Inhibitory Effect of (-)-myrtenol alone and in combination with antifungal agents on Candida spp

The aim of this study was to examine the effects of (-)-myrtenol alone and combined with antifungal agents against Candida spp. The Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration of (-)-myrtenol and fluconazole against C. albicans and C. parapsilosis strains was obtained using CLSI guidelines. Combination of (-)-myrtenol with antifungal drugs was determined by checkboard test. The (-) myrtenol showed MIC ranging from 256 to 512 μg/mL against both species assay. And the MFC was 512 μg/mL, demonstrated nature fungicidal (MFC/MIC < 4). In addition, combination of antifungal agents (amphotericin B and fluconazole) and (-) myrtenol showed synergistic and additive effects on strains assays. Based on these results, the present study demonstrates that () myrtenol showed strong fungicide activity against Candida spp. In addition, Combination of antifungal agents and () myrtenol reduces the effective concentrations of both the agents with synergistic to additive effects. Therefore, (-) myrtenol has potential to be developed into an antifungal agent.


Introduction
Fungal infections have achieved considerable importance over the last few decades, especially candidiasis, the most frequent opportunistic fungal infection, caused by strains of the Candida spp. With the development of superficial or problematic infections, such as invasive candidiasis, with a high mortality rate (Ben-Ami, 2018).
Invasive candidiasis affecting mainly immunocompromised patients in intensive care units (ICUs). Among the different Candida species isolated, C. albicans is the most common isolated, followed by non-albicans species such as C. glabrata, C. parapsilosis, C. krusei, C. tropicalis and the most recent C. auris, pathogen-resistant multidrug emerging (Jeffery-Smith et al., 2018;Pappas et al., 2018).
The strategy for the treatment of candidiasis depends on the patient's immune status, location, and severity of the infection, however a lack of rapid diagnostic assay, mainly invasive candidiasis, normally empiric antifungal therapy was initiated. This approach can lead to the unnecessary use of antifungal agents and promote the emergence of resistance in individuals (Bhattacharya et al., 2020).
A major factor that contributes to the high levels of morbidity and mortality of fungal diseases is the limited number of antifungal drugs. Currently, there are only three classes of antifungal drugs approved for the treatment of invasive mycoses, echinocandins, polyene and azoles. The efficacy of these agents is compromised by the development of drug resistance in pathogen populations, respective adverse effects and treatment costs (Lee et al., 2020;Wall & Lopez-Ribot, 2020). Therefore, it is necessary to search for alternatives to this scenario. Through the development of new drugs that are more effective and have fewer adverse effects or that can be used in combination with current antifungals. One of the main sources of search is natural products and their derivatives, such as bicyclic monoterpene alcohols that have been shown to have several biological effects in the literature. (Nikitina et al., 2021) (-) Myrtenol is an example of this group with several reported biological effects in literature: angiogenesis (Huang et al., 2021), gastric protector (Viana et al., 2019), anti-bacterial (Cordeiro et al., 2020). Thus, the aim of this study was to examine the effects of myrtenol alone and in combination with antifungal drugs against Candida spp.

Type of study
This is an experimental laboratory study of a quantitative nature (Tacconi, 2018)

Microorganisms:
Candida spp. used in the antifungal assay was obtained from the archival collection of the Federal University of

Minimum Inhibitory Concentration (MIC) and Minimum fungicide Concentration (CFM)
The MIC of myrtenol and fluconazole was carried out using the microdilution technique in a microplate containing 96 wells with a U-shaped bottom (ALAMAR ® ), described by M27 A2 document (CLSI, 2008), with some modifications. The myrtenol had their test concentrations ranging between 1024 µg/mL and 0.5 µg/mL, Fluconazole was used as a positive control in assays at concentrations ranging from 64 to 0.125 µg/mL. Tension viability controls, average sterility, DMSO and Tween 80%, solvents used to prepare myrtenol emulsion, were performed simultaneously with the assay. All the tests were performed in duplicate and the plates were incubated at 35 ± 2°C for 24 hours to be conducted later playback. MIC was defined as the lowest concentration of products capable of producing visible inhibition on fungal growth when compared to its control.
The result was expressed by the arithmetic mean of the MICs obtained in the three tests. According to Sartoratto et al., 2004, results is considered strong antifungal activity MIC values between 0.05-0.50 mg/mL, MIC values between 0.6 to 1.50 mg/mL are considered moderately active and values MIC higher than 1.50 mg/mL are related to a weak antifungal activity.
After determination of the MIC, 10 l aliquots of the supernatant from the wells corresponding to the MIC, MIC × 2 and MIC × 4 were subculture in an SDA containing plate, which was then incubated at 37∘C for 24-48h. The MFC was the lowest drug concentration that showed either no growth or fewer than three colonies. All assays were performed in triplicate, and the geometric mean values were calculated. The MFC/MIC ratio was calculated to determine if the substance had fungistatic (MFC/MIC ≥ 4) or fungicidal (MFC/MIC < 4) activity (Siddiqui et al., 2013)

Combination study (checkerboard assay)
The (-) myrtenol was used in combination with fluconazole and amphotericin, following a method described previously Ahmad et al., 2015. (-) Myrtenol was combined with the antifungal drugs in a 1:1 volume ratio to the first row of microtiter plate and were serially diluted. A 100 μl of culture inoculum was added into each well and the plates were then incubated at 37 °C for 24 h, followed by MICs recording as described above. The experiment was performed in triplicate to validate the results. Based on lower additivity zero-interaction theory, combination interaction was calculated by determining The interpretation was made as follows: synergistic (< 0.5), additivity (0.5-1.), indifferent (> 1) or antagonistic (> 4) (Ahmad et al., 2015)

Results and Discussion
The strategy for the treatment of candidiasis depends on the patient's immune status, location, and severity of the infection, however a lack of rapid diagnostic assay, mainly invasive candidiasis, normally empiric antifungal therapy was initiated. This approach can lead to the unnecessary use of antifungal agents and promote the emergence of resistance in individuals (Bhattacharya et al., 2020). Therefore, there is a dire need to discover novel antifungal agents.
The MIC and MFC values are presented in Table 1. The molecule showed inhibitory activity against all tested strains, with a MIC ranging from 256 to 512 µg/mL against both species assay. And the MFC was 512 µg/mL, demonstrated nature fungicidal (MFC/MIC < 4) (Siddiqui et al., 2013). This result showed that the (-) myrtenol has showed strong fungicide activity against Candida spp. with MIC: 512 μg/mL, according criteria marked by Sartoratto et al., 2004. Supporting these results, Nikitina et al., 2021 showed that (-)myrtenol has high antimycotic activity against all tested fungal species (yeast and filamentous fungi), MIC range (23.5 μg/mL -47 μg/mL).
The literature data suggest that antifungal activity of (−)-myrtenol, possibly, probably damages the fungal membrane, affecting the change in the functional state of integrin-like proteins, which can lead to disruption of the morphogenesis of the fungal cell (Gomes et al., 2017) In addition to their inherent antifungal properties, natural products and their derivatives may alter the effects of standard antifungal drugs. The potential benefits of combination therapy include: different mechanisms acting together allow complementary targeting within the fungal cells; expansion of the action spectrum, toxicity reduction due to lower dosage use and lower number of resistant organisms (Chang et al., 2017;Fuentefria et al., 2018).
In Tables 2 and 3, the results were observed to combinations of the (-) myrtenol with antifungal drugs (amphotericin B and fluconazole) against C. albicans ATCC 76485 and C. parapsilosis ATCC 22019. Additive effect (FIC: 1) was observed for the combinations of (-) myrtenol with fluconazole against C. albicans ATCC 76485. Synergistic effects (FIC: 0.187 and 0.625) were observed for the combinations of (-) myrtenol with amphotericin B against C. albicans ATCC 76485 and C.
parapsilosis ATCC 22019. The same synergistic effect was observed from the combinations of (-) myrtenol with fluconazole against C. parapsilosis ATCC 22019.  In this study, synergistic and additivity effects were demonstrated when was combinate the (-) myrtenol with the antifungal agents (amphotericin B and fluconazole) against Candida spp., depending on the strain assay, suggesting the (-) myrtenol positively modulated the in vitro action of this drugs, suggesting future pharmacological use as an adjuvant for these drugs.
Several reports have been made concerning different antifungal combinations assayed in vitro and applied in the clinic (Dąbrowska et al., 2021;Fuentefria et al., 2018;Perea et al., 2002;Shaban et al., 2020). However, combinations of the (-) myrtenol with antifungal agents against Candida spp. are reported here for the first time.

Conclusion
Based on these results, the present study demonstrates that (-) myrtenol showed strong fungicide activity against Candida spp. with MIC 512 μg/ml. In addition, Combination of antifungal agents and (-) myrtenol reduces the effective concentrations of both the agents with synergistic to additive effects. Therefore, (-) myrtenol has potential to be developed into an antifungal agent. However, future studies on this product are necessary, such as search to mechanism of action, toxicity tests, aiming their possible application in the clinic or as a source of new antimicrobial compounds.