In vitro synergy of eugenol and methyleugenol with fluconazole against clinical Candida isolates

Abstract

The species Candida is a group of opportunistic pathogenic commensals in immune-compromised patients. Treatment of Candida infections is becoming increasingly difficult due to antifungal drug resistance, especially with fluconazole (FLC), which is a commonly used azole. In the present study the in vitro antifungal activity of eugenol (EUG) and methyleugenol (MEUG) alone and in combination against 64 FLC-sensitive and 34 FLC-resistant clinical Candida isolates is highlighted. All the strains were susceptible to both the naturally occurring phenyl propanoids. The nature of the interaction was studied from fractional inhibitory concentration indices (FICIs) for both EUG plus FLC, and MEUG plus FLC combinations calculated from chequerboard microdilution assays. FICI values depicted a high synergism of FLC with both compounds, which was greatest with MEUG. FLC-resistant Candida isolates showed high sensitivity to both compounds. No antagonistic activity was seen in the strains tested in the present study. From these results we suggest that EUG and MEUG have great potential as antifungals, and that FLC can be supplemented with EUG and MEUG to treat FLC-resistant Candida infections.

↵†These authors contributed equally to this work.

INTRODUCTION

Candida, which includes around 200 yeast species (Eggimann et al., 2003), is an opportunistic commensal of the human oral, gastrointestinal, vaginal, cutaneous and mucosal surfaces. Candida albicans is the predominant causative organism of virtually all types of candidiasis (Filler & Sheppard, 2006), but other emerging Candida species, including Candida glabrata, Candida krusei, Candida tropicalis and Candida parapsilosis, are now posing serious nosocomial threats to patient populations (Koehler et al., 1999; Chakrabarti et al., 2009). The incidence of candidiasis and other fungal infections has increased in the past few decades. The currently available drugs are insufficient as they have undesirable side effects, are ineffective against new or re-emerging fungi and lead to the rapid development of resistance.

Azoles have been a predominant therapy drug for Candida infections for a long time now. Fluconazole (FLC) is the most widely used azole for systemic candidiasis due to its high solubility, low toxicity and wide tissue distribution (Brammer et al., 1990). It targets lanosterol 14α-demethylase in the ergosterol biosynthetic pathway (Vanden Bossche et al., 1987). However, treatment failures are increasing, especially in AIDS patients, as prolonged use of this drug has led to resistance in Candida spp. probably due to the fungistatic rather than fungicidal nature of its antifungal action (Sanglard et al., 2003; Uppuluri et al., 2008). New therapeutic strategies are required to reduce the toxicity of these drugs. The efficacy of FLC and other antifungal agents can be improved by using combination therapy (Pinto et al., 2009; Ghannoum et al., 1995; Mukherjee et al., 2005; Tariq et al., 1995).

Plant essential oils possess a broad spectrum of antimicrobial properties that have been described in several studies (Shin & Pyun, 2004; Rosato et al., 2008; Guo et al., 2009). Phytochemicals that could be developed into useful antimicrobials for the treatment of infectious diseases are yet to be exploited. Eugenol (EUG) [2-methoxy-4-(2-propenyl)phenol] and methyleugenol (MEUG) [1, 2-dimethoxy-4-(2-propenyl)phenol] are two microbiocidal phenyl propanoids (Fig. 1⇓). They are found as major components in the essential oils of many aromatic plants, such as clove (Eugenia caryophyllus) and basil (Ocimum sanctum). Both possess antimycotic properties (Pinto et al., 2006; Khan et al., 2010). In a recent study we showed that EUG and MEUG contribute largely to the antifungal activity of basil oil, and hence have therapeutic potential (Khan et al., 2010). We also suggested that the compounds exert their antifungal activity by targeting sterol biosynthesis (Ahmad et al., 2010). The present study was undertaken to evaluate the efficacy of EUG and MEUG as antifungal agents against standard Candida strains, as well as both FLC-susceptible and -resistant clinical isolates using the Clinical and Laboratory Standards Institute (CLSI) microdilution method and agar disc diffusion assay. The in vitro activity of EUG and MEUG in combination with FLC was also investigated against both FLC-susceptible and -resistant Candida isolates using the chequerboard microdilution assay. The present work highlights a promising role for essential oil components (EUG and MEUG) used in combination with the most widely used azole, FLC, to treat resistant Candida infections.

Figure image not available in archive

Fig. 1.

Chemical structures of EUG (a) and MEUG (b).

METHODS

Strains and media.

Sixty-four FLC-sensitive and thirty-four FLC-resistant Candida strains were used in this study (Tables 1⇓ and 2⇓). The strains were grown in YPD media containing (w/v): 1 % yeast extract, 2 % peptone and 2 % dextrose. Strains were maintained on YPD plates with 2.5 % (w/v) agar. FLC and DMSO were obtained from Sigma Chemicals. EUG and MEUG were purchased from Aldrich; whereas, all inorganic chemicals were of analytical grade and were procured from Merck. The clinical Candida isolates were collected and identified by the Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India, and the Department of Pathology, Safdarjung Hospital, New Delhi, India. The patients and sample details were noted in the records (as allotted registration numbers).

Table 1.

Susceptibilities of standard and clinical FLC-sensitive Candida isolates to EUG and MEUG alone and in combination with FLC

MIC and FICI values are shown as a mean of three independent experiments.

Table 2.

Susceptibilities of FLC-resistant clinical Candida isolates to EUG and MEUG alone and in combination with FLC

MIC and FICI values are shown as a mean of three independent experiments.

Antifungal-susceptibility test.

The MICs of EUG, MEUG and FLC were determined by the CLSI broth microdilution method (CLSI, 2002). Cultures, with a final inoculum size of 1×10³ c.f.u. ml⁻¹, were grown with and without the test compounds. Stock solutions of test compounds or FLC were prepared in 10 % DMSO and then further diluted in distilled water to the desired concentration ranges. DMSO was always ≤1 % of the total volume. The compounds were diluted twofold initially, with a final concentration range of 50–1600 μg ml⁻¹ for test compounds and 0.125–128 μg ml⁻¹ for FLC. Once the concentration resulting in no visible growth was observed, the MIC range was further refined to find exact MIC values. In the second stage, concentrations of compounds were directly introduced with an initial step difference of 5.0 μg ml⁻¹ (FLC) and 50 μg ml⁻¹ (EUG, MEUG); and a final step difference of 0.25 μg ml⁻¹ (FLC) and 5 μg ml⁻¹ (EUG, MEUG). The tests were carried out in 96-well flat-bottomed microtitration plates, which were incubated at 35 °C for 24 h. Optical density was recorded at 630 nm for each well with a microplate reader. Experiments were performed in triplicate and the mean MIC values were calculated. The MIC of each drug in combination or alone was defined as the lowest drug concentration that resulted in a 90 % decrease in absorbance compared with that of the control (no drug). The absence of a trailing effect enabled the use of MIC₉₀ as end point instead of the standard MIC₈₀.

Chequerboard microdilution assay.

Drug interaction was studied using the chequerboard microdilution assay in 96-well microtitre plates. Assays were performed on all the isolates (64 FLC-sensitive and 34 FLC-resistant Candida isolates) according to methods of the CLSI (2002). The initial concentration of cell suspensions in the medium was 1×10³ c.f.u. ml⁻¹. The final concentrations ranged from 50 to 800 μg ml⁻¹ for test compounds and 0.125 to 128 μg ml⁻¹ for FLC. The compounds were diluted twofold initially and once the concentration resulting in no visible growth was observed, the MIC range was further refined to get exact MIC values. In the second stage, concentrations of compounds were directly introduced with an initial step difference of 1.0 μg ml⁻¹ (FLC) and 50 μg ml⁻¹ (EUG, MEUG); and a final step difference of 0.1 μg ml⁻¹ (FLC) and 5 μg ml⁻¹ (EUG, MEUG). Plates were incubated at 35 °C for 24 h and the optical density was measured at 630 nm. Each isolate was tested in triplicate.

To assess the interaction of combinations of drugs (EUG plus FLC and MEUG plus FLC) the data obtained spectrophotometrically were further analysed using the fractional inhibitory concentration index (FICI), which is based on the zero-interaction theory of Loewe additivity (Berenbaum, 1989). This theory suggests that a drug cannot interact with itself and that the concentrations of the drugs, alone or in combination, that produce the same effect are compared. FICI was defined as: FICI=FIC_A+FIC_B=MIC_A in combination/MIC_A tested alone+MIC_B in combination/MIC_B tested alone, where MIC_A and MIC_B are the MICs of drugs A and B, respectively. Synergy and antagonism were defined by a FICI of ≤0.5 and >4, respectively. A FICI result of >0.5 but ≤4 was considered as a result of indifference (Cantón et al., 2005).

Disc diffusion assay.

The assay was performed as discussed previously (Ahmad et al., 2010). Briefly, strains were inoculated into liquid YPD media and grown overnight at 37 °C. Cells were then washed three times with distilled water and approximately 10⁵ cells ml⁻¹ were inoculated into molten YPD agar at 42 °C and poured into 100 mm diameter Petri plates. Sterile filter discs (4 mm) were impregnated with drugs alone, and in combination, and placed on the agar plates. The final concentrations for EUG, MEUG and FLC were 500, 350 and 10 μg per disc, respectively, corresponding to their MICs. To determine the combined effects, each sterile filter disc was impregnated with a mixture of both drugs (a combination of their MIC values). The test compounds were initially dissolved in 10 % DMSO and then further diluted in distilled water to the desired concentration ranges for their respective MICs. The diameters of the zones of inhibition (ZOIs) were measured in millimetres after an incubation period of 48 h at 37 °C. The experiment was repeated three times. The type of halo formed depicted fungicidal/static characteristic of the test compound (Onyewu et al., 2003). The sensitivity index (SI) was calculated for both the FLC-susceptible and -resistant Candida isolates and was defined as: diameter of ZOI (mm)/concentration of drug (mg ml⁻¹)=clearing (mm mg⁻¹). The SI values are shown as mean± sem.

RESULTS AND DISCUSSION

Treating Candida infections with monotherapy is becoming more difficult, a major problem being the emerging drug resistance during treatment with various antibiotics. Hence, there is a need to search for alternate safe and effective non-antibiotic agents that have few or no side-effects. The present study was undertaken to evaluate the efficacy of two bioactive components found in essential oils of some medicinal plants (EUG and MEUG) as antifungal agents against FLC-susceptible and -resistant Candida isolates, and analyse their synergy with FLC.

MIC of EUG and MEUG against Candida

The MICs and FICI values obtained for each of the isolates are shown in Tables 1⇑ and 2⇑. Table 1⇑ gives the values for standard and FLC-sensitive clinical strains, while Table 2⇑ gives the same for FLC-resistant isolates. The MICs of EUG and MEUG against various clinical FLC-sensitive and -resistant Candida strains including five American Type Culture Collection (ATCC) type strains (C. albicans ATCC 10261, C. albicans ATCC 90028, C. albicans ATCC 44829, C. tropicalis ATCC 750 and C. glabrata ATCC 90030) range from 475 to 500 μg ml⁻¹ and 340 to 350 μg ml⁻¹, respectively (Tables 1⇑ and 2⇑). The MIC of FLC against clinical FLC-sensitive Candida isolates was 2.5–7.5 μg ml⁻¹ and was within the reference ranges. It should be noted that isolates intrinsically resistant to FLC (MICs 80–110 μg ml⁻¹) also show sensitivity to both the compounds EUG and MEUG (Table 2⇑). However, it is clear that MEUG proved to be more active against all Candida strains than EUG. These findings validate our previous results (Khan et al., 2010; Ahmad et al., 2010).

Drug susceptibility in combination with FLC

The FICIs for EUG and its derivative MEUG in combination with FLC were calculated. As is evident from Tables 1⇑ and 2⇑, the results of the chequerboard microtitre assay indicated significant combined effects between EUG/MEUG and FLC calculated for FLC-sensitive (Table 1⇑) and FLC-resistant Candida isolates (Table 2⇑). FICI values for EUG plus FLC and MEUG plus FLC combinations against all FLC-sensitive Candida isolates studied here ranged from 0.31 to 0.55 and 0.24 to 0.58, respectively. Out of 64 FLC-susceptible Candida isolates tested (59 clinical isolates and 5 ATCC strains), the interaction between EUG and FLC was synergistic in 58, whereas for MEUG and FLC, 59 showed synergy and only 5 isolates showed indifference by the FICI method. Out of 34 FLC-resistant Candida strains tested, 29 and 31 isolates showed synergistic affects for EUG plus FLC and MEUG plus FLC, respectively.

In vitro activities of EUG and MEUG alone and in combination with FLC against all Candida isolates were determined by disc diffusion assay. The antifungal activity of both compounds increased with increasing concentration. All the FLC-susceptible and -resistant Candida isolates showed a high degree of sensitivity as was evident from large inhibition zones. DMSO (used on the control disc) showed no antifungal activity. Thus, both EUG and MEUG showed a powerful fungicidal effect, visible on the agar plate, when combined with FLC.

Table 3⇓ shows in vitro sensitivity of Candida isolates to EUG and MEUG alone and in combination with FLC. The SI was defined as the ratio of the diameter of the ZOI (mm) to the concentration of each drug (mg ml⁻¹). The SI values were greater for MEUG than EUG in all the tested Candida isolates indicating the higher potency of this compound. Standard and clinical isolates were, respectively, 1.39 times and 1.55 times more sensitive to FLC than to MEUG. Interestingly the strains that were resistant to FLC, and hence showed no sensitivity to it, showed an SI value of 1.693 and 1.739 with EUG and MEUG, respectively. From this result we can conclude that FLC-resistant strains show an even higher sensitivity to these essential oil components than standard or clinical strains. In standard strains, the SI increased 2.85-fold and 3.03-fold for FLC when used in combination with EUG and MEUG, respectively. In clinical strains these values were 2.75-fold and 2.87-fold, respectively. The SI for FLC in the resistant strains was 5.45 and 6.25 when used in combination with EUG and MEUG, respectively. Hence, it is very obvious (Table 3⇓) that FLC-resistant isolates show more sensitivity than the other isolates to EUG and MEUG alone, and even greater sensitivity when EUG and MEUG were used in combination with FLC. While variability may occur among the two essential oil components to the drug combinations, it is exciting that the fungicidal potential of normally fungistatic FLC can be revealed, as is evident from the clear zones of inhibition in the disc diffusion assay. Additional in vitro synergy tests are required on new fungal strains and other pathogens to evaluate the antimicrobial potentials of EUG and MEUG and other essential oil components. With this synergistic combinatorial approach many antifungal agents may find even broader therapeutic applications.

Table 3.

In vitro sensitivity of Candida isolates to EUG and MEUG alone and in combination with FLC as determined by disc diffusion assay

Each isolate was tested in duplicate. SI is expressed as mean±sd and was calculated as the diameter of the ZOI (mm)/concentration of drug (mg ml⁻¹). n, Number of isolates.

As shown previously, EUG and MEUG both caused only 5–7 % haemolysis at their respective MICs of 500 and 350 μg ml⁻¹, showing their low cytotoxic activity, while FLC and amphotericin B showed more than 80 % cell lysis at the same concentration ranges (Ahmad et al., 2010). In the combination experiments against Candida strains, EUG and MEUG were used at their MICs, which led to minimal haemolysis. Hence, our studies indicate that these compounds were able to induce a synergistic effect with FLC below their cytotoxic concentrations against all the tested Candida species and strains. Although these compounds appear to be weaker antifungals than FLC, they showed better activity against FLC-resistant Candida strains.

A number of plants and plant products are known to possess potent medicinal properties (Cowan, 1999; Arif et al., 2009). Synergism of natural products and antibiotics against pathogenic micro-organisms is a major thrust of research in medical microbiology, leading to the development of novel potential phytopharmaceuticals. Many studies have shown the synergistic action of essential oil fractions from different plants with synthetic drugs as antifungal agents. The role of the principal components of the essential oil fractions in such an interaction is less well understood and has not been explored thoroughly. Similar studies showing the synergistic effect of essential oils with amphotericin B and nystatin against Candida spp. have been reported elsewhere (Rosato et al., 2009; Hadizadeh et al., 2009). The present study, however, explores FLC, a member of a major class of antifungals in clinical practice, against a wide variety of Candida isolates. The importance of this study lies in the fact that EUG and MEUG were shown to be synergistically active against FLC-resistant Candida isolates. The study is significant as there is a rise in cases of antifungal resistance, especially FLC resistance in patients. Our findings suggest options for expanding the utility of essential oil components as antifungal agents.

Not much is known about the mode of action of EUG and MEUG. It has been reported that their lipophilic character enables them to enter between the fatty acyl chains making up membrane lipid bilayers, altering the fluidity and permeability of cell membranes. These compounds thus affect the activity and regulation of important membrane-bound enzymes that catalyse the synthesis of a number of major cell wall polysaccharide components, such as β-glucans, chitin and mannan, interfering with cell growth and envelope morphogenesis (Pina-Vaz et al., 2004; Braga et al., 2007). We have reported previously that they exert antifungal activity by targeting the sterol biosynthetic pathway (Ahmad et al., 2010). An investigation into their mode of action needs to be carried out.

In the present study we have demonstrated that essential oil components EUG and MEUG are effective antifungal agents showing potent in vitro antifungal activity against different species of Candida like C. albicans, C. tropicalis, C. parapsilosis, C. krusei and C. glabrata, and including Candida species that are intrinsically resistant to FLC. The interaction between EUG and FLC, and MEUG and FLC showed a high amount of synergism. No antagonistic interaction was observed in the strains tested. FLC administered in combination with essential oil components like EUG and MEUG will have augmented efficacy and thus have a reduced minimum effective dose. Our findings are encouraging in view of the growing treatment failures and antibiotic resistance in Candida and suggest a way of treating resistant Candida infections through a drug combination approach. Further in vivo studies with other fungi will assess the potential of these compounds for therapeutic applications.

Acknowledgments

This work was supported by the Indian Council of Medical Research, India (grant no. 59/24/2008/BMS/TRM [2008-04780]), to N. M. and L. A. K. The authors wish to thank Dr Malini R. Capoor, Safdarjung Hospital, New Delhi, India, and Dr I. Xess, All India Institute of Medical Sciences, New Delhi, India, for providing clinical Candida isolates.

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