Lemongrass Oil Components Synergistically Activates Fluconazole against Biofilm Forms of <em>Candida albicans</em>

Research Article

J Bacteriol Mycol. 2018; 5(3): 1069.

Lemongrass Oil Components Synergistically Activates Fluconazole against Biofilm Forms of Candida albicans

Pandurang MS, Devrao HS, Ganpatrao BR, Mohan KS*

¹School of Life Sciences (DST-FIST & UGC-SAP Sponsored), SRTM University, Nanded, India

*Corresponding author: Karuppayil Sankunny Mohan, School of Life Sciences, SRTM University, Nanded, India

Received: May 02, 2018; Accepted: June 04, 2018; Published: June 11, 2018


Background of Research: Device related infections caused Biofilm forms of Candida albicans are life threatening because of their increased tolerance of antifungal drugs. Combinatorial therapy involving phytochemicals and antifungal drugs would be an effective approach against drug resistant Candida biofilms. Some of the components of lemongrass oil were tested in combination with fluconazole against planktonic and biofilm forms of C. albicans ATCC 90028 and one clinical isolate.

Methods: Activities of various combinations were analyzed by chequerboard format. The interaction between lemongrass oil components with fluconazole were determined by calculating fractional inhibitory concentration indices (FICI) and results obtained by XTT metabolic assay were confirmed by scanning electron microscopy. Result: Components of lemongrass oil, like Nerol, Citral, Linalool, β-ionone, Terpinolene, 1,8 Cineol, Geraniol & Geranyl acetate in combination with fluconazole showed synergistic interaction against developing biofilm of C. albicans. Whereas Geraniol, Nerol and β-ionone with fluconazole combination showed synergistic interaction against mature biofilms of C. albicans.

Conclusion: This study for the first time indicates that lemongrass oil components increases efficacy of fluconazole against biofilms of C. albicans.

Keywords: Candida albicans; Synergistic; Biofilm; Combination; Drug resistance; Lemongrass oil


MIC: Minimum Inhibitory Concentration; SEM: Scanning Electron Microscopy; YPD: Yeast-Peptone-Dextrose, PBS: Phosphate Buffer Saline; MOPS: 3-[N-morpholine] Propane Sulphonic Acid; RPMI: Roswell Park Memordium Institute; DMSO: Dimethyl Sulphoxide; XTT: 2,3-bis (2-methoxy-4-nitro-sulfophenyl)- 2H-tetrazolium-5-carboxanilide; CLSI: Clinical and Laboratory Standards Institute; FLZ: Fluconazole; FICI: Fractional Inhibitory Concentration Indices.


Candida albicans, an important pathogen of the humans can form drug resistant biofilms on biotic as well as a biotic surfaces [1,2]. It is the most common hospital-acquired infectious agent. C. albicans may cause superficial and serious systemic mycosis. Biofilm formation on prosthesis devices like, heart valves, stents or indwelling medical devices is a serious clinical challenge. Many of the prescribed antifungal drugs are ineffective against biofilms [3,4]. Antifungal agents like azole group of drugs target ergosterol synthesis in the cell membrane of Candida albicans, while some studies showed that the level of ergosterol is significantly decreased in intermediate and mature phase of biofilm growth compared to those in developmental phase [5,6]. Antifungal agents available in the market are ineffective because of the development of multifactorial drug resistance mechanisms. The mature biofilm may act as a source of re-current infections, so there is a need to identify better drug targets for eradication of mature biofilm [7].

There is considerable interest in using essential oils and their components as alternatives to synthetic antifungal drugs. Lemongrass (Cymbopogoncitratus) is used since ancient times in traditional medicine in many countries. Lemongrass oil has different properties like antibacterial, antifungal activities as well as analgesic and antiinflammatory activities [8]. The antifungal activities of essential oil of Lemongrass (Cymbopogoncitratus) are reported against C. albicans [9]. Lemongrass oil contains 12 to 15 components of Terpenoids and Terpene in nature. The chemical composition of the Lemongrass oil is Geraniol (0.72%), Linalool (3.82%), Limonene (4.12%), Terpinolene (1.52%), 1-8cineole (0.12%), Myrcene (18.5%), Geranyl acetate (0.23%), Nerol (23.92%), Geranial (43.2%). β-Ionone (0.03%), β-citronellol (2.05%), a pinene (0.06%), β-caryophyllene (2.05%) and Citral is an combination of neral plus geranialreported by Farhang, et al (2013), Wagara, et al (2011) and Sulaiman, et al (2013) [10,11,8].

Many terpenoid molecules of plant origin have promising activity against growth and the virulence factors of C. albicans [12,13]. A main characteristic of C. albicans biofilm is resistance to various antifungal such as the widely used antifungal drug fluconazole. High dosages of antifungal drugs are not recommended due to increased toxicity as side effect. To limit the use of high concentration of antifungal drugs, drug combination strategy is explored against biofilm associated C. albicans infections [14,15]. To study the effect of these components with fluconazole a combination study is done. Phytochemicals are being proposed as candidates for Synergy research to generate new pharmaceuticals [16]. Combination therapy is very useful and effective therapy for increasing the efficacy of drug towards the resistant strain of pathogen. Rate and degree of microbial killing could be increased by using combinational approach [17].

Materials and Methods

A culture of Candida albicans (ATCC 90028), was obtained from the Institute of Microbial Technology, Chandigarh, India. One clinical isolates was obtained from Government Hospital, Parbhani, Maharashtra, India. Both the cultures were maintained on yeast extract peptone dextrose (YPD) agar slants at 40C (HiMedia laboratories Pvt. Ltd. Mumbai India). A single colony from YPD agar plate was inoculated in 50ml of YPD broth and incubated at 30°C temperature, on a shaking (120rpm) incubator for 24h. Cells from the activated culture were collected by centrifugation at 2000g speed for 5min, washed thrice and resuspended in PBS (pH 7.4) for further experimentation. RPMI-1640 medium (w/L-glutamine, w/o sodium bicarbonate; pH7, buffered with 165mM MOPS (3-[N-morpholine] propane sulphonic acid) was filter sterilized and used as the assay medium. Various concentrations of components of lemongrass oil were prepared in RPMI by double dilution method. Concentration of the solvent i.e. Dimethyl Sulphoxide (DMSO), never exceeded 1%. Fluconazole was used as a standard antifungal drug.

All media components and chemicals were purchased from HiMedia laboratories Pvt. Ltd. Mumbai. XTT [i. e. 2,3-bis (2-methoxy-4-nitro-sulfophenyl)-2H-tetrazolium-5-carboxanilide] and menadione were procured from Sigma-Aldrich Chem. Ltd. Components of lemongrass oil like Citral, Nerol, Linalool, 1,8 cineole, Geraniol, Β-citronellol, Myrcene, Geranyl acetate, limonene (all analytical grade) were obtained from Sigma-Aldrich Chemicals Ltd, Mumbai, India. β-Ionone as well as other media components was purchased from HiMedia laboratories Pvt. Ltd. Mumbai, India.

Minimum inhibitory concentration/minimum fungicidal concentration

The effect of drug combinations on the growth of C. albicans was studied using the standard broth micro dilution methodology based on the Clinical Laboratory Standards Institute guidelines [18]. Various concentrations of test molecules ranging from 0.031 to 4 mg/ ml were prepared in RPMI-1640 medium in 96 well plates (Costar, Corning Inc., USA). Wells without test compounds was served as controls, while Fluconazole (1–256µg/ml) was used as a standard antifungal agent. One hundred micro liter of inoculum was added to 100µl of RPMI-1640 medium in each well to obtain 1X103cells/ ml. The plates were incubated at 35°C for 48h. To analyze growth, the absorbance was read at 620nm using a microplate reader (Multiskan EX, Thermo Electron Corp., USA). The lowest concentration of the test compound which will cause a 50% reduction in the absorbance compared to the control was considered as the minimum inhibitory concentration (MIC) for growth of Candida albicans.

Molecule for which the MIC was achieved (in the range 0.031 to 4 mg/ml) was selected for minimum fungicidal concentration (MFC) testing. To determine the MFC with respect to Candida, cells from the MIC and wells containing concentrations above the MIC was used. Aliquots of 10µl from these wells were spread on YPD agar. These plates was incubated for 48h at 30°C and observed for the presence of colonies. No appearance of colonies on the agar plates was noted as a fungicidal effect [18]. The lowest concentration of the test molecule in the microplate well from which an aliquot showing no growth was considered as the MFC.

Biofilm Formation and drug susceptibility

For biofilm formation, 100μl of the cell suspension (1×107cells/ ml in PBS) were added to each well of 96 well microtitre plates. Plates were incubated at 370c on an orbital shaker for 90 minutes of adhesion phase. Wells were washed with sterile PBS to remove non-adhered cells and 200μl of RPMI for-1640 medium was added to adhered cells. To carry out susceptibility of biofilm development, medium containing various concentration of the drugs were added at the zero hour of biofilm formation i.e. immediately after adhesion phase and the plates were incubated at 370c for 48hr, at 100rpm in an orbital shaker. While, to analyse the effects on mature biofilms, medium with a range of drug concentration were added to the 24hr. mature biofilms the plates were further incubated for 48 hour at 370C. Density of the cells survived in biofilm forms were analysed through metabolic activity in XTT formazan reduction assay [19].

Biofilm Quantitation by XTT assay

Biofilm formation was quantitated using XTT [i.e. 2,3-bis (2- methoxy-4nitro-sulfophenyl)-2H-tetrazolium-5-carboxanilide] (sigma-Aldrich, India) reduction assay. Briefly, XTT solution was prepared by mixing 1mg/ml XTT salt in PBS and stored at -20c. Prior to use, menadione solution was prepared in acetone (sigma- Aldrich, India)was added to XTT to a final concentration of 4µM. The wells containing biofilm was washed with PBS to remove non adhered cells and incubated for 5 hour in 100µl of XTT–menadione solution in dark, at 370c at 100rpm. The colour formation by water soluble formazan product was measured at 450nm using a microplate reader. Wells without biofilms were served as a blank [19].

Checkerboard format for determination of FICI

Combinatorial efficacy of the components of lemongrass oil and Fluconazole were analyzed in terms of fractional inhibitory concentration indices (FICI) obtained in checkerboard assay. Dilutions of Fluconazole and components of lemongrass oil as well as their combinations were prepared in a checkerboard format as per standard methodology [17]. A two dimensional array of serial concentrations of test compounds was used for preparation of dilutions of the drugs. For planktonic growth, the micro plates were incubated at 35°C, while for biofilm growth the plates were incubated at 37°C for 48 hours. MICs for planktonic and biofilm growth were determined as mentioned in previous sections.

FICI values were calculated using formula:


Where FICA= (MIC of drug A in combination/MIC of drug A alone),

FICB= (MIC of drug B in combination/MIC of drug B alone).

When the value of £FIC = 0.5 it is the synergism; between 0.5 and 1.0 it is additive; and when £FIC > 4 it is known as the antagonism. A FIC result of > 1.0 but =4 is considered as indifference [20].

Microscopic analysis of Candida albicans and Biofilm

Cells were observed under an inverted light microscope (Metzer, India). Photographs were taken by a Labomed microphotography system (LabomedKorntal, Germany).For scanning electron microscopy (SEM), samples were fixed in 2.5% glutaraldehyde in 0.1mol 1-1 phosphate buffer (ph 7.2) for 24 hour at 4°C samples was post fixed in 2% aqueous solution of osmium tetra oxide for 4 hour, then dehydrated in a series of graded alcohols and finally dried stubs, and gold coating were performed using an automated gold coater for 3min. biofilms were observed under an inverted light microscope[19].

Statistical analysis

Values mentioned are the mean with standard deviations, obtained from three different observations. Values in the control and treatment groups for various molecules were compared using Student’s t -test. A value of P< 0.05 was considered statistically significant [18].


Lemongrass oil Components sensitizes Candida albicans to fluconazole

Components of lemongrass oil like, citral, Nerol, geraniol, linalool, 1,8cinole, terpinolene, ionone, and limonene and geranyl acetate showed MICs against planktonic growth of both the strains of C .albicans. At concentrations above 2mg/ml geraniol, citronellol and linalool exhibited inhibitory activity against C. albicans. 1,8-cineole, Nerol and terpinolene required high concentration for inhibition i.e. above 4mg/ml. Citral, ionone and geranyl acetate were highly effective at concentrations above 0.5mg/ml. A significant decrease in MICs of fluconazole was observed when cells were exposed to combination of Fluconazole with Lemongrass oil components against C albicans ATCC 90028 and one clinical strain (isolate). For example, the MIC of fluconazole in combination with limonene was 0.125mg/ml concentration and limonene was found to be 0.5mg/ml concentration. The FICI value for Limonene-fluconazole combination was calculated to be 0.4, which indicated that the combination is synergistic. Similarly as per observation table (Table 1) the calculated FICI value for the other oil components like Citral, Geraniol, Nerol, Linalool, Citronellol and 1,8 cineole with fluconazole in combination is below 0.5. It is indicated that the combination is Synergistic against C. albicans ATCC 90028.Similarly Citral, Nerol, Citronellol, β-ionone and Geraniol combination with fluconazole were showed synergistic interaction against Clinical isolate of C. albicans. Whereas the treatment of β-ionone & terpinolene with fluconazole together was not synergistic. FICI value of β-ionone fluconazole combination is 0.628. Terpinolene fluconazole combination showed FICI value of 0.6, indicating additive effect against C. albicans. 1,8 cineol, Terpinolene, Linalool and limonene in combination with fluconazole were additive effect. Geranyl acetate combination with fluconazole showed Indifference effect, FICI value for this combination is 1.064 against both strain of C. albicans (Table 1 and 2).