Synergistic Interaction of Certain Essential Oils and Their Active Compounds with Fluconazole against Azole-resistant Strains of Cryptococcus neoformans

Abstract

Objectives:

This study investigated the anti-cryptococcal potential of certain essential oils (EOs)/compounds alone and in combination with fluconazole.

Materials and Methods:

We investigated the antifungal activity of oils of Cinnamomum verum, Cymbopogon citratus, Cymbopogon martini, and Syzygium aromaticum, and their major active ingredients cinnamaldehyde, citral, eugenol, and geraniol against clinical and standard strains of Cryptococcus neoformans (CN). Disc diffusion, broth microdilution, checkerboard methods, and transmission electron microscopy were employed to determine growth inhibition, synergistic interaction, and mechanism of action of test compounds.

Results:

EOs/compounds showed pronounced antifungal efficacy against azole-resistant CN in the order of cinnamaldehyde > eugenol > S. aromaticum > C. verum > citral > C. citratus > geraniol ≥ C. martini, each exhibiting zone of inhibition >15 mm. These oils/compounds were highly cidal compared to fluconazole. Eugenol and cinnamaldehyde showed the strongest synergy with fluconazole against CN by lowering their MICs up to 32-fold. Transmission electron microscopy indicated damage of the fungal cell wall, cell membrane, and other endomembranous organelles.

Conclusion:

Test oils and their active compounds exhibited potential anti-cryptococcus activity against the azole-resistant strains of CN. Moreover, eugenol and cinnamaldehyde significantly potentiated the anti-cryptococcal activity of fluconazole. It is suggested that multiple sites of action from oils/compounds could turn static fluconazole into a cidal drug combination in combating cryptococcosis.

INTRODUCTION

The occurrence of microbial infections, especially invasive opportunistic mycoses, has increased in recent times. This is attributed to a mounting number of immunocompromised patients due to the HIV infections, recipients of hematopoietic stem cell or solid organ transplants, patients recuperating from major surgery or have undergone chemotherapy for cancer, premature newborns, and elderly patients.[1,2] Infections of the pulmonary and central nervous system (CNS) are primary cause of death among immunocompromised patients. Cryptococcal pneumonia is the most common manifestation of cryptococcosis in AIDS patients,[3,4] whereas cryptococcal meningitis is the fourth.[5]

Most antifungal drugs employed against these diseases are amphotericin B, flucytosine, fluconazole, ketoconazole, and terbinafine.[6] However, studies have shown host cell toxicities on the use of amphotericin B and azoles. Fluconazole, bestowing decreased toxicity after oral and intravenous administration, has become the drug of choice.[6,7] Although Cryptococcus neoformans (CN) strains have shown less common resistance to antifungals, there is a huge concern that less susceptible or resistant strains are emerging because of the use of fluconazole in long-term therapy.[8]

This scenario justifies the search for effective alternatives in the form of newer antifungal drugs with a wider spectrum of action and lesser dose-regulating side effects. Plant products, especially essential oils (EOs), have traditionally been used in ethnomedicine and several in vitro and in vivo studies have shown encouraging broad-spectrum antifungal activities without any side effects.[9,10,11] Even though the EOs are most promising groups of natural products showing broad-spectrum therapeutic activity, they have been little exploited against CN. In addition, the combination of drugs with different antifungal mechanisms results in the potentiation of conventional drugs. Studies have shown better therapeutics against the invasive and systemic mycoses when two or more drugs are combined.[6] Therefore, in the present study, antifungal activities of oils of Cinnamomum verum, Cymbopogon citratus, Cymbopogon martini, and Syzygium aromaticum, and their major active ingredients cinnamaldehyde, citral, eugenol, and geraniol were evaluated for anti-cryptococcus potential against the clinical and standard strains, one each, of CN. Furthermore, oils/compounds were tested for their synergistic interaction with fluconazole against the test strains.

MATERIALS AND METHODS

Organisms and media

The clinical isolate of CN was obtained from the Jawaharlal Nehru Medical College and Hospital, Aligarh, India. CN MTCC4424 was procured from the Microbial Type Culture Collection, India. The strains were characterized morphologically, and biochemical tests, i.e., India ink staining, phenol oxidase, sugar and nitrogen assimilation, urease, and nitrate reduction tests were carried out. The strains were identified as CN and kept on Sabouraud Dextrose Agar (SDA) slants at 4°C and subcultured in the Sabouraud Dextrose Broth (SDB) before use.

Plant essential oils and drugs

Oils of C. verum and S. aromaticum were obtained from Wyndmere Naturals, Inc., USA. Oils of C. citratus, C. martini, and pure compounds of cinnamaldehyde (95%), citral (96%), eugenol (99%), and geraniol (98%) were obtained from Merck-Sigma, USA. The sterile paper discs, antifungal susceptibility test discs, and antifungal drugs (fluconazole, itraconazole, and ketoconazole) were procured from Merck-Sigma, USA. Stock solutions of oils, compounds, and antifungals were prepared in 1% dimethyl sulphoxide (DMSO) and stored at − 20°C. All test oils were subjected to quality assessment using physicochemical tests such as refractive index, solubility in alcohol, and specific gravity at the Fragrance and Flavor Development Centre, India.

Gas chromatography and gas chromatography–mass spectrometry of essential oils

The identification of compounds in the oils was determined by GC-FID using a Shimadzu 2010 Gas Chromatograph equipped with an FID and WCOT column (25 m × 0.25 mm × 0.25 m) coated with diethylene glycol (AB-Innowax, 7031428, Japan). GC analysis was carried out on temperatures at 270°C (injector) and 280°C (detector), N₂ (carrier gas) at 3.0 mL/min, and 74.9 kPa (column pressure). A 0.2 L of test oils was injected into the column (split ratio of 90.0) and the linear temperature program (60°C–230°C) was set at 3°C/min withhold at 230°C for 10 min. Further to identify compounds, the samples were studied on the same Shimadzu instrument with MS parameters (ionization voltage [EI] 70eV, peak width 2 s, mass range 40–600 amu, and detector voltage 1.5 V). Peak identification was carried out by comparison of the mass spectra with the database of NBS75K, NIST05, and Wiley 8 libraries.

Disc diffusion assays

The susceptibility of test strains to drugs/oils/compounds was evaluated using the disc diffusion method as recommended by Clinical and Laboratory Standards Institute (CLSI) M44-A2[12] with a few modifications. Briefly, 100 µl of cell suspension (1.5 × 10⁸ cfu/ml) was spread onto SDA plates and spotted with antifungal discs (10–20 µg/disc). Whereas, for oils/compounds, paper discs (8 mm diameter) soaked with 10 µl of test agents were placed over the agar surface. The plates were incubated at 37°C for 48 h and the diameter of the zone of inhibition (mm) was noted. Each experiment was performed in triplicate and data were shown as mean ± standard deviation.

Determination of minimum inhibitory concentration by broth microdilution assays

The minimum inhibitory concentration (MIC) of drugs/oils/compounds against the test strains was determined using the microbroth dilution method, the CLSI reference method for broth macrodilution M27-A3[13] with some modifications. Briefly, cell suspension (1.5 × 10⁸ cfu/ml) was prepared in RPMI 1640 medium with L-glutamine but without bicarbonate and buffered to pH 7.0 with MOPS. Further, two-fold serial dilutions of test drugs were made in the broth in 96-well microtiter plates to obtain concentration ranging from 0.06 to 256 µg/ml. Next, 10 µl of inoculum was added to the 100 µl of diluted medium and incubated at 37°C for 48 h. Whereas, for the evaluation of MICs of oils/compounds, the test agents were two-fold serially diluted in 100 µl of SDB to achieve concentration range from 25 to 1600 µg/ml. Further, 10 µl of cell suspension (1.5 × 10⁸ cfu/ml) was added and incubated at 37°C for 24 h. Drug-free control was also included, and plates were read at 600 nm to determine the cell growth. The lowest concentration that inhibited 80% of the control growth is considered MIC of the test agents. Each experiment was performed three times, and the mean values were computed for MICs.

Time kill assays

The time-dependent killing by the test agents and fluconazole was assessed against the strain CN. For the assay, 2 × MIC concentrations of oils/compounds or drugs were selected. Briefly, 20 ml of phosphate buffer saline (PBS) solution comprising chosen concentrations of test agents were inoculated with 1 ml of cell suspension (1.5 × 10⁸ cfu/ml) and incubated at 37°C for 48 h. The control solution contained PBS with strain, but no test agents or drugs were also included. Viable counts were noted at 0, 2, 4, 6, 8, 12, 24, 36, and 48 h by plate inoculation method. The experiment was carried out in triplicate and to normalize the data, and adjusting the difference in starting inoculum concentrations, the mean colony count was converted to values relative to the mean colony count at 0 h. The relative viable count was plotted against the time on a log scale.

Cytotoxicity assays

The oils and active compounds exhibiting potential biological activities were evaluated for their toxicity by testing the hemolytic activity as described by Powell et al.,[14] with some modifications. A 4% red blood cell (RBC) suspension in 5% (w/v) glucose solution was prepared by adding the defibrinated blood, after washing with 1 mL of PBS (pH 7.0). Next, PBS having chosen concentrations of test agents was mixed in 1:1 with RBC suspension in Eppendorf tubes and incubated at 37°C for 2 h. Triton X-100 (0.1% [v/v] in PBS) was used as a positive control, whereas 1% DMSO and PBS were used as controls. Tubes were centrifuged for 10 min, at 2000 rpm, and the absorbance of the supernatant was read at 540 nm. % hemolysis was determined as: % hemolysis = ([A₅₄₀–B₅₄₀]/C₅₄₀) ×100.

Where A is the absorbance of RBC-treated sample and B is the absorbance of PBS (solvent control) alone. Whereas C is the absorbance of triton X-100 (1%) showing 100% lysis. The experiment was conducted in triplicate, and for the calculation of % hemolysis, the mean values were considered.

Interaction of oils/compounds with antifungal drugs

To examine the interaction of test compounds with fluconazole against the strains CN and CN MTCC4424, a checkerboard microtiter test was carried out. In a microtiter well plate, to obtain four times the final concentration, two-fold dilutions of test compounds were prepared in SDB (for oils or compounds) and DMSO (for drugs). To achieve the various combinations of oils/compounds and fluconazole, 50 µl each of oil/compound and fluconazole dilutions were added to microtiter plate wells in the vertical and horizontal directions, respectively. Next, each well was inoculated with 100 µl of cell suspension (1.5 × 10⁸ cfu/ml), and the plates were incubated for 48 h, at 37°C. The type of interaction was described quantitatively in terms of fractional inhibitory concentrations (FIC). Where fractional inhibitory concentration index (FICI) was calculated as FIC₁ (MIC of test oils or compounds in the presence of fluconazole/MIC of test oils or compounds alone) + FIC₂ (MIC of fluconazole in the presence of oils or compounds/MIC of fluconazole alone). The combination outcome was interpreted as follows: FICI ≤0.5, synergy; >0.5 to ≤1, additive; >1 to ≤4, no interaction; and >4.0, antagonistic.[15]

Transmission electron microscopy

To observe the effects of test agents on the structural integrity of fungal cells, transmission electron microscopy was performed. Briefly, CN cells (1.5 × 10⁸ cfu/ml) were treated with the subinhibitory concentrations of cinnamaldehyde at 50 μg/mL and eugenol at 200 μg/mL and allowed to incubate for 48 h, at 120 rpm and 37°C. Untreated samples were also included to compare the effects of compounds on the cellular structures. The cell pellets were obtained, and at room temperature, it was fixed with 2.5% glutaraldehyde (in 0.1 M cacodylate buffer, pH 7.2) for 24 h. After which, they were postfixed in 1% osmium tetroxide (in cacodylate buffer). The cells were then dehydrated in crescent concentrations of acetone and embedded in Spurr epoxy resin. Ultrathin sections were obtained and stained with 12.5% alcoholic uranyl acetate. The specimens were observed under Morgagni 268D transmission electron microscope at 80 kv.

RESULTS

Major active compounds of essential oils

As shown in Table 1, cinnamaldehyde was the major compound (79.10%) present in C. verum, whereas the oil of S. aromaticum was composed of 74.32% eugenol and 23.25% caryophyllenes. C. citratus was comprised of two major constituents’ α-citral (43.95%) and β-citral (28.87%). However, geraniol was the major constituent sharing 43.84% of C. martini oil followed by geraniol acetate at 26.91%.

Table 1.

Major chemical constituents of essential oils as identified by gas chromatography/gas chromatography–mass spectrometry

Compounds	Percentage amount	Retention time
C. verum
o-cymene	5.43	3.94
Cinnamaldehyde	79.10	9.32
Cinnamic aldehyde	5.81	10.26
Cinnamic acid	8.91	13.13
α-thujene	0.75	19.34
S. aromaticum
Eugenol	74.32	6.28
β-caryophyllene	4.92	7.03
Iso-caryophyllene	5.96	7.09
β-caryophyllene	7.04	7.14
α-caryophyllene	1.28	7.34
α-caryophyllene	4.05	7.40
Caryophyllene oxide	2.41	8.61
C. citratus
α-limonene	2.74	11.00
α-linalool	2.77	21.58
β-citral	28.87	25.97
α-terpineol	1.55	26.16
α-citral	43.95	27.45
trans-geraniol	4.0	30.24
C. martini
α-terpinene	0.26	9.15
trans-ocimene	5.44	11.97
β-ocimene	3.24	12.48
β-citronellal	1.32	19.63
α-linalool	2.96	21.57
Geranyl acetate	26.91	27.96
Geraniol	43.84	30.43
Neryl acetate	2.07	31.55

C. verum=Cinnamomum verum, S. aromaticum=Syzygium aromaticum, C. citratus=Cymbopogon citratus, C. martini=Cymbopogon martini

Susceptibility of Cryptococcus neoformans strains to antifungal drugs

The susceptibility of CN strains to antifungal drugs varied from nil to 19.33 mm in terms of zone of diameter of inhibition [Table 2]. The strains were considered resistant at the MIC value ≥1.0 µg/ml for itraconazole, ≥1.0 µg/ml for ketoconazole, and ≥64.0 µg/ml for fluconazole as established by CLSI breakpoints and by Milan et al.[16] As presented in Table 3, strain CN was resistant to fluconazole, whereas strain CN MTCC4424 was intermediate resistant to fluconazole. However, both the test strains were found to be resistant to itraconazole (64–256 µg/ml) and ketoconazole (1–32 µg/ml).

Table 2.

Sensitivity of Cryptococcus neoformans strains to antifungal drugs in terms of zone of inhibition, minimum inhibitory concentration, and minimum fungicidal concentration

Strains	Diameter of the zone of inhibition (mm)			KTC		ITC		FLC
	KTC	FLC	ITC	MIC	MFC	MIC	MFC	MIC	MFC
C. neoformans	19.33±1.24	-	-	0.25	0.50	64	128	128	256
C. neoformans MTCC4424	-	14.33±1.24	-	64	128	128	256	8	32

MIC and MFC values are presented in μg/mL=No zone of inhibition, FLC=Fluconazole (10 μg/disc), ITC=Itraconazole (10 μg/disc), KTC=Ketoconazole (10 μg/disc), MIC=Minimum inhibitory concentration, MFC=Minimum fungicidal concentration, C. neoformans=Cryptococcus neoformans

Table 3.

Sensitivity of Cryptococcus neoformans strains to essential oils and active compounds

Strains	Diameter of zone of inhibition (mm)
	Essential oils				Active compounds
	CL	CM	LG	PR	EL	CD	CT	GR
C. neoformans	19.00±0.81	15.66±1.24	21.00±1.63	18.33±1.24	22.66±1.24	48.33±3.39	25.33±2.05	19.33±1.24
C. neoformans MTCC4424	32.33±2.86	23.33±2.05	16.66±1.24	20.00±1.41	40.33±1.69	45.66±2.49	22.66±1.24	19.33±1.24

Each disc contains 10 μL of essential oils or active compounds=No zone of inhibition, C. neoformans=Cryptococcus neoformans, S. aromaticum=Syzygium aromaticum, C. verum=Cinnamomum verum, C. citratus=Cymbopogon citratus, C. martini=Cymbopogon martini, CL=Clove oil (S. aromaticum), CM=Cinnamon oil (C. verum), LG=Lemongrass oil (C. citratus), PR=Palmrosa oil (C. martini), EL=Eugenol, CD=Cinnamaldehyde, CT=Citral, GR=Geraniol

Susceptibility of Cryptococcus neoformans strains to essential oils and active compounds

As depicted in Table 3, the test oils and their major active compounds exhibited a zone of inhibition ≥15 mm against the test strains. The test oils/compounds were active in the order of cinnamaldehyde > eugenol > S. aromaticum > citral > C. verum > C. citratus > C. martini > geraniol. As evident from Table 4, cinnamaldehyde and C. martini each exhibited MIC of 100 µg/ml against the test strains, whereas eugenol, C. citratus, C. verum, and S. aromaticum showed MIC of 200 µg/ml. Geraniol and citral could show MIC in the range of 400–1600 µg/ml.

Table 4.

Minimum inhibitory concentration and minimum fungicidal concentration of essential oils and active compounds against Cryptococcus neoformans

Strains	Essential oils
	Essential oils								Active compounds
	CL		CM		LG		PR		EG		CD		CT		GR
	MIC	MFC	MIC	MFC	MIC	MFC	MIC	MFC	MIC	MFC	MIC	MFC	MIC	MFC	MIC	MFC
C. neoformans	200	400	200	400	200	400	100	200	200	400	100	200	800	1600	400	800
C. neoformans MTCC4424	200	400	200	400	200	400	100	200	200	400	100	200	1600	3200	800	1600

MIC and MFC values are presented in μg/mL. C. neoformans=Cryptococcus neoformans, S. aromaticum=Syzygium aromaticum, C. verum=Cinnamomum verum, C. citratus=Cymbopogon citratus, C. martini=Cymbopogon martini, CL=Clove oil (S. aromaticum), CM=Cinnamon oil (C. verum), LG=Lemongrass oil (C. citratus), PR=Palmrosa oil (C. martini), CD=Cinnamaldehyde, CT=Citral, GR=Geraniol, MIC=Minimum inhibitory concentration, MFC=Minimum fungicidal concentration

Time kill assays

The oils/compounds were evaluated for their time-dependent killing ability against the CN strain. As presented in Figure 1a and b, treatment of eugenol and cinnamaldehyde for 7 h resulted in a decrease of >1log₁₀ in the viable count compared to the untreated control. For C. verum (8 h), geraniol (9–10 h), S. aromaticum, C. citratus, C. martini (16 h), and citral (19–20 h). However, fluconazole could not show this decrease even up to 48 h.

Figure 1.

Time-dependent killing potential of oils (a) and compounds (b) against Cryptococcus neoformans

Toxicities of test oils and compounds

No test oils or their active ingredients showed hemolysis at their respective MFCs to the test strain. At 2 to 4 × of MFCs (3200 µg/ml), partial hemolysis (15%–20%) could be observed. However, 100% hemolysis was resulted in 0.1% (v/v) Triton X-100 used as a positive control, but 1% DMSO and PBS used as solvent controls demonstrated no hemolysis [Table 5].

Table 5.

Hemolytic properties of essential oils and active compounds

Essential oils and active compounds	Percent hemolysis of RBC
	Concentrations of essential oils and active compounds (μg/mL)
	25	50	100	200	400	800	1600	3200
S. aromaticum	1.90±0.09	2.20±0.19	2.50±0.13	3.09±0.17	4.26±0.09	5.63±0.18	6.05±0.12	18.78±0.16
C. verum	1.88±0.31	2.14±0.21	2.57±0.26	3.41±0.15	4.21±0.32	4.61±0.28	6.81±0.29	22.89±0.51
C. citratus	1.45±0.10	1.92±0.10	2.54±0.15	3.39±0.09	4.56±0.20	4.81±0.12	6.68±0.17	19.08±0.24
C. martini	1.58±0.14	1.84±0.14	2.74±0.19	3.51±0.07	4.27±0.17	4.62±0.27	5.98±0.37	18.61±0.26
EL	1.65±0.14	1.98±0.09	3.02±0.08	3.72±0.16	4.26±0.16	4.78±0.25	5.60±0.63	19.03±0.30
CD	1.84±0.19	2.87±0.07	3.39±0.12	3.96±0.05	4.40±0.17	5.06±0.20	6.22±0.19	18.11±0.40
CT	1.78±0.16	2.86±0.15	3.80±0.18	4.29±0.12	4.51±0.13	5.98±0.21	6.96±0.10	17.61±1.04
GR	1.33±0.17	2.16±0.25	2.31±0.26	2.87±0.23	3.36±0.18	3.85±0.11	4.74±0.34	14.34±0.23

All the experiments were performed in triplicate, and data are presented as mean±SD. RBC=Red blood cell, SD=Standard deviation, EL=Eugenol, CD=Cinnamaldehyde, CT=Citral, GR=Geraniol, S. aromaticum=Syzygium aromaticum, C. verum=Cinnamomum verum, C. citratus=Cymbopogon citratus, C. martini=Cymbopogon martini

Synergistic interaction of compounds with antifungal drugs

The combinational effects of oils/compounds with fluconazole against CN and CN MTCC4424 strains are presented in Table 6. All the tested combinations were synergistic except C. citratus/fluconazole and C. martini/fluconazole, which were indifferent.

Table 6.

Interaction of eugenol and cinnamaldehyde with fluconazole against Cryptococcus neoformans strains

Combination (μg/mL)	C. neoformans CN				C. neoformans MTCC4424
	MIC of agent alone	MIC of agent in combination	FICI	Nature of interaction	MIC of agent alone	MIC of agent in combination	FICI	Nature of interaction
EL-FLC
EL	200	12.50	0.125	Synergy	200	12.50	0.187	Synergy
FLC	128	8			8	1
CD-FLC
CD	100	12.50	0.187	Synergy	100	12.50	0.250	Synergy
FLC	128			8	8	1

EL=Eugenol, CD=Cinnamaldehyde, FLC=Fluconazole, FICI=Fractional inhibitory concentration index, MIC=Minimum inhibitory concentration

Cinnamaldehyde (FICI value 0.156) and eugenol (FICI value of 0.187) exhibited the maximum synergy by lowering the MICs of fluconazole up to 32-fold and their own by 8 folds against CN. However, oils of C. verum and S. aromaticum followed by gerniol and citral were less synergistic with fluconazole against strains tested. In our study, none of the tested combinations showed antagonistic interaction.

Effects of subinhibitory concentrations of test compounds on cellular structures of Cryptococcus neoformans

In an untreated sample of Cryptococcus test strain, organelles, cell wall, cell membrane, and capsules appeared to be normal [Figure 2a]. Treatment of cells with cinnamaldehyde/eugenol showed numerous changes to the ultrastructures such as thickening and disorganization of cell wall integrity, loosening and disintegration of cell membrane, and abnormal distribution of polysaccharides, leading to the deterioration of cytoplasmic organelles [Figure 2b-e].

Figure 2.

Transmission electron micrographs of Cryptococcus neoformans. (a) untreated control, showing intact cell and its ultra-structures (b and c) treated with cinnamaldehyde at 50 μg/ml; (d and e) treated with eugenol at 100 μg/ml. (1) loss of capsule, thickening of cell wall; (2) excessive vacuolization; (3) loosening and disintegration of cell membrane; (4) loosening of cell wall and capsule and their disintegration; (5) disruption of cell membrane; (6) excessive deposition of lipid globules; (7) stripping of cell membrane; (8) abnormal distribution of polysaccharides in cytoplasm and disorganization or degradation of organelles; (9) disruption of capsule and cell wall thickening

DISCUSSION

Despite the availability of anti-cryptococcal agents, the mortality rates and treatment failures for cryptococcosis treatment have remained disappointingly high due to the toxicities and development of resistant strains.[8] Therefore, the incompetence of existing antifungal drugs for the treatment of cryptococcosis, and, their pharmacological restrictions, have led to the necessity to search for newer and alternative strategies to overcome these infections.

In our study, CN strains have displayed a wide range of multidrug resistance (1–128 µg/ml) to various azoles such as itraconazole, ketoconazole, and fluconazole. Although the clinical isolates of CN have not commonly shown resistance to antifungals, the long-term use of antifungal agents has resulted in the emergence of drug resistance in CN.[17]

Because of disappointments with conventional antifungal drugs, the public acceptance and effectiveness of plant-based natural alternatives is increasing. Therefore, considering the consequences of reduced susceptibility of Cryptococcus strains to conventional azoles, and in the hope of alternative remedy, we screened CN strains for their susceptibility to a variety of EOs and their active compounds. Cinnamaldehyde, eugenol, and oils of C. citratus, C. martini, C. verum, and S. aromaticum were very active against azole-resistant strains of CN with the MIC ranging from 100 to 200 µg/ml. Our studies find support from other reports on the antifungal efficacy of oils of oregano, lemongrass, clove, peppermint, thyme, tulsi, villos, and their major compounds against the CN.[18,19] The gas chromatography and gas chromatography–mass spectrometry (GC/GC-MS) analysis of test oils revealed that the major active ingredients for C. verum are cinnamldehyde (79%), for S. aromaticum is eugenol (74%); for C. citratus are α- and β- citral (73%); and for C. martini are geraniol (44%) and geranyl acetate (27%). Considering the chemical constituents of oils and their anti-cryptococcal efficacy, we could decipher that cinnamaldehyde (MIC, 100 µg/ml) compound is the highest antifungal component of C. verum, whereas other minor ingredients, when interacted, could slightly reduce the antifungal potential of its oil (MIC, 200 µg/ml). Whereas the antifungal efficacy of eugenol (MIC, 200 µg/ml) is not affected by its interaction with the other constituents of S. aromaticum as the oil showed the similar MIC, 200 µg/ml. On the other hand, geraniol and citral exhibited least antifungal efficacy (MICs, ranging from 400 to 1600 µg/ml). However, the minor compounds of their respective oils could enhance the antifungal potential of oils of C. martini (100 µg/ml) and C. citratus (200 µg/ml).

Furthermore, as evident from the time-kill assays, these oils/compounds showed higher cidal activity than fluconazole against azole-resistant strains of CN. It is argued that the fungistatic nature of fluconazole is attributed to its low killing potential.[16] It is to be mentioned that the fungicidal property of an agent in the initial phase of therapy is associated with better clinical outcomes.[20] Moreover, since Cryptococcus facilitates invasive and systemic infections, it is required that antifungal agent should be nontoxic to host cells. Therefore, we tested the cellular toxicity of the test oils/compounds and were found to be nontoxic when assayed for RBC lysis. Usually, fluconazole is nontoxic but higher clinical doses of fluconazole are required for invasive cryptococcosis that can induce host toxicity.[21] This high dose-related toxicity of fluconazole can be addressed in combination therapy as combined doses of antifungal agents are lowered compared to individual doses.

Moreover, despite diverse biological activities of EOs/their active constituents, they have been less investigated in combination with antifungal drugs. Some researchers have shown the synergy of plant products with azoles or amphotericin B against filamentous fungi, Candida spp., and other dermatophytes,[22,23] but investigations against Cryptococcus spp. are scarce. Therefore, we attempted to evaluate the interaction of test oils/compounds with fluconazole against the drug-resistant strain of CN. To the best of our knowledge, the tested combinations have not been conducted earlier. We found that cinnamaldehyde and eugenol, in addition to, being potential antifungal agents alone, they also demonstrated synergistic interaction with fluconazole against both the test strains. MICs of eugenol, cinnamaldehyde, and fluconazole were lowered markedly up to 32-fold and hence highlighted the usefulness of these combinational approaches. However, the oils containing these compounds, i.e., C. verum and S. aromaticum, in combination with fluconazole, could result in up to 8-fold reduction in their MICs. This could be because that other minor components of these oils such as cymene, cinnamic acid, and caryophyllenes are lowering the synergistic interaction between major compounds and fluconazole. On the other hand, geraniol and citral despite being the least antifungal alone, showed synergy with fluconazole against Cryptococcus strains and reduced the MICs up to 8-fold. On the contrary, their oils, despite being active antifungal alone, exhibited indifferent interaction with fluconazole against the test strains. We speculate that other minor constituents of oils of C. martini and C. citratus such as limonene, linalool, ocimene, and neryl acetate are interacting negatively toward the synergy between major compounds of respective oils and fluconazole. It may be justified by the reason that components of oils with various modes of action may interact either alone or among themselves in oil, in one or other way, with antifungal drugs depending on the proportions and doses used.

Antifungal agents in combination have shown superior action due to the enhanced killing potential because of multiple target sites of action. Furthermore, combination therapy results in a broad spectrum of action toward different pathogens, reduction in duration of therapy and relapses, and thereby decreased likelihood of emergence of resistance.[24] To discover the sites of action of active compounds, strain CN was examined by transmission electron microscopy after treating with subinhibitory concentration of cinnamaldehyde (50 μg/mL) and eugenol (100 μg/mL). TEM studies of treated cells revealed interference with the cell wall and membrane structure as evidenced by its thickening, stretching, and disruption. The disorganization of cytoplasmic organelles could be because of the abnormal distribution of polysaccharides.[25] Whereas an increase in vacuolization and endoplasmic reticula is the result of cell response to stress. Such kind of structural changes are shown by a cell to detoxify the drugs or pesticides.[26] Furthermore, the augmentation in the quantity of cytoplasmic vacuoles could be associated with changes in lipid metabolism tempted by the treatment with antifungals. This, in turn, may promote the buildup of biosynthesis pathway intermediates such as squalene, as shown in terbinafine/amphotericin B-treated cells of CN reported from Guerra et al.[7] Moreover, the observed thickening of the cell wall in our study could be because of the deposition of electron-dense vesicles, and it has highlighted that the eugenol/cinnamaldehyde acts through interference with ergosterol biosynthesis. Similar ultrastructural modifications have also been reported by Ishida et al.[27] in C. albicans cells when treated with the compounds that target the ergosterol biosynthesis pathway. Interruption with ergosterol, a vital lipid component of the fungal cell membrane, is considered a prime target for antifungals.[28,29] Moreover, it is observed that treatment of CNS infections by Cryptococcus becomes difficult as the blood–brain barrier (BBB) restricts the diffusion of drug compounds to the brain tissues.[30] However, it is reported that EOs and their components can easily penetrate through the skin and across the BBB depending on their lipophilicity, molecular size, and shape.[31] This characteristic could be an extra advantage in developing phytocompounds as anti-cryptococcal drug.

Currently, combination therapy has spawned sizeable appeal with respect to difficult-to-treat fungal infections.[32] In the clinical settings, echinocandins (targeting cell wall) have been combined with azoles (targeting cell membrane) for better efficacy against fungal infections.[33] It appears that eugenol and cinnamaldehyde are targeting cell membranes as fluconazole targets ergosterol synthesis may be targeting different biosynthetic pathway. In addition, these compounds are also interrupting cell wall-like echinocandins. The treatment strategies for invasive cryptococcal infections are restricted and Cryptococcus spp. are intrinsically resistant to echinocandins and utilize an armory of defenses enabling resistance to the azoles.[34] Therefore, when used in a combination approach, these agents could potentially complement each other by showing multiple sites of action, leading to a broad spectrum of anti-cryptococcal activity, and an improved safety profile with reduced dosages. It is anticipated that fluconazole is better than amphotericin B, being less toxic, but underperformed in action compared to amphotericin B. It is known that the fungistatic nature of fluconazole may lead to relapses of infections and the development of resistance.[20] However, its suboptimal exploitation could be converted into a potential drug candidate in combination with oils/compounds. It is suggested that the synergistic interaction of oils/compounds, especially eugenol and cinnamaldehyde with fluconazole, due to resulted simultaneous multiple sites of action, could turn static azole to a cidal combination in combating cryptococcosis caused by drug-resistant strains.

CONCLUSION

Our findings suggest that EOs especially C. citratus, C. martini, C. verum, and S. aromaticum and active compounds eugenol and cinnamaldehyde, are more cidal and safe compared to conventional antifungal drugs. It could be used either alone or in combination with fluconazole to deliver an enhanced and safe clinical strategy in managing the opportunistic fungal infections caused by azole-resistant strains of CN. In vivo experimentations are needed to address pharmacological questions regarding their efficacy against mycoses caused by the CN in mono and combinational therapy with antifungal drugs.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.