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 . 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%) . 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%) . 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% . Other species of Candida like C. tropicalis, C. parapsilosis and C. glabrata are also increasingly reported responsible for candidemia and other conditions . Furthermore, biofilm formation by Candida species has been documented on variety of medical devices such as catheters, dialysis, joint devices etc . 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 . 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 . T. vulgaris essential oil and its active constituent, thymol have been used as an antioxidant, anti-inflammatory, local anaesthetic, antiseptic, antibacterial and antifungal agent . 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  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 . 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 . 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  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 . 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 . 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 . 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.  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
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
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 . 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 .
The strains of C. albicans were also previously studied for their antifungal susceptibility
profile and found resistant to common antifungal drugs . 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 . 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 .
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
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 subMICs. Different concentrations including sub-MICs of T. vulgaris and thymol tested in our
previous study showed no cellular toxicity to red blood cells .
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
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 .
Page 13 of 18
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
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 . 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
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.
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