Cytotoxicity and anti-Sporothrix brasiliensis activity of the Origanum majorana Linn. oil

Abstract The study aimed to evaluate the anti-Sporothrix sp. activity of the essential oil of Origanum majorana Linn. (marjoram), its chemical analysis, and its cytotoxic activity. A total of 18 fungal isolates of Sporothrix brasiliensis (n: 17) from humans, dogs and cats, and a standard strain of Sporothrix schenckii (n: 1) were tested using the broth microdilution technique (Clinical and Laboratory Standard Institute - CLSI M27-A3) and the results were expressed in minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC). The MIC50 and MIC90 of itraconazole against S. brasiliensis were 2 µg/mL and 8 µg/mL, respectively, and the MFC50 and MFC90 were 2 µg/mL and >16 µg/mL, respectively, with three S. brasiliensis isolates resistant to antifungal. S. schenckii was sensitive at MIC of 1 µg/mL and MFC of 8 µg/mL. For the oil of O. majorana L., all isolates were susceptible to MIC of ≤2.25-9 mg/mL and MFC of ≤2.25-18 mg/mL. The MIC50 and MIC90 were ≤2.25 mg/mL and 4.5 mg/mL, respectively, and the MFC50/90 values were twice more than the MIC. Twenty-two compounds were identified by gas chromatography with a flame ionization detector (CG-FID) and 1,8-cineole and 4-terpineol were the majority. Through the colorimetric (MTT) assay, the toxicity was observed in 70-80% of VERO cells between 0.078 and 5 mg/mL. For the first time, the study demonstrated the satisfactory in vitro anti-Sporothrix sp. activity of marjoram oil and further studies are needed to ensure its safe and effective use.

Anti- Sporothrix spp. activity of medicinal plants

ABSTRACT Cases of sporotrichosis in humans and animals without satisfactory clinical response have increased, a warning sign of strains resistant to conventional antifungal agents. The urgent search for alternative therapies was an incentive for research on medicinal plants with anti-Sporothrix spp. properties. A bibliographic survey was performed based on scientific papers about in vitro and in vivo antifungal activity of essential oils and extracts of plants in differents solvents against the fungal of the Sporothrix schenckii complex. The study methodology consisted of a literature review in Google Scholar, Science Direct, Pubmed, Bireme and Springer link with papers from 1986 to 2015. We found 141 species of plants that were investigated, of which 100 species were concentrated in 39 botanical families that had confirmed anti-Sporothrix activity. Combretaceae, Asteraceae and Lamiaceae represented the botanical families with the greatest number of plants species with antifungal potential, using different methodologies. However, there are few studies with medicinal plants in experimental infection in animals that prove their activity in the treatment of sporotrichosis. It reinforces the need for further research related to standardization of in vitro methodologies and in vivo studies related to safety and to toxicity potential of these plants with anti-Sporothrix spp. activity.

RESUMO Casos de esporotricose em humanos e animais sem resposta clínica satisfatória têm aumentado, sinal de alarme para o surgimento de cepas resistentes aos antifúngicos convencionais. A urgente busca por alternativas terapêuticas tem incentivado as pesquisas em plantas medicinais com atividade anti-Sporothrix spp. Um levantamento bibliográfico foi realizado com base em artigos científicos sobre a atividade antifúngica in vitro e in vivo de óleos essenciais e extratos de plantas preparados em diferentes solventes contra o complexo Sporothrix schenckii. A metodologia do estudo consistiu em uma revisão bibliográfica em Google Scholar, Science Direct, Pubmed, Bireme e Springer link com artigos desde 1986 até 2015. Foram encontradas 141 espécies de plantas já investigadas, das quais 100 espécies concentradas em 39 famílias botânicas apresentaram atividade anti-Sporothrix spp. confirmada. Combretaceae, Asteraceae e Lamiaceae representaram as famílias botânicas com maior número de espécies vegetais com potencial antifúngico, empregando diferentes metodologias. Entretanto, há poucos estudos com plantas medicinais em infecção experimental animal comprovando sua atividade no tratamento da esporotricose. Reforça-se a necessidade de mais pesquisas relacionadas à padronização de metodologias in vitro e a estudos in vivo relacionados à segurança e potencial tóxico dessas plantas com atividade anti-Sporothrix spp.

Susceptibility of dermatophytic fungi to commonly used disinfectants / Suscetibilidade de fungos dermatófitos a desinfetantes comumente utilizados

This study aimed evaluate the antidermatophytic activity of three commercial disinfectants commonly used for environmental control of microorganisms in veterinary medicine. Sodium hypochlorite at 40 µL/mL, chloro-phenol derived at 30 µL/mL and chlorhexidine digluconate at 66.7 µL/mL were tested against 14 strains of dermatophytes, identified as Microsporum canis (n: 3) and Microsporum gypseum (n: 11). The tests was performed in accordance with guidelines of the Clinical and Laboratory Standards Institute (CLSI), documents M38-A2 and M51-A, adapted to disinfectants. In the microdilution broth test, chlorhexidine digluconate had MIC values (Minimum Inhibitory Concentration) of 4.16 µL/mL and MCF (Minimum Fungicidal Concentration) from 4.16 to 8.33 µL/mL, while chloro-phenol derived obtained MIC and MCF of 1.87 µL/mL, and both disinfectants had fungicidal activity at concentrations below the recommended. Sodium hypochlorite obtained MIC from 10 to 80 µL/mL and MFC of 40 to 80 µL/mL, requiring at most isolates twice the recommended concentration to achieve same activity. In the disc diffusion test, the mean inhibition zones for chlorhexidine digluconate was 10.53 mm, for chloro-phenol of 9.9 mm and for sodium hypochlorite was 6.2 mm. Chlorhexidine digluconate and chloro-phenol presented a significant reduction in the growth of dermatophytes, while sodium hypochlorite in concentration recommended showed a low antifungal activity against tested isolates.

O objetivo do estudo foi avaliar a atividade antidermatofítica de três desinfetantes comerciais frequentemente utilizados no controle ambiental de micro-organismos em medicina veterinária. Hipoclorito de sódio a 40 µL/mL, derivado de clorofenol a 30 µL/mL e digluconato de clorexidine a 66.7 µL/mL foram testados contra 14 cepas de dermatófitos, identificados como Microsporum canis (n: 3) e Microsporum gypseum (n: 11). Foram utilizadas as diretrizes do Clinical and Laboratory Standard Institute (CLSI), documentos M38-A2 e M51-A, com adaptações para desinfetantes. Na microdiluição em caldo, digluconato de clorexidine apresentou valores de CIM (Concentração Inibitória Mínima) de 4.16 µL/mL e CFM (Concentração Fungicida Mínima) entre 4.16 a 8.33 µL/mL; derivado de clorofenol obteve CIM e CFM de 1.87 µL/mL, demonstrando que ambos os desinfetantes apresentaram atividade fungicida em concentrações inferiores às recomendadas. O hipoclorito de sódio demonstrou CIM entre 10 a 80 µL/mL e CFM de 40 a 80 µL/mL, requerendo duas vezes a concentração recomendada pelo fabricante para obter atividade fungicida frente a maioria dos isolados fúngicos testados. No teste de disco-difusão, a média das zonas de inibição do digluconato de clorexidine foi de 10.53 mm; do derivado clorofenol 9.9 mm e do hipoclorito de sódio 6.2 mm. O digluconato de clorexidine e o derivado cloro-fenol apresentaram redução significante no crescimento dos dermatófitos testados, enquanto o hipoclorito de sódio, na concentração recomendada, demonstrou baixa atividade antifúngica contra os dermatófitos testados.

Clinical and mycological analysis of dog's oral cavity

The oral microbiota of humans and animals is made up of a wide variety of yeasts and bacteria, but microbiota of dogs is not totally described. Although such identification is an important step to establish the etiopathogenesis and adequate therapy for the periodontal disease The aim of this study was to evaluate and correlate oral alterations with the presence of yeasts in oral cavity of female dogs. After clinical evaluation samples from healthy and from dogs with oral diseases were obtained from three different oral sites by swabs, curettes, millimeter periodontal probes and HA membrane tip in cellulose ester. Yeast identification was performed through macroscopic and microscopic colony features and biochemical tests. Dental calculus was the most prevalent occurrence in the oral cavity of 59 females. However, the isolation of yeasts was significantly higher (p < 0.05) in animals suffering from halitosis. Eleven yeast species were identified, namely: Malassezia pachydermatis, Rhodotorula spp., Candida albicans, C. catenulata, C. famata, C. guilliermondii, C. parapsilosis, C. intermedia, Trichosporon asahii, T. mucoides and Cryptococcus albidus. It could be concluded that the yeasts are part of the microbiota from the different sites of the oral cavity of the female canines studied without causing any significant alterations except halitosis.

Efeito antifúngico de agentes químicos sobre leveduras com potencial patogênico isoladas de ambiente hospitalar veterinário / Antifungal effect of chemical agents in yeasts with pathogenic potential isolated from veterinary hospital environment

Os ambientes hospitalares podem albergar micro-organismos patogênicos e oportunistas, capazes de infectar, particu- larmente, os indivíduos imunossuprimidos. O processo de limpeza e desinfecção assume importância capital para o controle de tais doenças. O presente trabalho avaliou a suscetibilidade de leveduras isoladas de superfícies de ambiente hospitalar veterinário frente a quatro desinfetantes/antissépticos comumente utilizados na rotina de desinfecção. O teste de microdiluição em caldo foi realizado com 24 isolados leveduriformes frente ao hipoclorito de sódio 4%, cloreto de benzalcônico 2%, clorexidina-cetrimida 6,6% e derivado de cloro-fenol 3%. A clorexidina-cetrimida, cloreto de ben- zalcônio e derivado de cloro-fenol foram eficazes em todos os isolados com concentração inibitória mínima e concen- tração fungicida inferiores à concentração recomendada pelo fabricante. Por outro lado, a ação fungicida do hipoclorito de sódio em 79,1% dos isolados testados foi obtida na concentração recomendada pelo fabricante, com desempenho inferior aos demais desinfetantes avaliados.

Hospital environments can have pathogens and opportunistic pathogens, important for immunocompromising individuals. The process of cleaning and disinfection constitutes an important measure for the control of these diseases. This study evaluated the sensitivity of yeasts isolated from surfaces of nosocomial veterinary environment to four disinfectants/antiseptics used in the routine of disinfection. The test of broth microdilution was carried in 24isolates of yeasts against 4% sodium hypochlorite, 2% benzalkonium chloride, 6.6% chlorexidine-cetrimide and 3% chloro-phenol derivate. Chlorexidine-cetrimide, benzalkonium chloride and chloro-phenol derivate were efficient in all isolates with minimal inhibitory concentration and minimal fungicide concentration lower than the concentra- tion recommended by manufacturer. By other hand, fungicide action of the sodium hypochlorite in the concentration recommended by the manufacturer was verified in 79.1% of the isolated tested, with the lower performance of than the others evaluated disinfectants.

Alterações hematológicas em felinos com esporotricose cutânea / Hematological alterations of cats with cutaneous sporotrichosis / Alterações hematológicas en felinos con esporotricosis cutânea

A esporotricose é a micose subcutânea de maior ocorrência em pequenos animais, especialmente felinos e caninos. Esta doença causa lesões cutâneas ulceradas de caráter subagudo ou crônico que podem resultar no comprometimento do estado geral do animal. Este estudo avaliou alterações hematológicas em gatos com esporotricose relacionando-as com a forma clínica da doença. As alterações hematológicas foram observadas em 73,3% dos animais, sendo caracterizadas principalmente por leucocitose por neutrofilia e anemia. Os resultados demonstram que animais com a forma cutânea fixa da micose não apresentam alterações no hemograma, enquanto animais com a forma cutânea disseminada desenvolvem importantes alterações que indicam o agravamento do quadro clínico com possibilidade de evolução da micose para uma forma sistêmica.

Sporotrichosis is the most frequent subcutaneous mycosis of small animals, specially cats and dogs. This disease leads toof the mycosis do not present alterations in the blood count. However, animals with the cutaneous disseminated form develop important alterations that can aggravate the disease and evolve the mycosis to a systemic form.

La esporotricosis es la micosis subcutánea de mayor ocurrencia en pequeños animales, específicamente felinos y caninos. Esta enfermedad produce lesiones cutáneas ulceradas de carácter sub agudo o crónico que pueden resultar en el comprometimiento del estado general del animal. Este estudio evalúo alteraciones hematológicas en gatos con esporotricosis, relacionándolas con la forma clínica de la enfermedad. Se ha observado alteraciones hematológicas en 73,3% de los animales, siendo caracterizadas principalmente por leucocitosis por neutrofilia y anemia. Los resultados demuestran que animales con la forma cutánea fija de la micosis no presentan alteraciones en el hemograma, mientras que los animales con la forma cutánea diseminada desarrollan importantes alteraciones que indican el comprometimiento del cuadro clínico con posibilidad de evolución de la micosis para una forma sistémica.

Ultrastructural study of the mycelial phase of clinical isolates of Sporothrix schenckii obtained from feline, canine and human cases of sporotrichosis

Using transmission electron microscopy, we studied the presence of melanin and cell wall thickness of clinical isolates of Sporothrix schenckii obtained from cats, dogs and humans as compared to reference strains. We detected differences regarding presence of the melanin among the clinical isolates of S. schenckii and a correlation between presence of melanin and cell wall thickness.

Efficacy of terbinafine and itraconazole on a experimental model of systemic sporotrichosis / Eficiência de terbinafina e itraconazol em um modelo experimental de esporotricose sistêmica

Itraconazole is currently considered the drug of choice to treat the diverse clinical presentation of sporotrichosis. On the other hand terbinafine by virtue of its excellent in vitro activity is under comparative evaluation for its therapeutic potential for a wide range of fungal infections. In this study, our aim was to determine the in vivo efficacy of terbinafine and itraconazole on a experimental model of systemic sporotrichosis. 120 rats Wistar received an injection of 2x10³ S. schenckii cells by via the lateral tail vein. After 3 days the animals were treated with terbinafine (250mg/kg) and itraconazole (100 mg/kg) and their respective diluents. In our model, terbinafine and itraconazole were effective in reducing the number of clinical lesions and positive organ cultures. There was statistical difference between the groups treated with the antifungals in relation to the control groups (p<0,05) concerning the clinical alterations, anatomic-pathological findings and in the positive organ cultures of the agent, being that the treated animals resulted in the absence and/or reduction of all the evaluated parameters. As for the treatments, terbinafine showed similar or higher activity that itraconazole in the evaluation of the testicle alteration (p=0,0004), as well as in the positive organ cultures of microorganism from the organ (p=0,0142). With these results it is possible to conclude that the antifungals studied are effective in the treatment of experimental systemic sporotrichosis.

Itraconazol é atualmente considerado a droga de escolha para o tratamento das diferentes formas clínicas da esporotricose. Por outro lado a terbinafina devido a sua excelente atividade in vitro está sendo avaliada quanto ao seu potencial terapêutico frente a diversas infecções fúngicas. O objetivo deste estudo foi determinar a eficácia in vivo da terbinafina e itraconazol em um modelo de esporotricose experimental sistêmica. 120 ratos Wistar receberam uma injeção de 2x10³ células de S. schenckii pela veia lateral da cauda. Após 3 dias os animais foram tratados com terbinafina (250mg/kg) e itraconazol (100mg/kg) e os seus respectivos diluentes. No modelo experimental estudado, a terbinafina e itraconazol se mostraram efetivos reduzindo o número de sintomas clínicos e retroisolamento positivo para o agente. Houve diferenças estatísticas entre os grupos tratados com os antifúngicos em relação aos grupos controle (p<0,05) nas alterações clínicas, achados anatomopatológicos e no retroisolamento do agente, sendo que os animais tratados resultaram na ausência e/ou diminuição de todos os parâmetros avaliados. Quanto aos tratamentos a terbinafina se mostrou com atividade similar ou superior ao itraconazol quando avaliado as alterações anatomopatológicos do testículo (p=0,0004), assim como no retoisolamento do órgão (p=0,0142). Com estes resultados permite-se concluir que os antifúngicos estudados são efetivos no tratamento da esporotricose sistêmica experimental.

Contaminação do ar por Aspergillus em ambiente de reabilitação de animais marinhos / Air contamination with Aspergillus species at an indoor rehabilitation centre for sea animals

Espécies fúngicas do gênero Aspergillus são frequentemente associadas com alta mortalidade de aves marinhas em cativeiro. Tendo em vista que a aspergilose geralmente é adquirida pela inalação dos propágulos fúngicos presentes no ar, o estudo objetivou avaliar a qualidade do ar quanto às espécies de Aspergillus, das instalações internas de um centro de reabilitação de animais marinhos que frequentemente recebe pingüins, gaivotas, albatrozes e petréis acometidos por alguma moléstia. Oitenta e um dias de colheitas foram realizados distribuídos em um período de aproximadamente dois anos, através da exposição de placas de Petri contendo Agar Sabouraud dextrose acrescido de cloranfenicol no ambiente, as quais foram posteriormente incubadas a 25ºC. As colônias identificadas como pertencentes ao gênero Aspergillus, foram classificadas quanto à espécie através da avaliação macro e micro morfológica associada a uma chave de identificação. Foram obtidos 43 isolados classificados em sete espécies distintas, sendo A. fumigatus a predominante correspondendo a 27,9%, seguida de A. niger, A. flavus e outras quatro espécies de Aspergillus sp., demonstrando que as aves marinhas estão expostas a espécies fúngicas com potencial patogênico, o que enfatiza a necessidade de um controle microbiológico no ambiente onde são mantidos os animais em cativeiro.

Aspergillosis in captivity seabirds is often associated with elevated rates of mortality. The infection is usually acquired by inhalation of airborne fungal conidia. The aim of this study was to evaluate the presence of Aspergillus species in the indoor environment of a rehabilitation centre for marine animals in Southern Brazil. This centre continuously receives injured penguins, seagulls, albatrosses and petrels. Petri dishes plates with Agar Sabouraud dextrose and chloramphenicol were left open for 15 minutes in 3 distinct points in the rehabilitation centre and then incubated at 25ºC. During a period of two years the indoor air was sampled in 81 occasions. A total of 43 isolates belonging to 7 different Aspergillus species were recovered. Aspegillus fumigatus was the predominant species (27.9%), followed by A. niger (25.6%), and A. flavus (16.3%). Four other Aspergillus species were isolated. This study demonstrates that seabirds were exposed to pathogenic Aspergillus species in our rehabilitation centre, reinforcing the need for a strict microbiology control of the indoor air in the captivity environment.

Esporotricose óssea e cutânea em canino / Cutaneos and osseous sporotrichosis in dog

A esporotricose é uma micose subcutânea causada pelo fungo dimórfico Sporothrix schenckii. Este artigo descreve o primeiro caso de esporotricose óssea e cutânea, em canino, na cidade de Pelotas, Rio Grande do Sul. O animal apresentava lesões ulceradas e crostosas, há aproximadamente três anos no plano nasal e membro torácico direito, dispnéia e apatia. Para confirmação do diagnóstico, foram realizados exames micológico, histopatológico, radiológico e hematológico. O animal foi tratado durante três meses com 10mg/kg de itraconazol,por via oral, obtendo-se a cura das lesões. Este estudo alerta clínicos de pequenos animais para a ocorrência desta micose em caninos na região de Pelotas, RS.

The sporotrichosis is a subcutaneous mycosis caused by dimorphic fungus Sporothrix schenckii. This article describes the first cutaneous and osseous sporotrichosis case in canine in the Pelotas city, Rio Grande do Sul. The animal presented crusts and ulcerated lesions,approximately the three years in the nasal plan and right hind-foot,dispneic and apathy. Mycological, histopathological, radiological and hematological diagnosis was realized. The animal was treated by three months with itraconazole, administered orally at a dosage of 10mg/kg, until lesions disappeared. This study alert small animals clinicians for the occurrence of this mycosis in dogs in the region of Pelotas, RS.