A3 document.In brief, 2 fold serial dilutions of EOs (15.6, 31.2, 62.5, 125, 175,250, 350, 500, 700, 1000 and 2000 µg/mL) and 3 azole agents (0.031-64 µg/mL) were prepared in 96-well plates using RPMI-1640 media (Sigma, St. Louis, MO, USA) buffered with MOPS (Sigma, St. Louis, MO, USA).Candida inoculums were adjusted to density of 1 × 10 3 -5 × 10 3 CFU/mL with the help of a hemocytometer and 0.1 mL of each inoculum was added to microplate wells which were incubated at 30°C for 24-48 hours.Non-inoculated medium (200 mL) was considered as negative control (blank), and the well with no EO/azole was spotted as positive control.The growth in each well was compared with the growth in the control well.Minimum inhibitory concentrations (MICs) were visually determined and defined as the lowest concentration of the EO/azole which inhibited growth in comparison with positive control.All tests were done in replicates of three (16).Determination of minimum fungicidal concentration Wells with no visible growth after 48 hours of incubation were cultured on Sabouraud Dextrose Agar by surface plate count method.Plates were incubated at 30°C for 10 days.Minimum fungicidal concentration (MFC) was determined as the lowest concentration with no growth on solid media.All experiments were done with 3 replicates (17).

MFC/MIC ratio
EOs were considered fungicidal if the ratio of MFC to MIC was less or equal to 4 and if the ratio was higher than four the EO was deemed fungistatic (18).

Statistical analysis
Data were analyzed using SPSS version 21.Analysis of variance (ANOVA) and Dunnett post hoc test were done to compare different data.A P value of less than 0.05 was considered significant.

Results
Isolation of Candida species Specimens were taken from upper respiratory tract, urine and blood.Candida isolates were initially tested for susceptibility to FLC by broth microdilution method.Isolates with MIC range of 64 or greater were considered resistant to FLC.A total of 12 Candida species that were resistant to FLC were selected for further studies.These isolates included C. albicans (6 isolates), Candida glabrata (3 isolates), Candida krusei (2 isolates) and Candida tropicalis (1 isolate).
Analysis of essential oils by Gas chromatography/ Mass spectrometry EOs were analyzed on a 6890 N Agilent Gas chromatograph coupled to a 5975 C Agilent mass-selective detector (Agilent Technologies, Avondale, PA, USA) with a 7683 Agilent auto sampler.In brief, 1 µL of each sample was injected in the split less mode at 250°C/into a 30 m × 0.25 mm × 0.5 µm DB-5 MS capillary column and operated by MSD Chemstation software (Agilent Technologies).The temperature program used for the chromatographic separation was 50°C/2 min; 25 • C/1 min; 100°C/2 min; 5°C/1 min and 290 • C/5 min.The carrier gas was helium (99.999%) with a constant flux of 1.0 mL/min.Mass spectrometry was operated in an electron impact ionization mode with the energy of 70 eV.Several unknown peaks were observed after injection.Peaks were identified using computer library.The main components of the EOs are summarized in Table 1

Determination of fungicidal activity
The ration of MIFC/MIC of studied EOs against Candida isolates is shown in Figure 1.Since the MIC/MFC ratios of all EOs in this study were less than 4, they could be considered fungicidal.C. cayennense MIC/MFC ratio was similar for all Candida species.While A. aromatica A. Nelson had the nearest ratio against C. krusei and other essences had the lowest ratio for C. tropicalis.

Discussion
Candida species cause a variety of fungal infections from superficial candidiasis of oral and vaginal mucosa to disseminated and deep tissue infections.These infections are most often caused by C. albicans followed by C. glabrata, C. parapsilosis/C.tropicalis and C. krusei according to epidemiological studies in different geographical regions (19).FLC, an azole antifungal, is the most common antifungal used for treatment of candidiasis.It inhibits lanesterol 14 α demethylase enzyme encoded by ERG11 gene.However, some non albicans Candida species have intrinsic resistant to azoles.In addition, Candida spp.are able to develop resistant to azoles (20,21).Azole resistance is seen especially in patients receiving azole prophylaxis (22).In this study we examined the fungicidal activities of 5 Iranian EOs on FLC resistant to Candida species.Candida yeasts were isolated from candidiasis cases.Resistant to other conventional systemic azoles in Iran was investigated as well.In our study only one isolate of C. glabrata was resistant to VRC and one isolate of C. glabrata as well as C. krusei were dose dependently susceptible to ITC.According to recent studies, resistance to azole antifungals has increased in recent years.Widespread use of triazoles has resulted in emergence of resistant to non albicans Candida species including C. glabrata and C. krusei (23,24).These species have intrinsic resistant to FLC and it is said that this inherited resistance could be seen in VRC as well (23).In our study only one isolate of C. glabrata was resistant to VRC.Studies have shown that mutations in ERG11 gene could induce azole resistance in C. albicans (24).Stephanie et al demonstrated that there is a combination effect between FLC and VRC susceptibility in C. albicans strains, while ITC susceptibility is not affected (24).We did not observe any correlation between FLC and VRC susceptibility among our C. albicans isolates.It might be due to different mutation regions in our isolates.Biological activity of EOs is highly related to their chemical compounds (25).In our study, C. cayennense was the most effective one against Candida isolates.GS/MS analysis revealed cinnamaldehyde, cinnamaldehyde dimethyl acetal and eugenol as its main ingredients.Other studies have demonstrated that the amount of cinnamaldehyde affect antifungal activity of Cinnamomum spp.The higher cinnamaldehyde content the higher antifungal activity would be (26).O. majorana var.majoranoides and A. citratus had the best anti Candida activity following C. cayennense.GC/MS analysis revealed that isothymol and thymol compromised approximately 50% of A. citratus.These compounds are known for Trans-Caryophyllene is known for its anti-inflammatory and pain killer effects (29).S. aromaticum activity against FLC resistant Candida species was also suitable (MIC = 700 µ/mL).Only one isolate of C. glabrata and C. krusei had the MIC of 1000 µg/mL.Other studies have demonstrated a suitable antifungal activity for eugenol in vivo and in vitro (30). A. aromatica A. Nelson was found to have the lowest antifungal activity against the studied Candida species in our study.Since the numbers of isolates were not similar in our study, we could not predict if the tested EOs act differently on various Candida species, although according to our results there was no difference between FLC resistant Candida species.All EOs in our study had fungicidal properties.It has been documented that fungicidal activity of antifungals is related to more therapeutic success and less recurrent and persistent infection especially in invasive candidiasis and candidemia (12,13).It has been shown that fungicidal compounds inhibit morphogenic transformation of Candid spp while fungistatics do not affect morphogenesis (18).

Conclusion
In conclusion, EOs in this study have potential antifungal effects against Candida species and there is no known resistant mechanism in Candida species against studied EOs.These EOs are probably suitable to be used as supplementary agents along with conventional antifungals.However more studies are needed to evaluate their safety on host cells and to examine the effects of their chemical compounds alone and/or in combination with Data are shown as mean (µg/mL).

Figure 1 .
Figure 1.(A) Comparison the MIC/MFC ratio of different essential oils against C. albicans, C. glbrata, C. krusei and C. tropicalis.Data are shown as mean±SE.(B) a diagram on relative MIC/MFC ration of studied essential oils.
Determination of MIC and MFCAntifungal susceptibility test of EOs in comparison with ITC and VRC against FLC resistant Candida species was done using broth micro dilution method (Table 2).All C. albicans and C. tropicalis isolates were susceptible to both ITC and VRC.Whereas, isolates of C. krusei and C. glabrata were resistant to ITC.ITC resistant C. glabrata was resistant to VRC as well.Among EOs, MICs of A. aromatica A. Nelson, A. citratus, O. majorana var.O. majorana var.majorana had significantly higher MICs for C. glabrata than other examined Candida species (P<0.05).The MIC ranges of other EOs against Candida species were not statistically significant (P > 0.05).C. cayennense could significantly inhibit growth of C. albicans in lower concentrations than O. majorana var.majoranoides, S. aromaticum and A. aromatica A. Nelson (P < 0.05).Whilst it was only more effective than O. majorana var.majoranoides against C. glabrata isolates (P < 0.05).The most effective EOs on C. krusei isolates were A. citratus and C. cayennense (P < 0.05); and O. majorana var.majoranoides was significantly more effective against C. tropicalis than A. aromatica A. Nelson and C. cayennense (P < 0.05).

Table 1 .
Summary of the chemical composition (%) of essentials oils evaluated by GC/MS