molecules Article Antifungal Activity of Melaleuca alternifolia Essential Oil (TTO) and Its Synergy with Itraconazole or Ketoconazole against Trichophyton rubrum Janira Roana 1 , Narcisa Mandras 1, Daniela Scalas 2, Paolo Campagna 3 and Vivian Tullio 1,* 1 Department of Public Health and Pediatrics, Microbiology Division, University of Turin, via Santena 9, 10126 Turin, Italy; [email protected] (J.R.); [email protected] (N.M.) 2 Department of Veterinary Sciences, University of Turin, Grugliasco, 10095 Turin, Italy; [email protected] 3 Società Italiana per la Ricerca Sugli Oli Essenziali, 00161 Rome, Italy; [email protected] * Correspondence: [email protected]; Tel.: +39-011-670-5637 Abstract: Over the past 20–30 years, Trichophyton rubrum represented the most widespread der- matophyte with a prevalence accounting for 70% of dermatophytosis. The treatment for cutaneous infections caused by Trichophyton spp. are imidazoles (ketoconazole (KTZ)) and triazoles (itraconazole (ITZ)). T. rubrum can develop resistance to azoles after prolonged exposure to subinhibitory concen- trations resulting in therapeutic failures and chronic infections. These problems have stimulated the search for therapeutic alternatives, including essential oils, and their potential use in combination with conventional antifungals. The purpose of this study was to evaluate the antifungal activity of tea tree oil (TTO) (Melaleuca alternifolia essential oil) and the main components against T. rubrum and to assess whether TTO in association with KTZ/ITZ as reference drugs improves the antifungal activity of these drugs. We used a terpinen-4-ol chemotype (35.88%) TTO, and its antifungal properties were evaluated by minimum inhibitory and minimum fungicidal concentrations in accordance with the Citation: Roana, J.; Mandras, N.; Scalas, D.; Campagna, P.; CLSI guidelines. The interaction between TTO and azoles was evaluated through the checkerboard Tullio, V. Antifungal Activity of and isobologram methods. The results demonstrated both the fungicide activity of TTO on T. rubrum Melaleuca alternifolia Essential Oil and the synergism when it was used in combination with azoles. Therefore, this mixture may reduce (TTO) and Its Synergy with the minimum effective dose of azole required and minimize the side effects of the therapy. Synergy Itraconazole or Ketoconazole against activity offered a promise for combination topical treatment for superficial mycoses. Trichophyton rubrum. Molecules 2021, 26, 461. https://doi.org/10.3390/ Keywords: Trichophyton rubrum; essential oils; tea tree oil; Melaleuca alternifolia; azoles; checker- molecules26020461 board; synergism Academic Editor: Francesca Mancianti Received: 30 December 2020 Accepted: 13 January 2021 1. Introduction Published: 17 January 2021 Dermatophytes are the most common fungal agents leading to superficial skin in- Publisher’s Note: MDPI stays neu- fections. During the last century, their spread changed under the influence of several tral with regard to jurisdictional clai- elements, such as socioeconomic factors, practices bound to life-style, increasing travel, ms in published maps and institutio- and migration from southern to northern countries and vice versa [1]. Trichophyton rubrum, nal affiliations. frequently causing tinea pedis, tinea unguium, and tinea corporis, has increased steadily over this period and became the main agent isolated worldwide [2–4]. Over the past 20–30 years, T. rubrum has represented the most widespread dermatophyte in Central and North Europe with a prevalence accounting for about 70% of dermatophytosis. This species is often the Copyright: © 2021 by the authors. Li- key etiological agent of chronic infections with a slow progression, suggesting that the censee MDPI, Basel, Switzerland. fungus has adapted to the human host [5]. Sometimes it is responsible for a deeper and This article is an open access article disseminated infection, particularly in immunocompromised patients [3]. distributed under the terms and con- Azoles and allylamines (such as terbinafine) (Figure1) are the most important oral and ditions of the Creative Commons At- topical antifungal drugs used to treat dermatophytosis: they interfere with the ergosterol tribution (CC BY) license (https:// creativecommons.org/licenses/by/ biosynthesis pathway causing structural modifications in the lipid membrane. 4.0/). Molecules 2021, 26, 461. https://doi.org/10.3390/molecules26020461 https://www.mdpi.com/journal/molecules Molecules 2020, 25, x; doi: FOR PEER REVIEW 2 of 10 sponsible for a deeper and disseminated infection, particularly in immunocompromised patients [3]. Azoles and allylamines (such as terbinafine) (Figure 1) are the most important oral Molecules 2021, 26, 461 2 of 10 and topical antifungal drugs used to treat dermatophytosis: they interfere with the er- gosterol biosynthesis pathway causing structural modifications in the lipid membrane. FigureFigure 1.1. Chemical structures of of azoles azoles (itraconazole, (itraconazole, ketoconazole) ketoconazole) and and terbinafine. terbinafine. The preferred treatment for cutaneous infections caused by Trichophyton spp. are The preferred treatment for cutaneous infections caused by Trichophyton spp. are imidazoles, such as ketoconazole (KTZ), and triazoles, such as itraconazole (ITZ), often imidazoles, such as ketoconazole (KTZ), and triazoles, such as itraconazole (ITZ), often resulting in a complete clearance of the lesions. However, inappropriate prescribing, and resulting in a complete clearance of the lesions. However, inappropriate prescribing, and the increased use of systemic antifungal agents in recent years are the main factors leading the increased use of systemic antifungal agents in recent years are the main factors lead- toing the to emergence the emergence of resistance of resistance to antifungal to antifungal drugs drugs (i.e., azoles) (i.e., azoles) [6]. In [6]. fact, In if notfact, properly if not treated,properly dermatophytosis treated, dermatophytosis may become may chronic, become requiringchronic, requiring oral antifungal oral antifungal drugs, which drugs, are oftenwhich associated are often associated with side effectswith side such effects as hepatotoxicity. such as hepatotoxicity.T. rubrum T.can rubrum develop can develop resistance toresistance azoles after to azoles prolonged after exposureprolonged to exposure subinhibitory to subinhibitory concentrations concentrations of these drugs of leadingthese todrugs therapeutic leading failuresto therapeutic and to thefailures persistence and to andthe persistence chronicity ofand the chronicity infections. of Furthermore, the infec- complicationstions. Furthermore, like bacterial complications superinfections like bacterial can superinfections occur [7]. can occur [7]. TheseThese problems have stimulated the the search search for for therapeutic therapeutic alternatives, alternatives, including including naturalnatural compoundscompounds suchsuch as as essential essential oils, oils which, which have have been been increasingly increasingly used used in recent in recent years foryears their for biologicaltheir biological and antimicrobial and antimicrobial activities. activities. Furthermore, Furthermore, a recent a recent focus focus of research of re- onsearch essential on essential oils is oils to investigate is to investigate their potentialtheir potential use inuse combination in combination with with conventional conven- antimicrobialtional antimicrobial drugs drugs [8,9]. [8,9]. In fact, In fact, essential essential oils oils are are one one of theof the most most evident evident promising prom- sourcesising sources for pharmacological for pharmacologic researchal research and theand development the development of new of new natural natural antimicrobial antimi- agentscrobial againstagents against many microbialmany microbial pathogens pathogens such assuch bacteria, as bacteria, yeasts, yeasts, filamentous filamentous fungi, includingfungi, including dermatophytes, dermatophytes, and viruses and viruses [10]. Among [10]. Among the best the known best known oils, teaoils, tree tea oiltree (TTO), oil the(TTO), essential the essential oil of Melaleuca oil of Melaleuca alternifolia alternifolia(Myrtaceae), (Myrtaceae), often used often as used an antiseptic as an antiseptic to treat skinto infections,treat skin infections, has recently has received recently much received attention much forattention its antifungal for its antifungal properties. properties Because. of itsBecause lipophilic of its nature, lipophilic which nature, facilitates which skin facilitates penetration, skin itpenetration is used as, ait local is used formulation as a local for dermatologicalformulation for disordersdermatological [11]. disorders [11]. Hence,Hence, thethe purposepurpose ofof thisthis studystudy waswas toto evaluateevaluate thethe antifungalantifungal activityactivity ofof TTOTTO and and its mainits main components components against againstT. rubrum T. rubrumand assessand assess for the for first the timefirst whethertime whether TTO inTTO association in as- withsociation ITZ orwith KTZ ITZ improves or KTZ the improves antifungal the activityantifungal of these activity drugs of againstthese drugsT. rubrum against. T. rubrum. 2. Results 2.1. TTO Composition The phytochemical composition of TTO used for this study was determined by GC-MS, and the results are reported in Table1. Molecules 2020, 25, x; doi: FOR PEER REVIEW 3 of 10 2. Results 2.1. TTO Composition Molecules