Essential Oils in Respiratory Mycosis: A Review

Respiratory mycosis is a major health concern, due to the expanding population of immunosuppressed and immunocompromised patients and the increasing resistance to conventional antifungals and their undesired side-effects, thus justifying the development of new therapeutic strategies. Plant metabolites, namely essential oils, represent promising preventive/therapeutic strategies due to their widely reported antifungal potential. However, regarding fungal infections of the respiratory tract, information is disperse and no updated compilation on current knowledge is available. Therefore, the present review aims to gather and systematize relevant information on the antifungal effects of several essential oils and volatile compounds against the main type of respiratory mycosis that impact health care systems. Particular attention is paid to Aspergillus fumigatus, the main pathogen involved in aspergillosis, Candida auris, currently emerging as a major pathogen in certain parts of the world, and Cryptococcus neoformans, one of the main pathogens involved in pulmonary cryptococcosis. Furthermore, the main mechanisms of action underlying essential oils’ antifungal effects and current limitations in clinical translation are presented. Overall, essential oils rich in phenolic compounds seem to be very effective but clinical translation requires more comprehensive in vivo studies and human trials to assess the efficacy and tolerability of these compounds in respiratory mycosis.


Introduction
Respiratory infections have increased over the past decades, becoming important causes of morbidity among immunocompromised and immunosuppressed patients and accounting for 4.3 million annual deaths worldwide [1]. Among these, fungal infections are underestimated, despite being responsible for mortality rates above 50% [2]. Although exposure to these respiratory pathogens occurs regularly during a lifetime, healthy individuals rarely develop symptomatic infections. However, in individuals with impaired defenses-for example, due to the use of chemotherapeutic and immunosuppressive agents, and antibiotics, or having prosthetic devices, grafts, burns, neutropenia or HIV-systemic life-threatening fungal infections may occur [3].
Overall, two main types of infections can occur: endemic (primary) or opportunistic. The first occur primarily in patients living or traveling in developing countries and, despite being mostly asymptomatic or causing mild infections, they account for huge health-care costs in these regions. Importantly, an exposure to high concentrations of inoculum or in patients with compromised immune defenses, life-threatening infections or the reactivation of latent foci may occur. The most common fungal infections include blastomycosis, are capable of biofilm growth, forming highly organized communities that are resistant to antifungals and are responsible for recurrent infections [14]. Table 1 summarizes the main fungi responsible for relevant respiratory mycoses and points out for each pathogen (divided into endemic or opportunistic) the main source of infection, relevant pathological manifestations, diagnostic methods, and current therapy. The majority of information on some of the pathogens included in the table was based on the online textbook Microbiology, an OpenStax resource [15], the information on the remaining fungi being completed with other bibliographic sources such as clinical guidelines [16], centers for disease and health control [17] and professional manuals [18] available online.

In Vitro and In Vivo Models to Assess Antifungal Properties
The search for new antifungal agents involves in vitro susceptibility tests. Reference methods, namely the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standard methods, are available enabling more practical, reliable, and reproducible protocols for antifungal susceptibility testing. Both resort to the broth microdilution method and assess the minimal inhibitory concentration (MIC) of an antifungal drug, which indicates the minimal drug concentration that inhibits fungal growth. Although these tests present some slight methodological differences, MICs are comparable [19]. Commercial antifungal susceptibility tests are also available. Examples include broth microdilution methods such as the Sensititre TM or YeastOne TM that use color endpoints based on metabolic dyes such as AlamarBlue, incorporated into the growth media or agar-based methods, such as the Etest ® and the automated system VITEK ® 2 [20].
In addition to in vitro testing, pre-clinical animal models have enabled us to increase the knowledge on fungal infections and putative therapeutic strategies. In these assays it is important to consider the model species and its immune status, the route of infection, and the fungal strain used, as these aspects can impact experimental outcomes. Overall, inbred strains of laboratory mice are the most common models mainly due to their relatively low cost and the wide availability of immunological reagents. However, other vertebrates such as rats, guinea pigs, rabbits, and zebrafish have gained popularity [21]. Recently, the Galleria mellonella model has been one of the most used as it is inexpensive, easy to use, and does not require a dedicated infrastructure, the antifungal efficacy of the drug being estimated by fungal burden or mortality rate in infected and treated larvae [22]. To mimic respiratory mycosis, an intranasal or intratracheal injection of a liquid fungal suspension or the inhalation of dry fungal cells can be used. A catheter can be inserted beyond the vocal cords to facilitate intratracheal delivery of fungal cells and their dispersion into the pulmonary parenchyma may be enhanced by mechanical ventilation [23] or by using a microsprayer attached to the syringe tip [24]. Classical readouts include organ fungal burden and histopathology.

The Relevance of Essential Oils
Essential oils are mixtures of volatile compounds present in various organs of aromatic plants and obtained from the plant by hydrodistillation, steam distillation or dry distillation or, in the case of Citrus fruits, by a suitable mechanical process [25,26]. Essential oils generally present a strong odor and high lipophilicity, being primarily composed of terpenic compounds, such as monoterpenes and sesquiterpenes. In some cases, phenylpropanoids may also occur in high amounts and more rarely nitrogen and sulphur derivatives can be found. An important aspect to consider is their high chemical variability mainly due to environmental factors or genetic variations [27]. The latter may result in the expression of different metabolic pathways with significant variations in essential oils' composition that justify the identification of chemotypes. To assure high quality of the commercialized essential oils, analytical guidelines published by several institutions, such as the European Pharmacopoeia, the International Standard Organization (ISO), and the World Health Organization (WHO), should be followed.
Essential oils play relevant natural roles primarily in plant defense and signaling processes and have been explored by several industries due to particular features such as aroma, taste and bioactive potential. Indeed, they are valuable raw materials in the pharmaceutical, agronomic, food, sanitary, cosmetic, and perfume industries [28]. In what concerns bioactive potential, several studies have highlighted the antifungal and anti-inflammatory potential of these compounds as reviewed elsewhere [29,30]. These properties are quite relevant in the context of respiratory mycosis as fungal infections are associated with acute inflammation, which exacerbates the infection and delays its eradication. Therefore, extracts or compounds that combine both antifungal and anti-inflammatory activities, at concentrations devoid of toxicity, emerge as suitable preventive/therapeutic agents.

Antifungal Effect of Essential Oils on Respiratory Mycosis
Essential oils and their volatile compounds have shown promising effects against fungi involved in infections of the respiratory tract. Overall, Aspergillus spp., Candida albicans, and Cryptococcus neoformans are by far the most assessed strains, with only a few studies being carried out on other relevant pathogens referred in Table 1. Next, a compilation of selected studies is presented, the assortment criteria being defined in each section.

Essential Oils in Aspergillosis
Regarding Aspergillus pathogens, A. fumigatus is the most ubiquitous strain and the major causal agent of aspergillosis, followed by A. flavus, A. niger, A. terreus, and A. nidulans [31]. Besides species diversity, several types of aspergillosis are known, namely pulmonary aspergillosis that generally develops in patients with underlying lung pathology; allergic bronchopulmonary aspergillosis, an allergic reaction that results from hypersensitivity to Aspergillus colonization and is generally exclusive to asthma and cystic fibrosis patients and invasive aspergillosis, the most severe type that occurs when the infection travels from the lungs into the bloodstream [32]. As A. fumigatus is the main pathogen involved in aspergillosis, a compilation of the studies showing minimal inhibitory (MIC) or minimal fungicidal/lethal concentrations (MFC/MLC) is presented in Table 2. Only essential oils presenting MICs lower than 10 mg/mL were considered, being the plant name, part of the plant used to obtain the essential oils and antifungal effect highlighted, whenever this information was available in the original study. Broader studies, gathering more than three species, are not included in the table, and are discussed next. Furthermore, since only a few studies assessed a possible mechanism of action underlying these effects, this topic is discussed in Section 4.2.   As previously mentioned, some studies have included several essential oils. For example, in a study involving fifteen species from the Asteraceae family, only Achyrocline alata and Baccharis latifolia essential oils were effective against A. fumigatus (geometric mean MIC value of 78.7 and 157.4 µg/mL, respectively) [62]. Moreover, in some cases both essential oil direct contact or vapor phase are assessed, thus foreseeing different modes of administration and distinct applications. For example, resorting to these two strategies, the essential of Eugenia caryophyllata, Lavandula angustifolia, Origanum vulgare, Salvia sclarea, Thuja plicata and Thymus vulgaris were assessed, being Origanum vulgare essential oil the most effective, through direct contact, with a MIC value of 0.025%. However, the volatile vapor of 0.075% of the majority of the oils was also able to exert a fungicidal effect on A. fumigatus, proving that these oils can be used, for example, for environmental disinfection [63].
Synergistic studies between essential oils and isolated compounds or essential oils with conventional antifungals are also relevant, thus reducing the effective dose needed and improving therapeutic outcomes [64]. Indeed, a chessboard assay using twenty-five essential oils showed that a combination of Cymbopogon citratus and Thymus serpyllum oils was the most potent against A. fumigatus (fractional inhibitory concentration index -FICI-of 0.1875; with a total synergistic effect considered for values ≤ 0.5) [65]. Additionally, Thymus vulgaris essential oils and thymol showed significant levels of synergistic interaction with fluconazole presenting an FICI value of 0.187 [40].
Moreover, mixed microbial infections are known to cause significant health care burdens and are commonly found in patients with chronic infections. Therefore, some studies have also considered this reality by assessing the polymicrobial antibiofilm effect of essential oils. A study carried out by Pekmezovic and colleagues addressed this ability using selected Citrus essential oils and showed that 10 mg/L of the oils were able to both inhibit a mixed biofilm formation composed of Pseudomonas aeruginosa and Aspergillus fumigatus and affect quorum sensing [66].
Finally, in vivo validations are of utmost importance but are quite sparse regarding respiratory mycosis. A pre-clinical study confirmed the potential of Leptospermum petersonii essential oil by showing a significant reduction of fungal infection in the lungs of animals that completed the treatment regimen and the effect was strikingly superior to that reported for conventional antifungal drugs [48].
Regarding the potential of isolated compounds, very few studies have been performed. Once again phenolic compounds stand out as very effective, namely eugenol-MIC = 250 ug/mL [49], thymol-MIC = 192 µg/mL [40] and carvacrol-MIC = 0.16 µL/mL [51]. Indeed, this could explain the excellent activity of some of the essential oils pointed out above such as Thymus vulgaris (rich in thymol) and Lavandula multifida (with high amounts of carvacrol). Besides phenolic compounds, other essential oils constituents such as safrole have shown very effective antifungal abilities against A. fumigatus, with a MIC of 39.1 µg/mL [42]. Interestingly, although the essential oil obtained from the roots of Cinnamomum camphora had a high concentration of this compound (57.6%), the antifungal potential of the whole extract was not so prominent (MIC = 312.5 µg/mL), thus showing that, in some cases, antagonistic effects may also take place between different compounds present in the mixture. Additionally, 1,8-cineole, a compound widely found in many essential oils, showed promising effects with a MIC value of 156.3 µg/mL [42].

Essential Oils in Respiratory Candidiasis
Candida infections are quite difficult to detect as Candida spp. inhabit the normal microflora of the skin, oral cavity, gastrointestinal mucosa, respiratory tract, and genitourinary tract [67]. Moreover, despite covering more than two-thirds of fungal infections, invasive candidiasis (defined as the presence of Candida in the blood-candidemia-or firmly established Candida infections) rarely manifest as Candida pneumonia and antifungal treatments in these cases is a matter of ongoing debate [68]. Nevertheless, Candida albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei have been pointed out as the most prevalent strains in invasive candidiasis. Since several reviews have largely covered the antifungal potential of essential oils on these pathogens [69][70][71][72][73], we now focus on a once considered rare species, Candida auris, that is currently emerging as a major pathogen in certain parts of the world. C. auris infections are difficult to treat as this pathogen does not respond to conventional antifungal drugs. This fungus also presents a high risk of transmission as it persists on several equipment and medical devices [74]. Importantly, some essential oils have shown promising antifungal potential against this species. Table 3 compiles this information by pointing out the plant species, part of the plant used to obtain the essential oil and its main compounds, whenever referred to in the original study. The antifungal potential of the essential oil is also highlighted, as well as the mechanism of action underlying the observed effect, if assessed. Although C. auris is an emergent species, several studies have already shown the potential of essential oils as effective antifungal agents, as highlighted in Table 3. Nevertheless, as different susceptibility tests are used and units lack uniformity among studies, it is quite difficult to perform comparisons between studies and understand which essential oil is the most effective. Even so, it seems that essential oils rich in phenolic compounds exert a more potent inhibitory effect. Indeed, the phenolic compound carvacrol assessed alone has already shown promising effects. A median MIC of 125 µg/mL was reported as well as synergistic and additive effects in combination with the conventional antifungals fluconazole, amphotericin B, nystatin and caspofungin [82].

Essential Oils in Cryptococcosis
Pulmonary cryptococcosis, contrarily to cryptococcal meningitis, remains underdiagnosed mainly due to limitations in diagnostic tools. Indeed, the infection presents similar clinical and radiological features to lung cancer, pulmonary tuberculosis, bacterial pneumonia, and other pulmonary mycoses. The genus Cryptococcus consists of more than 70 species, the two main human pathogens being C. neoformans and C. gattii [83]. Several studies have also pointed out the antifungal potential of essential oils, mainly for C. neoformans. Table 4 compiles these studies referring to the plant name, part of the plant used to obtain the essential oils, and antifungal effect. Since the mechanisms of action underlying these effects are poorly explored, this topic will be discussed collectively in Section 4.2.      Besides the studies referred to in Table 4, others were performed gathering a higher number of species, with bigger data approaches enabling more extensive analysis. For example, using this approach, eighty-two essential oils were analyzed, with fifteen being highlighted as very potent (MIC ≤ 100 µg/mL), and from these Cedrus atlantica standing out as the most effective, with a MIC value of 20 µg/mL [116]. In another extensive study, the antifungal potential of sixty commercially-available essential oils was also assessed, with Cinnamomum cassia, C. zeylanicum, Coriandrum sativum, Pogostemon cablin, Santalum album, S. austrocaledonicum, S. paniculatum and Vetiveria zizanoides being very effective (MIC = 20 µg/mL) [117].
Some studies also resort to ethnopharmacological evidence and tend to assess the potential of essential oils based on their traditional uses, thus recovering relevant knowledge that tends to be lost over time. For example, the study carried out by Lawson and colleagues considered a specific group of plants from the Asteraceae family used in Chero-kee and other Native American traditional medicines, and showed a very potent effect of Eupatorium serotinum essential oil against Cryptococcus neoformans with a MIC value of 78 µg/mL [118].
Regarding synergistic studies between essential oils and conventional drugs, interesting results against Cryptococcus neoformans have also been reported. For example, Scalas and colleagues, through checkerboard testing and isobolographic analysis, showed synergistic effects (FICI ≤ 0.5) between itraconazole and Origanum vulgare, Pinus sylvestris or Thymus vulgaris essential oils. Importantly, a synergistic effect was also observed with itraconazole and Thymus vulgaris essential oil (chemotype: thymol 26.52%; carvacrol 7.85%) on an azole not susceptible strain of Cryptococcus neoformans, thus confirming the potential of essential oils as cost-effective adjuvants in antifungal therapy [119]. Using similar methodology, the combination of Mentha x piperita essential oil with itraconazole also exerted a synergistic effect (FICI ≤ 0.5), with a decrease in the MIC. Nevertheless, on the azole-resistant strain, the binary combination of itraconazole and the oil yielded additive effects [97]. Furthermore, the combination of Ocimum basilicum var. Maria Bonita (a genetically improved cultivar) essential oil with fluconazole enhanced the antifungal activity, especially against the resistant strain of Cryptococcus neoformans, with MIC being reduced from 1250 µg/mL to 625 µg/mL [120].
Combinations between essential oils have also shown positive effects on Cryptococcus neoformans. For example, combining the essential oils of Boswellia rivae, B. neglecta and B. papyrifera with Commiphora guidotti or C. myrrha oils displayed synergistic, additive and noninteractive properties with MICs ranging from 0.5-5.3 mg/mL [121].

Essential Oils on Other Respiratory Infections
Concerning other fungal strains involved in respiratory mycosis, namely those referred to in Table 1, in vitro studies have been performed, although to a much lesser extent. Table 5 compiles these studies and points out their main findings.   [126] Regarding isolated compounds, several studies have been performed. Overall, farnesol, a sesquiterpenic compound, and terpinene-4-ol, a terpene alcohol, were the most assessed and showed very promising effects. For example, farnesol was tested against Coccidioides posadasii-MIC = 0.002-0.01 mg/L [127], Histoplasma capsulatum var. capsulatum-MIC = 0.008-0.003 µM [128] and Sporothrix schenckii-MIC = 0.003 to 0.222 µg/mL [129]. Importantly, in the first two pathogens, synergistic effects with amphotericin B, itraconazole, voriconazole and caspofungin or itraconazole, respectively were observed [127,128], thus confirming its potential as an adjuvant in fungal infections. On the other hand, terpinene-4ol was tested against Sporothrix schenckii (MIC = 4-32 mg/L) and was able to decrease its cellular ergosterol content. In combination with itraconazole or terbinafine, it also exerted a synergistic effect [130]. Furthermore, this compound was also effective against Coccidioides posadasii (MIC = 350-1420 µg/mL) and Histoplasma capsulatum [MIC = 20-1420 µg/mL (filamentous phase) and 40-350 µg/mL (yeast phase)], although to a lesser extent [131].
Other compounds have also been tested, such as the monoterpene p-cymene (MIC = 1024 µg/mL) and the phenolic compound thymol (MIC = 128-256 µg/mL), against Rhizopus oryzae [124]. Furthermore, the sesquiterpene dialdehyde polygodial was also very effective against Penicillium marneffei with a MIC value of 3.3 µg/mL [132]. These studies, although still limited in number, show that other fungal pathogens are being considered for antifungal susceptibility testing, besides the gold standard strains, thus opening new avenues for the development of new preventive/therapeutic strategies.

Mechanism of Action Underlying Essential Oils Antifungal Effects
The majority of the in vitro studies performed tend to identify the MIC and MLC/MFC of the volatile extract/compound and in some cases, the putative mechanism of action underlying the antifungal effect. Indeed, several methods have been proposed to elucidate the target site or the mechanism of action of the essential oils, in fungal cells [133]. Generally, mechanistic studies are performed on Candida albicans and the main effects considered are biofilm disruption, cell morphology and plasma membrane integrity. In less extent mitochondrial enzymes, reactive oxygen species (ROS) and gene expression are also assessed. Next, studies performed on other pathogens rather than Candida albicans are highlighted, namely for biofilm and cell wall/membrane integrity.
Biofilms are important virulence factors for pathogenic fungi naturally formed when fungi change from a planktonic to a sessile state and attach to surfaces and to each other, being involved and protected by a polymeric extracellular matrix. The fungi also secrete quorum-sensing molecules that play a relevant role in fungal resistance and pathogenicity [134] and, therefore, constitute a very promising therapeutic target.
Kumari and colleagues carried out a study using several compounds present in essential oils and confirmed their anti-biofilm effect on Cryptococcus laurentii and Cryptococcus neoformans in the following order: thymol > carvacrol > citral > eugenol = cinnamaldehyde > menthol. Indeed, for the most effective compounds, a potent effect on biofilm morphology was confirmed by scanning electron microscopy and confocal laser scanning microscopy that showed the absence of extracellular polymeric matrix, reduction in cellular density and alteration in the surface morphology of biofilm cells [122].
Other relevant therapeutic targets that are highly assessed are the structural elements of fungi cell walls and membranes, as their inhibition can affect cell wall maturation, septum formation, and bud ring formation, by damaging cell division and cell growth. Essential oils are able to disrupt the cell wall, leading to cytoplasm leakage and compromise fungi membrane permeability and fluidity by altering its properties and compromising membrane-associated functions. Indeed, morphological and ultrastructural alterations were observed in Aspergillus fumigatus exposed to Cuminum cyminum, Nigella sativa and Ziziphora clinopodioides essential oil that interfered with the enzymes involved in cell wall synthesis, caused high vacuolation of the cytoplasm, detachment of the fibrillar layer of cell wall, and plasma membrane disruption. Additionally, disorganization of nuclear and mitochondrial structures was observed [44]. Similarly, Leptospermum petersonii essential oil disturbed Aspergillus fumigatus cell membrane with alterations observed in hyphal morphology, susceptibility of spheroplasts and uptake of propidium iodide following exposure to the oil [48]. Regarding isolated compounds, eugenol reduced the cell diameter and capsule size of Cryptococcus gattii and C. neoformans. The compound was also able to increase the levels of ROS, leading to increased lipid peroxidation, mitochondrial membrane depolarization and reduction of lysosomal integrity in these fungi [135]. Another relevant target for essential oils is ergosterol, a compound present in the fungal cell membrane whose biosynthesis can be altered by disturbing sterol biosynthetic pathways. Importantly, the absence or reduced presence of ergosterol in fungal membranes results in osmotic and metabolic instability of the fungal cell, compromising reproduction and infectious activity [136]. Indeed, it was shown that Thymus vulgaris essential oil and its main compound, thymol, were effective against Rhizopus oryzae due to their interaction with ergosterol, thus supporting their use in the management of mucormycosis [124].

Translation to the Clinic: Limitations and Future Perspectives
Studies comparing different treatments for respiratory mycosis, namely cryptococcal meningitis, candidemia, endemic mycoses, and invasive aspergillosis, are among the most cited clinical trials and are very relevant for the development of treatment guidelines for these infections [137]. Nevertheless, regarding essential oils as anti-infective agents, clinical trials are lacking. Moreover, essential oils present several features such as hydrophobicity, instability, high volatility, and possible toxicity that compromise their use. Therefore, to overcome these limitations, encapsulation resorting to delivery systems, namely lipidbased carriers, have been developed to stabilize these compounds, improve the shelf-life of the formulated products and prolong the biological effect of the active molecules [138]. For example, Lavandula angustifolia essential oil encapsulated in liposomes was effective against persistent biofilms of Candida auris [78] while Lippia sidoides essential oil encapsulated in nanostructured lipid carriers showed anti-Candida auris potential and low toxicity, suggesting a new strategy to overcome multidrug-resistant pathogens [79]. On the other hand, due to their volatility, essential oils can easily reach the upper and lower parts of the respiratory tract via active or passive inhalation. In the first case, an inhalation device is needed for the patients to directly inhale the volatile compounds whereas in the latter, heating, vaporization or ventilation is used to deliver these compounds to the environment [139]. Interestingly, over the last years, several patents on portable inhalation devices have been registered and have been shown to be suitable delivery systems for these volatile compounds. Another possible form of administration resorts to patches, the volatiles being released to the skin and/or inhaled by the patient [140].

Final Remarks
The present review provides, for the first time, an updated compilation of relevant information on the antifungal potential of essential oils and their volatile compounds on respiratory mycosis. Overall, our bibliographic search showed that the majority of the studies are performed on strains involved in opportunistic infections, namely aspergillosis, candidiasis and cryptococcosis, with the present review focusing on strains involved in lifethreatening invasive diseases such as Aspergillus fumigatus and Cryptococcus neoformans and relevant emergent strains such as Candida auris. Moreover, it is quite evident that essential oils rich in phenolic compounds, namely thymol and carvacrol, are very effective, and therapeutic improvements can be achieved by combining essential oils and/or their volatile compounds with conventional antifungal drugs. Furthermore, several administration strategies and devices have been designed to effectively deliver these volatile compounds but clinical translation still requires in vivo validations and human trials to confirm the efficacy and tolerability of these extracts/compounds in respiratory mycosis.