Susceptibility of Malassezia pachydermatis to azole antifungal agents evaluated by a new broth microdilution method *

* This manuscript is based on a thesis submitted by the senior author in partial fulfillment of requirements for the degree of Master of Veterinary Sciences from the Postgraduate Program in Veterinary Sciences Faculdade de Veterinária (FAVET) de Porto Alegre, Universidade Federal do Rio Grande do Sul (UFRGS), Brazil. 1 Programa de Pós-Graduação em Ciências Veterinárias, FAVET/UFRGS. 2 Fellowship students. 3 Laboratório de Micologia Especial da Escola Paulista de Medicina, São Paulo, Brazil. 4 Laboratório de Pesquisas Micológicas (LAPEMI) da Universidade Federal de Santa Maria (UFSM), Santa Maria, Brazil. 5 Setor de Micologia do Departamento de Patologia Clínica Veterinária (DPCV) FAVET/ UFRGS. CORRESPONDENCE: L. Ferreiro [laerte.ferreiro@ufrgs.br ; FAX: + 55 51 3316 7305]. Received: June 2002 Accepted: March 2003 ABSTRACT


INTRODUCTION
The yeast Malassezia pachydermatis is a common inhabitant of the ear canal of dogs, cats, and other animals, but it can also be found in the in the skin, rectum, anal sacks and vagina.This yeast can also become an opportunistic pathogen [3,6,14].An infectious state caused by the M. pachydermatis usually parallels or follows a primary established illness in the animal [2][3][4]11,13,20,22,23,26,29].Recently, an increase in the incidence of canine otitis by M. pachydermatis has been reported in the region covered by this study [12,18].
The treatment of yeast infections has been rather empirical and is most often unsuccessful.In aggravation, susceptibility tests are seldom performed [8].The in vitro susceptibility tests for Malassezia pachydermatis was first described in 1976 by Maestrone et al. [19].In Brazil, Coutinho & Paula [6,7] described the results of similar test in 1997, utilizing an expensive and non-standardized method, named ETEST.Several other studies have reported distinct procedures and non-standard methods [7,8,15,17,24,26,31,32]. Clearly, a standardization of procedures currently used in veterinary mycology laboratories is still lacking.
The aim of this study was to determine the susceptibility profile of 82 Malassezia pachydermatis cultures obtained from the ear canal of dogs and cats to the common antifungal agents Ketoconazole, Fluconazole and Itraconazole, using a Broth Microdilution Method modified from procedures described by the National Committee for Clinical Laboratory Standards (NCCLS, USA) [25].

Isolates and culture conditions: Eighty-two
Malassezia pachydermatis cultures obtained from the ear canal of dogs and cats were used in this study.Initially, the samples were isolated on Sabouraud dextrose agar supplemented with chloramphenicol 1 (500 mg/L), following incubation at 35°C during four to seven days.All cultures were maintained on the same medium described above, at room temperature, with subcultures being carried out on a monthly basis.Prior to the assays, isolates were subcultured twice on the same medium above, and incubated at 35°C for 48h to ensure purity and optimal growth.Antifungal agents: Media containing each azole agent used in these experiments were prepared in stock solutions.Fluconazole 2 and Itraconazole 3 were dissolved in sterile distilled water and in polyethylene glycol 400 4 , respectively, to obtain stock solutions containing 500µg/mL.The stock solution of Ketoconazole 3 was diluted in a hydrochloric acid solution (0.2N) to a final concentration of 10.000µg/ mL, to be subsequently aliquoted and stored frozen at -70°C until use.
Assay medium: Based on preliminary studies with RPMI 1640 5 and Yeast Nitrogen Base 6 media (data not shown), Sabouraud dextrose broth, supplemented with 1% Tween 80 [16] was chosen for the assays.
Broth microdilution method: Testing was performed in 96-well round-bottom microtiter plates.Cell suspensions were prepared in Sabouraud dextrose supplemented with 1% Tween 80, and inoculum concentrations were adjusted to approximately 0.5 x 10 6 to 3.0 x 10 6 cells/mL.Stock solutions were diluted with Sabouraud dextrose broth supplemented with 1% Tween 80.The final antifungal agent concentrations ranged from 0.125 to 64 µg/mL for Fluconazole, 0.078 to 4.0µg/mL for Itraconazole, and 0.015 to 8.0µg/mL for Ketoconazole.Volumes of 100µL of each antifungal concentration were dispensed in each well, following procedures described elsewhere (M27 NCCLS protocol).Plates were frozen until assayed, when 100µL of the adjusted inoculum were enclosed.Subsequently, plates were incubated at 35°C, to be read after 48h.The MICs (minimal inhibitory concentrations) were defined as the lowest azole concentration at which there was 50% inhibition of growth (MIC-50) compared with a drug-free growth control.A culture of Malassezia pachydermatis with a known clinical sensibility to Itraconazole treatment was employed as a control reference.

RESULTS
A total of 246 MICs for the azoles used in this study were determined from the isolates of Malassezia pachydermatis obtained from clinical cases of canine otitis (Table 1).The MICs varied from 0.015 to 0.25 mg/mL (mean of 0.08 mg/mL), 1.0 to 32.0 mg/mL (mean of 9.22 mg/mL), and 0.007 to 0.125 mg/mL (mean of 0.05 mg/mL) to Ketoconazole, Fluconazole, and Itraconazole, respectively.The variation observed for the MIC to Ketoconazole was greater than to the other azoles used in these experiments.
The patterns of susceptibility for the 82 isolates of M. pachydermatis, considering the MIC values (in mg/mL) for Ketoconazole (MIC-50 ≤ 0.06 and MIC-90 ≤ 0.125), Fluconazole (MIC-50 ≤ 8 and MIC-90 ≤ 16), and Itraconazole (MIC-50 ≤ 0.06 and MIC-90 ≤ 0.125), are presented in Table 2. Overall, for the range of azole concentrations used in the susceptibility study, a very low frequency of resistance was observed in the isolates, with all samples being susceptible to Itraconazole.

DISCUSSION
A better understanding of the fungal susceptibility profiles to antifungal agents is essential for therapeutics, providing epidemiological elements to unravel the reality of mycotic infections.
After intensive investigations by research groups [5,9,10,21,27,28,30,31], in vitro susceptibility tests have been recently standardized (NCCLS M27-A document) [25] for Candida spp.and Cryptococcus neoformans, with some still being currently underway.However, the M27-A technique should not be readily applied to Malassezia pachydermatis, since this yeast has distinct biochemical demands than former species.Due to its excellent growth pattern demonstrated in preliminary studies [33], Sabouraud dextrose broth supplemented with 1% Tween 80, was chosen as the assay medium to be used in these experiments.
The inoculum concentrations recommended by the M27-A document for Candida sp. and Cryptococcus neoformans did not confer an adequate pathogen growth in culture when applied for Malassezia pachydermatis.An optimal growth pattern was obtained only after a minimum of 100-fold increase in cell counts (0.5-3.0 x 10 6 cfu/mL).
The MIC patterns observed in this study displayed an acceptable profile if confronted with assays carried out with those described for Candida sp. and Cryptococcus neoformans.Based on the results obtained, it appears that important variables such as medium, inoculum concentrations, pH, and temperature and time of incubation were adequately selected for the execution of these experiments.
The interpretation of the observed MIC values also requires caution because the classification as susceptible, susceptible-dose depending (SDD), or resistant, as recommended by the M27-A document, applies specifically to Candida sp. and Cryptococcus neoformans.After adapting this guideline to Malassezia pachydermatis, the results showed 28 (34.1%)SDD to Fluconazole, 3 (3.7%)resistant to Ketoconazole, with all isolates being susceptible to Itraconazole.The significance of these findings, however, can only be validated when standardized techniques become available.Further studies on Malassezia pachydermatis, as also valid for others species of importance in veterinary mycology, are still required for the development of standard methodologies that could be useful for comparison studies among MICs.Recently, molecular studies on clinical M. pachydermatis isolates from dogs and cats have demonstrated the existence of some genetic diversity, with strains being classified in subgroups named A, B, C and D [1].The epidemiological implications of this classification regarding strain virulence, clinical presentation and susceptibility to antifungal agents are still unknown.Studies proposed to approach these questions must be encouraged.
Finally, it must be emphasized that the methodology employed in this study is easy to perform, has a very low cost, and could be readily adopted by veterinary laboratories currently investigating clinical and biological features of Malassezia pachydermatis.