Use of Pinus sylvestris L. (Pinaceae), Origanum vulgare L. (Lamiaceae), and Thymus vulgaris L. (Lamiaceae) essential oils and their main components to enhance itraconazole activity against azole susceptible/not-susceptible Cryptococcus neoformans strains

Systemic fungal infections caused by Cryptococcus neoformans, an encapsulated pathogenic yeast, are a serious health concern for immunocompromised patients worldwide, and occasionally in immunocompetent subjects [1, 2]. Cryptococcal infection occurs by inhalation of dried yeast cells from avian excreta, especially from Columba livia pigeons, causing potentially severe pulmonary infection, followed by haematogenous spread to the central nervous system, with meningoencephalitis being the predominant clinical presentation in human HIV-infected patients [1]. The currently recommended therapy for cryptococcosis is amphotericin B (AMB), due to its high fungicidal activity in the central nervous system, usually in combination with 5-flucytosine. However, long-term treatment with AMB has certain drawbacks due to toxic side effects (i.e. nephrotoxicity and hepatotoxicity). In addition to AMB formulations, low-dose fluconazole (FLC) and itraconazole (ITC) are used as long-term maintenance therapy of cryptococcosis, whereas voriconazole (VRC) and posaconazole (POS) are used as consolidation therapy. Compared to other azoles, ITC has a lower toxicity, and a better therapeutic index, which allow using this drug even for organ transplant, and AIDS patients [3]. Despite the effectiveness of these drugs, many recent studies indicate that the widespread use of azoles, mainly FLC, is associated with the emergence of drug-resistant isolates, and related treatment failures and infection relapses, during long-time or repeated treatment [4]. To date, there are few other molecules with any activity towards C.neoformans, leading to heightened interest in finding new alternatives to conventional drugs for the treatment of mycosis caused by this yeast [1]. Particularly, essential oils (EOs) have emerged in recent years as potential natural and economic alternatives or as adjuvants in combination with conventional antifungal agents and continue to be of great interest [5‐10]. A large number of EOs and their major components have shown to exhibit a wide range of biological properties (antibacterial, antifungal, antiviral, anti-inflammatory, anticancer) [11‐16]. Moreover, as EOs are multicomponent, there is a low probability by microorganisms to develop resistance to this mixture of substances than to a single target.

Notably, Pinus sylvestris L. (Pinaceae), Origanum vulgare L. (Lamiaceae), and Thymus vulgaris L. (Lamiaceae) EOs, as well as their main components (α-pinene, carvacrol, and thymol) have long been known for their wide use in phytomedicine, thanks to their numerous beneficial properties, including antibacterial and antifungal activities [16, 17]. A smaller number of comprehensive studies have focused on interaction of these EOs and their major components with available antifungal drugs against C.neoformans [18]. Conversely, these EOs and main components have been widely investigated against a wide range of bacteria [19, 20], yeasts, especially Candida spp. [12, 13, 21], moulds [15], but to a lesser extent on C.neoformans [22, 23].

In this study, we evaluated the antifungal activity of P.sylvestris (pine), O.vulgare (oregano), and T.vulgaris (thyme red) EOs, and their main components (α-pinene, carvacrol, and thymol), in comparison with that exerted by FLC, ITC and VRC against C.neoformans clinical isolates from HIV-infected patients with cryptococcosis. Then, we investigated the effect of EOs and EO components in combination with ITC against C.neoformans isolates. Unlike FLC, ITC is a very lipophilic drug, which may enhance penetration into the yeast cell, allowing its use also in combination with other high lipophilic compounds, such as EOs.

Over the last decade, cryptococcosis has increased and has become one of the major fungal diseases of medical importance in both immunocompromised and immunocompetent individuals. Unfortunately, treatment failures, due to the emergence of yeast strains resistant to azole drugs, and mortality remain high [1]. Currently, EOs are considered as a potential rich source of bioactive antimicrobial compounds to improve antifungal treatment. Moreover, synergy research is actively studying efficacy of EOs and their main components in combination with already existing drugs, so that drug dosages and treatment-related adverse side effects can be significantly reduced, preventing or delaying the development of drug resistant strains [6‐10, 18]. To date, few studies have evaluated the combinatorial effects of EOs with azoles against C. neoformans using in vitro synergy [18]. Therefore, in the present study we tested three EOs from Lamiaceae (O. vulgare and T. vulgaris) and Pinaceae (P. sylvestris) plant families, some of the best inhibitors of fungal pathogens, and single main components (α-pinene, carvacrol and thymol), against C.neoformans clinical strains from HIV-infected patients with cryptococcosis with a different pattern of susceptibility to azoles (FLC, ITC, and VRC). Moreover, we evaluated whether these natural compounds may enhance the activity of ITC against azole susceptible and not-susceptible C.neoformans isolates.

Based on antifungal susceptibility testing, pine EO displayed the highest inhibitory activity (MIC = 0.07–0.27 mg/ml) on six C. neoformans isolates susceptible to azole drugs (FLC, ITC, and VRC), whereas oregano EO was found to be the most effective against the azole not-susceptible isolate (MIC = 0.3 mg/ml; Table 2). As previously seen with C. albicans by Khan et al. [21], high antifungal activities of oregano and thyme EOs may be ascribed to the presence of phenolic components such as carvacrol and thymol, which may determine membrane deterioration through oxidative stress and alteration of the antioxidant defence system even at low concentrations. Interestingly, other authors reported that thyme EO may induce phenotypic switching on the polysaccharide capsule of C. neoformans, decreasing capsule size and leading to alteration of yeast virulence [29].

According to our MIC/MFC results, thymol, the main component of thyme red EO, was the most effective pure compound, exhibiting yeast growth inhibition stronger than thyme EO alone against all C. neoformans isolates at low MIC values ranging from 0.02 to 0.08 mg/ml (Table 2). Thymol was also more effective than carvacrol and α-pinene, the main bioactive components of oregano and pine EOs, respectively. In fact, α-pinene and carvacrol showed a good antifungal activity against all C. neoformans isolates, though at higher MIC concentrations (Table 2). These findings on α-pinene are consistent with previous studies and may be related to significant inhibition of C. neoformans phospholipase and esterase activities caused by both α- and ß positive enantiomers of pinene [30]. On the contrary, MIC results of carvacrol and thymol differ to some extent from those previously reported by other authors [22, 23, 31, 32]. However, we would like to highlight that the methodological approaches for MIC determination used in these studies were clearly different, rather not standard, and therefore susceptibility results were difficult to compare.

This study showed, for the first time, synergistic and additive effects of combinations of pine, oregano, and thyme red EOs with ITC against both azole susceptible and not-susceptible C. neoformans clinical isolates. According to FICI results and isobolographic analysis, pine, oregano, and thyme red EOs in combination with ITC showed a synergistic action against azole susceptible yeast strain. Among main components, carvacrol showed a synergistic profile when combined with ITC, while ITC/α-pinene and ITC/thymol combinations resulted in either additive or indifferent interactions (Table 3). In this study binary mixtures of ITC with our thyme EO chemotype (thymol 26.52%; carvacrol 7.85%), and carvacrol were found to be synergistic against azole not-susceptible C. neoformans strain. On the contrary, oregano EO and α-pinene were found to exert additive effects when used in combination with ITC, whereas pine EO and thymol displayed indifferent interactions in association with ITC. Antagonism was never observed.

Finally, the time-kill method revealed a more detailed insight into the antifungal activities of the synergistic combined mixtures. In agreement with checkerboard data, ITC at sub-MIC concentration (1/4 MIC) in combination with oregano and thyme red EOs (1/8 MIC) confirmed to be highly effective against C. neoformans azole susceptible strain (Fig. 3). Particularly, these binary combinations produced the same antifungal effects obtained when testing the singular drug at MIC level by time-kill assay (data not shown). The synergistic combination of ITC with our tested EO chemotypes (oregano and thyme red) might be explained by the EOs promoting the effects of antifungal drugs, mainly on the cell wall, plasma membrane and other membrane structures of C. neoformans. In addition, EO damage on yeast cell wall and membrane may facilitate ITC entry into the cell, probably leading to a greater effect on ergosterol biosynthesis inhibition and adding to C. neoformans membrane destruction. Overall, this issue might also turn the fungistatic action of ITC into a fungicidal action [7, 9].

Conversely, time-kill results with binary combinations of ITC (1/8 MIC) plus pine EO (1/4 MIC) and ITC (1/8 MIC) plus carvacrol (1/8 MIC) were not really consistent with those from checkerboard assays, displaying additive effects towards C. neoformans ADS 37 isolate (Fig. 4). Likewise, synergistic binary mixtures of ITC (1/8 MIC) with thyme red EO (1/4 MIC) and carvacrol (1/4 MIC) yielded additive interactions on azole not-susceptible C. neoformans strain (Fig. 5). Despite these apparently contradictory results between checkerboard and time-kill assays, these differences may be ascribed to the different methods used, as time-kill assay records a fungicidal effect, whereas the checkerboard titration reveals at least inhibition of fungal growth [27]. According to literature data, an indifferent effect was detected against both C. albicans and C. neoformans strains when α- and ß- pinene positive enantiomers of pinene were combined with AMB [30]. Moreover, other authors have found that thymol may exert synergistic interactions with AMB, FLC and ITC towards C. albicans, whereas it may display either synergistic or additive, or indifferent activities when combined with AMB, ITC, and FLC against C. neoformans strains, respectively [18].

Terpenoids/monoterpenes are the main constituents of plant-derived EOs, frequently used in folk medicines, pharmaceutical industry and cosmetics. Among monoterpenes tested in our study, both carvacrol and thymol have been classified as GRAS (generally recognized as safe) for human consumption at low concentrations, as they do not exhibit systemic toxicity. Furthemore, their use in food has been also approved by European Parliament and Council, making them a potential option for developing anti-cryptococcal drugs [31]. In addition, α, and β pinene are generally considered as safe at low concentrations and are commonly used to produce balsamic candies and fumigations [30]. Notably, thymol is also widely used in dental practice, and has shown to interact positively with the GABA(A) receptor in mouse cortical neurons [33]. Recently, some authors demonstrated through cytotoxicity assays and keratinocyte- Cryptococcus spp. co-culture infection models that thymol and carvacrol were efficient in terms of human safety, suggesting that these pure compounds can be further exploited as cost-effective and non-toxic anti-cryptococcal drugs [31]. Nevertheless, in human organism, other monoterpenes (e.g., pulegone, menthofuran, camphor, and limonene) have been reported to exhibit toxic effects in various organs, mostly in liver [34]. Therefore, it is always advisable that EOs should be used carefully, as they may be toxic and show adverse effects on humans when overdosed [35].

Synergistic and additive EO/azole drug combinations may be potential strategies to counteract antifungal resistance, since use of multiple compounds may disrupt several fungal functions and thus minimize selection of resistant fungal strains. In this context, our findings represents an original proof-of-concept, as they highlight the potential effectiveness of oregano, thyme red EOs and carvacrol as natural and cost-effective adjuvants, at low doses (i.e., < 0.1 mg/ml) [31], when used in combination with ITC for cryptococcosis treatment.

Further in-depth studies of the modes of action for synergistic combinations, as well as in vivo preclinical models are encouraged to predict the translational use of these data for clinical settings.