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Antifungal, anti-biofilm and adhesion activity of the essential oil of Myrtus communis L. against Candida species a

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Sara Cannas , Paola Molicotti , Donatella Usai , Andrea Maxia & Stefania Zanetti

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Microbiologia Sperimentale e Clinica, Dipartimento di Scienze Biomediche, Università degli Studi di Sassari, Viale San Pietro 43/ b, 07100 Sassari, Italy b

Divisione di Botanica e Orto Botanico, Dipartimento di Scienze della Vita e dell'Ambiente, Università degli Studi di Cagliari, Viale Sant'Ignazio, da Laconi 13, 09127 Cagliari, Italy Published online: 24 Jun 2014.

To cite this article: Sara Cannas, Paola Molicotti, Donatella Usai, Andrea Maxia & Stefania Zanetti (2014): Antifungal, anti-biofilm and adhesion activity of the essential oil of Myrtus communis L. against Candida species, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.925892 To link to this article: http://dx.doi.org/10.1080/14786419.2014.925892

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Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.925892

SHORT COMMUNICATION Antifungal, anti-biofilm and adhesion activity of the essential oil of Myrtus communis L. against Candida species Downloaded by [UZH Hauptbibliothek / Zentralbibliothek Zürich] at 06:29 01 July 2014

Sara Cannasa*, Paola Molicottia, Donatella Usaia, Andrea Maxiab and Stefania Zanettia a

Microbiologia Sperimentale e Clinica, Dipartimento di Scienze Biomediche, Universita` degli Studi di Sassari, Viale San Pietro 43/b, 07100 Sassari, Italy; bDivisione di Botanica e Orto Botanico, Dipartimento di Scienze della Vita e dell’Ambiente, Universita` degli Studi di Cagliari, Viale Sant’Ignazio, da Laconi 13, 09127 Cagliari, Italy (Received 6 February 2014; final version received 15 May 2014) Candida species belong to the normal microbiota of the oral cavity, gastrointestinal tract and vagina. The increasing incidence of drug-resistant pathogens and the toxicity of the antifungal compounds have drawn the attention towards the antimicrobial activity of natural products, an inexpensive alternative. The aim of this work was to evaluate the adhesion activity, the biofilm formation and the action of the Myrtus communis L. essential oil (EO) on the biofilm formation towards three species isolated from clinical samples: Candida albicans, Candida parapsilosis and Candida tropicalis. Furthermore, we evaluated the antimycotic activity of the EO towards the three species, and the results were compared with the minimum inhibitory concentration of six antimycotics. The activity of the EO against C. albicans and C. parapsilosis was better than that obtained against C. tropicalis; moreover, the strains used in the assay were adhesive and biofilm producer, and the effect of myrtle EO on the biofilm formation yielded encouraging results. Keywords: Candida spp; Myrtus communis L; biofilm; adhesion

Introduction The genus Candida is composed of a heterogeneous group of organisms belonging to the normal microbiota of the mucosa of oral cavity, gastrointestinal tract and vagina (Shao et al. 2007). They are responsible for clinical manifestations from mucocutaneus overgrowth to blood-stream infections (Eggimann et al. 2003); more than 90% of invasive infections are caused by Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei (Pfaller & Diekema 2007). The incidence of fungal infections has increased significantly, especially in immunocompromised patients and those hospitalised (Espinel-Ingroff 2009). In European countries, more than half of the cases of candidaemia were caused by C. albicans, and the incidence rates for non-albicans candidaemia infections were 14% each for C. glabrata and C. parapsilosis, 7% for C. tropicalis and 2% for C. krusei (Tortorano et al. 2006). The pathogenicity of Candida species is attributed to different virulence factors, such as the ability to evade host defences, adherence and biofilm formation (Martinez & Fries 2010). Biofilm-related infections are difficult to treat and the production of biofilm is also associated with a high level of antimicrobial resistance of the associated organisms (Rautemaa & Ramage 2011). The increasing incidence of drug-resistant pathogens and the toxicity of existing antifungal compounds have drawn attention towards the antimicrobial activity of natural products encouraging the development of alternative treatments (de Oliveira et al. 2006; Sardi

*Corresponding author. Email: [email protected] q 2014 Taylor & Francis

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S. Cannas et al.

et al. 2011). Plants are good options for obtaining a great variety of drugs; they are extensively used in folk medicine because they represent an inexpensive alternative, are easily accessible and can be applicable to various pathologies (Sardi et al. 2011). They constitute an excellent source for substances that can be used in the formulation of new antifungal agents. In particular, we focused our attention on Myrtus communis L. in Italy, it grows along the coasts and in the internal hills, and it is spread especially in the islands, where it is one of the most characteristic species. The essential oil (EO) obtained from leaves and, sometimes, flowers and berries has been used for its tonic, and balsamic properties, and it is used in flavour and fragrance industries. EOs are gaining remarkable interest for potential multi-purpose use as antioxidant, antibacterial and antiseptic agents (Alem et al. 2008). In our previous studies, we evaluated the antimicrobial properties of Myrtle EO against several clinical strains and in particular against Helicobacter pylori and Mycobacteria, and we obtained encouraging results (Deriu et al. 2007; Zanetti et al. 2010). The aim of this work was to evaluate the adhesion activity, the biofilm formation and the action of the Myrtle EO on the biofilm formation towards three species of yeasts isolated from clinical samples: C. albicans (five strains), C. parapsilosis (six strains) and C. tropicalis (five strains). Furthermore, we evaluate the antimycotic activity of Myrtle EO towards three species of yeasts isolated from clinical samples. The results obtained at 24 and 48 h were compared with the MIC of six antimycotics chosen: amphotericin B, fluconazole, voriconazole, anidulafungin, micafungin and caspofungin. Results and discussion Determination of minimum bactericidal concentration MIC and minimum bactericidal concentration (MBC) data for the Candida spp. are summarised in Table 1. The results obtained exhibited a considerable activity of EO. In particular, the C. parapsilosis strains showed an MBC, which was as follows: four with an MBC , 0.125%, one strain with an MBC of 2% and one at 8%. C. albicans strains exhibited the following values: four strains with an MBC , 0.125% and one strain with 1%. C. tropicalis strains exhibited the highest MBC; in particular, only one strain had an MBC , 0.125%, two strains 0.25%, one strain 16% and last one . 16%. All the yeasts tested were susceptible to amphotericin B, fluconazole, voriconazole, anidulafungin, micafungin and caspofungin. Adhesion assays Regarding the C. parapsilosis strains assessed, four displayed a weak adhesive capacity, one strain had no adhesive capacity and only one strain exhibited a considerable adhesive capacity. Among the C. albicans strains assessed, one exhibited a strong adhesive capacity, one a weak adhesion and three strains were adherent. As regards C. tropicalis: one strain displayed no adherence capacity, three strains exhibited a strong adherence and the last one was adherent. Biofilm formation The results are summarised in Figure 1. The strains that produced a medium-level biofilm were as follows: two strains of C. parapsilosis, one strain of C. albicans and one strain of C. tropicalis. A weak biofilm production was displayed by one strain of C. parapsilosis, four strains of C. albicans and three strains of C. tropicalis. Only three strains of C. tropicalis were no biofilm producers. The EO of 0.125% on the C. parapsilosis and C. tropicalis strains exhibited an excellent activity against biofilm production, while on C. albicans strains, the results were as follows: no activity on two strains, a weak activity on one strain and a medium activity on the two remaining strains. Statistical analysis and, in particular, Student’s t-test revealed a p ¼ 0.0009 for C. parapsilosis and p ¼ 0.0004 for C. tropicalis demonstrating an extremely

Range

MBC

Range 0.125– 0.5 0.25– 1 0.5– 1 0.5– 2 0.5– 2 0.5– 4

0.5 1 0.5 1

2 4

1 2

0.5 1 0.03 0.03

0.03 0.03

0.03– 0.125 0.125 0.06– 0.25 0.25

0.008– 0.03 0.016– 0.03

0.008– 0.03 0.008– 0.03

MIC90

Range

Range

MIC90

Voriconazole (mg/mL)

Fluconazole (mg/mL)

0.5 0.5

MIC90

Amphotericin B (mg/mL)

C. albicans (5) 24 h , 0.125 –1 0.25 0.125–0.5 48 h 0.125 –1 0.5 0.25–0.5 C. tropicalis (5) 24 h , 0.125 –. 16 6 0.125–0.5 48 h , 0.125 –. 16 6 0.25–1 C. parapsilosis (6) 24 h , 0.125 –8 2 0.25–0.5 48 h , 0.125 –16 4 0.25–1

Organism (n= 16)

Myrtle % (v/v)

Table 1. In vitro susceptibility of Candida spp. isolates to Myrtle oil and six antifungal drugs.

MIC90

Range

0.25– 1 0.5– 1

1 1

0.03– 0.06 0.06 0.03– 0.06 0.06

0.25– 1 0.5– 2

1 2

MIC90

0.125– 1 0.25– 1

1 1

0.25 0.25

0.03– 0.125 0.125 0.06– 0.125 0.125

Range

Caspofungin (mg/mL)

0.06– 0.125 0.125 0.125– 0.25 0.06– 0.125 0.125 0.125– 0.25

0.03 0.03

MIC90

Micafungin (mg/mL)

0.008– 0.03 0.03 0.008– 0.03 0.008– 0.03 0.03 0.008– 0.03

Range

Anidulafungin (mg/mL)

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S. Cannas et al. Biofilm productions Biofilm Candida spp. Biofilm Candida spp. and Myrtus communis EO

OD 595 nm

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2.5 2 1.5 1 0.5 0 1

2

3

4

C. parapsilosis

5

6

7

8

9

10 11 12 13 14 15 16

C. tropicalis

C. albicans

Strains

Figure 1. Biofilm production of Candida species and effects of Myrtle oil.

statistically significant difference, while the p value of 0.245 for C. albicans indicated a nonstatistically significant difference. The increasing incidence of drug-resistant pathogens and the toxicity of existing antifungal compounds have drawn the attention towards the antimicrobial activity of natural products. The small number of drugs available for fungal treatment, most of which are fungistatic, and the emerging resistance to antifungal agents have encouraged the search for alternative treatments (de Oliveira et al. 2006). Several studies have been conducted using natural products to evaluate interference in C. albicans biofilm and anticandidal activity on planktonic and biofilm cultures of the C. parapsilosis complex (Furletti et al. 2011; Pires et al. 2011). The interest in natural products has increased regardless of whether they are associated with other therapies. There is a need to search for new products with effective antifungal abilities because of the adverse side effects of existing medications, the increasing emergence of strains that are resistant to conventional antifungal agents and the biofilm formation in medical devices and tissues. Conclusion The results obtained exhibited a considerable activity of the extract against C. albicans and C. parapsilosis after 24 – 48 h, while against C. tropicalis, a higher MBC was observed. At first sight, the results obtained appear higher than those obtained with the conventional antimycotics tested, but lower than those reported in the literature. It is also important to stress that these strains were adhesive and biofilm producers, and the effect of myrtle EO on the biofilm production had yielded encouraging results. Our assays included not only C. albicans, but also non-albicans strains of Candida, with promising results. These data are very interesting considering that these yeasts are resistant to the conventional antifungal drugs, as it can be seen also from our results, making them difficult to be treated. Further studies are necessary to evaluate the exact chemical composition and toxicity of the Myrtle EO.

Supplementary material Supplementary material relating to this article is available online, alongside Table S1.

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Conflict of interest The authors declare that there is no conflict of interest.

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Antifungal, anti-biofilm and adhesion activity of the essential oil of Myrtus communis L. against Candida species.

Candida species belong to the normal microbiota of the oral cavity, gastrointestinal tract and vagina. The increasing incidence of drug-resistant path...
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