The MICs and SICs of the azoles and statins were assessed using the herein proposed agar-well diffusion bioassay prior to testing their synergism. According to the azole MICs, C. utilis was more sensitive than S. cerevisiae to clotrimazole, fluconazole and itraconazole (Table 1). On the contrary, the MICs for atorvastatin, lovastatin, rosuvastatin and simvastatin were 40μg/ml against S. cerevisiae whilst 200μg/ml in the case of C. utilis, with corresponding doses of 1 and 5μg per well, respectively. Pravastatin showed the lowest yeast inhibition power, with a MIC of 200μg/ml against both yeasts. Based on these first results, the SICs of statins used for synergism tests were 4μg/ml for S. cerevisiae and 20μg/ml for C. utilis, whereas in the case of pravastatin, 20μg/ml was applied for both yeast strains.

When azoles were applied in combination with atorvastatin, lovastatin, rosuvastatin and simvastatin, inhibition haloes became increased in comparison to the independent azole effect thus giving evidence of their synergism. The growth inhibition potentiation ranged between 25.3 and 77.5% for S. cerevisiae (Fig. 1a), and between 7.0 and 43.2% for C. utilis (Fig. 1b). Pravastatin exhibited the mildest synergism in both yeasts, with increment values around 0–7.6% for S. cerevisiae, and between 0 and 4% against C. utilis. The best combination according to the in vitro activity resulted from the association between the SIC of simvastatin and the MIC of miconazole, displaying a 77.5% inhibition enhancement against S. cerevisiae, and 43.2% against C. utilis (Fig. 1a and b).

Fig. 1.

Synergism between clotrimazole (CLT), fluconazole (FLC), itraconazole (ITC), ketoconazole (KTC) and miconazole (MCZ) at MIC, in combination with SICs of lovastatin, pravastatin, simvastatin, rosuvastatin and atorvastatin, as witnessed by the agar-well diffusion bioassay with S. cerevisiae ATCC 32051 (a) and C. utilis Pr 1–2 (b). Results are expressed as the percent increase in growth inhibition haloes in comparison to the individual azole inhibitory effects and interaction ratios (IRs) are also annotated. MIC, minimal inhibitory concentration; SIC, sub inhibitory concentration.

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According to the IR criterion, the best combinations against S. cerevisiae were those between either atorvastatin, rosuvastatin or simvastatin at SIC with MICs of clotrimazole (IR, 2.2–2.6), itraconazole (IR, 2.3–2.6) or miconazole (IR, 2.6–3). Usually, the highest values corresponded to simvastatin associations. In the case of C. utilis, the most significant synergistic effects were observed for miconazole–rosuvastatin (IR, 1.8) and miconazole–simvastatin (IR, 2.2). For both yeasts, the highest IR values corresponded to miconazole–simvastatin associations (Fig. 1a and b).

In order to confirm the presence of a synergistic effect between statins and azoles and, to study the synergism influence on total cellular sterols content, the tested yeasts strains were grown in liquid culture media amended with either miconazole, or miconazole plus simvastatin. All of them were added at the SIC value previously assayed in liquid cultures (data not shown), which corresponded to 2.5μg/ml simvastatin plus 0.5μg/ml miconazole for S. cerevisiae, and 12.5μg/ml simvastatin plus 1.0μg/ml miconazole for C. utilis. The simultaneous use of azole and statin SIC values complied with the necessity of obtaining a suitable amount of yeast biomass for sterol extraction.

As it could be noted, the SICs of both miconazole and simvastatin were lower than those required on solid cultures (see Table 1 and results above), a fact not surprising considering the closest contact of yeasts and drugs, and the better diffusion rate under submerged culture conditions. Data concerning the sterol content reduction is displayed in Figs. 2 and 3. A preliminary indication was provided by TLC (Fig. 2), and these results were further confirmed by RP-HPLC analyses (Fig. 3) by using an ergosterol calibration curve (0–500μg/ml; R2=0.994).

Fig. 2.

Effects of miconazole (MCZ), simvastatin (Sim) and their synergic combination (MCZ+Sim) on the cellular sterols content of S. cerevisiae ATCC 32051 (a) and C. utilis Pr 1–2 (b) after growth in liquid cultures, as detected by TLC. (c) Graphical representation of TLC data, as analyzed by means of the ImageJ program. Percentages of reduction are referred to the control growth with no MCZ or Sim. Results from independent triplicate culture/extractions are displayed.

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Fig. 3.

Influence of miconazole (MCZ), simvastatin (Sim) and their synergic combination (MCZ+Sim) on the cellular ergosterol content of S. cerevisiae ATCC 32051 (a) and C. utilis Pr 1–2 (b) after growth in liquid cultures, according to RP-HPLC. (c) Graphical representation of RP-HPLC data. Percentages of reduction are referred to the control growth with no MCZ or Sim (7.68 and 12.71mg ergosterol/g biomass wet weight for S. cerevisiae or C. utilis, respectively). Results correspond to independent triplicate culture/extractions. AU: absorbance units. Inset in (a) corresponds to UV–vis spectrum of ergosterol peak.

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Analytical data revealed that the exposure of S. cerevisiae to simvastatin plus miconazole resulted in a 40%-higher ergosterol level reduction than the accumulated miconazole and simvastatin individual effects (Fig. 3a and c). Likewise, in the case of C. utilis, the same drugs association led to a 22%-enhanced ergosterol level drop as compared to the summation of simvastatin and miconazole separate effects (Fig. 3b and c).

Finally, in order to confirm the implication of a blocked ergosterol biosynthesis in the growth inhibition of tested yeast cells, cultures were supplemented with increasing concentrations of ergosterol. In general, yeast cell growth could be progressively restored as ergosterol concentration increased (Fig. 4). In reversion tests, ergosterol could be incorporated in ethyl acetate solutions with no need of combination with Tween 80, as previously indicated.23 Ergosterol rescue could be detected at concentrations between 5 and 25mmol/l.

Fig. 4.

Ergosterol rescue of statin-induced growth inhibition in S. cerevisiae ATCC 32051 (a) and C. utilis Pr 1–2 (b). Wells were filled in with either solutions containing statins at MIC, or a combination of statin (at MIC) plus variable concentrations of ergosterol (1–3) dissolved in ethyl acetate (see Results section). Ato: atorvastatin, Lov: lovastatin, Pra: pravastatin, Ros: rosuvastatin, Sim: simvastatin. Ergosterol concentrations: 0, none, 1=5mmol/l, 2=10mmol/l, 3=25mmol/l. Controls: (–)1, ethyl acetate; (–)2, ethyl acetate-dissolved ergosterol. MIC: minimal inhibitory concentration.

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The obtained results confirmed the rescue of statin-induced growth inhibition by means of ergosterol supplementation, both in S. cerevisiae and C. utilis. However, differences in ergosterol reversion could be observed among tested statins, pravastatin being the easiest inhibition to be abolished and rosuvastatin being the most ergosterol–refractory inhibition in both yeasts (Fig. 4). It should be also noted that, despite inhibition could be reverted to a higher or lesser extent, particularly for some statins, yeast growth could not be completely restored.

This work represents a practical approach for testing the synergistic antifungal interaction between several statins and azoles by means of the use of a straightforward agar-well diffusion bioassay. According to this methodology, the effects of five statins (atorvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin) and five azoles (clotrimazole, fluconazole, itraconazole, ketoconazole and miconazole) in twenty-five different antifungal combinations could be tested in a time-saving, simple, low-cost and reproducible bioassay.

Previous reports emphasized the relevance of studying the in vitro sensitivity to antifungals in order to obtain relatively reliable results to select the most appropriate drug for fungal treatment. To this end, continuous trials to normalize and standardize in vitro tests have been performed.9

Up to date studies published about the effect of statins, azoles or their combinations against yeasts were mostly performed in liquid cultures.11,13,21–23,27,32 Only Chin et al.11 mentioned in their work a preliminary test carried out on solid medium to determine antifungal activity of simvastatin, lovastatin, fluvastatin and pravastatin, but only fluvastatin was active under those conditions. That may have been due to the use of inactive-statin lactone forms, whose activation via hydrolysis is probably not possible under solid culture conditions. More recent work also briefly mentioned the growth inhibition effects of atorvastatin and simvastatin on C. glabrata during growth on solidified minimal media.40 However, statin–azole combined effects do not seem to have been previously assessed according to the herein proposed methodology.

The statin MIC values obtained in the present work would be in the order of those elsewhere reported by other researchers. The higher pravastatin concentrations eventually found may be related to the high hydrophilic nature of this statin, the most polar between natural statins. Lovastatin values ranging from 5 up to 200μg/ml have been already described for testing inhibitory effects and sterols levels modifications on yeasts.3,12,16,21 Macreadie et al.23 found MIC values higher than 40μg/ml for simvastatin and 100μg/ml for atorvastatin against Candida strains. Also for fluvastatin, a potent synthetic statin, complete inhibition of C. albicans at physiological pH would require around 50μg/ml,32 whilst MIC values above 128μg/ml were described for different Candida species and Cryptococcus neoformans.11 Distinct sensitivity to statins was also described by Lukács et al. in Mucor circinelloides,22 with MIC values in the order of those herein reported and being fluvastatin much more effective than lovastatin and simvastatin.

Controversy is frequently observed when comparing statins susceptibility data. That may be due to differences in the applied methodology and the strains involved.22 Divergence in susceptibility among fungal genera and species has been already demonstrated, although the underlying molecular background for this distinctive behaviour has not been discovered yet. A possible explanation may be a difference in HMG-CoA reductase gene copy numbers. Lukács et al. have elegantly demonstrated a decreased sensitivity to statins in M. circinelloides when the hmgR gene dose became increased by transformation.22 Likewise, differences on the effectiveness among statins have been correlated to structural divergences, which may influence the interaction with the catalytic site of HMG-CoA reductase.13

As also pointed out for azolic antifungals, variables such as culture medium, pH, inoculum size or reading criteria provide a wide range of sensitivities depending on the method used or the laboratory involved.9 For comparison purposes, the MIC definition may also account for differences. Some works, normally referring to liquid cultures, described MIC as the drug dose at which growth became reduced by 50%, as witnessed by optical density measurements.32,40 Nevertheless, there are authors that defined MIC as herein applied, i.e. the minimal concentration at which no visible growth occurred.3,21

By means of a different mechanism, statins by inhibiting HMG-CoA reductase, and azoles by blocking sterol C-14 α-demethylation, both interfere with sterol biosynthesis. These effects were herein confirmed by sterols qualitative evaluation and ergosterol quantification. Nevertheless, in comparison, azoles exhibit a certain degree of toxicity that has been already demonstrated.5,19 In this context, the possibility to evaluate the combined antifungal effects of statins with azoles may provide relevant information for alternative treatments.

As previously speculated, the statin-driven increase in cellular permeability to exogenous sterols may additionally augment the permeability, susceptibility or both, to antifungal azoles.21 Changes in the lipid structure and plasma membrane dynamics in lovastatin-treated Candida cells have been recently confirmed.16 As a further hypothesis for the enhancing effect observed, the ability of statins to reduce sterols esterification may lead to the accumulation of unsterified C-14 methyl sterols consequent on the azoles action, and the impossibility to accommodate them in cellular membranes would then inhibit yeast growth.21

Agar-well diffusion bioassays demonstrated that SICs of statins significantly potentiated the activity of azoles against yeast growth, except for pravastatin. Similarly, Nash et al. found no synergism between pravastatin and fluconazole against C. albicans isolates.27 The existing synergism between tested sterol biosynthetic inhibitors (SBIs), which would exert an attractive multiplying effect,12 might be applied for instance, to reduce the required doses of azoles for fungal treatments or eventually, for increasing their potency in the case of refractory infections.

Yeast strains herein tested have been already considered as low-virulent opportunistic pathogens.26,34 However, the bioassay herein developed could be also useful for testing sensitivity to new azole–statin combinations of more invasive or virulent pathogen fungal isolates. This work thus describes a technical tool with potential application for clinical purposes. Emergent resistance to conventional antifungal treatments has also reinforced the idea of continuing with sensitivity tests not only against new triazolic derivatives but also any other antifungal agent.9

It is currently known that statins alone are prescribed and administered despite already known eventual side effects such as rhabdomyolisis (0.3 fatal cases per 100,000 person-years). As it occurs with many other currently administered drugs, the knowledge on their side effects is under permanent investigation, and the results on this aspect are taken into consideration in order to evaluate the risk–benefit relationship of the drug when a highly refractory disease has to be attacked. In the case of statins, although the known side effects, they are widely recognized as the best and most effective drugs for treating unmanageable hypercholesterolemia when other resources fail.15

With respect to S. cerevisiae, it has been also described as an attractive experimental system for studying azole toxicity mechanisms.19 In the case of C. utilis, infections caused by non-albicans Candida species are progressively increasing and usually the cases are resistant to common antifungals, which emphasizes the need for novel therapies for their effective management.35 In addition, the use of antifungal azoles for prophylaxis or empirical therapy was shown to promote a shift to non-albicans Candida infections in immunocompromised or cancer patients.16 From another point of view, yeasts isolates currently in use for industrial or biotechnological processes may be potentially pathogenic.20,26,34 It would be therefore suitable to count with standardized and straightforward methodology for testing their sensitivity to currently available antifungal drugs and combinations.

Concerning ergosterol recovery from statin-induced inhibition, present results were in agreement with previous observations with reference to the sterol dose-dependent mode of action.23 Complete growth recovery could not always be achieved, even at the highest ergosterol concentrations. A slow sterol uptake as well as the deleterious statin-induced depletion of many other endproducts from the mevalonate pathway may account for the rescue fail by the sole ergosterol supplementation.40

In conclusion, the proposed agar-well diffusion bioassay showed to be a rapid, simple, low-cost and reliable approach to test antifungal activity of different statin–azole associations as compared to assays in liquid media. Synergism could be assessed with two different yeast species giving an idea of the not circumscribed potentiality of this evaluation tool.

The co-administration of statins may be useful not only for increasing the efficacy of the antifungal treatment or for decreasing the required doses of toxic azoles, but also for eventually obtaining desirable side effects such as tumour-abating properties.16,32,41 In the light of the obtained results, investigations to determine the applicability of these antifungals combinations to in vivo systems would deserve particular attention.

The authors declare no conflict of interest.