Journal Of Medical Mycology

Thymus vulgaris essential oil and thymol inhibit biofilms and interact
synergistically with antifungal drugs against drug resistant strains of
Candida albicans and Candida tropicalis


Role of biofilm in disease development and enhance tolerance to antifungal drugs among

Candida species has necessitated search for new anti-fungal treatment strategy. Interference

in pathogenic biofilm development by new antifungal compounds is considered as an

attractive anti-infective strategy. Therefore, the objective of this study was to evaluate

Thymus vulgaris essential oil and its major active compound, thymol for their potential to

inhibit and eradicate biofilms alone and in combination with antifungal drugs against

Candida spp. with especial reference to Candida tropicalis.

Anti-candidal efficacy of T. vulgaris and thymol in terms of minimum inhibitory

concentration (MIC) was first determined to select the sub-MICs against C. albicans and C.

tropicalis. Biofilm formation in the presence and absence of test agents was determined in

96-well microtiter plate by XTT reduction assay and effect of essential oils at sub-MICs of

the test agents on biofilm development on glass surface was analysed by light and scanning

electron microscopy. Synergistic interaction between essential oils and antifungal drugs were

studied by checkerboard method.

Effect of sub-MIC of T. vulgaris (0.5 × MIC) and thymol (0.5 × MIC) on biofilm formation

showed a significant reduction (p <0.05) in biofilms. Light microscopy and SEM studies revealed disaggregation and deformed shape of C. albicans biofilm cells and reduced hyphae formation in C. tropicalis biofilm cells at sub-MICs of thymol. Significant effect of T. Page 2 of 18 Journal Pre-proof 2 vulgaris and thymol was also recorded on pre-formed biofilms of both C. albicans and C. tropicalis. T. vulgaris and thymol also showed synergy with fluconazole against both in planktonic and biofilm mode of growth of C. albicans and C. tropicalis. However, synergy with amphotericin B is clearly evident only in planktonic Candida cells. Thyme oil and thymol alone or in combination with antifungal drugs can act as promising antibiofilm agent against drug resistant strains of Candida species and needs further in vivo study to synergise its therapeutic efficacy. Keywords: Antibiofilms; T. vulgaris; Thymol; Synergy; C. albicans; C. tropicalis; synergistic interaction; biofilm inhibition; antifungal drugs 1. Introduction Candida albicans and non-albicans Candida species are associated with superficial and systemic infections in critically ill patients with weakened immunity [1]. Candida species have been emerged as a significant cause of morbidity and mortality and accounting for approximately 72% of all nosocomial fungal infections, 15% of all hospital acquired infections and blood stream infections (8-15%) [2]. According to Brazilian Network of Candidemia study, about 40.9% cases of infections are caused by C. albicans followed by C. tropicalis (20.9%), C. parapsilosis (20.5%) and least with C. glabrata (4.9%) [3]. C. albicans is very common opportunistic pathogen producing oral, vaginal and or systemic candidiasis [4,5] and also the most common agent of hospital acquired bloodstream infections [6,7]. About 8 to 9% of all the blood stream infections with the crude mortality rate of 40% [8]. Other species of Candida like C. tropicalis, C. parapsilosis and C. glabrata are also increasingly reported responsible for candidemia and other conditions [3]. Furthermore, biofilm formation by Candida species has been documented on variety of medical devices such as catheters, dialysis, joint devices etc [4]. Fungal implant infections are less common than bacterial infections but tend to be more serious or problematic. In the biofilm mode of growth, yeast cells display characteristic traits different with planktonic state. Biofilms provide several folds increase in resistance to antifungal drugs as well as resist host defence which lead to failure of conventional antifungal therapy [9]. Antifungal drugs mainly polyenes and azoles are commonly used to treat Candida infections. However, efficacy of these drugs is limited in many cases due to development of resistance, poor penetration power in biofilm and undesirable side effects [9,10]. Page 3 of 18 Journal Pre-proof 3 In this perspective, search for alternative mode of therapy and discovery of new anti-candidal compounds/combinations with improved mode of action with no or least toxicity from various sources including medicinal plants are needed. Plant derived products and essential oils are used against various ailments including infectious diseases since long in traditional system of medicine [11]. T. vulgaris essential oil and its active constituent, thymol have been used as an antioxidant, anti-inflammatory, local anaesthetic, antiseptic, antibacterial and antifungal agent [12]. Antibiofilm activity of essential oils have been subject of recent investigation [13,14, 15]. However, little work has been reported on T. vulgaris and thymol against drug resistant Candida species especially C. tropicalis. In this study, the antibiofilm activity was investigated against the strong biofilm forming strains of C. albicans and C. tropicalis at sub-MICs in vitro. Furthermore, combination of oils with antifungal drugs (fluconazole and amphotericin B) was determined to explore the synergistic interaction against the strains of C. albicans and C. tropicalis. 2. Materials and methods 2.1. Candida strains used Four clinical isolates of Candida were obtained from Department of Microbiology, King George Medical University, Lucknow, India, two C. albicans strains [CAJ-01, CAJ-12 (KGMU028)] and two strains of C. tropicalis (CT-03 and CT-04) were further characterized and identified in our laboratory. Reference strains such as C. albicans MTCC3017 and C. tropicalis NRLLY12968 were obtained from Microbial Type Culture Collection, CSIR, IMTECH, Chandigarh, India and Fungal Culture Collection of the Agricultural Research Service, USDA at Peoria, USA respectively. The strains were tested for their morphological and biochemical characteristics such as growth on Hicrome Candida differential agar, germ tube formation, nitrate reduction and urease production using standard method [16. 17]. All strains of Candida were maintained in the laboratory on Sabouraud dextrose agar (SDA) slants at 4°C. The strain CAJ-01(Accession number: KY884676), CAJ-12 (KGMU028) (Accession number: MN263238) and CT-04 (Accession number: KY033482) were characterized by 18S rRNA gene sequence analysis by Macrogen, South Korea. Medium, Sabouraud dextrose broth/agar (SDB/SDA) and Hicrome Candida differential agar were obtained from Hi-Media Laboratory, Mumbai, India. 2.2. Antifungal drugs and essential oils Page 4 of 18 Journal Pre-proof 4 Drug powder of amphotericin B (Hi-Media, India), fluconazole (Pfizer Co., India), ketoconazole (Hi-Media, India) and itraconazole (Jansen Co., Mumbai, India) were tested for antifungal activity against the test strains. Stock solutions of antifungal drugs were prepared in dimethyl sulphoxide (DMSO) at a concentration of 25 mg/ml and stored at -4°C until use. Essential oils of Thymus vulgaris and thymol (99% purity) were purchased from Aroma Sales Corporation, New Delhi, India and Hi-Media Laboratory, Mumbai, India respectively. DMSO (1%) was used to dilute essential oil and thymol. Fresh antifungal drug solution was prepared in DMSO before use. 2.3. Gas chromatography and high resolution gas chromatography-mass spectrometry analysis of plant essential oils The composition of T. vulgaris oils was identified by GC-HRMS analysis. GC-MS analysis was carried out on JEOL AccuTOF GCV equipped with FID detector and separation was attained in column of 30 m × 0.22 mm × 0.25 µm (Fischer Scientific, UK) at IIT Bombay, Mumbai India. Helium gas was the carrier gas and the flow rate of mobile phase was set at 1.21ml/min. The sample was injected into the column with a split ratio of 1:10. The linear temperature was set at 60 ºC to 230 ºC with the hold time at 60 ºC for 2 min. The peak of the samples was identified by using the above system with the reference database NIST libraries. The relative retention indices of the compounds were compared with the reports in the literature to identify the compound. 2.4. Determination of minimum inhibitory concentration (MIC) of antifungal drugs and essential oils against planktonic growth The Clinical Laboratory Standards Institute (CLSI) method M27-A3 [18] with some modifications was used to determine MIC/MFC of antifungal drugs and essential oils against Candida strains. Briefly, overnight grown culture of yeast strains (0.5 McFarland) prepared in SDB. A hundred microliter of two-fold dilution of test agent were made in RPMI 1640 medium (Sigma, India) and 100 µl of inoculum (2.5 × 103 CFU/ml) was added in each well. Plates were incubated for 48 h at 37°C. Agent free control was included. MIC was defined as the lowest concentration of agents inhibited the visible growth of Candida strains. Minimum fungicidal concentration (MFC) was defined as the concentration completely inhibited the growth of Candida. Based on MIC values, the strains were designated as resistant if MIC values are ≥1.0µg/ml, ≥64 µg/ml, ≥2.0 µg/ml and ≥1.0 µg/ml for ketoconazole, fluconazole, amphotericin B and itraconazole respectively [15]. Page 5 of 18 Journal Pre-proof 5 2.5. Biofilm formation assay using microtiter plate Biofilm forming ability of test isolates was screened by 96-well microtiter plate method as previously described [19]. In brief, Candida strains were grown in SDB (glucose 8% w/v) at 37°C for 24 h. Harvested Candida cells were re-suspended in RPMI 1640 medium containing L-glutamine and bicarbonate absence. A hundred microliter of Candida cell suspension (1.5 × 106 CFU/ml) after standardization was added to the wells of microtiter plates and incubated at 37°C for 48 h followed by gently aspiration of the medium. The wells were washed three times to remove non-adherent cells. Next, XTT was used to assay biofilm formation as described earlier [15] by taking absorbance at 490 nm using MTP reader. Each experiment was conducted at least two times in triplicate and data was recorded as the mean absorbance values. 2.6. Inhibition of biofilm formation The biofilm cells of test strains were treated with different concentrations of test agent in 96- well microtiter plate was analysed as described above [15]. Briefly, biofilm was formed using RPMI 1640 medium in the presence and absence of sub-MICs of test agents (essential oil, thymol and antifungal drugs). On the basis of MIC value of Candida, sub-MICs (0.25 × MIC and 0.5× MIC) of test agents (fluconazole, amphotericin B, T.vulgaris and thymol) were diluted in RPMI 1640 medium to prepare final concentrations. Then, 0.1 ml of test agents (2 × final concentrations) and 0.1 ml of standardized cell suspension were added to each well of microtiter plates. The mixture was incubated at 37°C for 48 h. Test agent free wells and biofilm free well serve as a positive and negative control respectively. Subsequently non adherent cells were removed by washing wells with PBS. The experiment was performed three times in triplicates and mean absorbance values were used to measure the inhibition of biofilm formation as follows: (mean OD490 of treated well/ mean OD490 of untreated control well) × 100 2.7. Light microscopy of biofilms developed on glass surface C. albicans (CAJ-01) and C. tropicalis (CT-04) were allowed to grow in the presence of thymol on cover slip in 12-well tissue culture plate using similar conditions as described for microtiter plate method [15]. In brief, Candida strains was grown in SDB (glucose 8% w/v) at 37°C for 24 h. Candida cells were harvested and re-suspended in RPMI 1640 medium. Two-fold serial dilution of thymol were prepared in RPMI 1640 medium and one ml was added to each well of plate containing sterile glass coverslips (diameter 15 mm). Subsequently, 1 ml of standardised cell suspension was inoculated and incubated at 37°C for 48 h. At the end of incubation, medium was discarded and glass coverslips were washed 2-3 Page 6 of 18 Journal Pre-proof 6 times with sterile PBS and stained with 0.1% crystal violet and incubated at 37°C for 10 min. The glass cover slip was viewed under light microscope (Olympus, Japan). 2.8. Scanning electron microscopy of biofilms developed on glass surface C. albicans (CAJ-01) and C. tropicalis (CT-04) biofilms were formed on glass coverslips at sub-MICs of thymol at 37°C for 48 h as described above. Biofilm cells were washed with PBS and fixed with 5% glutaraldehyde in cacodylate buffer in a graded concentration of ethanol (25, 50, 75, 95 and 100%), immersed in hexamethyldisilazane and dried under air for overnight at room temperature. The glass coverslips were then mounted on aluminium stubs with silver paint, sputter coated with gold and subjected to SEM analysis (JSM 6510, LV, JEOL, JAPAN). 2.9. Determination of biofilm eradication by essential oils and antifungal drugs Candida biofilms were allowed to form in 96-well microtiter plate as mentioned above. Next, 0.1 ml of two-fold serial dilutions of test agents (fluconazole, amphotericin B, T. vulgaris and thymol) made in RPMI 1640 medium were added to each biofilm well of microtiter plates and further incubated at 37℃ for 48 h. A series of drug-free wells and biofilm-free wells (medium broth) were also included to serve as positive and negative control respectively. Biofilm eradication was determined as sessile MIC (SMIC) by XTT reduction assay. Each experiment was conducted at least two times in triplicate and SMIC was determined by comparing the reduction in the mean absorbance of the test agents treated biofilm to the untreated control and expressed as the MIC of agent that eradicated ≥ 80% of the sessile cells. 2.10. Kinetics of inhibition of sessile cells Time dependent killing assay was performed to determine the potency of essential oils and antifungal drugs using a standardized method [19]. Briefly, pre-formed biofilm in 96-well plate was challenged with 2 × MIC of test agents. After incubation wells were washed to remove non-adherent cells and biofilm mass was scraped off the well using a sterile scalpel. Subsequently, the biofilm cells were added to PBS and vortexed gently to disrupt the aggregates, serially diluted in normal saline solution (NSS) and spread on SDA plates. The plates were incubated at 37°C for 24 h. Viable count of Candida was determined and data was presented as log10 CFU/ml. 2.11. In vitro synergy assay between essential oil and antifungal drugs in planktonic mode of growth A checkerboard microtiter assay was adopted to assess the synergy between test agents against the test strains of C. albicans (CAJ-01, CAJ-12 (KGMU028) and C. albicans Page 7 of 18 Journal Pre-proof 7 MTCC3017) and C. tropicalis (CT-03, CT-04 and C. tropicalis NRLLY12968) by using the method as described by Vitale et al. [20] with little modifications. Briefly, two-fold serial dilutions of test agents were prepared in RPMI 1640 medium in 96-well microtiter plate. Further, 50 µl from each dilution of essential oils were added to the 96-well microtiter plates in the vertical direction and same amount of antifungal drugs were added in horizontal direction to obtain the various combinations of test compounds. Subsequently, 100 µl of inoculum suspension (0.5 McFarland) of Candida strains was added to each well followed by incubation at 37°C for 48 h. The interaction was determined as fractional inhibitory concentrations index (FICI) which was calculated as follows MIC of the combination of essential oils or active compounds with fluconazole or amphotericin B divided by the MIC of essential oils or active compounds or fluconazole or amphotericin B alone. FICI was determined by adding both FICIs. The FICI result was interpreted as follows: FICI ≤ 0.5: synergistic, > 0.5-4.0: no interaction, > 4.0: antagonistic.

2.12. In vitro synergy assay between essential oils and antifungal drugs in sessile mode of


A checkerboard microtiter assay was performed to evaluate the interaction of thyme oil,

thymol with fluconazole and amphotericin B against the test Candida strains. In brief,

biofilms of test strains were formed in the wells of microtiter plates and treated with various

combinations of test agents (essential oils and drugs) by adding 50 µl of each prepared

dilution of essential oils and drugs in the vertical and horizontal direction of plate. The plates

were incubated at 37°C for 48 h.The extent of synergy was determined in terms of FICI index

3. Statistical analysis

Statistical analysis was determined by one way ANOVA using Duncan’s method (IBM SPSS

Statistics, version 20). The data with p value <0.05 was considered significant. 4. Results 4.1 Phytochemical analysis of essential oil by GC-MS analysis The chemical composition of T. vulgaris essential oil is presented in table 1 and figure 1. Various volatile compounds mainly mono-terpenes and sesqui-terpenes were identified. The major components of T. vulgaris essential oil were thymol (54.73%), carvacrol (12.42%), terpineol (4.00%), nerol acetate (2.86%) and fenchol (0.5%). 4.2. Minimum inhibitory concentration of antifungal drugs and essential oils Page 8 of 18 Journal Pre-proof 8 MIC and MFC of antifungal drugs were determined against C. albicans and C. tropicalis as presented in Table 2. MICs of fluconazole and itraconazole ranged from 8-1024 µg/ml and 256-1024 µg/ml respectively. In contrast, MICs of amphotericin B and ketoconazole was found to be in the range of 2-16 µg/ml and 16-1024 µg/ml respectively. Based on the MIC values of antifungal drugs presented in table 2 it is evident that all strains of C. albicans and C. tropicalis showed variation in MIC values against antifungals being maximum tolerant to itraconazole. While CAJ-12 (KGMU028) also exhibited high MIC (1024 µg/ml) against fluconazole. Antifungal activity of T. vulgaris and thymol are presented in table 3 against the Candida strains. Planktonic MIC (PMIC) of T. vulgaris and thymol were exhibited ranging from 1.56- 50 µg/ml against the test strains of C. albicans and C. tropicalis. MICs of T. vulgaris essential oil were 25, 3.12 and 1.56 µg/ml against the C. albicans (CAJ-01), CAJ-12 (KGMU028) and C. albicans MTCC3017 respectively. In contrast, MICs of thyme oil were 25, 50 and 25 µg/ml against CT-03, CT-04 and C. tropicalis NRLLY12968 respectively. 4.3. Biofilm formation by Candida strains The biofilm forming ability on polystyrene microtiter plate was determined on the basis of absorbance in XTT reduction assay. The strains were divided as strong (OD490 >0.800),

moderate (OD490 >0.4 to 0.8) and weak (OD490 <0.4) [15, 21]. All the strains of C. albicans

(CAJ-01, CAJ-12 (KGMU028) and C. albicans MTCC3017) and C. tropicalis (CT-04 and C.

tropicalis NRLLY12968) formed strong biofilms except CT-03. The biofilm forming ability

in terms of absorbance was found 1.369 ± 0.02, 1.145 ± 0.08, 0.973 ± 0.13, 0.433 ± 0.03,

1.338 ± 0.02 and 0.931 ± 0.03 for CAJ-01, CAJ-12 (KGMU028), C. albicans MTCC3017,

CT-03, CT-04 and C. tropicalis NRLLY12968 respectively (Table 4).

4.4. Inhibition of biofilm formation

Data presented in table 5 shows ability of T. vulgaris and thymol to inhibit biofilm

development in Candida strains. At 0.5 × MIC of T. vulgaris (12.5 µg/ml) and thymol (3.12

µg/ml), the biofilm formation in CAJ-01 was found to be 26.30 and 16.93% respectively.

Similarly, CAJ-12 (KGMU028) cells also displayed noticeable reduction in biofilm

formation at sub-MICs of T. vulgaris and thymol. At 0.5 × MIC of thymol, the biofilm

forming ability of C. tropicalis (CT-04) strain was 20%. Similarly, T. vulgaris also showed

significant (p <0.05) reduction in the biofilm formation.

4.4.1. Light microscopy of biofilm cells

Inhibition of biofilm formation by thymol was also analysed on glass coverslips and

visualized under light microscope. Untreated control C. albicans (CAJ-01) and C. tropicalis

(CT-04) biofilms of 48 h exhibited multi-layered yeast cells with substantial amount of

extracellular matrix. C. tropicalis also formed hyphae in the sessile mode (Fig. 2A). Biofilm

formation was inhibited to varying extent at sub-MICs of thymol. CAJ-01 and CT-04 biofilm

showed disaggregation of cells and reduced matrix production (Fig. 2). At sub-MIC (1.56

µg/ml) of thymol, there were reduction in hyphae production in CT-04 strain.

4.4.2. Scanning electron microscopy of biofilm cells

The above study also analysed the ultra structural changes in the thymol treated biofilm cells

by electron microscopy. Distinct morphological changes were also observed in the sessile cells

of CAJ-01 and CT-04 at sub-MICs of thymol (Fig. 3 and 4). Untreated CAJ-01 cells exhibited

multilayer of yeast cells with substantial amount of matrix whereas CT-04 exhibited dense

network of cells with hyphae formation. Thymol treated CAJ-01cells showed various

morphological changes. There were reduced numbers of Candida biofilm cells after treatment

with thymol as compared to control. There were also shrinkage of cell membrane and leakage

of intracellular material [Fig. 4 (C1 and C2)]. Microscopy revealed distorted cell shape as well

as reduced hyphae formation in CT-04 after treatment with thymol as shown in Fig. 3(C1 and

C2). Treated Candida biofilm cells had scattered aggregation. Shrinkage of the cells and

permeabilization of cell membrane was also observed in CAJ-01 and CT-04 at sub-MIC of


4.5. Eradication of pre-formed biofilms

Sessile MIC (SMIC) of test compound was considered as the concentration eradicating 80%

of pre-formed biofilms. SMIC of T. vulgaris and thymol varied from 6.25- 100 µg/ml and

3.12-25 µg/ml respectively, against one or other Candida strains. The test agents (T. vulgaris

and thymol) showed 2-4 folds increased in SMIC against the strains of C. albicans (CAJ-01,

CAJ-12 (KGMU028 and C. albicans MTCC3017). Thymol showed no increase in SMIC

compared to PMIC against CAJ-12 (KGMU028), CT-03 and CT-04. Whereas SMIC of T.

vulgaris against CT-04 was found to be 100 µg/ml. SMICs of T. vulgaris and thymol were

increased only 2-folds against C. tropicalis NRLLY12968 respectively (Table 3).

4.8. Kinetics of inhibition of sessile cells

The time dependent killing of CAJ-01, CAJ-12 (KGMU028), CT-04 and C. tropicalis

NRRLY12968 by the T. vulgaris, thymol, fluconazole and amphotericin B is shown in Fig. 5.

Treatment of pre-established biofilms with 2 × SMIC of test oils showed strong fungicidal

effect on the strains of C. albicans and C. tropicalis biofilms. Within 24 h of treatment with

T. vulgaris, log10 CFU count was reduced from 7.2 to 3.8 against the strains of CAJ-01.

Similarly, CAJ-12 (KGMU028), CT-04 and C. tropicalis NRRLY12968 also showed

significant reduction in viable count within 24 h of treatment of T. vulgaris. All the test

strains showed significant reduction in log10 CFU count within 12 h of treatment of thymol.

However, amphotericin B and fluconazole could not produce killing effect even upto 48 h.

4.9. Synergistic interaction of essential oils with antifungal drugs in planktonic mode

The synergistic effect of T. vulgaris and thymol with fluconazole and amphotericin B were

evaluated against CAJ-01, CAJ-12(KGMU028), C. albicans MTCC3017, CT-03, CT-04 and

C. tropicalis NRLLY12968 strains as shown in Table 6a and 6b. T. vulgaris and thymol

exhibited synergy with fluconazole and amphotericin B against all the tested strains. Thymol

showed highest synergy with fluconazole (FICI values 0.156) against CAJ-01, C. albicans

MTCC3017, CT-03 and CT-04. T. vulgaris also exhibited highest synergy with fluconazole

(FICI values 0.140) against C. tropicalis NRLLY12968. MICs of fluconazole and

amphotericin B were reduced upto 32-folds against the strains of C. albicans and C.

tropicalis whereas reduction in T. vulgaris and thymol MICs were 8 to 16-folds against the

test strains.

4.10. Synergistic interaction of essential oils with drugs in sessile mode

The synergistic effect of T. vulgaris and thymol with fluconazole and amphotericin B were

evaluated against CAJ-01, CAJ-12(KGMU028), CT-04 and C. tropicalis NRLLY12968

sessile cells. Table 7a and 7b revealed the synergistic interaction of essential oils (T. vulgaris

and thymol) with fluconazole against the above test strains. Thymol exhibited highest

synergy with fluconazole against CAJ-01 (FICI values 0.187) and CT-04 (FICI values 0.125).

Interestingly, SMICs of fluconazole with thymol were reduced upto 16-folds against all the

test strains of Candida species. There was also reduction in SMICs of thymol upto 8-folds

against the test strains of C. albicans (CAJ-01 and CAJ-12(KGMU028)). SMIC of thymol

5. Discussion

Variation in biofilm forming ability under in vitro condition is commonly observed among

Candida species. However, the ability to form biofim in vivo condition may not be directly

correlated with in vitro ability. Different factors are known to influence the biofilm formation

under in vivo as well as in vitro condition [22]. Such variations have also been previously

reported [15, 21]. However in vitro biofilm study is important to evaluate the relative

characteristics of cell growth in planktonic and sessile mode and provide a platform to asses

an antibiofilm activity of bioactive compounds. Biofilm formation by C. albicans and C.

Page 11 of 18

tropicalis has been documented by many researchers [4, 22, 23, 24]. In vitro strong biofilm

formation by C. albicans and C. tropicalis has formed the basis of selection of these strains in

the test system for antibiofilm screening. Role of biofilm in virulence and pathogenicity of

Candida is well documented which provides several advantages to the organisms such as

enhance resistance level and protection against host defence system [22].

The strains of C. albicans were also previously studied for their antifungal susceptibility

profile and found resistant to common antifungal drugs [15]. The MIC values of fluconazole,

amphotericin B and ketoconazole against C. albicans showed variation from 4 to 32 µg/ml

except CAJ-12 (KGMU028) strain where it was ranged from 2 to 1024 µg/ml. Relatively

higher MIC values of above antifungal drugs was recorded against C. tropicalis.

Interestingly, all the test strains of C. albicans and C. tropicalis showed high level of MIC

(1024 µg/ml) against itraconazole. Similar level of variation in MIC values of antifungal

drugs was also recorded in Candida species by other researchers [21, 25, 26].

Azoles are fungistatic rather than fungicidal so the treatment provides the opportunity for

acquired resistance to develop in the presence of these drugs [27]. Amphotericin B has been

used as the drug of choice when acquired drug resistance emerges to azoles. However,

resistance to amphotericin B has been attributed to absence of ergosterol in the cell

membrane, activation of antioxidant mechanisms and decrease in mitochondrial activity [28].

Antifungal activities of plant essential oils and active constituents and their mode of action

are documented [29, 30]. The antifungal activity of essential oil is attributed due to the

presence of functional groups such as phenols, aldehydes, ketones, alcohols, esters,

hydrocarbons [31, 32].

Considering the problem of drug resistance to conventional antimicrobials in pathogenic

strains of fungi, T. vulgaris and thymol were screened for their efficacy against the drug

resistant strains of Candida species. Anti-candidal activity of T. vulgaris and thymol against

drug resistant strains of C. albicans and C. tropicalis demonstrated promising anti-candidal

activity of thymol as compared to thyme oil as shown by their MIC values. Similar activity of

T. vulgaris and thymol were also reported by many other researchers [33, 34].

Furthermore, T. vulgaris constitutes high percentage of phenolic compound such as thymol.

Thus, it is speculated that the fungicidal and/or fungistatic activity of T. vulgaris can be

attributed to its main component, thymol whereas role of other compounds might be

contributing in nature.

Page 12 of 18

Journal Pre-proof


Inhibition of biofilm and eradication of pre-formed pathogenic biofilm by anti-infective

agents are considered as an effective approach to combat biofilm associated infections.

Therefore, T. vulgaris essential oil and thymol was evaluated for biofilm inhibition at sub￾MICs. Different concentrations including sub-MICs of T. vulgaris and thymol tested in our

previous study showed no cellular toxicity to red blood cells [35].

Varying level of attenuation of C. albicans and C. tropicalis biofilms in the presence of T.

vulgaris and thymol indicated that these agents inhibited biofilm either by preventing

adherence or subsequent biofilm development.

Further to assess the structural changes in the biofilm development, light and scanning

electron microscopy were conducted on glass coverslip surface. Thymol treated cells

exhibited disorganization of C. albicans and C. tropicalis biofilm cells. There were also

reduced number of C. albicans and C. tropicalis biofilm cells with the increase in

concentration of thymol. Light microscopy examination also revealed reduced hyphae

formation in CT-04 with the increase in concentration of thymol compared to control

(untreated biofilms).

Further, SEM examination of C. albicans and C. tropicalis biofilm cells revealed the dense

cell architecture with huge amount of matrix in the control sets. Whereas treated cells

exhibited unorganised biofilm cells at sub-MICs of oil. Similar changes in cell morphology of

C. albicans in the presence of thymol were also reported [34, 36]. SEM images of C. albicans

and C. tropicalis biofilm cells also revealed distorted cell shape, permeabilization of cell

membrane and contraction of cell wall that may caused leakage of intracellular material at

sub-inhibitory concentrations of thymol.

SEM analysis clearly demonstrated the mode of action of thymol in yeast which showed

interaction with cell envelop and intracellular targets as evident from disruption of the cell

membranes. Many authors have suggested the similar mechanism of disruption of cell

membrane integrity [21, 37, 38].

In the present study, T. vulgaris and thymol showed the potential to eradicate the sessile cells

of C. albicans as well as C. tropicalis. The sessile MICs of antifungal drug against the test

strains exhibited several folds increase in MICs as compared to PMIC. SMIC of fluconazole

and amphotericin B was raised upto 1000-folds in the test strains whereas sessile MIC of T.

vulgaris and thymol was raised only 2-4 folds against the test strains. Planktonic cells shed

from the biofilm surface may get killed by conventional antimicrobial drug therapy however

they fail to eradicate sessile cells that are embedded within the EPS matrix [22].

Page 13 of 18

Journal Pre-proof


Furthermore, EPS production is considered as an important virulence factor of Candida

species. It is also responsible for persistence, colonization and firm adherence of pathogen in

the host tissues. It is reported that metabolically inactive non dividing persister cells within

biofilms may be present. These persister cells are tolerant to a number of antimicrobial drugs

despite the fact that they are genetically identical to the rest of the microbial population. It is

believed that these cells are responsible for recurring of biofilms on treatment with

antimicrobial drugs [22, 39, 40].

Furthermore, efficacy of T. vulgaris and thymol were investigated in terms of the time

dependent killing of established C. albicans and C. tropicalis biofilms. Interestingly, test oil

and its active compound showed good fungicidal activity against the test isolates. In contrast,

antifungal drugs were showing least activity. The present data indicates that test oils

exhibited fungicidal activity rather than fungistatic which is very important to combat with

recalcitrant infections.

The present study highlights the synergistic interaction between the test essential oil and

active compounds with antifungal drugs against the test strains. In our study, interaction of T.

vulgaris and thymol with fluconazole and amphotericin B is clearly indicated in FICI index

against planktonic and sessile mode. Antifungal drugs activity is greatly increased with

thymol against the C. albicans and C. tropicalis strains. The FICI index also revealed the

synergy of thyme oil with fluconazole or amphotericin B in planktonic mode. Furthermore,

T. vulgaris and thymol has anti-candidal activity alone as well as in combination with drugs.

Thyme oil and its major component thymol, also showed significant synergy with

fluconazole in sessile mode. However, thymol showed more synergy with fluconazole against

C. albicans and C. tropicalis strains in sessile mode. These findings are encouraging and

could be exploited in combination therapy as also suggested by other authors [21, 35, 41, 42].

The two antifungal drugs were selected based on their different mode of action and their

associated side effects or toxicity. Unfortunately, these drugs may not be used alone to

combat fungal infections caused by drug resistant strains of Candida species which may

require higher doses application resulting in increasing adverse side effects [43]. To

overcome such problems, combination therapy is advantageous over monotherapy as it can

exhibit more effective way of killing or attenuating pathogenic organisms. Such

synergistic/combinational interaction might results in enhanced efficacy of drugs, decreased

chances of resistance emergence as well as reducing dose related toxicity [20, 21].

Page 14 of 18

Journal Pre-proof


6. Conclusions

The findings of the present study highlight the promising role of T. vulgaris and thymol as

alternative agents in the treatment of biofilm associated with C. albicans and C. tropicalis

infections. Further, their synergistic interaction with antifungal drugs could be exploited

against infection caused by the drug resistant Candida species.

Disclosure of interest

The authors declare that they have no conflict of interest.


One of the author HJ is thankful to UGC- New Delhi for granting Non-Net Research

Fellowship through AMU, Aligarh. We are grateful to Sophisticated Analytical Instrument

Facility (SAIF) at Indian Institute of Technology, Bombay for GC-HRMS analysis. We are

also thankful to Chairman, Department of Agricultural Microbiology for providing support

for this work. Facility extended by University Sophisticated Instrumentation Facility (USIF),

AMU, Aligarh is thankfully acknowledged.


[1] Maheronnaghsh M, Tolouei S, Dehghan P, Chadeganipour M, Yazdi M. Identification

of Candida species in patients with oral lesion undergoing chemotherapy along with

minimum inhibitory concentration to fluconazole. Adv Biomed Res 2016; 5:132.

[2] Amran F, Aziz MN, Ibrahim HM, Atiqah NH, Parameswari S, Hafiza MR, Ifwat M. In

vitro antifungal susceptibilities of Candida isolates from patients with invasive candidiasis in

Kuala Lumpur Hospital, Malaysia. J Med Microbiol 2011; 60(9):1312-1316.

[3] Nucci M, Queiroz-Telles F, Tobon AM, Restrepo A, Colombo AL. Epidemiology of

opportunistic fungal infections in Latin America. Clin Infect Dis 2010; 51: 561–570.

[4] Sardi JC, Scorzoni L, Bernardi T, Fusco-Almeida AM, Mendes Giannini MJ. Candida

species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products

and new therapeutic options. J Med Microbiol 2013; 62(1):10-24.

[5] Singh A, Verma R, Murari A, Agrawal A. Oral candidiasis: an overview. J Oral

Maxillofac Pathol 2014; 18, Suppl S1: 81-85.

[6] Citak S, Ozeelik B, Cesar S, Abbasoglu U. In-vitro susceptibility of Candida species

isolated from blood cultures to some antifungal agents. Jpn J Infect dis 2005; 56:44-46.

[7] Yapar N. Epidemiology and risk factors for invasive candidiasis. Ther Clin Risk Manag

2014; 10: 95–105.

Page 15 of 18

Journal Pre-proof


[8] Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: a persistent public health

problem. Clin Microbiol Res 2007; 20:133–163.

[9] Silva S, Rodrigues CF, Araújo D, Rodrigues ME, Henriques M. Candida species biofilms’

antifungal resistance. J Fungi 2017; 3(1):8.

[10] Bondaryk M, Kurzątkowski W, Staniszewska M. Antifungal agents commonly used in

the superficial and mucosal candidiasis treatment: mode of action and resistance

development. Adv Dermatol Allergol 2013; 30(5): 293–301

[11] Saviuc CM, Drumea V, Olariu L, Chifiriuc MC, Bezirtzoglou E, Lazăr V. Essential oils

with microbicidal and antibiofilm activity. Curr Pharm Biotechnol 2015; 16(2):137-51.

[12] Marchese A, Orhan IE, Daglia M, Barbieri R, Di Lorenzo A, Nabavi SF, Gortzi O, Izadi

M, Nabavi SM. Antibacterial and antifungal activities of thymol: A brief review of the

literature. Food Chem 2016; 210: 402-414.

[13] Souza CM, Pereira Junior SA, Moraes TD, Damasceno JL, Amorim Mendes S, Dias HJ,

Stefani R, Tavares DC, Martins CH, Crotti AE, Mendes-Giannini MJ. Antifungal activity of

plant-derived essential oils on Candida tropicalis planktonic and biofilms cells. Sabouraudia.

2016; 54(5):515-23.

[14] Budzyńska A, Różalska S, Sadowska B, Różalska, B. Candida

albicans/Staphylococcus aureus dual-species biofilm as a target for the combination of

essential oils and fluconazole or mupirocin. Mycopathologia 2017: 182; 989–995.

[15] Jafri H, Khan MSA, Ahmad I. In vitro efficacy of eugenol in inhibiting single and

mixed-biofilms of drug-resistant strains of Candida albicans and Streptococcus mutans.

Phytomedicine 2018; 10.1016/j.phymed.2018.10.005

[16] Barnett, James Arthur, Roger William Payne, and David Yarrow. Yeasts: characteristics

and identification. Cambridge University Press, 1983.

[17] Madhavan P, Jamal F and Chong RP. Laboratory isolation and identification of Candida

spp. J Appl Scien 2011:2870-2871.

[18] CLSI. Reference method for broth dilution antifungal susceptibility testing of yeasts: 3rd

informational supplement. CLSI document M27–A3. Wayne, PA: CLSI, 2008.

[19] Khan MSA and Ahmad I. Antibiofilm activity of certain phytocompounds and their

synergy with fluconazole against Candida albicans biofilms. J Antimicrob Chemother 2012;

67: 618 –621.

Page 16 of 18

Journal Pre-proof


[20] Vitale RG, Afeltra J, Dannaoui E. Antifungal combinations (Methods in molecular

medicine) in: E.J. Ernst, P.D. Rogers (Eds.) Antifungal Agents: Methods and Protocols.

Humana Press Inc, Totowa, New Jersey 2005; 143–152.

[21] Khan MSA, Ahmad I. Anti-candidal activity of essential oils alone and in combination

with amphotericin B or fluconazole against multi-drug resistant isolates of Candida albicans.

Med Mycol 2012; 50(1):33-42.

[22] Cavalheiro M, Teixeira MC. Candida biofilms: threats, challenges, and promising

strategies. Front Med 2018; 5:28.

[23] Silva DR, Endo EH, Filho BP, Nakamura CV, Svidzinski TI, de Souza A, Young MC

Ueda-Nakamura T, Cortez MC. Chemical Composition and Antimicrobial Properties of Piper

ovatum Vahl. Molecules 2009; 14: 1171.

[24] Tati S, Davidow P, McCall A, Hwang-Wong E, Rojas IG, Cormack B, Edgerton M.

Candida glabrata binding to Candida albicans hyphae enables its development in

oropharyngeal candidiasis. PLoS pathogens 2016; 12(3):e1005522.

[25] Linares CE, Giacomelli SR, Altenhofen D, Alves SH, Morsch VM, Schetinger MR.

Fluconazole and amphotericin-B resistance are associated with increased catalase and

superoxide dismutase activity in Candida albicans and Candida dubliniensis. Rev Soc Bras

Med Trop 2013; 46(6):752-758.

[26] Castanheira M, Deshpande LM, Davis AP, Rhomberg PR, Pfaller MA. Monitoring

antifungal resistance in a global collection of invasive yeasts and moulds: application of

CLSI epidemiological cut off values and whole genome sequencing analysis for detection of

azole resistance in Candida albicans. Antimicrob Agents Chemother 2017; 61(10): e00906-


[27] Berkow EL, Lockhart SR. Fluconazole resistance in Candida species: a current

perspective. Infect Drug Resist 2017; 10:237-245.

[28] Mesa-Arango AC, Rueda C, Román E, Quintin J, Terrón MC, Luque D, Netea MG, Pla

J, Zaragoza O. Cell wall changes in AmB-resistant strains from Candida tropicalis and

relationship with the immune responses elicited by the host. Antimicrob Agents Chemother

2016: AAC-02681.

[29] Ahmad A, Khan A, Akhtar F, Yousuf S, Xess I, Khan LA, Manzoor N. Fungicidal

activity of thymol and carvacrol by disrupting ergosterol biosynthesis and membrane

integrity against Candida. Eur J Clin Microbiol Infect Dis 2011; 30(1): 41-50.

[30] Nazzaro F, Fratianni F, Coppola R, De Feo V. Essential oils and antifungal

activity. Pharmaceuticals 2017; 10(4):86.

Page 17 of 18

Journal Pre-proof


[31] Kalemba D, Kunicka A. Antibacterial and antifungal properties of essential oils. Curr

Med Chem 2003; 10(10):813-29.

[32] Mota ML, Lobo LT, Costa JM, Costa LS, Rocha HA, Rocha e Silva LF, Pohlit AM,

Neto VF. In vitro and in vivo antimalarial activity of essential oils and chemical components

from three medicinal plants found in north eastern Brazil. Planta Med 2012; 78(7): 658-64.

[33] Innsan MF , Shahril MH , Samihah MS , Asma OS , Radzi SM, Jalil AKB and Hanina

MN Pharmacodynamic properties of essential oils from Cymbopogon species. Afr J Pharm

Pharmacol 2011; 5(24): 2676-2679.

[34] de Castro RD, de Souza TMPA, Bezerra LMD, Ferreira GLS, Costa EMMDeB,

Cavalcanti AL. Antifungal activity and mode of action of thymol and its synergism with

nystatin against Candida species involved with infections in the oral cavity: an in vitro study.

BMC Complement Altern Med 2015; 15: 417.

[35] Khan MS, Ahmad I, Cameotra SS. Carum copticum and Thymus vulgaris oils inhibit

virulence in Trichophyton rubrum and Aspergillus spp. Braz J Microbiol 2014;45(2):523–


[36] Sharifzadeh A, Khosravi AR, Shokri H, Shirzadi H. Potential effect of 2-isopropyl-5-

methylphenol (thymol) alone and in combination with fluconazole against clinical isolates of

Candida albicans, C. glabrata and C. krusei. J Mycol Med 2018; 28(2):294-299.

[37] Cristani M, D’Arrigo M, Mandalari G,Castelli F, Sarpietro MG, Micieli D, Venuti V,

Bisignano G, Saija A, Trombetta D. Interaction of four monoterpenes contained in essential

oils with model membranes: implications for their antibacterial activity. J Agric Food Chem

2007; 55: 6300–6308.

[38] Chouhan S, Sharma K, Guleria S. Antimicrobial activity of some essential oils—present

status and future perspectives. Medicines 2017; 4(3):58.

[39] Fauvart M, De Groote VN, Michiels J. Role of persister cells in chronic infections: clinical

relevance and perspectives on anti-persister therapies. J Med Microbiol 2011; 60 (6): 699-709.

[40] Wood TK, Knabel SJ, Kwan BW. Bacterial persister cell formation and dormancy.

Appl Environ Microbiol 2013; 79:237116-237121.

[41] Guo N, Liu J, Wu X, Bi X, MengR, Wang X, Xiang H, Deng X, YuL.Antifungal

activity of thymol against clinical isolates of fluconazole-sensitive and -resistant Candida

albicans. J Med Microbiol 2009; 58(8):1074-1079.

[42] Faria NC, Kim JH, Gonçalves LA, Martins MdeL, Chan KL, Campbell BC. Enhanced

activity of antifungal drugs using natural phenolics against yeast strains of Candida and

Cryptococcus. Lett Appl Microbiol 2011; 52(5):506-13.

Page 18 of 18

Journal Pre-proof


[43] Mourad A, Perfect JR. Tolerability profile of the current antifungal armoury. J

Antimicrob. Chemother 2018; 73(suppl_1):i26-32.

[44] Benameur Q, Gervasi T, Pellizzeri V, Pľuchtová M, Tali-Maama H, Assaous F, Guettou

B, Rahal K, Gruľová D, Dugo G, Marino A. Antibacterial activity of Thymus vulgaris

essential oil alone and in combination Fluconazole with cefotaxime against bla ESBL producing multidrug

resistant Enterobacteriaceae isolates. Nat. Prod. Res. 2019; 33(18):2647-2654.

[45] Nowak A, Kalemba D, Piotrowska M, Czyżowska A. Effects of thyme (Thymus vulgaris

L.) and rosemary (Rosmarinus officinalis L.) essential oils on growth of Brochothrix

thermosphacta. Afr. J. Microbiol. 2013; 7(26):3396-404.

[46] Satyal P, Murray B, McFeeters R, Setzer W. Essential oil characterization of Thymus

vulgaris from various geographical locations. Foods 2016; 5(4):70.