Susceptibility variation of Malassezia pachydermatis to antifungal agents according to isolate source Caroline Borges Weiler1, Francielli Pantella Kunz de Jesus2, Graziela Habib Nardi3, Érico Silva Loreto2, Janio Morais Santurio2, Selene Dall’Acqua Coutinho3, Sydney Hartz Alves1,2 1Programa de Pós-graduação em Ciências Farmacêuticas, Universidade Federal de Santa Maria, Santa Maria, RS, Brasil. 2Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Maria, Santa Maria, RS, Brasil. 3Laboratório de Biologia Molecular e Celular, Universidade Paulista, São Paulo, SP, Brasil. Submitted: August 18, 2011; Approved: July 02, 2012. Abstract Malassezia pachydermatis is associated with dermatomycoses and otomycosis in dogs and cats. This study compared the susceptibility of M. pachydermatis isolates from sick (G1) and healthy (G2) ani- mals to azole and polyene antifungals using the M27-A3 protocol. Isolates from G1 animals were less sensitive to amphotericin B, nystatin, fluconazole, clotrimazole and miconazole. Key words: Malassezia pachydermatis, susceptibility, antifungal agents. Currently, the genus Malassezia includes fourteen species, thirteen of which are lipid-dependent and are most frequently recovered from humans, ruminants and horses (Malassezia furfur, M. globosa, M. obtusa, M. restricta, M.slooffiae, M. sympodialis, M. dermatis, M. nana, M. ja- ponica, M. yamatoensis, M. equina, M. caprae and M. cuniculi); the non-lipid-dependent species, M. pachydermatis, is commonly recovered from dogs and cats (Cabañes et al., 2011). The distribution of different species, the colonization prevalence and the population density of Malassezia spp. varies by carrier and affected sites of the body, which are influenced by the skin lipid composition and the presence of competitive microbiota (Sugita et al., 2010). M. pachydermatis is the species most adapted to ani- mals and is often recovered as part of the microbiota of the ear canal and skin of dogs, cats and other species of domes- tic and wild animals. In dogs and cats, M. pachydermatis has been linked to both localized (otitis externa and derma- titis) and systemic disease (Guillot and Bond, 1999). Anti- fungal triazoles are commonly used for treatment, and treatment should be continued until the clinical signs re- solve and yeast are no longer observed during direct examination (Daigle, 2007). The aim of this study was to compare the susceptibil- ity of M. pachydermatis isolates recovered from healthy an- imals and animals with otitis to ketoconazole, fluconazole, itraconazole, voriconazole, clotrimazole, miconazole, nys- tatin and amphotericin B. We studied two groups of M. pachydermatis isolates taken from dogs and cats at the Cellular and Molecular Bi- ology Laboratory of the Paulista University (UNIP, São Paulo, Brazil). Group 1 (G1) was comprised of 40 isolates recovered from the ear canals of animals with otitis externa; group 2 (G2) was comprised of 40 isolates recovered from the ear canals of healthy animals. Isolate identification was confirmed by randomly amplified polymorphic DNA using the Mpa-F (CTGCCATACGGATGCGCAAG) and 58S-R (TTCGCTGCGTTCTTCATCGA) primers (Sugita et al., 2003). Stock solutions of antifungal agents were obtained from the dilution of each antifungal drug in dimethyl sulfo- xide or sterile distilled water for fluconazole. The drugs were serially diluted in RPMI 1640 broth (GIBCOTM) to obtain the following final concentrations: ketoconazole Brazilian Journal of Microbiology 44, 1, 174-178 (2013) Copyright © 2013, Sociedade Brasileira de Microbiologia ISSN 1678-4405 www.sbmicrobiologia.org.br Send correspondence to C.B. Weiler. Programa de Pós-graduação em Ciências Farmacêuticas, Universidade Federal de Santa Maria, Santa Maria, RS, Brasil. E-mail: carolweiler@hotmail.com. Short Communication (16 �g/mL-0.007 �g/mL) (Janssen Beerse), itraconazole (16 �g/mL-0.007 �g/mL) (Janssen Beerse), clotrimazole (64 �g/mL-0.125 �g/mL) (Bayer), voriconazole (16 �g/mL- 0.007 �g/mL) (Pfizer), miconazole (64 �g/mL- 0.125 �g/mL) (Labware), nystatin (64 �g/mL- 0.125 �g/mL) (Bristol-Myers), amphotericin B (16 �g/mL- 0.007 �g/mL) (Bristol-Myers) and fluconazole (64 �g/mL-0.125 �g/mL) (Pfizer). Inocula were obtained from 48-h pure Dixon agar cultures and consisted of micro- organism suspensions in sterile saline (0.85%) plus Triton X-100 (0.05%) (Merck), whose turbidity was adjusted to 0.5 on the McFarland scale. Inocula were diluted 1:50 in sterile distilled water and then at 1:20 in RPMI 1640 broth. For each isolate, microdilution plates containing 10 �Lof antifungals in RPMI 1640 diluted in different concentra- tions were inoculated with 10 �Lof the standardized ino- cula. As positive control, the standardized inocula were cultured alone; the negative control was the antifungal alone diluted in RPMI 1640 broth. Culture plates were in- cubated at 37 °C for 48 h. Minimum inhibitory concentra- tions (MICs) were recorded following the M27-A3 protocol (CLSI, 2007). All tests were performed in dupli- cate. The Mann-Whitney test was used to compare the two groups of isolates to determine whether they had similar susceptibility patterns to the antifungal agents tested. Because no standardized susceptibility testing proce- dures exist for M. pachydermatis, the present study was based on the M27-A3 protocol (CLSI, 2007); in the absence of specific breakpoints for Malassezia spp., we utilized breakpoints described for Candida spp. In this work, the lowest MICs were observed with ketoconazole, itraconazole and voriconazole (Table 1). Voriconazole showed the smallest variations of MICs with the MICs for G1 isolates ranging from 0.01-0.25 �g/mL, while the MICs for G2 isolates ranged from 0.01 to 0.125 �g/mL. These data are consistent with findings from Gupta et al. (2000) that reported MICs ranging from 0.03 to 0.25 �g/mL. Hammer et al. (2000) reported the lowest MICs with ketoconazole, with MIC50 and MIC90 values similar to values we recorded for G2 isolates (Table 2). Al- though the susceptibility testing for itraconazole, fluco- nazole and amphotericin B showed that 100% of the isolates were susceptible to these antifungals, the MIC range was higher in G1 isolates compared to G2 isolates (Table 2). Nakamura et al. (2000) tested seven Malassezia species and all were susceptible to itraconazole; however, M. pachydermatis exhibited the highest MIC range among the species evaluated. Statistical analysis revealed significant differences in susceptibility between G1 and G2 isolates. In the suscepti- bility tests with amphotericin B, nystatin, fluconazole, miconazole and clotrimazole, G1 isolates had significantly higher MICs compared to G2 isolates (p < 0.05). The exis- tence of a different susceptibility profile between the two groups revealed higher antifungal resistance in the isolates obtained from infected animals (G1). The study by 176 Weiler et al. Table 1 - Distribution of MICs (�g/mL) for M. pachydermatis isolated from ear canals of animals with and without otitis. Groups No. (%) of isolates sensitive to concentrations of: 0.01 0.03 0.06 0.125 0.25 0.5 1.0 2.0 KTZa G1 19 (47.5) - 3 (7.5) 8 (20) 2 (5) 8 (20) - - G2 17 (42.5) 2 (5) 5 (12.5) 10 (25) 4 (10) 2 (5) - - FLZb G1 2 (5) 2 (5) 3 (7.5) 9 (22.5) 5 (12.5) 15 (37.5) 4 (10) - G2 3 (7.5) 2 (5) 2 (5) 11 (27.5) 5 (12.5) 7 (17.5) - - ITZc G1 22 (55) 2 (5) 4 (10) 5 (12.5) 3 (7.5) 4 (10) - - G2 17 (42.5) 3 (7.5) 10 (25) 9 (22.5) 1 (2.5) - - - VRZd G1 10 (25) 10 (25) 9 (22.5) 7 (17.5) 4 (10) - - - G2 16 (40) 9 (22.5) 6 (15) 9 (22.5) - - - - CTZe G1 5 (12.5) 4 (10) 1 (2.5) 7 (17.5) 7 (17.5) 12 (30) 4 (10) - G2 6 (15) 11 (27.5) 4 (10) 9 (22.5) 5 (12.5) 5 (12.5) - - MCZf G1 6 (15) 6 (15) 2 (5) 4 (10) 8 (20) 14 (35) - - G2 9 (22.5) 10 (25) 5 (12.5) 5 (12.5) 4 (10) 7 (17.5) - - NYSg G1 3 (7.5) 6 (15) 3 (7.5) 5 (12.5) 7 (17.5) 11 (27.5) 1 (2.5) 4(10) G2 7 (17.5) 8 (20) 5 (12.5) 6 (15) 5 (12.5) 7 (17.5) 2 (5) - AMBh G1 4 (10) 4 (10) 1 (2.5) 7 (17.5) 7 (17.5) 13 (32.5) 4 (10) - G2 12 (30) 3 (7.5) 2 (5) 8 (20) 3 (7.5) 12 (30) - - aKetoconazole, bFluconazole, cItraconazole, dVoriconazole, eClotrimazole, fMiconazole, gNystatin, hAmphotericin B. G1: isolated from animals with otitis; G2: saprophytic isolates. Lyskova et al. (2007) using M. pachydermatis isolates from animals with and without otitis media found that all isolates exhibited high susceptibility to all of the antifungal agents tested in this study, except for fluconazole, to which 4.4% of the isolates were resistant. However, in this same study, significant differences in susceptibility between the two isolate groups were not observed. Fluconazole resistance has been described previously as Eichenberg et al. (2003) observed fluconazole resistance in 2.4% of the 82 isolates tested. Further, Jesus et al. (2011) induced resistance to fluconazole in vitro, demonstrating that M. pachydermatis isolates can become resistant during treatment with this antifungal. Bernardo et al. (1998) found a M. pachydermatis isolate that was resistant to several anti- fungals, including amphotericin B, nystatin and micona- zole. Malassezia species have a structure resembling a cap- sule around the cell wall that provides protection and is similar in form to the capsule of Cryptococcus spp. (Ashbee and Bond, 2010). In saprophytic strains, this capsule con- tains high levels of lipids, blocking the exposure of fungal antigenic proteins. Factors such as humidity, high tempera- ture and high fat environment favor rapid M. pachydermatis multiplication, which is accompanied by decreased lipid content and exposure of antigenic particles. The reduced susceptibility observed in G1 isolates against azole antifungal agents might be linked to variations in lipid composition. The main mechanism of action of azoles is to inhibit ergosterol synthesis. However, these drugs also act on the cell membrane through direct interaction with its lipid components (Hitchcock et al., 1986). Thus, during in- fection, the body’s immune cells release substances that promote a lipid imbalance in the constitution of the fungal membrane, affecting membrane permeability (De Kruyff et al., 1973) and interfering with the action of azole antifungal agents. In the case of polyene antifungal agents, the re- duced susceptibility of the G1 isolates may be related to the high activity of fungal intracellular enzymes such as cata- lase and/or superoxide dismutase. In microorganisms, catalase plays an important role in the detoxification of re- active oxygen species that are released by phagocytic cells during the host immune response (Hampton et al., 1998). Because polyenes act directly on fungal ergosterol causing direct oxidative damage to the cell membrane, the high con- centration of catalase in fungal cells could protect the fun- gal cells from the oxidative action of polyene antifungal agents, possibly explaining the higher MICs in isolates ob- tained from animals with malasseziosis. This study demonstrated that M. pachydermatis iso- lated from animals with otitis are less sensitive to some antifungal agents than yeasts isolated from animals without otitis. This finding may explain because some malassezio- sis treatment failure and emphasizes the importance to evaluate the susceptibility of this pathogenic fungi. References Ashbee HR, Bond R (2010) Malassezia Species and Immunity: Host-Pathogen Interactions. In: Boekhout T, Guého- Kellermann E, Mayser P, Velegraki A (eds) Malassezia and Susceptibility of M. pachydermatis to antifungal agents 177 Table 2 - In vitro susceptibility of M. pachydermatis isolates to antifungal agents. Antifungal Groups MIC rangea (�g/mL) MIC50 b (�g/mL) MIC90 c (�g/mL) GMd (�g/mL) Ketoconazole G1 0.01-0.5 0.06 0.5 0.048 G2 0.01-0.5 0.06 0.25 0.041 Fluconazole G1* 0.01-1.0 0.25 0.5 0.219 G2** 0.01-0.5 0.125 0.5 0.068 Itraconazole G1 0.01-0.5 0.01 0.25 0.032 G2 0.01-0.25 0.03 0.125 0.032 Voriconazole G1 0.01-0.25 0.03 0.125 0.042 G2 0.01-0.125 0.03 0.125 0.029 Clotrimazole G1* 0.01-1.0 0.25 0.5 0.163 G2** 0.01-0.5 0.06 0.5 0.069 Miconazole G1* 0.01-0.5 0.25 0.5 0.124 G2** 0.01-0.5 0.06 0.5 0.061 Nystatin G1* 0.01-2.0 0.25 1.0 0.181 G2** 0.01-1.0 0.06 0.5 0.084 Amphotericin B G1* 0.01-1.0 0.25 0.5 0.180 G2** 0.01-0.5 0.125 0.5 0.081 G1: isolated from animals with otitis; G2: saprophytic isolates; aInterval between the lowest and highest MICs; bMinimum concentration of the antifungal agent able to inhibit the growth of 50% of the isolates; cMinimum concentration of the antifungal agent able to inhibit the growth of 90% of the isolates; dGeometric mean of MICs. 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