CLSI BROTH MICRODILUTION METHOD FOR TESTING …

1 Brazilian Journal of Microbiology (2009) 40:222-226 ISSN 1517-8382222 CLSI BROTH MICRODILUTION METHOD FOR TESTING susceptibility OFMALASSEZIA PACHYDERMATIS TO THIABENDAZOLEPatr cia da Silva Nascente1*; Ana Raquel Mano Meinerz1; Renata Os rio de Faria 1; Luiz Filipe Dam Schuch2;M rio Carlos Ara jo Meireles2; Jo o Roberto Braga de Mello11 Programa de P s Gradua o em Ci ncias Veterin rias, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil;2 Departamento de Veterin ria Preventiva, Faculdade de Veterin ria, Universidade Federal de Pelotas, Pelotas, RS, BrasilSubmitted: October 18, 2007; Returned to authors for corrections: January 10, 2008; Approved: April 08, , classified as antiparasitic and also used as an antifungal drug, can be found as otologicalsolution indicated for treatment of parasitic and fungal external otitis in small animals. Malasseziapachydermatis is a yeast recognized as a normal inhabitant on the skin and mucous membranes of dogs andcats.

2 However, it is considered an opportunistic agent that causes external otitis and dermatitis in theseanimals. The aim of this study was to evaluate the in vitro effect of thiabendazole against 51 isolates of using the CLSI BROTH MICRODILUTION METHOD that has been adapted for this yeast species(NCCLS, 2002). Based on this test, the Minimum Inhibitory Concentrations (MIC) of thiabendazol wascalculated. Subsequently, the susceptibility of each isolate against this antifungal was determined. It wasobserved that the MIC of thiabendazole against M. pachydermatis ranged from to > 4 g/mL. A totalof of the isolates were found to be resistant, were intermediate and were sensitive tothe drug. The rate of resistance of the yeasts against thiabendazole was similar to the results previouslyobtained with other antifungals, while the adapted BROTH MICRODILUTION technique used in this study provedto be words: Malassezia pachydermatis, Minimum Inhibitory Concentration, thiabendazole*Corresponding Author.

3 Mailing address: Universidade Federal de Pelotas, Faculdade de Veterin ria, Campus Universit rio s/n. CEP: 96010-900. Cap o doLe o, RS, Brasil. Tel.: +55 53 32279876; Fax: +55 53 32759004. E-mail: need for an antihelmintic, with a broad spectrum ofantiparasitic effect, highly efficient, with good safety limits andthat would also be versatile in its administration, promoted astudy of hundreds of benzimidazole compounds. Thiabendazolewas the first compound that fulfilled all of those requirementswith an extraordinary performance (26,27). Its in vitroantifungal activity was observed against Blastomyces spp,Aspergillus spp, Fusarium spp, Penicillium spp andTrichophyton spp, although there have been controversiesregarding its effect against some yeasts, such as Candida sp.(26,30).Malassezia pachydermatis is a yeast that is considered anormal inhabitant and an opportunistic pathogen of the externalear canal of dogs and cats, and it can also be found on the skinand in the rectum, anal sacs and vagina of these animals(2,3,4,5).

4 This yeast is one of the most frequent microorganismsassociated with external otitis in dogs (9,19). In recent years,studies have also reported M. pachydermatis as the cause ofcanine dermatitis (4,6,21,24,28).The appearance of the clinical manifestations ofmalasseziosis indicates an alteration in the balance betweenthe commensal yeast and its host. The onset of clinical diseaseoccurs when there is excessive multiplication of due to alterations in the defense mechanismsof the host. An increase in the number of M. pachydermatiscells is favored when there is a modification in themicroenvironment, such as excessive production of cerumen,after therapy with antibiotics or corticoids (17,24) and also withSusceptibility of M. pachydermatis to thiabendazole223concomitant primary diseases, such as hypersensitivity to food,flea bites and/or atopy, pyodermitis, demodicosis and endocrineproblems (hyperadrenocorticism, hyperthyreodism, diabetesmellitus).

5 Therefore, malasseziosis is an infection frequentlyassociated with concomitant primary diseases (3,17).Only one study of the in vitro activity of thiabendazoleagainst M. pachydermatis has been carried out (18). This studyused the agar diffusion METHOD for TESTING this and otherantifungal drugs against M. pachydermatis. Thiabendazole hadits in vitro inhibitory activity confirmed by this technique,without the calculation of the Minimum InhibitoryConcentration (MIC).This drug has been used in suspension as a fungicide forpoultry breeding and livestock farming, mainly in the preventionof aspergilosis, without showing any incompatibility. Currently,an otological solution of thiabendazole is recommended forthe treatment of mycoses caused by Microsporum canis, , Trichophyton mentagrophytes and T. equinum. InBrazil, the otological solution of this drug is found in productssuch as Otoden plus (Vetbrands) and Otoneodexa (UsinasQuimicas Brasileiras) for the treatment of canine external drug is used in association with neomycin anddexametazone, which amplify the spectrum of action ofThiabendazole, enabling it to be effective for the treatment ofbacterial, fungal and inflammatory canine external otitis (12),including those infections caused by M.

6 Pachydermatis, whichis recognized as a normal inhabitant and is also considered anopportunistic agent that causes external otitis and dermatitis(1,5,16,23).In vitro TESTING of antimicrobial susceptibility is of greatimportance. Some authors recommend the evaluation ofantifungal sensitivity through the use of antimycograms inspecial clinical conditions, such as cases of therapeutic failure,which is quite common in cases of canine external otitis anddermatitis exacerbated by M. pachydermatis (1,8,14).The aim of this study was to use the BROTH microdilutionmethod (22) to test the activity of thiabendazole against 51isolates of M. pachydermatis obtained from clinical cases ofcanine external AND METHODSAll strains, collected with sterile swabs from canine earcanals, were cultured in Sabouraud dextrose agar medium withchloramphenicol and cycloheximide, kept at 32 C for ten days,and their macroscopic characteristics were observed daily.

7 Cellmorphology was observed through the use of colony smears,which were stained with Gram stains and then characterized(15).The National Committee for Clinical Laboratory Standards(22), currently known as the Clinical and Laboratory StandardsInstitute (CLSI) recently published two standardized methodsfor BROTH MICRODILUTION for use in antifungal TESTING , the M27A2and the M38P. The NCCLS BROTH MICRODILUTION (BM) methodadapted for M. pachydermatis was performed by Eichenberget al. and Nascente et al. (9,19) according to the M27A2document (22) and thiabendazole recommendations. Thismethodology was used to evaluate the susceptibility of 51isolates of M. pachydermatis against thiabendazole, with a fewmodifications in the METHOD for this specific antifungal. Theantifungal susceptibility test by BM was carried out in sterilemicrodilution plates (Nuclon ) with 96 flat bottomed wells,composed of eight series identified from A to H, each one withtwelve M.

8 Pachydermatis isolates were suspended in salinesolution with the turbidity adjusted to level 1 of the McFarlandscale. After that, they were diluted in Sabouraud BROTH mediumand a volume of 100 l was dispensed into the wells, whichcontained 100 l of twice the final concentrations of theantifungal. Thus, the final dilution of the starting inoculum was1 the stock-solution of the drug ( mg/mL), dilutedaccording to the methodology recommended by the NCCLS/CLSI (22), ten successive dilutions were prepared with aconcentration 10 times greater than the final solution ofthiabendazole. The dilutions were further diluted to 1:5 inSabouraud dextrose BROTH with chloranphenicol and 100 laliquots were sequentially dispensed into the microdilutionplates, filling the wells in the columns numbered from 1 to columns 11 and 12 were reserved as controls. Thepositive control was composed of 100 l of Sabouraud dextroseagar and 100 l of the half-inoculum solution.

9 The negativecontrol was composed of 200 l of the same culture plates were incubated at 37 C for 72h. The readings weremade visually, comparing the growth of the yeast in the wellsnumbered one to ten (with varying concentrations of antifungal)with the wells that had the positive control (cavity 11). Thelowest concentration that produced a significant inhibition(around 50%) of the growth of the yeast in comparison withthe positive control was identified as the AND DISCUSSIONThe MIC of thiabendazole, observed through the BMmethod against 51 isolates of M. pachydermatis ranged to >4 g/mL (Fig. 1) with a mean MIC of g/mL anda mode of 4 mg/mL. In the current literature there are no studiesof thiabendazole and the adapted BM METHOD to compare observation of the MIC, M. pachydermatis isolateswere classified as sensitive (S), intermediate (I) and resistant(R), according to the criteria proposed by Colombo et al.

10 (7)and the results of the MIC50 and MIC90: S = MIC-strain <MIC-50%; I = MIC-50% > MIC- strain < MIC-90%; R = MIC-224 Nascente, et > MIC-90%. Resistance was verified in seven ( )isolates of the yeast, intermediate sensitivity in 24 ( )isolates and sensitivity in 20 ( ) strains (Table 1).However, when comparing the susceptibility of against thiabendazole with other antifungals,the MIC has different values and the percentage of resistantisolates to this NCCLS/CLSI methods are indicated for some yeastsand filamentous fungi against the antifungals ketoconazole,fluconazole, itraconazole, amphotericine and flucitosine. TheNCCLS (22) recommends the use of RPMI 1640 in antifungaltests with yeasts (Candida spp. and Cryptococcus spp.), althoughit has not yet been used with the Malassezia species (25).Other authors (9,19) used Sabouraud dextrose liquid insteadof RPMI 1640 in the BM METHOD , and reported a better growthof the M27A2 is a reference protocol that was written toprovide consensus on the evaluation of the susceptibility ofyeasts against antifungals.