Synergic effect of simvastatin in combination with amphotericin B against environmental strains of Cryptococcus neoformans from northeastern Brazil: a prospective experimental study

BACKGROUND: Statins are used as cholesterol-lowering drugs and may also have direct antimicrobial effects. OBJECTIVE: To evaluate synergic interactions between simvastatin and both amphotericin B and fluconazole, against environmental strains of Cryptococcus neoformans isolated from captive birds’ droppings. DESIGNAND SETTING: Experimental study conducted at Federal University of Piauí, Parnaíba, in collaboration with Federal University of Triângulo Mineiro, Uberaba, Brazil. METHODS: Statin susceptibility tests of Cryptococcus neoformans samples were performed as prescribed in standards. Interactions of simvastatin with amphotericin and fluconazole were evaluated using the checkerboard microdilution method. Presence of these interactions was quantitatively detected through determining the fractional inhibitory concentration index (FICI). RESULTS: Isolates of Cryptococcus neoformans were obtained from 30 of the 206 samples of dry bird excreta (14.5%) that were collected from pet shops and houses. Ten isolates were selected for susceptibility tests. All of them were susceptible to amphotericin and fluconazole. All presented minimum inhibitory concentration (MIC) > 128 µg/ml and, thus, were resistant in vitro to simvastatin. An in vitro synergic effect was shown through combined testing of amphotericin B and simvastatin, such that six isolates (60%) presented FICI < 0.500. Two isolates showed considerable reductions in MIC, from 1 µg/ml to 0.250 µg/ml. No synergic effect was observed through combining fluconazole and simvastatin. CONCLUSION: These results demonstrate that simvastatin should be considered to be a therapeutic alternative, capable of potentiating the action of amphotericin B. However, further studies are necessary to clarify the real effect of simvastatin as an antifungal agent.


INTRODUCTION
Cryptococcus neoformans is an encapsulated and opportunistic yeast fungus that has worldwide distribution. In the urban environment, it is often found in soil contaminated with dried bird excreta, and thus can infect humans and other animals through inhalation. 1,2 Currently, the treatment for cryptococcosis is based on use of amphotericin B alone or in combination with 5-flucytosine or azoles, in the early stages of the disease. However, the high toxicity associated with these antifungals may restrict their use in special clinical settings, such as in cases of chronic kidney diseases. In situations of secondary prophylaxis, use of fluconazole is indicated to minimize the risk of recurrence of infection in patients who are still in an immunosuppressive state. 3 Treatment of cryptococcosis using azoles (e.g. fluconazole) requires long periods and can favor the emergence of microbial resistance. Although azoles are less toxic than polyenes, they present low efficacy for the initial treatment of the disease. In contrast, amphotericin B provides a more effective therapeutic response against the fungus, although its use should be limited because of its toxicity. 2 Emergence of antifungal resistance among environmental and clinical isolates of Cryptococcus neoformans has been described over the last few decades. Prolonged  patients with immunosuppressive disorders such as acquired immune deficiency syndrome (AIDS) may contribute towards antifungal resistance. [4][5][6] Development of new therapeutic strategies is therefore extremely important, given the low number of antifungals available and the scarcity of these drugs in limited-resource settings. In this context, new drugs can contribute towards treatment of cryptococcosis through enhancing the effect of traditional antifungals. 7 Statins are drugs that are used to treat cardiovascular diseases relating to high cholesterol levels in humans. The mechanism of action of statins is based on inhibition of 3-hydroxy-3-methyl-gl- Therefore, combining statins with antifungal agents could decrease the length of time for which infected patients are exposed to toxic drugs. Consequently, this would reduce the side effects from use of antifungal agents, especially in cases of emergence of fungal strains that are resistant to conventional treatment. 9,10 In this context, given the increasing numbers of cases of antifungal resistance and the toxicity presented by the conventional treatment scheme, the antifungal potential of simvastatin alone and in association with amphotericin B and fluconazole was tested on Cryptococcus isolates that were recovered from the dried feces of captive birds in northeastern Brazil.

Ethics statement
This study was approved by the Research Ethics Committee of the feces were picked up from the birdcages using sterile swabs and were inoculated into tubes containing 10 ml of sterile saline solution with 0.4 g/l of chloramphenicol. It was not possible to determine how long these samples had been exposed before the cleaning procedures were performed.
Isolates of Cryptococcus neoformans were obtained from 30 of the 206 samples of dry bird excreta (14.5%) that had been collected from pet shops and houses. Ten of these isolates were selected for susceptibility tests because all the 30 isolates belonged to the same molecular type.
Each of the 206 feces samples was placed in a separate tube (thus, there were 206 tubes). The tubes were shaken to mix the contents for two minutes and the samples were then left to settle at room temperature for 30 minutes. Next, 100 ml of the supernatant was inoculated onto Niger seed (Guizotia abyssinica) agar plates supplemented with 0.4 g/ml of chloramphenicol. Each sample was spread on four plates, incubated at 35 °C and examined daily for 10 days, macroscopically, in order to identify any presence of smooth, beige to dark brown colonies suggestive of Cryptococcus spp. 11 These colonies were streaked onto Sabouraud agar at 35 °C for 48 hours and were identified using the India ink test and urease.
The isolates thus obtained (which were clones) were then stored until the tests were performed. To ensure preservation, some of the cell masses were stored in distilled water at 4 °C, while others were stored in yeast peptone dextrose broth (Difco Laboratories, Sparks, MD, USA) with 30% glycerol at 4 °C.

Deoxyribonucleic acid extraction
Deoxyribonucleic acid (DNA) extraction was performed as previously described by Bolano et al. 12 Briefly, a single colony was spread on yeast malt agar (Difco Laboratories) at 37 °C for 72 hours. Then, a loopful of cells from the culture was transferred to Eppendorf tubes and incubated at -20 °C overnight. To precipitate the genomic DNA, the aqueous phase was transferred to a new tube and 500 ml of isopropanol alcohol was added and this mixture was incubated at -20 °C overnight. The solution was centrifuged at 4 °C for 15 minutes at 8150 g to pellet the DNA and was then washed with ice-cold 70% ethanol. It was again centrifuged as above and was then air-dried.

Minimum inhibitory concentration (MIC) and fractional inhibitory concentration index (FICI)
The broth microdilution test was performed in accordance with the prescriptions of the Clinical and Laboratory Standards Institute (CLSI). The incubation temperature was changed from 37 °C to 33 °C in order to standardize the growth of the strains.  Cryptococcus neoformans were identified as VNI (Figure 1).

Isolates of
Ten isolates were selected to be subjected to the susceptibility tests. All of them presented susceptibility to amphotericin and fluconazole. On the other hand, all of these isolates presented MIC > 128 µg/ml in relation to simvastatin and, thus, were considered resistant in vitro to this drug.  19,20 However, in other regions of Brazil, the prevalence has differed, ranging 25.3% in the southern region to 50% in the central region of this country. 21 The variation in the isolation rates can be related to climatic factors, since high temperatures inhibit fungal growth. 22 Additionally, the methodology     There is increasing interest in evaluating the antifungal activity of antifungal drugs, especially in the field of combined therapy. 25 In addition, evidence demonstrating the potential use of statins for preventing and treating infections has been reported. Statins have been shown to attenuate the pathogenicity of microorganisms through modulating the signaling and other regulatory pathways that are involved in the infection. 26,27 The activity of statins against Cryptococcus and Candida species, with particular emphasis on simvastatin, used in isolation or in combination with classical antifungals such as amphotericin B and fluconazole, was previously described by Brilhante et al. 10 Chin et al. 9  It is important to note that, in the present study, two isolates had a MIC of 1.0 μg/ml for amphotericin B, which was the highest MIC among the strains evaluated. These isolates presented a considerable reduction in MIC (0.250 μg/ml) when the combination of amphotericin B and simvastatin was tested, which again emphasizes the potential use of this combination of these drugs.
On the other hand, among the four isolates presenting indifferent results regarding the association between amphotericin B and simvastatin, three of them demonstrated values that were slightly above the cutoff that had been adopted.
Given that all of the isolates of the present study belonged to the same molecular type, it is possible that a greater number of samples

CONCLUSION
The data of this study demonstrate that simvastatin should be considered to be a possible therapeutic alternative, with the capacity to potentiate the action of amphotericin B. Through using this drug, the duration of cryptococcosis treatment could potentially become shorter and, consequently, the time for which patients are exposed to the toxic effects of this antifungal could be reduced. In addition, statins may have an important role in the future, as a new treatment alternative in situations of resistance to antifungals.