For this study, 51 strains of Candida spp. (16 Candida albicans; 12 Candida tropicalis; 11 Candida krusei; 12 Candida parapsilosis sensu lato), and 25 strains of Cryptococcus spp. (13 C. neoformans – serotypes A, D and AD; and 12 Cryptococcus gattii – serotypes B and C) isolated from animals were used. The isolates belong to the culture collection of the Specialized Medical Mycology Center, Brazil. The purity and identity of the Candida spp. strains were confirmed by growth on chromogenic medium and microscopical and biochemical features.20 For the Cryptococcus spp. strains, capsule formation, melanin production, and biochemical testing were evaluated and the serotype of each strain was assessed by PCR.21

Susceptibility assays were performed using the broth microdilution method, as described by the document M27-A3 of the Clinical and Laboratory Standards Institute.22 The tests were performed in duplicate and visually read after 48h of incubation at 35°C. The strains C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 were included as quality control for all tests.22

Inocula were prepared to obtain a final concentration of 0.5–2.5×103cellsmL−1.22 The statins simvastatin (Medley Indústria Farmacêutica Ltda, Campinas, SP, Brazil), atorvastatin (Laboratórios Pfizer Ltda, São Paulo, SP, Brazil), and pravastatin (Bristol-Myers-Squibb, Nova York, NY, USA) and the antifungal drugs amphotericin B (Sigma Chemical Corporation, St Louis, USA), itraconazole (Janssen Pharmaceutica, Beerse, Belgium), and fluconazole (Pfizer Pharmaceuticals, New York, USA) were tested against all strains.

To obtain the stock-solutions of each drug, atorvastatin, pravastatin, and fluconazole were diluted with sterile distilled water, and amphotericin B and itraconazole were diluted with dimethylsulfoxide (DMSO). Simvastatin was activated from its lactone prodrug form through hydrolysis in ethanolic NaOH (15% (v/v) ethanol, 0.25% (w/v) NaOH), at 60°C, for 1h.15 The concentration range tested was 3.9–2000μgmL−1 for simvastatin, 19.5–10,000μgmL−1 for atorvastatin, 97.6–50,000μgmL−1 for pravastatin, 0.0312–64μgmL−1 for amphotericin B and itraconazole, and 0.25–256μgmL−1 for fluconazole. The minimum inhibitory concentration (MIC) was defined as the lowest drug concentration able to inhibit 100% of fungal growth for amphotericin B and 50% inhibition of fungal growth when compared to the free-drug azoles22 and statins control.

As simvastatin provided the best antifungal results, we evaluated the interaction between this drug and the antifungal drugs against the tested yeasts. For drug interaction studies, simvastatin was tested with each azole by broth microdilution method, using the MIC of each tested drug alone as the highest concentrations tested in combination. The concentrations of the drugs in combination ranged from 0.03 to 1000, 0.00024 to 2, 0.00006 to 64 and 0.00048 to 256μgmL−1 for simvastatin, amphotericin B, itraconazole and fluconazole, respectively. The reading criteria were the same as for the antifungal drugs alone, namely 100% inhibition when combined with amphotericin B and 50% inhibition when combined with azoles. The interaction between the drugs was analyzed by calculating the fractional inhibitory concentration index (FICI), with values ≤0.5 indicating synergism.23

Simvastatin was tested against growing biofilms and mature biofilms of Candida spp. and Cryptococcus spp. Amphotericin B and itraconazole were used in all tests as control drugs for biofilm inhibition. The tests were performed in triplicate using one biofilm-producing strain of each tested fungal species (C. albicans, C. tropicalis, C. parapsilosis, C. krusei, C. neoformans and C. gattii), according to the methodology described by Chatzimoschou et al.,24 with some modifications. Briefly, strains were grown on Sabouraud dextrose agar for 48h at 30°C and then subcultured into Sabouraud dextrose broth for 24h, at 30°C, under agitation at 150rpm. After this period, the suspensions were centrifuged at 3000rpm for 10min, the supernatant was discarded, and the pellet was washed twice with sterile PBS. Then, the pellet was resuspended in RPMI 1640 medium (Gibco-BRL, USA), reaching a concentration of 1×106cellsmL−1. Tests were performed in 96-well polystyrene plates.

To evaluate the effect of simvastatin, amphotericin B, and itraconazole on growing biofilm, 100μL of the fungal suspension was exposed to 100μL of simvastatin and incubated at 35°C for 48h. The tested concentrations were based on the MIC obtained for each drug against fungal planktonic growth, including MIC, 10xMIC and 50xMIC. On the other hand, to evaluate the effect of simvastatin and the antifungals alone against mature biofilm, 100μL of the fungal suspension was added to 100μL of RPMI 1640 medium and incubated at 35°C for 48h. Then, the mature biofilms were exposed to simvastatin, amphotericin B, and itraconazole and incubated at 35°C for 48h. The tested concentrations against mature biofilms were 10xMIC, 50xMIC and 100xMIC. For all tests, drug-free growth control for each strain was included.

After 48h of drug exposure, the growing and mature biofilms were submitted to the following procedures: supernatants were collected and reserved for further analysis, and plates were washed twice with sterile PBS Tween 20 (0.05%, v/v) solution to remove non-adhered cells. Then, the biofilm viability was evaluated through XTT assay, according to Martinez and Casadevall,25 with modifications. Stock solutions of XTT (1mgmL−1) were previously prepared, filtrated and stocked at −20°C, until used. Menadione (Sigma) (0.4mM in acetone) was prepared at the moment of use. Afterwards, 50μL of sterile PBS, 75μL of XTT solution, and 6μL of menadione solution were added to each well. Plates were incubated at 35°C during 5h, in the dark, and then XTT was all transferred to a new plate and read in a spectrophotometer at 492nm.

For analysis of the MIC data of drugs against planktonic cells, Student's t test for independent and paired samples was used. To check the variation of the MIC values of the drugs in combination, as well as the FICI value, Student's t test for paired samples was also used. Regarding the biofilm assay, all tests were made in triplicate and results were evaluated by ANOVA and Tukey's multiple comparison post-test. p-Values<0.05 were considered statistically significant.

Among the tested statins, simvastatin showed the lowest MIC, with geometric means varying from 29.45 to 567.16 and from 62.5 to 500mgL−1 against the genera Candida and Cryptococcus, respectively (Table 1). Atorvastatin showed better results against Candida species, when compared to Cryptococcus spp., with MIC geometric means varying from 52.06 to 1682.37mgL−1 against Candida spp. and from 3886.02 to >10,000μgmL−1 against Cryptococcus spp. (Table 1). As for pravastatin, the MIC geometric means varied from 2159.24 to >50,000mgL−1 against Candida spp. and from 44079.56 to >50,000mgL−1 against Cryptococcus (Table 1). MIC geometric means for the classic antifungals against Candida spp. varied from 0.343 to 1.624mgL−1 for amphotericin B, from 0.046 to 15.021mgL−1 for itraconazole, and from 0.659 to 82.346mgL−1 for fluconazole. MIC geometric means for classic antifungals against Cryptococcus spp. varied from 0.125 to 0.735mgL−1 for amphotericin B, from 0.189 to 1mgL−1 for itraconazole, and from 6.498 to 64mgL−1 for fluconazole (Table 1).

Results for the in vitro interaction between simvastatin and antifungal drugs against these yeasts are shown in Table 2. In general, a synergistic interaction was observed between simvastatin and both azoles against C. albicans (n=10/10), C. tropicalis (n=11/11) and C. parapsilosis sensu lato (n=12/12) (p<0.05). Concerning C. krusei, only the combination of simvastatin and itraconazole was synergistic (n=9/10) (p<0.05). As for Cryptococcus spp., synergistic interactions were observed between simvastatin and the three antifungals tested against C. gattii (simvastatin/amphotericin B and simvastatin/fluconazole: n=7/7; simvastatin/itraconazole: n=5/7) and C. neoformans (simvastatin/amphotericin B: n=11/11; simvastatin/itraconazole: n=9/11; simvastatin/fluconazole: n=10/11) (p<0.05).

Regarding the action of simvastatin against biofilm of Candida spp., simvastatin inhibited growing biofilms at concentrations greater than 10xMIC (Fig. 1). Amphotericin B caused significant decrease in metabolic activity of growing biofilms at 10xMIC and 50xMIC, while itraconazole caused inhibition at all tested concentrations (p<0.05). As for mature biofilms, simvastatin caused significant decrease in metabolic activity (Fig. 1) (p<0.05), at concentrations above 50xMIC. Amphotericin B inhibited mature biofilms at all tested concentrations, while itraconazole only at 50xMIC and 100xMIC (p<0.05).

Fig. 1.

In vitro effect of simvastatin (SIM), amphotericin B (AMB), and itraconazole (ITC) at 3 different concentrations on biofilm formation (A) and mature biofilm (B) of Candida spp. Black bars: positive control; MIC: minimum inhibitory concentration. Absorbance of XTT (492nm). *Represents statistically significant difference (p<0.05) when compared to the positive control. Data expressed as mean±SEM.

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Regarding the genus Cryptococcus, simvastatin inhibited growing biofilms at all tested concentrations (Fig. 2) (p<0.05), similar to what was observed when amphotericin B and itraconazole were used (p<0.05). Concerning mature biofilms, simvastatin caused significant decrease in metabolic activity of Cryptococcus biofilm at 50xMIC and 100xMIC (Fig. 2) (p<0.05). Amphotericin B inhibited mature biofilms at all tested concentrations (p<0.05), while itraconazole did not decrease biofilm metabolic activity at any tested concentration.

Fig. 2.

In vitro effect of simvastatin (SIM), amphotericin B (AMB), and itraconazole (ITC) at 3 different concentrations on biofilm formation (A) and mature biofilm (B) of Cryptococcus spp. strains. Black bars: positive control; MIC, minimum inhibitory concentration. Absorbance of XTT (492nm). *Represents statistical significant difference (p<0.05) when compared to the positive control. Data expressed as mean±SEM.

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