Nos últimos anos, várias espécies de fungos, até então conhecidos como saprófitas do ambiente, têm emergido como importantes patógenos na prática clínica, associados muitas vezes com a resistência aos antimicrobianos disponíveis comercialmente. Dessa forma, o conhecimento de aspectos relacionados à patogenicidade desses micro-organismos, como a formação de biofilme, assim como o perfil de susceptibilidade aos antifúngicos e a novas alternativas terapêuticas faz-se necessário. Uma importante tecnologia neste contexto é a utilização de sistemas nanoestruturados a partir de polímeros biodegradáveis para a veiculação de fármacos. Este trabalho visa verificar em isolados fúngicos emergentes a expressão fenotípica de biofilme através do teste em microplaca de poliestireno e o perfil de susceptibilidade frente a antifúngicos na forma livre e incorporados em sistemas nanoestruturados, através da técnica de microdiluição em caldo. Foram selecionados para o estudo 82 isolados fúngicos potencialmente patogênicos, sendo 26 oriundos do ambiente e 56 procedentes de espécimes clínicos. Quanto à capacidade de formação de biofilme verificou-se que 68 (82,9%) isolados produziram biofilme pelo teste da microplaca, sendo 38 classificados como fortes, 17 como moderados e 13 como fracos produtores. Além disso, observou-se que os isolados clínicos apresentaram maior capacidade de formação de biofilme do que os isolados ambientais. Não houve correlação entre a produção de biofilme e a susceptibilidade aos antifúngicos testados nas células planctônicas. De um modo geral, os fungos foram sensíveis à maioria dos antifúngicos testados neste trabalho. Entretanto, o fluconazol apresentou baixa atividade (CIM≥64μg/mL) para 45% dos isolados avaliados. Com o intuito de investigar o efeito de cetoconazol e de fluconazol associados a nanoestruturas sobre os diferentes fungos apresentados no estudo, também foram desenvolvidas nanocápsulas de poli-ɛ-caprolactona contendo estes fármacos. Análises físico-químicas das nanoestruturas revelaram tamanho médio de partícula variando de 206 nm para as nanocápsulas de cetoconazol (CN) e de 211 nm para as nanocápsulas de fluconazol (FN). Ambas as formulações apresentaram características homogêneas e demonstraram estruturas monodispersas. A eficiência de encapsulação do cetoconazol e do fluconazol nas formulações foi de 86,35 e de 80,5%, respectivamente. Os diversos gêneros investigados parecem se comportar de maneira diferente quando expostos às nanoestruturas dos dois fármacos em estudo, sugerindo que as espécies de Candida poderiam apresentar uma maior susceptibilidade in vitro frente a estas nanoestruturas quando comparadas à forma livre dos antifúngicos em questão. Os resultados obtidos demonstram a potencialidade destas formulações para a veiculação de cetoconazol e fluconazol. Como perspectivas, sugere-se a realização de estudos adicionais para evidenciar o efeito in vitro das nanopartículas sobre os diferentes fungos de importância clínica e ambiental. / In recent years, several fungal species known as environmental saprophytes have emerged as important pathogens in clinical practice, often associated with antimicrobial resistance commercially available. Thus, the knowledge of aspects related to these pathogenic micro-organisms, such as biofilm formation, as well as the profile of susceptibility to antifungal agents and new therapeutic options is necessary. An important technology in this context is the use of nanostructured materials from biodegradable polymers for the placement of drugs. This work aims to examine the emerging fungal isolates in the phenotypic expression of biofilm through the test on polystyrene microplate and the susceptibility profile to antifungals front in the free trade and in the nanostructured systems by the broth microdilution method. In this study, 82 potentially pathogenic fungal isolates were selected, being the 26 from environment originating and the 56 from the clinical specimens. Regarding the ability of biofilm formation was found that 68 (82,9%) isolates produced biofilm by the microplate assay. The isolates were classified as 38 strong, 17 moderate and 13 weak biofilm producers. Moreover, it was noted that clinical isolates showed greater capability for the biofilm formation than the environmental isolates. There was no correlation between the biofilm production and the susceptibility to antifungal agents tested in planktonic cells. In general, the fungi were susceptible of the majority of the antifungal agents tested in this study. However, the fluconazole showed low activity (MIC≥64μg/mL) for 45% of the isolates. In order to investigate the effect of the ketoconazole and the fluconazole associated with nanostructures against different fungi contained in the study, polymeric nanocapsules have also been developed containing these drugs. Physicochemical analyzes showed an average size of nanostructures ranging from 206 nm for the nanocapsules of ketoconazole (CN), and 211 nm for the fluconazole (FN). The formulations showed homogeneous characteristics and demonstrated monodisperse structures. The encapsulation efficiency of ketoconazole and fluconazole in the formulations was 86,35 and 80,5%, respectively. The diverse genera investigated appear to behave differently when exposed to the nanostructures of the two drugs have being studied, suggesting that Candida species could exhibit a greater sensitivity to these nanostructures compared to the free form of antifungal concerned. The results demonstrate the potential of the formulations for the delivery of fluconazole and ketoconazole. As perspectives, we suggest further studies to demonstrate the in vitro effect of the nanoparticles against different fungi from the clinical and the environmental importance.

Charles University, Faculty of Pharmacy, Hradec Králové Field of study: Specialist in laboratory methods Author: Bc. Tomáš Hampl Supervisor: PharmDr. Ondřej Janďourek, Ph.D. Title of diploma thesis: Mycobacterium tuberculosis as causative agent of grave illness The aim of this diploma thesis is to deal with tuberculosis, describe the pathogen, taxonomic classification, describe the origin, course and clinical manifestations of the disease, diagnosis, including laboratory diagnostics, epidemiology of tuberculosis, treatment and prevention. Tuberculosis (TBC) is one of the deadliest infectious diseases in the world caused by bacterium M. tuberculosis in humans. TBC is sometimes referred as a disease of the past, but the recent rise in cases of the disease, and especially the spread of forms of drug-resistant TBC, makes the disease very dangerous. Currently, a major problem is the alarming rise in resistant and multi-resistant tuberculosis, which is not counteracted by commonly used first-line drugs. Therefore, there is a constant emphasis around the world on discovering and testing new potential antituberculotics that could help patients with resistant and multidrug-resistant TBC. In the experimental part, the thesis deals with screening of antimycobacterial activity of potential antituberculotics...

The thermophilic Campylobacters, Campylobacter jejuni and Campylobacter coli are found as commensals in the intestinal tract of healthy mammals and birds. Campylobacter jejuni is one of the leading causes of sporadic food-borne bacterial disease in humans which is predominantly contracted from poultry products. Although the vast majority of these infections are mild, life-threatening complications should be treated with antimicrobials. Patients are usually treated with either macrolides of fluoroquinolones. However, globally there is an increased trend in the development of resistance to these antibiotics. This trend has also been observed in infection of poultry and pigs. The aim of this investigation was to determine antimicrobial sensitivity of thermophilic Campylobacters isolated from pigs and poultry by broth microdilution minimum inhibitory concentration testing. A total of 482 samples of the small intestinal content from poultry and pigs from the Western Cape and Gauteng Provinces were collected and analysed. Thirty-eight Campylobacter isolates were obtained. Statistical analyses included percentage resistance, minimum inhibitory concentrations (MIC50 and MIC90) as well as the distribution percentages of the MICs. The non-parametric Mann-Whitney U test was used to establish any significant differences at an interspecies, interhost and interprovincial level. Analyses of the data obtained revealed indications of decreasing susceptibility to several antibiotic groups including the tetracyclines, macrolides, erythromycin and tylosin, as well as the lincoasamides, and fluoroquinolones. It was found that isolates from the Western Cape were more likely to be resistant to the fluoroquinolones (p = 0.0392), macrolides (p = 0.0262), and lincoasmides (p = 0.0001) and, as well as to a certain extent the pleuromutulins (p = 0.0985), whereas isolates from Gauteng were more resistant to the tetracycyclines (p = <.0001). Poultry Campylobacter spp. were more prone to be resistant to enrofloxacin (p = 0.0021). Campylobacter jejuni, mainly isolated from poultry, was more liable to be resistant to the tetracyclines (chlrotetracycline p= 0.0307), whereas C. coli, predominatly isolated from pigs was more likely to be resistant to the macrolides (tylosin p= 0.063). Four of the bacteria isolated from the Western Cape were resistant to three or more antibiotic classes, namely; tetracyclines, macrolides, lincosamides, pleuromutulins and fluoroquinolones. No multi-resistant Campylobacter spp. were isolated from the flocks in Gauteng. With the exception of tiamulin, the bacterial populations could clearly be divided into resistant and susceptible populations. As consequence of the increased resistance to the antimicrobial classes used for human therapy and the geographical differences in antimicrobial susceptibility, it is recommended that an antimicrobial resistance monitoring system for the thermophilic Campylobacter spp. be initiated in the South Africa National Veterinary Surveillance and Monitoring Programme for Resistance to Antimicorbial Drugs (SANVAD) Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Veterinary Tropical Diseases / unrestricted

Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Study program: Specialist on Laboratory Methods Autor: Bc. Marcela Vízková Supervisor: PharmDr. Ondřej Janďourek, Ph.D. Title: In vitro screening of novel potentially active antibacterial compounds III Since the discovery of penicillin, antibiotics have become part of modern therapeutic approaches. But the global spread of resistance makes their future uncertain. It is necessary to find new antibacterial substances useful in practice. As resistance is a global threat, the theoretical part deals with this issue. In addition to resistance, the theoretical part also briefly describes the antibiotics used, including new antibiotics, which were recently registered or likely to be registered. For selected groups, their mechanism of action is described in detail. Sensitivity should be determined to correctly indicate the antibiotic treatment. For this reason, the theoretical part also describes methods by which the sensitivity of a microbe to an antibiotic can be determined qualitatively or quantitatively. The theoretical part does not neglect the history of the development of antibacterial substances and familiarization with antibiotic policy in the Czech Republic. This diploma thesis is based on...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Study program: Specialist in Laboratory Methods Candidate: Bc. Aneta Janků Supervisor: PharmDr. Ondřej Janďourek, Ph.D. Title of diploma thesis: In vitro screening of novel potentially active antimycotic compounds III Key words: yeasts, moulds, antimycotics, mechanism of action of polyenes, microdilution broth method, MIC - minimal inhibition concentration. The main topic of this diploma thesis is in vitro screening of new, potentially active antifungal compounds. The introductory chapter in the theoretical part of the work is devoted to the epidemiology of fungal infections. The classification of micromycetes and the characteristics of the fungal eukaryotic cell are given below, focusing on composition of the plasma membrane and the cell wall. A separate chapter also deals with specific species of fungi and the diseases they cause. The main topic is drugs from the group of antifungals, where more attention is focused on polyene antifungals, their mechanism of action in detail and the emergence of resistance. An integral part are also the stages of development of new drugs and a list of novels developing antifungals. The final chapter describes the types of methods designed for in vitro evaluation...

Charles University Faculty of Pharmacy in Hradec Kralove Department of Biological and Medical Sciences Study program: Specialist on Laboratory Methods Author: Bc. Tereza Pchálková Supervisor: PharmDr. Ondřej Janďourek, Ph.D. Title of diploma thesis: In vitro screening of potential antimycobacterial compounds active against fast growing strains of Mycobacterium genus II Key words: Mycobacteria, Tuberculosis, Antituberculotics, Microdilution broth method, Minimum inhibition concentration Background: The aim of this diploma thesis has been screening of in vitro antimycobacterial activity of novel compounds against fast growing strains of the genus Mycobacterium (Mycobacterium smegmatis and Mycobacterium aurum). Another aim has been predicting the structure-activity relationships for tested compounds. Methods: The technique used for activity determination was microdilution broth method. The value of minimum inhibition concentration for each compound was determined. The evaluation was performed visually and spectrophotometrically using the Alamar blue indicator. Results: A total of 79 compounds were tested. 22 tested compounds showed significant activity against mycobacteria. The substances were sorted according to the similarity in chemical structure into 10 groups. From a chemical point of view, these...

Ökande antibiotikaresistens utgör en allvarlig samhällsutmaning. För resistensbestämning av bakterier används standardmetoden buljongspädning. Då bestäms minsta inhiberande koncentration (MIC) som den lägsta koncentration av antibiotika som kan förhindra tillväxt. Examensarbetets syfte var att undersöka om val av odlingsbuljong påverkar MIC-värdet för Gramnegativa bakterier. En litteraturundersökning genomfördes av studier som jämfört resistensbestämning i standardbuljong med alternativ buljong. Därutöver gjordes en experimentell jämförelse. Litteraturstudien visade att buljongens sammansättning kan ha betydelse för MIC-värdet, men resultatet varierade med både bakteriearter och typer av antibiotika. Viktigt att beakta är det begränsade materialet i studien, möjliga förklaringar till MIC-varianser är exempelvis bikarbonat och antibiotikums olika känslighet för ämnet. Den experimentella studien visade att buljongvalet inte hade någon inverkan på MIC-värdet när det undersöktes för Klebsiella pneumoniae och antibiotikan meropenem. Det kan möjligen förklaras av att effekten av meropenem inte påverkas av buljongvalet. Slutsatsen är att många faktorer påverkar MIC-värdet och att standardbuljongen är en tillgång med stor mängd historiska data som kan nyttjas för att följa resistensutvecklingen, även om alternativa buljonger i vissa fall kan vara bättre lämpade för att förutsäga behanlingsbarheten. Buljongval bör väljas med noggrannhet efter rådande omständigheter. / Increasing antibiotic resistance is a concerning societal challenge. The standard test for measuring antibiotic susceptibility of bacteria is the method of broth microdilution. This method is used to determine the minimum inbibitory concentration (MIC), defined as the lowest concentration of an antibiotic that can inhibit the growth of a specific strain of a bacterium. This study aimed to investigate whether the choice of broth affects the MIC-value for Gram-negative bacteria. A literature review was conducted to examine studies comparing standard broth against alternative broth, alongside an experimental comparison of the same. The result of the literature review indicated that the composition of the broth does have an effect. However, the effect varies depending on the bacterial species and type of the antibiotic that was analyzed. Its important to note the limited amount of data from both prior work and this study, but one possible explanation to the variance in MIC-values could be e.g., bicarbonate and the specific antibiotic´s sensitivity to this substance. The experimental part of this study concluded that the choice of broth did not affect the resulting MIC-value when the antibiotic susceptibility of Klebsiella pneumoniae to meropenem was analyzed. This could be attributed to the efficacy of meropenem being unaffected by the broths used in this study. The conclusing of this study is that multiple factors contribute to the resulting MIC-value, and that the standard broth with its large volume of historical data, is a valuable asset to use when tracking the evolution of antibiotic resistance, even though an alternative broth may be better suited to predict treatability. The choice of broth should be made with great care and be adapted to actual circumstances and conditions.

A candidíase é uma infecção fúngica oportunista freqüente, causada por leveduras do gênero Candida. A utilização de anti-sépticos como medida complementar na higiene oral é cada vez mais difundida. Sendo assim, é de grande importância a avaliação das metodologias que são utilizadas para avaliação da atividade antifúngica. A avaliação in vitro da sensibilidade de leveduras a anti-sépticos tem sido pouco relatada e os resultados nem sempre são concordantes, pois as metodologias são divergentes. O objetivo desse estudo foi avaliar três metodologias: macro e microdiluição em caldo e difusão em ágar por meio de poços, com utilização dos meios de cultura RPMI 1640 e Mueller-Hinton para suscetibilidade dos anti-sépticos bucais Listerine®, Malvatricin® Dentes Sensíveis, Noplak®Max, Oral B®, Periogard®, Plax® e Reach® frente a C. albicans ATCC 64548, C. glabrata ATCC 90030, C. krusei ATCC 6258, C. parapsilosis ATCC 22019 e C. tropicalis ATCC 750. Em relação à comparação das concentrações inibitórias mínimas de cada meio de cultura, os índices kappa foram bons ou excelentes (0,73 a 1,00). Na metodologia da difusão em ágar o coeficiente de correlação de concordância entre os resultados dos meios de cultura também foi excelente (0,95 a 0,99). Concluímos que ambos os meios de cultura podem ser utilizados na avaliação da suscetibilidade de anti-sépticos bucais frente espécies de Candida, pois apresentaram índices de concordância e correlação de bom à excelente. Quanto à correlação entre as metodologias de macro e microdiluição, o kappa foi excelente (1,00) para todas as cepas no meio Mueller-Hinton e variou de 0,42 a 0,72 (bom a excelente) no meio RPMI-1640. As CIMs geradas pela macro e microdiluição em caldo são concordantes possibilitando a utilização de ambas as metodologias na avaliação de anti-sépticos bucais, no entanto a microdiluição detectou a ação inibitória de anti-sépticos voláteis em menores concentrações. A utilização da difusão em ágar é discutível visto que os anti-sépticos voláteis Listerine® e Malvatricin® não apresentaram atividade inibitória nesta metodologia. Todos os anti-sépticos avaliados apresentaram atividade fungistática e fungicida in vitro pelas metodologias de diluição frente a cepas ATCC de Candida quando avaliado na formulação disponibilizada pelos fabricantes. Os anti-sépticos bucais Oral B® e Periogard® apresentaram ações inibitórias (CIM) e fungicidas (CFM) de 1,25% para todas as cepas, meios e metodologias. O Noplak® e o Plax®, CIM e CFM variando de 1,25% a 2,5% para todas as cepas, meios e metodologias. Os anti-sépticos Listerine® e Malvatricin® apresentaram CIM de 5% a 50% e CFM de 5% a 100% dependendo do meio e metodologia. O anti-séptico Reach® apresentou CIM e CFM variando de 1,25% a 50% dependendo da cepa avaliada. Estudos com maior número de isolados, maior diversidade de espécies, concentrações intermediárias de anti-séptico e resultados interlaboratoriais poderão contribuir na padronização de métodos para avaliação de anti-sépticos bucais. / Candidosis is a frequent opportunistic fungal infection caused by yeasts of the gender Candida. The use of mouthwashes as a preventive measure in oral hygiene has been more and more diffused. Therefore it is of great importance the evaluation of the methodologies that are used on the antifungal evaluation. The in vitro evaluation of yeast susceptibility to mouthwashes has been little reported and the results are not always in agreement. The goal of this study was to evaluate three methodologies: broth macro and microdilution and agar well diffusion, using the media RPMI 1640 and Mueller-Hinton on the susceptibility of the mouthwashes Listerine®, Malvatricin® Dentes Sensíveis, Noplak®Max, Oral B®, Periogard®, Plax® e Reach® against C. albicans ATCC 64548, C. glabrata ATCC 90030, C. krusei ATCC 6258, C. parapsilosis ATCC 22019 e C. tropicalis ATCC 750. The kappa indexes for the comparison between the minimal inhibitory concentrations obtained with each culture medium were good to excellent (0,73 to 1,00). On the agar well diffusion method, the concordance correlation coefficients between the results obtained with the different media were also excellent (0,95 to 0,99). We concluded that both culture media can be used to evaluate the susceptibility of mouthwashes against species of Candida because they have concordance and correlations that go from good to excellent. The kappa indexes for the correlation between the broth macro and microdilution methodologies were excellent (1,00) for all the isolates in the Mueller-Hinton media and varied from 0,42 to 0,72 (good to excellent) with RPMI 1640. The MICs generated by broth macro and microdilution agree which makes it possible to use both methodologies on the evaluation of mouthwashes; however the microdilution detects the inhibitory action of volatile mouthwashes at smaller concentrations. The use of the agar well diffusion method is not advised since the volatile mouthwashes Listerine® and Malvatricin® have not presented inhibitory action in that methodology. All the evaluated mouthwashes presented fungistatic and fungicide activity at the formulation available in the market. The mouthwashes Oral B® and Periogard® presented MICs and MFCs of 1,25% for all strains, methodologies and media. Noplak® and Plax®, MIC and MFC that ranged from 1,25% to 2,5% for all strains, methodologies and media. The mouthwashes Listerine® and Malvatricin® MICs ranged from 5% to 50% and MFCs from 5% to 100% depending on media and methodology. The mouthwash Reach® presented MICs and MFCs ranging from 1,25% to 50% depending on the strain evaluated. Studies with a higher number of strains, bigger variety of species, intermediary concentrations of mouthwashes and interlaboratory results can contribute to the standardization of methods to evaluate mouthwashes.

A candidíase é uma infecção fúngica oportunista freqüente, causada por leveduras do gênero Candida. A utilização de anti-sépticos como medida complementar na higiene oral é cada vez mais difundida. Sendo assim, é de grande importância a avaliação das metodologias que são utilizadas para avaliação da atividade antifúngica. A avaliação in vitro da sensibilidade de leveduras a anti-sépticos tem sido pouco relatada e os resultados nem sempre são concordantes, pois as metodologias são divergentes. O objetivo desse estudo foi avaliar três metodologias: macro e microdiluição em caldo e difusão em ágar por meio de poços, com utilização dos meios de cultura RPMI 1640 e Mueller-Hinton para suscetibilidade dos anti-sépticos bucais Listerine®, Malvatricin® Dentes Sensíveis, Noplak®Max, Oral B®, Periogard®, Plax® e Reach® frente a C. albicans ATCC 64548, C. glabrata ATCC 90030, C. krusei ATCC 6258, C. parapsilosis ATCC 22019 e C. tropicalis ATCC 750. Em relação à comparação das concentrações inibitórias mínimas de cada meio de cultura, os índices kappa foram bons ou excelentes (0,73 a 1,00). Na metodologia da difusão em ágar o coeficiente de correlação de concordância entre os resultados dos meios de cultura também foi excelente (0,95 a 0,99). Concluímos que ambos os meios de cultura podem ser utilizados na avaliação da suscetibilidade de anti-sépticos bucais frente espécies de Candida, pois apresentaram índices de concordância e correlação de bom à excelente. Quanto à correlação entre as metodologias de macro e microdiluição, o kappa foi excelente (1,00) para todas as cepas no meio Mueller-Hinton e variou de 0,42 a 0,72 (bom a excelente) no meio RPMI-1640. As CIMs geradas pela macro e microdiluição em caldo são concordantes possibilitando a utilização de ambas as metodologias na avaliação de anti-sépticos bucais, no entanto a microdiluição detectou a ação inibitória de anti-sépticos voláteis em menores concentrações. A utilização da difusão em ágar é discutível visto que os anti-sépticos voláteis Listerine® e Malvatricin® não apresentaram atividade inibitória nesta metodologia. Todos os anti-sépticos avaliados apresentaram atividade fungistática e fungicida in vitro pelas metodologias de diluição frente a cepas ATCC de Candida quando avaliado na formulação disponibilizada pelos fabricantes. Os anti-sépticos bucais Oral B® e Periogard® apresentaram ações inibitórias (CIM) e fungicidas (CFM) de 1,25% para todas as cepas, meios e metodologias. O Noplak® e o Plax®, CIM e CFM variando de 1,25% a 2,5% para todas as cepas, meios e metodologias. Os anti-sépticos Listerine® e Malvatricin® apresentaram CIM de 5% a 50% e CFM de 5% a 100% dependendo do meio e metodologia. O anti-séptico Reach® apresentou CIM e CFM variando de 1,25% a 50% dependendo da cepa avaliada. Estudos com maior número de isolados, maior diversidade de espécies, concentrações intermediárias de anti-séptico e resultados interlaboratoriais poderão contribuir na padronização de métodos para avaliação de anti-sépticos bucais. / Candidosis is a frequent opportunistic fungal infection caused by yeasts of the gender Candida. The use of mouthwashes as a preventive measure in oral hygiene has been more and more diffused. Therefore it is of great importance the evaluation of the methodologies that are used on the antifungal evaluation. The in vitro evaluation of yeast susceptibility to mouthwashes has been little reported and the results are not always in agreement. The goal of this study was to evaluate three methodologies: broth macro and microdilution and agar well diffusion, using the media RPMI 1640 and Mueller-Hinton on the susceptibility of the mouthwashes Listerine®, Malvatricin® Dentes Sensíveis, Noplak®Max, Oral B®, Periogard®, Plax® e Reach® against C. albicans ATCC 64548, C. glabrata ATCC 90030, C. krusei ATCC 6258, C. parapsilosis ATCC 22019 e C. tropicalis ATCC 750. The kappa indexes for the comparison between the minimal inhibitory concentrations obtained with each culture medium were good to excellent (0,73 to 1,00). On the agar well diffusion method, the concordance correlation coefficients between the results obtained with the different media were also excellent (0,95 to 0,99). We concluded that both culture media can be used to evaluate the susceptibility of mouthwashes against species of Candida because they have concordance and correlations that go from good to excellent. The kappa indexes for the correlation between the broth macro and microdilution methodologies were excellent (1,00) for all the isolates in the Mueller-Hinton media and varied from 0,42 to 0,72 (good to excellent) with RPMI 1640. The MICs generated by broth macro and microdilution agree which makes it possible to use both methodologies on the evaluation of mouthwashes; however the microdilution detects the inhibitory action of volatile mouthwashes at smaller concentrations. The use of the agar well diffusion method is not advised since the volatile mouthwashes Listerine® and Malvatricin® have not presented inhibitory action in that methodology. All the evaluated mouthwashes presented fungistatic and fungicide activity at the formulation available in the market. The mouthwashes Oral B® and Periogard® presented MICs and MFCs of 1,25% for all strains, methodologies and media. Noplak® and Plax®, MIC and MFC that ranged from 1,25% to 2,5% for all strains, methodologies and media. The mouthwashes Listerine® and Malvatricin® MICs ranged from 5% to 50% and MFCs from 5% to 100% depending on media and methodology. The mouthwash Reach® presented MICs and MFCs ranging from 1,25% to 50% depending on the strain evaluated. Studies with a higher number of strains, bigger variety of species, intermediary concentrations of mouthwashes and interlaboratory results can contribute to the standardization of methods to evaluate mouthwashes.

Malassezia pachydermatis is an opportunistic fungus associated to dermatomycoses and otopathies in domestic and wild animals. The aim of this study was to compare the susceptibility profile of clinical isolates of M. pachydermatis against topic and systemic antifungals, through two standardized CLSI techniques: broth microdilution (M27-A3, 2008) and disk diffusion (M44-A, 2004). A standard Candida albicans strain (ATCC 28367) was used as quality control. M. pachydermatis isolates assayed through the broth microdilution method showed susceptibility to anfotericina-B (100%), fluconazole (97,83%), ketoconazole (95.66%), itraconazole (93,48%), followed by clotrimazole and miconazole (86.96%). The disk diffusion method showed susceptibility of 97,83% to nystatin, 95,66% to itraconazole and to amphotericin B, 91,32% to ketoconazole, 89,14% to fluconazole and of 86.96% to miconazole and clotrimazole. Through the in vitro induction of resistance to fluconazole (100%), cross-resistance was observed through the broth microdilution method among the 30 M. pachydermatis isolates against the azoles itraconazole (93%), ketoconazole (97%) and voriconazole (100%). The study of the activities of the essential oils obtained through the broth microdilution, based on geometric means of the minimum fungicidal concentration, showed that concentrations above 195.42 μg/mL, 332.49 μg/mL and 448.8 μg/mL of oregano, Mexican oregano and cinnamon essential oils, respectively, have fungicidal action against M. pachydermatis, independently of the sensitivity or resistance to fluconazole. The fungicidal activity of the essential oils against fluconazole-resistant M. pachydermatis strains is important for further therapeutic studies of this mycosis. Despite the susceptibility observed among sensitive M. pachydermatis strains suggesting fluconazole as a safe drug for the treatment of most cases of superficial and invasive malasseziosis, it is essential continuous surveillance studies to detect changes in the microbiological profile due to therapeutic practices. / Malassezia pachydermatis é um fungo oportunista, associado à dermatomicoses e otopatias em animais domésticos e selvagens. O objetivo deste estudo foi comparar o perfil de susceptibilidade de isolados clínicos de M. pachydermatis frente antifúngicos tópicos e sistêmicos, através de duas técnicas padronizadas pelo CLSI: microdiluição em caldo (M27- A3, 2008) e disco-difusão (M44-A, 2004). Para todos os testes foi utilizada uma cepa padrão de Candida albicans (ATCC 28367) como controle de qualidade. Isolados de M. pachydermatis testados através do método de microdiluição em caldo apresentaram em ordem decrescente de susceptibilidade: anfotericina-B (100%), fluconazol (97,83%), cetoconazol (95,66%), itraconazol (93.48%), seguido de clotrimazol e miconazol (86,96%). Pelo método de disco-difusão 97,83% dos isolados foram suscetíveis a nistatina, 95,66% ao itraconazol e a anfotericina-B; 91,32% ao cetoconazol, 89,14% ao fluconazol; e 86,96% ao miconazol e clotrimazol. Através da indução de resistência in vitro, de 30 isolados de M. pachydermatis, ao fluconazol (100%) foi possível evidenciar resistência cruzada pelo método de microdiluição em caldo frente aos antifúngicos azólicos, itraconazol (93%), cetoconazol (97%) e voriconazol (100%). O estudo da atividade de óleos essenciais gerados pelo método de microdiluição em caldo, baseado nas médias geométricas das CFMs, evidenciou-se que concentrações acima de 195.42 μg/mL de óleo essencial de orégano, 332.49 μg/mL de óleo essencial de orégano mexicano e 448.8 μg/mL de óleo essencial de canela, possuem ação fungicida in vitro sobre M. pachydermatis, independente da sensibilidade ou resistência ao fluconazol. A atividade de óleos essenciais frente M. pachydermatis fluconazol resistente, é um achado que consolida a importância destes para estudos futuros voltados a terapêutica desta micose. Apesar dos dados de susceptibilidade apresentados pelas cepas sensíveis de M. pachydermatis sugerirem que o fluconazol ainda é uma droga segura para terapêutica da maioria dos casos de malasseziose superficial ou invasiva, é fundamental que estudos de vigilância epidemiológica sejam realizados continuamente para evidenciar mudanças no perfil microbiológico em função de práticas terapêuticas.