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Background: Due to its ocular microflora, the equine species is predisposed to develop mycotic ulcers which, when notproperly treated, can lead to the formation of a stromal abscess. A stromal abscess occurs through the introduction ofmicroorganisms into the corneal stroma. During re-epithelialization, the foreign body is encapsulated, thus creating abarrier that protects bacteria or fungi from treatment with antimicrobial medication. This framework can end up resultingin blindness due to chronic iridocyclitis, putting the animal’s vision at risk. The current work aims to report a case of corrective surgery for stromal abscess in a mare with the administration of intraoperative intrastromal fluconazole, in orderto corroborate the effectiveness of the technique.Case: A 9-year-old mare was evaluated, with the complaint that her right eye was closed and “yellowish” and that she hadalready been treated with intramuscular injectable anti-inflammatory drugs based on flunexin meglumine (Banamine® -50 mg) for 15 days, referring to a possible ulcer in the right eye. Ophthalmic screening resulted in a negative direct reflexand no threat response in the right eye. Examination of the conjunctiva showed congestion and chemosis. Examination ofthe cornea of the right eye was negative for Fluorescein and Green Lissamine tests, and opacity and corneal neovascularization were noted. The final diagnosis was a corneal abscess of probable fungal origin secondary to a keratomycosis. Afterthe consultation, complementary blood and biochemical tests were performed, which showed normal results for the speciesin question, and treatment was started with eye drops based on atropine 1% (Fagra® - 20 mL), ciprofloxacin antimicrobialeye drops (Ciprovet Colirio® - 5 mL), and antifungal eye drops based on ketoconazole...

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Abstract Fungal infections have become a concern for health professionals, and the emergence of resistant strains has been reported for all known classes of antifungal drugs. Among the fungi causing disease, we highlight those that belong to the genus Aspergillus. For these reasons, the search for new antifungals is important. This study examines the effects of a coumarin derivative, 4-acetatecoumarin (Cou-UMB16) both alone and together with antifungal drugs, and its mode of action against Aspergillus spp. Cou-UMB16 was tested to evaluate its effects on mycelia growth, and germination of Aspergillus spp. fungal conidia. We investigated its possible action on cell walls, on the cell membrane, and also the capacity of this coumarin derivative to enhance the activity of antifungal drugs. Our results suggest that Cou-UMB16 inhibits Aspergillus spp. virulence factors (mycelia growth and germination of conidia) and affects the structure of the fungal cell wall. When applying Cou-UMB16 in combination with azoles, both synergistic and additive effects were observed. This study concludes that Cou-UMB16 inhibits mycelial growth and spore germination, and that the activity is due to its action on the fungal cell wall, and that Cou-UMB16 could act as an antifungal modifier.

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Abstract Since, there is no study reporting the mechanism of azole resistance among yeasts isolated from aquatic environments; the present study aims to investigate the occurrence of antifungal resistance among yeasts isolated from an aquatic environment, and assess the efflux-pump activity of the azole-resistant strains to better understand the mechanism of resistance for this group of drugs. For this purpose, monthly water and sediment samples were collected from Catú Lake, Ceará, Brazil, from March 2011 to February 2012. The obtained yeasts were identified based on morphological and biochemical characteristics. Of the 46 isolates, 37 were Candida spp., 4 were Trichosporon asahii, 3 were Cryptococcus laurentii, 1 Rhodotorula mucilaginosa, and 1 was Kodamaea ohmeri. These isolates were subjected to broth microdilution assay with amphotericin B, itraconazole, and fluconazole, according to the methodology standardized by the Clinical and Laboratory Standards Institute (CLSI). The minimum inhibitory concentrations (MICs) of amphotericin B, itraconazole, and fluconazole were 0.03125–2 µg/mL, 0.0625 to ≥16 µg/mL, and 0.5 to ≥64 µg/mL, respectively, and 13 resistant azole-resistant Candida isolates were detected. A reduction in the azole MICs leading to the phenotypical reversal of the azole resistance was observed upon addition of efflux-pump inhibitors. These findings suggest that the azole resistance among environmental Candida spp. is most likely associated with the overexpression of efflux-pumps.

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Abstract Objective Candida albicans is the primary causative agent of oral candidosis, and one of its key virulent attributes is considered to be its ability to produce extracellular phospholipases that facilitate cellular invasion. Oral candidosis can be treated with polyenes, and azoles, and the more recently introduced echinocandins. However, once administered, the intraoral concentration of these drugs tend to be sub-therapeutic and rather transient due to factors such as the diluent effect of saliva and cleansing effect of the oral musculature. Hence, intra-orally, the pathogenic yeasts may undergo a brief exposure to antifungal drugs. We, therefore, evaluated the phospholipase production of oral C. albicans isolates following brief exposure to sub-therapeutic concentrations of the foregoing antifungals. Materials and methods Fifty C. albicans oral isolates obtained from smokers, diabetics, asthmatics using steroid inhalers, partial denture wearers and healthy individuals were exposed to sub-therapeutic concentrations of nystatin, amphotericin B, caspofungin, ketoconazole and fluconazole for one hour. Thereafter the drugs were removed and the phospholipase production was determined by a plate assay using an egg yolk-agar medium. Results The phospholipase production of these isolates was significantly suppressed with a percentage reduction of 10.65, 12.14, 11.45 and 6.40% following exposure to nystatin, amphotericin B, caspofungin and ketoconazole, respectively. This suppression was not significant following exposure to fluconazole. Conclusions Despite the sub-therapeutic, intra oral, bioavailability of polyenes, echinocandins and ketoconazole, they are likely to produce a persistent antifungal effect by suppressing phospholipase production, which is a key virulent attribute of this common pathogenic yeast.

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MENDONÇA, Estela Cristina Lima. Universidade Federal do Acre, março de 2020. Perfil de susceptibilidade in vitro da Malassezia pachydermatis isolada de quadros otológicos caninos a diferentes extratos de plantas e ao mel de Melipona eburnea. Orientador: Acácio Duarte Pacheco, Coorientadora Luciana dos Santos Medeiros. A Malassezia pachydermatis é uma levedura lipofílica não lipodependente, comensal e oportunista da pele de caninos doméstico. O tratamento convencional contra malasseziose é baseado no uso de antifúngicos tópicos ou sistêmicos, principalmente por antifúngicos da classe dos azóis. O uso indevido destes medicamentos torna a formação de microrganismos resistentes mais recorrente, isto influencia no aparecimento de otopatias crônicas ou recidivantes. Desta forma, o objetivo deste estudo foi a avaliação da susceptibilidade in vitro de cepas de Malassezia pachydermatis isoladas de quadros otológicos caninos, atendidos na Unidade de Ensino e Pesquisa em Medicina Veterinária da UFAC a diferentes extratos de plantas e ao mel de Melipona eburnea. Foram incluídos no presente estudo 32 cães atendidos na Unidade de Ensino e Pesquisa em Medicina Veterinária, o projeto teve a aprovação do CEUA da UFAC, sob protocolo de autorização nº 51/2018. Ao todo 23 isolados de Malassezias pachydermatis da orelha de cães com otopatias foram armazenados em meio BHI (Brain Heart Infusion) com glicerol a 20%. Cada inóculo gerou uma suspensão de 1-5x10 CFU/Ml na escala de 0,5 McFarland, todos foram testados através difusão em poço e microdiluição em caldo para determinar a CIM (Concentração inibitória Mínima) e CFM (Concentração Fungicida Mínima) a diferentes extratos de plantas (Carapa guianensis, Copaifera sp., Momordica charantia Linn, Croton sp., Arrabidaea chica, Bertholletia excelsa, Euterpe oleracea, Oenocarpus bataua Mart) e mel de Melipona eburnea, utilizando como controle positivo o Fluconazol. Durante o teste de difusão em poço apenas o óleo de Copaifera sp. teve efeito inibitório sobre a Malassezia pachydermatis, apresentando diferentes tamanhos de halos (mm). A CIM do óleo foi de 0,61% e a CFM de 0,61% na maior parte dos inóculos. Tal resultado é favorável no que diz respeito a elaboração de novos tratamentos que têm como base o uso de fitoterápicos contra o fungo.

MENDONÇA, Estela Cristina Lima. Universidade Federal do Acre, March 2020. In vitro susceptibility profile of Malassezia pachydermatis isolated from canine otological pictures to different plant extracts and to Melipona eburnea honey. Advisor: Acácio Duarte Pacheco, Co-supervisor Luciana dos Santos Medeiros. Malassezia pachydermatis is a non-lipid-dependent, commensal and opportunistic lipophilic yeast from domestic canine skin. Conventional treatment against malasseziosis is based on the use of topical or systemic antifungals, mainly by azole class antifungals. The improper use of these drugs makes the formation of resistant microorganisms more recurrent, this influences the appearance of chronic or recurrent diseases. Thus, the aim of this study was to evaluate the in vitro susceptibility of strains of Malassezia pachydermatis isolated from canine otological conditions, treated at the UFAC Teaching and Research Unit in Veterinary Medicine to different plant extracts and Melipona eburnea honey. In the present study, 32 dogs treated at the Teaching and Research Unit in Veterinary Medicine were included in the study. The project was approved by CEUA at UFAC, under authorization protocol nº 51/2018. Altogether 23 isolates of Malassezias pachydermatis from the ear of dogs with ear diseases were stored in BHI (Brain Heart Infusion) medium with 20% glycerol. Each inoculum generated a suspension of 1-5x10 CFU / Ml on the scale of 0.5 McFarland, all were tested by diffusion in well and microdilution in broth to determine the MIC (Minimum inhibitory concentration) and CFM (Minimum fungal concentration) to different extracts plants (Carapa guianensis, Copaifera sp., Momordica charantia Linn, Croton sp., Arrabidaea chica, Bertholletia excelsa, Euterpe oleracea, Oenocarpus bataua Mart) and Melipona eburnea honey, using Fluconazole as a positive control. During the well diffusion test only Copaifera sp. had an inhibitory effect on Malassezia pachydermatis, presenting different sizes of halos (mm). The MIC of the oil was 0.61% and the CFM of 0.61% in most inocula. This result is favorable with regard to the development of new treatments based on the use of herbal medicines against the fungus.

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In vitro interaction between tacrolimus (FK506) and four azoles (fluconazole, ketoconazole, itraconazole and voriconazole) against thirty clinical isolates of both fluconazole susceptible and -resistant Candida glabrata were evaluated by the checkerboard microdilution method. Synergistic, indifferent or antagonism interactions were found for combinations of the antifungal agents and FK506. A larger synergistic effect was observed for the combinations of FK506 with itraconazole and voriconazole (43%), followed by that of the combination with ketoconazole (37%), against fluconazole-susceptible isolates. For fluconazole-resistant C. glabrata, a higher synergistic effect was obtained from FK506 combined with ketoconazole (77%), itraconazole (73%), voriconazole (63%) and fluconazole (60%). The synergisms that we observed in vitro, notably against fluconazole-resistant C. glabrata isolates, are promising and warrant further analysis of their applications in experimental in vivo studies.

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Background: Reports of yeast isolates resistant to traditional antifungal drugs have become common. Similarly, refractory clinical cases treated with these traditional antifungal drugs have also been reported. These cases ‘may or may not be related to pregnancy. Newly developed therapeutic approaches, such as the immunostimulant β-glucan combined with the traditional antifungal agents show promising results. Therefore, knowledge of the effects of these new associations is essential. The aims of this study were to evaluate the effects of the combination of itraconazole and β (1-3) glucan on fertility in female rats and its interference in the development of their offspring, including teratogenic potential.Materials, Methods & Results: A total of 180 female Wistar rats (90 days old) separated into six groups were used (n = 30 per group): Negative Control - treated daily with the volume corresponding to 10 mL.kg-1 of sterile distilled water orally, and 0.25 mL of sterile 0.9% NaCl solution subcutaneously weekly; IT - treated daily with itraconazole at a dose of 10 mg.kg-1 orally, and 0.25 ml of sterile 0.9% NaCl solution subcutaneously weekly; Beta - treated daily with the volume corresponding to 10 mL.kg-1 of sterile distilled water orally, and 0.5 mg of β (1-3) glucan subcutaneously weekly; DT - treated daily with itraconazole at a dose of 10 mg.kg-1 orally, and 0.5 mg of β (1-3) glucan subcutaneously weekly; DT5x - treated daily with itraconazole at a dose of 50 mg.kg-1 orally, and 0.5 mg of β (1-3) glucan subcutaneously weekly; DT10x - treated daily with itraconazole at a dose of 100 mg.kg-1 orally, and 0.5 mg of β (1-3) glucan subcutaneously weekly. The rats were treated before (14 days) and during the mating period (up to 21 days), pregnancy (21 days) and lactation (21 days).[...]

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A heterogeneidade genética de Candida parapsilosis ocasionou a sua reclassificação taxonômica em Complexo C. parapsilosis, composto por C. parapsilosis sensu stricto, C. orthopsilosis e C. metapsilosis, cujas diferenças biológicas estão sendo elucidadas. O objetivo desse estudo foi caracterizar o Complexo C. parapsilosis abordando as estratégica de identificação fenotípica, cinética de crescimento, os atributos de virulência e a sensibilidade aos antifúngicos clássicos e aos flavonoides kaempferol e quercetina. No primeiro momento, cepas foram submetidas a testes fenotípicos para triagem à identificação molecular das espécies crípticas; no segundo momento, a cinética de crescimento das espécies foi avaliada em diferentes condições de salinidade e pH. No terceiro momento, a produção de fatores de virulência, a patogenicidade e a sensibilidade aos antifúngicos clássicos, foram analisados. Por fim, as atividades antifúngica e antibiofilme dos flavonoides foram investigadas. Como resultados, os métodos fenotípicos são concordantes (>80%) com a identificação molecular do Complexo C. parapsilosis, e o crescimento de C. orthopsilosis foi reduzido na presença de 15% de NaCl ou pH 7,0. Além disso, foram observados elevados valores de concentração inibitória mínima (CIM) para azóis e caspofungina (CAS) contra C. orthopsilosis, e para azóis contra C. metapsilosis. A anfotericina B e a CAS causaram maior redução da atividade metabólica e da biomassa dos biofilmes maduros. Todas as três espécies produziram fosfolipases, proteases, hemolisinas e biofilme, e um aumento da taxa de mortalidade de Caenorhabditis elegans. Finalmente, os valores de CIM para kaempferol (32-128g/mL) e quercetina (0,5-16g/mL) foram encontrados. No biofilme em formação, os flavonoides reduziram a atividade metabólica das espécies. No biofilme maduro, o kaempferol aumentou a atividade metabólica de C. parapsilosis sensu stricto e C. orthopsilosis, mas reduziu a biomassa de C. orthopsilosis e C. metapsilosis, e a quercetina apenas aumentou a atividade metabólica de C. parapsilosis sensu stricto. A microscopia mostrou uma reestruturação dos biofilmes após exposição aos flavonoides. Os achados reforçam a heterogeneidade das espécies quanto às características fenotípicas, e às caracteríscticas de sensibilidade antifúngica e patogenicidade, enfatizando a relevância do monitoramento desses patógenos. Ademais, os efeitos dos flavonoides reiteram o potencial uso dos recursos naturais como ferramentas sutentáveis a serem aplicadas nas áreas da ciências médica e veterinária.

The genetic heterogeneity of the Candida parapsilosis caused the taxonomic reclassification in C. parapsilosis complex, composed by C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis, whose biological differences that are being elucidated. This study aimed to characterize the C. parapsilosis complex by means of approaching the phenotipical identification strategies, growth kinetics differences, virulence attributes and susceptibility to classical antifungals and flavonoids kaempferol and quercetin. At first, strains were phenotypically screened for identification of cryptic species by molecular identification; and then, the growth kinetics of the species were evaluated in hypersaline and pH media. At a third time, a production of virulence factors, phatogenicity and antifungal susceptibility were analyzed. Finally, the antifungal and antibiofilm of flavonoids were investigated. The phenotypic methods were agreement (>80%) with the molecular identification of cryptic species, and the C. orthopsilosis growth at 15% NaCl or pH 7.0 medium was reduced. In addition, minimal inhibitory concentration (MIC) values of azole and caspofungin (CAS) were found to C. orthopsilosis and of azole against C. metapsilosis. The amphotericin and CAS caused descrease the metabolic activity and biomass of mature biofilms. All cryptic species produced phospholipases, proteases, hemolysins and biofilm, and high mortality of Caenorhabditis elegans were observed. Finally, the MIC values of kaempferol (32-128 g/mL) and quercetin (0.5-16 g/mL) were found. The flavonoids descreased the metabolic activity of growing biofilms of the species. As for mature biofilms, kaempferol increased the metabolic activity of C. parapsilosis sensu stricto and C. orthopsilosis, but reduced the metabolism and biomass of C. orthopsilosis and C. metapsilosis. Quercetin, in turn, only increased the metabolic activity of C. parapsilosis sensu stricto. The microscopic analyses showed restructuring of the cryptic species biofilm after flavonoids exposure. The results reinforce the heterogeneity of these cryptic species for their phenotypic characteristics, antifungal susceptibility, virulence and pathogenicity potential, and emphasize the relevance of monitoring these emerging pathogens. In addition, the effects of flavonoids highlight the potential use of natural resources as tools to be applied for medical and veterinary sciences.

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O complexo Cryptococcus neoformans é formado por basidiomicetos encapsulados, cosmopolitas, que causa criptococose tanto em humanos quanto em animais. No tratamento das infecções por C. neoformans tem-se utilizado derivados azólicos e anfotericina B na rotina clínica humana e veterinária. No entanto, o arsenal terapêutico disponível é limitado a poucas drogas e, além disso, tem surgido relatos de cepas com baixa sensibilidade a essas drogas. Estratégias como a investigação de compostos com potencial antifúngico e a associação de drogas com sinergismo farmacológico tem sido utilizadas. Nesse contexto, as antraquinonas formam um grupo de metabólitos secundários de plantas que apresentam diversas atividades biológicas, dentre elas atividade antimicrobiana. Tendo em vista a necessidade de compostos com ação antifúngica contra C. neoformans e a ação antimicrobiana das antraquinonas, o presente trabalho objetivou investigar a sensibilidade desses micro-organismos às antraquinonas aloe emodina, barbaloína e crisofanol. Para tanto, foi avaliada a ação desses compostos contra formas planctônicas de C. neoformans in vitro, e foi avaliada a interação das antraquinonas com as drogas antifúngicas. Inicialmente, as concentrações inibitórias mínimas (CIMs) dos compostos foram determinadas frente a 17 cepas de C. neoformans pelo método de microdiluição em caldo do documento M27-A3 do CLSI. Posteriormente, foi avaliada a interação de aloe emodina e barbaloína com itraconazol e anfotericina B por método de tabuleiro de xadrez (checkerboard) contra duas cepas de C. neoformans. Assim, aloe emodina, barbaloína e crisofanol mostraram intervalos de CIMs de 64 128 g/mL, 64 128 g/mL e 256 >256 g/mL, respectivamente. Em relação aos testes de interação, barbaloína apresentou sinergismo farmacológico com anfotericina B, enquanto aloe emodina apresentou interações aditivas com itraconazol e anfotericina B. Assim, antraquinonas possuem ação antifúngica contra C. neoformans e a barbaloína potencializa o efeito da anfotericina B in vitro, relevando a importância desses compostos como perspectiva de agentes antifúngicos contra sobre C. neoformans.

The Cryptococcus neoformans complex is a worldwide distributed encapsulated basidiomycete that causes cryptococcosis in both humans and animals. In the treatment of C. neoformans infections, azole derivatives and amphotericin B have been used in the human and veterinary clinical routine, however the available therapeutic arsenal is limited to few drugs and, in addition, reports of strains with low susceptibility to these drugs have been reported. Strategies such as the investigation of drugs with antifungal potential and the association of drugs with pharmacological synergism have been used. In this context, anthraquinones form a group of secondary plant metabolites that presents several biological activities, among them antimicrobial activity. Due to the searching for compounds that having antifungal action on C. neoformans and the antimicrobial action of anthraquinones, the susceptibility of this microorganism to these phytochemicals was evaluated. To this aim, the action of aloe emodin, barbaloin and chrysophanol on planktonic forms of C. neoformans in vitro was evaluated, and anthraquinone interaction with antifungal drugs was performed. Initially, the Minimum Inhibitory Concentrations (MICs) of compounds were determined against 17 strains of C. neoformans by broth microdilution method of CLSI document M27-A3. Subsequently, the interaction of aloe emodin and barbaloin with itraconazole and amphotericin B was evaluated by checkerboard method against 2 strains. Thus, aloe emodin, barbaloin and chrysophanol showed MICs ranged 64 - 128 g.ml-1, 64 - 128 g.ml-1, 256 -> 256 g.ml-1 respectively. Regarding interaction tests, barbaloin showed pharmacological synergism with amphotericin B. Moreover, aloe emodin showed additive interactions with itraconazole and amphotericin B. Thus, anthraquinones have antifungal action against C. neoformans and barbaloin potentiate the effect of amphotericin B in vitro, highlighting the importance of these compounds for perspectives of antifungal agents against C. neoformans.

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Malassezia pachydermatisis associated with dermatomycoses and otomycosis in dogs and cats. This study compared the susceptibility of M. pachydermatis isolates from sick (G1) and healthy (G2) animals 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.

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Leveduras do gênero Candida constituem os principais patógenos fúngicos para homens e animais. Embora Candida albicans ainda ocupe uma posição de destaque entre os casos de infeções, espécies não-albicans vem tendo importância aumentada nas últimas décadas. Além disso, isolados de C. albicans resistentes a derivados azólicos tem sido descritos na prática clínica humana e em cepas de origem veterinária. O presente estudo teve como objetivo reabordar a identificação laboratorial de isolados veterinários de C. albicans resistentes a derivados azólicos, propondo combinação de métodos fenotípicos para distinguir as espécies crípticas C. albicans e Candida dubliniensis. Além disso, almejou-se investigar os mecanismos de resistência das cepas estudadas. Para tanto, trinta e sete cepas veterinárias de C. albicans resistentes a derivados azólicos e três cepas humanas de C. dubliniensis foram submetidas à identificação fenotípica por prova do tubo germinativo, microcultivo em ágar cornmeal, semeadura em ágar cromogênico, semeadura em ágar semente de girassol, prova de opacificação em ágar Tween 80 e reação em cadeia da polimerase (PCR) espécie-específica. Para estudo dos mecanismos de resistência, cepas de C. albicans foram estudadas com testes de efluxo de rodamina 6G (n = 11), determinação do conteúdo de ergosterol (n = 6) e testes de expressão dos genes CDR1, CDR2, MDR1 e ERG11 (n = 30). As cepas estudadas apresentaram prova do tubo germinativo positiva e produção de clamidoconídios em ágar cornmeal. Trinta e cinco cepas (35/37) apresentaram coloração verde em ágar cromogênico. Em ágar semente de girassol, 36 cepas (36/37) apresentaram padrão de colônia lisa, e no ágar Tween 80, 34 (34/37) apresentaram opacificação em menos de 5 dias. Todas as cepas testadas apresentaram produto específico na PCR, sendo confirmadas como C. albicans. As cepas de C. dubliniensis apresentaram colônias de coloração verde no ChromoAgar Candida, aspecto rugoso no ágar girassol, não opacificaram o meio em ágar Tween 80 em até 12 dias e não apresentaram produto específico na PCR. Nos estudos de resistência, as cepas resistentes aos azólicos apresentaram valores de diferença de unidade de fluorescência relativa maiores que os isolados sensíveis nos estudos com rodamina 6G, demonstrando assim maior atividade de bombas de efluxo ATP-dependentes. Não houve diferença significativa no conteúdo de ergosterol entre as cepas estudadas. Foi verificado que 73,3% (22/30) dos isolados apresentaram superexpressão de um ou mais genes, sendo 40,9%, 18,2%, 59,1% e 54,5% superexpressão de CDR1, CDR2, MDR1 e ERG11, respectivamente. Nenhum isolado superexpressou os quatro genes simultaneamente. Por fim, conclui-se que a combinação de técnicas fenotípicas leva à distinção confiável entre C. albicans e C. dubliniensis, reservando as técnicas moleculares para contextos mais específicos. O padrão de resistência a derivados azólicos apresentado pelas cepas estudadas é multifatorial, incluindo, no mínimo, atividade elevada de bombas de efluxo e a superexpressão gênica.

Yeasts of genus Candida are the main fungal pathogens for humans and animals. Although Candida albicans still occupies a prominent position among cases of infections, non-albicans species have increased in the last decades. In addition, azole-resistant C. albicans have been described in human clinical practice and in strains of veterinary origin. The objective of this study was to reevaluate laboratory identification of azole-resistant C. albicans from veterinary isolates, proposing a combination of phenotypic methods for the reliable identification of the species. In addition, this study aimed to investigate the resistance mechanisms of the strains. Thirty-seven veterinary strains of azole-resistant C. albicans and three human strains of C. dubliniensis were submitted to phenotypic identification by germ tube test, micromorphological analysis on cornmeal agar, growth on chromogenic agar, growth on sunflower seed agar, opacification test on Tween 80 agar and species-specific PCR. For the study of resistance mechanisms, C. albicans strains were evaluated for efflux of 6G rhodamine (n=11), determination of ergosterol content (n=6) and expression of CDR1, CDR2, MDR1 and ERG11 genes (n=30). All studied strains presented positive germ tube test and production of chlamydoconidia on cornmeal agar. Thirty five strains (35/37) showed green colonies on chromogenic agar. On sunflower seed agar, 36 strains (36/37) presented a smooth colony pattern, and on Tween 80 agar, 34 (34/37) had opacification in less than 5 days. All strains tested showed specific product on PCR, which confirmed their identity as C. albicans. C. dubliniensis strains showed green colonies on ChromoAgar Candida, a rough appearance on sunflower agar, did not form opacification zone on Tween 80 agar, after up to 12 days, and did not present specific products in PCR. In resistance studies, azole resistant strains showed higher relative fluorescence unit difference values than sensitive isolates in efflux of 6G rhodamine tests, thus demonstrating increased activity of ATP-dependent efflux pumps. There was no significant difference in ergosterol content among the studied strains. It was verified that 73.3% (22/30) of the isolates presented overexpression of one or more genes, of which 40.9%, 18.2%, 59.1% and 54.5% strains overexpressed CDR1, CDR2, MDR1 and ERG11, respectively. No isolate overexpressed simultaneously the four genes. Finally, we conclude that the combination of phenotypic techniques leads to reliable differentiation of C. albicans and C. dubliniensis, reserving molecular techniques for more specific contexts. Resistance mechanisms to azole derivatives of the studied strains is multifactorial, including, at a minimum, the enhanced activity of efflux pump activity and the gene overexpression.

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COSTA, G. C. S. USO DA TERBINAFINA COMPARADO AO IODETO DE POTÁSSIO E ITRACONAZOL NO TRATAMENTO DA ESPOROTRICOSE FELINA. 2017, 47p. Dissertação (Mestrado em Medicina Veterinária Patologia e Ciências Clínicas), Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, Seropédica RJ, 2017. A esporotricose é uma infecção fúngica de tecido cutâneo e subcutâneo causada por fungos do complexo Sporothrix schenckii. É uma doença de caráter zoonótico, com transmissão principalmente por arranhadura, mordedura ou contato com exsudato de lesões de gatos contaminados. Apresenta alta prevalência na América Latina e grande incidência no Brasil, daí a importância e necessidade de mais estudos relacionados a tratamento dessa micose. O projeto teve como objetivo verificar a eficácia da terbinafina e do iodeto de potássio comparado ao itraconazol, por apresentarem a vantagem de causar menos efeitos colaterais e menor resistência no tratamento de gatos com a doença. Para a realização do presente trabalho foram incluídos 18 animais apresentando esporotricose cutânea, diagnosticada através da visualização da levedura na citologia e por cultura fúngica. Os animais foram distribuidos em três grupos de seis animais cada: o grupo TB foi medicado com a terbinafina manipulada na dose de 60 mg/gato a cada 24 horas por via oral, o grupo IO foi medicado com iodeto de potássio na dose de 10 mg/kg por via oral a cada 24 horas, e o grupo IT (grupo controle) medicado com itraconazol na dose de 100 mg/gato por via oral a cada 24 horas. Foi mensurado o diâmetro das lesões através de um paquímetro para avaliar a evolução e resposta terapêutica e realizado exames laboratoriais como hemograma, bioquímica renal e hepática quinzenalmente. Concluiu-se que a terbinafina, na dose utilizada, não apresentou eficácia na redução do número de lesões e na redução do tamanho das mesmas, e tanto o iodeto de potássio quanto o itraconazol foram eficazes no tratamento. Palavras-chave: Sporothrix schenckii, azólicos, zoonose

COSTA, G. C. S. USE OF TERBINAFINE COMPARED TO POTASSIUM IODIDE AND ITRACONAZOLE FOR FELINE SPOROTRICHOSIS TREATMENT. 2017. 47p. Dissertação (Mestrado em Medicina Veterinária Patologia e Ciências Clínicas), Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, Seropédica RJ, 2017. Sporotrichosis is a fungal infection of the cutaneous and subcutaneous tissues caused by fungi of the Sporothrix schenckii complex. It is a zoonotic disease transmitted mainly through bites, scratches or by direct contact with exsudates from lesions of sick cats. This disease is highly prevalent in Latin America and has a great incidence in Brazil, hence the importance and necessity of more studies related to the treatment of this mycosis. The project aims to verify the efficacy of terbinafine and potassium iodide, when compared to itraonazole because these have less side effects and less antifungal resistance. For the completion of the present work, 18 animals presenting with cutaneous sporotrichosis and diagnosed through fungal culture were included. They were distributed into three groups of six animals each; group TB was medicated with manipulated terbinafine at the dose of 60mg/cat every 24 hours orally, group IO received potasssium iodide at a dose of 10mg/kg orally every 24 hours and group IT (control) was given itraconazole at a dose of 100mg/cat orally every 24 hours. The size of the lesions was measured with the aid of a pachymeter to evaluate the evolution and therapeutic response of the patients as well as blood counts and serum biochemistry biweekly. We concluded that terbinafine at the given dose was not effective on reducing the number and the size of the lesions. Potassium iodide and itraconazole were effective on treatment of feline cutaneous sporotrichosis. Keywords: Sporothrix schenckii, azole, zoonosis

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Candida tropicalis, uma levedura comensal presente na microbiota de homens e outros animais, é uma das principais espécies de Candida associadas a infecções invasivas graves em humanos, podendo também causar unfecções em outros animais. O presente estudo objetivou analisar fatores de virulência, perfil de sensibilidade e mecanismos de resistência a azólicos em cepas de C. tropicalis de origem humana (n = 24) e veterinária (n = 28), bem como avaliar o efeito da prometazina sobre células de C. tropicalis resistente a derivados azólicos. Na primeira parte da pesquisa, foi avaliada a produção in vitro de fosfolipase e protease, atividade hemolítica, produção de biofilme e infecção experimental utilizando o nematódeo Caenorhabditis elegans, além de avaliar a sensibilidade de células planctônicas e sésseis frente aos antifúngicos fluconazol, itraconazol, voriconazol, caspofungina e anfotericina B. Na segunda parte, foram avaliados a quantidade de esteróis totais, o efluxo de rodamina 6G e a expressão dos genes CDR1, MDR1 e ERG11 em cepas de origem humana (n = 13) e veterinária (n = 13). Por fim, na terceira parte, a sensibilidade de células planctônicas e sésseis frente à prometazina sozinha e em combinação com antifúngicos foi analisada utilizando 2 cepas resistentes a azólicos e uma cepa sensível de C. tropicalis. A atividade antifúngica da prometazina foi investigada através da curva de tempo de morte e observando o efeito da droga sobre o efluxo da rodamina 6G, tamanho/granulosidade celular, integridade de membrana e potencial transmembrana mitocondrial, utilizando citometria de fluxo. Quanto à produção de fatores de virulência in vitro, 21,15% das cepas apresentaram a produção de fosfolipase, 44,23% produziram protease e 96% foram positivos para a atividade hemolítica. Quanto a produção de biofilme, 98% das cepas foram capazes de produzir essa formação fúngica, a maioria mostrando forte produção (65,38%). Altas taxas de mortalidade de C. elegans foram observadas quando os vermes foram expostos a cepas de C. tropicalis, atingindo 96% de nematódeos mortos após 96 h da exposição. No tocante à sensibilidade antifúngica, 10 isolados foram resistentes para pelo menos 1 azólico, sendo que 5 isolados foram resistentes a todos os azólicos testados. Caspofungina e anfotericina B apresentaram os melhores resultados de inibição frente ao biofilme maduro de C. tropicalis quando comparados com os derivados azólicos. Efeito paradoxal no biofilme foi observado em concentrações elevadas de caspofungina (8-64 µg/mL). Em relação ao estudo dos mecanismos de resistência a azólicos, não foi observado diferença entre os conteúdos de esteróis totais dos isolados testados, porém foi observado que 6 isolados não-sensíveis ao fluconazol apresentaram aumento do efluxo de rodamina 6G. Isolados não-sensíveis e pouco-sensíveis ao fluconazol apresentaram uma média de expressão para os genes CDR1, MDR1 e ERG11 maior que os isolados sensíveis, sendo que 2 isolados não-sensíveis apresentaram expressão elevada para todos os três genes. A prometazina inibiu as células planctônicas e sésseis nas concentrações 64 e 128 µg/mL, respectivamente. A adição de concentração sub-inibitórias de prometazina reduziu as CIMs dos azólicos testados para as cepas resistentes, alterando o fenótipo de resistência. Pré-incubação com prometazina reduziu o efluxo de rodamina 6G, além de causar alteração no tamanho/granulosidade celular, dano na membrana plasmática e despolarização da membrana mitocondrial. O presente trabalho mostrou que cepas humanas e veterinárias de C. tropicalis apresentam similar produção de fatores de virulência e patogenicidade. Além disso, a resistência a azólicos em cepas veterinárias está relacionada, em parte, à expressão elevada do gene ERG11 e de bombas de efluxo. Por fim, prometazina apresenta sinergismo com azólicos frente a cepas resistentes de C. tropicalis, causando danos na membrana celular e atividade mitocondrial da célula fúngica.

Candida tropicalis, an yeast isolated as comensal from humans and other animals, is a major Candida species associated with serious invasive infections in humans, being also able to cause infection in several animal species. The present study aimed to analyze virulence factors, the susceptibility profile and mechanisms of resistance to azoles in human (n = 24) and veterinary (n = 28) isolates of C. tropicalis, as well as to evaluate the effect of promethazine on cells of C. tropicalis isolates resistant to azoles. In the first part of this research, the in vitro production of phospholipases and proteases, hemolytic activity, biofilm production and experimental infection using the nematode Caenorhabditis elegans was evaluated, as well as the susceptibility of planktonic and sessile cells against the antifungals fluconazole, itraconazole, voriconazole, caspofungin and amphotericin B. Total sterols content, efflux of rhodamine 6G and expression levels of CDR1, MDR1 and ERG11 genes in human (n = 13) and veterinary (n = 13) isolates were also assessed. Finally, the susceptibility of planktonic and sessile cells to promethazine alone and in combination with antifungals was analyzed using 2 strains resistant to azoles and a sensitive strain of C. tropicalis. Antifungal activity of promethazine was investigated through the time-kill curve and assessing the effects of the drug on the efflux of 6G rhodamine, cell size/granularity, membrane integrity and mitochondrial transmembrane potential, through flow cytometry. Regarding the in vitro production of virulence factors, 21.15% of the strains showed phospholipase production, 44.23% produced protease and 96% were positive for hemolytic activity. 98% of the strains were able to produce biofilm, most showing strong production (65.38%). High mortality rates of C. elegans were observed when worms were exposed to C. tropicalis strains, reaching 96% of death after 96 h of exposure to the strains. Concerning the antifungal susceptibility profile, 10 isolates were resistant to at least 1 azole and 5 isolates were resistant to all azoles tested. Caspofungin and amphotericin B showed the best inhibition results against C. tropicalis mature biofilm when compared to the azole antifungals. Paradoxical effect on mature biofilm was observed at high concentrations of caspofungin (8-64 g/mL). Regarding the study of mechanisms of azole resistance, no difference was observed between total sterols contents of the groups analyzed, however, 6 fluconazole-non-susceptible isolates showed increased efflux of 6G rhodamine. Fluconazole-non-susceptible and fluconazole-less-susceptible isolates showed a mean expression for CDR1, MDR1 and ERG11 genes greater than the susceptible isolates, with 2 fluconazole-non-susceptible isolates showing high expression for all three genes. Promethazine showed inhibition of planktonic and sessile cells at concentrations of 64 and 128 g/mL, respectively. The addition of sub-inhibitory concentrations of promethazine reduced the MIC of the azoles tested against the resistant strains, altering the resistance phenotype. Pre-incubation with promethazine reduced the efflux of 6G rhodamine, in addition to causing changes in cell size/granularity, plasma membrane damage and mitochondrial membrane depolarization. The present work showed that human and veterinary isolates of C. tropicalis presented similar production of virulence factors and pathogenicity. In addition, azole resistance in veterinary C. tropicalis strains is related, in part, to elevated ERG11 gene expression and efflux pump activity. Finally, promethazine shows synergism with azoles against resistant strains of C. tropicalis, causing cell membrane and mitochondrial activity impairment on fungal cells.

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Malassezia pachydermatis é uma levedura componente da microbiota de cães e gatos, no entanto há trabalhos relatando casos de otite e dermatite seborreica nesses animais. O objetivo deste estudo foi determinar a sensibilidade antifúngica e avaliar a virulência e a patogenicidade de 25 cepas de M. pachydermatis de origem animal. A sensibilidade de células planctônicas ao cetoconazol, fluconazol, itraconazol, voriconazol, terbinafina e anfotericina B foi avaliada através do ensaio de microdiluição em caldo. Ademais, foi investigada a produção de biofilme, proteases, fosfolipases, hemolisinas, produção de melanina e adesão a células epiteliais por essas leveduras. Por fim, foi avaliada a patogenicidade das cepas frente ao Caenorhabditis elegans. Quanto à sensibilidade planctônica, as concentrações inibitórias mínimas variaram de <0,03 a >64 g/mL para os derivados azólicos, 0,03 a 0,25 para a terbinafina e 1 a >16 g/mL para anfotericina B. Todas as cepas foram classificadas como fortes produtoras de biofilme. Cetoconazol, fluconazol e anfotericina B apresentaram os melhores resultados de redução da atividade metabólica frente aos biofilmes maduros. Todos os isolados fúngicos foram positivos para a produção de proteases, enquanto 14/25 cepas foram positivas para fosfolipases. Não foi observado atividade hemolítica e 18/25 cepas apresentaram uma pigmentação escura, após exposição à L-DOPA, sugestivo de melanina. No tocante à adesão a células epiteliais, uma baixa adesão foi observada em 10/12 cepas avaliadas. A taxa de mortalidade de C. elegans chegou a 95,9%, após 96h de exposição a M. pachydermatis. Em suma, foram encontrados baixos valores de CIMs para terbinafina contra M. pachydermatis. Ademais, essa espécie apresenta produção de importantes fatores de virulência e alta patogenicidade para o modelo C. elegans, evidenciando sua importância clínica.

The yeast Malassezia pachydermatis is a component of the microbiota of dogs and cats, however it can cause otitis and seborrheic dermatitis in these animals. The objective of this study was to determine the antifungal susceptibility, and evaluate virulence and pathogenicity of 25 M. pachydermatis strains from animals. Planktonic cells susceptibility to ketoconazole, fluconazole, itraconazole, voriconazole, terbinafine, and amphotericin B was evaluated by broth microdilution assay. In addition, the biofilm-forming ability, protease, phospholipase, hemolysin, melanin production and adhesion to epithelial cells by this yeast species were assessed. Finally, strain pathogenicity was investigated using the nematode Caenorhabditis elegans. Concerning the planktonic susceptibility, minimum inhibitory concentrations varied from <0.03 to >64 g/mL for azole derivatives, 0.03 to 0.25 for terbinafine and 1 to >16 g/mL for amphotericin B. All strains were classified as strong biofilm producers. Ketoconazole, fluconazole and amphotericin B significantly decreased the metabolic activity of mature biofilms. All fungal isolates produced proteases, whereas 14/25 strains were positive for phospholipase production. Hemolytic activity was not observed and 18/25 strains showed dark pigmentation, in the presence of L-DOPA, which suggests melanin production. Regarding adhesion to epithelial cells, a low adhesion rate was observed in 10/12 evaluated strains. C. elegans mortality rate reached 95.9% after 96 h of exposure of the worms to M. pachydermatis. In summary, low MICs were found for terbinafine against M. pachydermatis. In addition, this yeast species produces important virulence factors and presents high pathogenicity to the C. elegans model, corroborating its clinical importance.

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Dermatophytosis is caused by a group of pathogenic fungi namely, dermatophytes, is among the most prevalent infectious diseases worldwide. Azole drugs are widely used in the treatment of dermatomycosis, but can cause various side effects and drug resistance to the patients. Hence, for solving this problem can be used from the plant extract as alternative for chemical drugs. Allicin is a pure bioactive compound isolated from garlic was tested for its potential as a treatment of dermatomycosis in this study. This study evaluated the in vitro efficacy of pure allicin against ten isolates of Trichophyton rubrum and the MIC50 and MIC90 ranged from 0.78-12.5 µg/ml for allicin. The results revealed that the order of efficacy based on the MICs values, all isolates showed almost comparable response to allicin and ketoconazole except for some isolates, at 28 ºC for both 7 and 10 days incubation. Mann-Whitney test indicate that MICs at 7 days incubation was not observed a significant difference between the effects of allicin and ketoconazole (p > 0.05), but MICs at 10 days incubation, a significant difference was observed (p < 0.05). On the other side, time kill studies revealed that allicin used its fungicidal activity within 12-24 h of management in vitro as well as ketoconazole. In conclusion, allicin showed very good potential as an antifungal compound against mycoses-causing dermatophytes, almost the same as the synthetic drug ketoconazole. Therefore, this antifungal agent appears to be effective, safe and suitable alternative for the treatment of dermatomycosis.

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Background: Sporotrichosis is a fungal infection caused by Sporothrix schenckii species complex, which is distributed worldwide, especially in regions of tropical and subtropical climates. It can affect both humans and a great variety of animals, among which, the domestic dog and cat. Today is considered the subcutaneous mycosis more frequent in Brazil, due to the progressive increase of zoonotic transmission of the disease in the metropolitan region of Rio de Janeiro. Review: In the endemic region of zoonotic sporotrichosis in Rio de Janeiro,veterinarians, their assistants and owners of cats with sporotrichosis are considered risk groups for acquiring the infection. Of the human cases of sporotrichosis in this endemic region that were accompanied by the Clinical Research Institute Evandro Chagas (IPEC / Fiocruz) in the period from 1998 to 2001, 5% of the patients diagnosed for the disease were veterinarians, demonstrating the occurrence of the infection in this occupational group. Biosafety is defined as a condition of security achieved by a set of measures aimed at the prevention, control, reduction or elimination of risks involved in activities that could endanger the health of humans, animals, plants and the environment. However, small animal veterinary pratictioners from the endemic region of Rio de Janeiro usually do not follow biosafety measures, which increases the risk of acquisition of sporotrichosis. In most cases, the infection results from traumatic inoculation of the fungus in skin and in humans, clinical forms may be: fixed or localized cutaneous, lymphatic-cutaneous, spread-cutaneous, mucosal and extra-cutaneous or systemic. In the endemic form of this disease that occurs in Rio de Janeiro, the cat is seen as a source of infection. The zoonotic potential of the cat is characterized by the abundance of yeasts found in their skin lesions and proximity with humans. Cats acquire the disease after fights with other infected cats and the skin lesions more frequently found are nodules and ulcers, covered or not by crusts, which can progress to necrosis with exposure of bones and muscles. The presence of respiratory signs, especially sneezing, with or without lesions in the mucosal and in the nasal region is common. The azole itraconazole is considered the drug of choice for humans and cats. However, unlike humans, the treatment is considered difficult in cats. The definitive diagnosis of sporotrichosis is obtained by isolation of Sporothrix in culture. Nonetheless, in cats, due to the great quantity of yeasts in their lesions, cytopathological test is strongly indicated in the presumptive diagnosis because of the speed in processing, low cost and no requirement of sophisticated technical training or complex laboratory structure. Discussion: A zoonotic form of sporotrichosis has become endemic in the metropolitan region of Rio de Janeiro and the veterinarians are a high risk group for acquiring the disease as well as have a very important role in the application of measures for the prevention and control of sporotrichosis. Therefore, in this review, specific biosafety procedures to reduce risks during the handling of cats with suspected sporotrichosis by veterinarians, technicians, caretakers and owners of cats were described. The topics aproached were: clinical care of the cat (where were appointed the recommended personal protective equipment, animal restraint and good practices), decontamination of the environment, equipment and items used in the cat care and management of waste. Aspects related to the fungus and the disease itself were also discussed.

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Espécies de fungos são isoladas com frequência da microbiota de diversos sítios anatômicos de animais sem causar danos ao hospedeiro, podendo ser encontradas também em ambientes terrestres e aquáticos, especialmente em áreas de cultivo animal. Espécies de Candida são comumente isoladas destes ambientes e de animais, sendo frequente a resistência a azólicos nestes isolados, por vezes, maior que o observado em isolados humanos. Este aparente paradoxo é mal compreendido, uma vez que a exposição de cepas ambientais e animais a estes antifúngicos não ocorre por tratamento/profilaxia dos hospedeiros animais. O presente estudo verificou a atividade antifúngica de extratos de Moringa oleifera frente 14 cepas de Candida spp. (3 C. ciferrii; 6 C. famata; 2 C. guilliermondii; 2 C. parapsilosis; e 1 C. tropicalis) e 10 cepas de Hortaea werneckii isolados da carcinicultura de M. amazonicum, assim como avaliou a toxicidade destes extratos in vitro. Ademais, foi investigada a expressão gênica das bombas de efluxo CDR1 e CDR2 em cepas de C. albicans resistentes a azólicos isoladas de animais (n=16) e humanos (n=2) e também a influência da exposição ao tetraconazol (azólico agrícola) e malathion (organofosforado) na ocorrência de resistência ao fluconazol, itraconazol e voriconazol em C. parapsilosis ATCC 22019. A partir dos dados obtidos no teste de microdiluição in vitro, evidenciou-se que o extrato clorofórmico de flores inibiu o crescimento de todas as cepas fúngicas associadas à carcinicultura de M. amazonicum. O extrato etanólico de folhas, flores e sementes inibiu o crescimento de 22/24, 21/24 e 20/24 cepas, respectivamente. O extrato de vagens inibiu somente cepas de Candida spp. (14/14) e, por sua vez, o extrato de caule inibiu apenas quatro cepas de H. werneckii (4/10). Os extratos de sementes, flores (fração clorofórmica), caules e folhas apresentaram baixa ou nenhuma toxicidade em larvas de M. amazonicum, enquanto que extratos de vagens e flores (fração etanólica) apresentaram toxicidade moderada. Quanto à expressão gênica analisada por qPCR, verificou-se que há correlação entre a resistência a fluconazol e a expressão de CDR1 em C. albicans resistentes a azólicos, uma vez que a expressão de CDR1 estava aumentada em 56,25% (9/16) destas cepas. Foi demonstrado ainda que a exposição, in vitro, ao tetraconazol, mas não ao malathion, diminui a sensibilidade aos azólicos clínicos, especialmente ao fluconazol. Este fenômeno é mediado, pelo menos em parte, pela atividade de bombas de efluxo dependentes de ATP, haja visto o aumento da sensibilidade das réplicas de C. parapsilosis, com o uso de prometazina; assim como o aumento no efluxo de rhodamina 6G e o aumento de expressão dos genes das bombas de efluxo CDR. Também há indícios de envolvimento de bombas MDR nas réplicas resistentes a fluconazol, sem alteração na biossíntese de ergosterol nem na expressão do gene ERG11. Portanto, ficou demonstrado na primeira parte deste trabalho, a potencialidade do uso de extratos de M. oleifera no controle de fungos associados a carcinicultura de M. amazonicum. Ademais, ficou evidente, na análise molecular, o envolvimento de bombas de efluxo CDR em C. albicans de origem animal resistentes a azólicos. Por fim, demonstrou-se que a exposição de C. parapsilosis ao tetraconazol, in vitro, induz resistência a azólicos de uso clínico também via superexpressão de CDR.

Fungal species are often isolated from microbiota of several anatomical sites from animals without causing harm to the host, and can also be found in terrestrial and aquatic environments, especially in animal farming areas. Candida species are opportunistic pathogens commonly isolated from these environments and animals, and azole resistance are frequent in these isolates, sometimes greater than that observed in human isolates. This apparent paradox is poorly understood, since exposure of environmental and animal strains to these antifungals does not occur during treatment/prophylaxis of animal hosts. This study investigated the antifungal activity of M. oleifera extracts against 14 strains of Candida spp. (3 C. ciferrii; 6 C. famata; 2 C. guilliermondii; 2 C. parapsilosis; and 1 C. tropicalis) and 10 strains of Hortaea werneckii isolated from shrimp Macrobrachium amazonicum, as well as the in vitro toxicity of these extracts. In addition, we investigated the gene expression of efflux pumps CDR1 and CDR2 in azole-resistant C. albicans strains from animals (n = 16) and humans (n = 2) and also the influence of exposure to tetraconazole (agricultural azole) and malathion (organophosphate) in the occurrence of resistance to fluconazole, itraconazole and voriconazole in C. parapsilosis ATCC 22019. Data from in vitro microdilution test demonstrated that the chloroform flower extract inhibits the growth of all tested fungal strains from shrimp M. amazonicum. The ethanolic extract of leaves, flowers and seeds inhibited the growth of 22/24, 21/24 and 20/24 of strains, respectively. The ethanolic extract of pods inhibited Candida spp. strains (14/14), in its turn, extracts of stem inhibited only four strains of H. werneckii (4/10). The extract of seed, flowers (chloroform fraction), stems and leaves showed low or no toxicity, while ethanolic pods and flowers extracts showed moderate toxicity. Concerning efflux pumps gene expression by qPCR, it was observed correlation between resistance to fluconazole and CDR1 expression in azole resistant C. albicans, since the CDR1 expression was increased in 56.25% (9/16) in these strains. Additionally, it was shown that exposure to tetraconazole, but not to malathion, decreases susceptibility to clinical azoles, particularly to fluconazole. This phenomenon is mediated, at least partially, by the increased activity of ATP-dependent efflux pumps, considering the increase in antifungal susceptibility after the addition of the efflux pump inhibitor promethazine, the increase in efflux of rhodamine 6G and in CDR gene expression. There is also evidence of involvement of MDR pumps in fluconazole-resistant replicas, with no change in ergosterol biosynthesis or in the expression of ERG11 gene. Therefore, the first part of this study showed the potential use of M. oleifera extracts in M. amazonicum shrimp farming fungal control. Moreover, in the molecular analysis, it was evident the involvement of CDR efflux pumps in azole resistance in C. albicans strains from animals. Finally, it was shown that in vitro exposure to tetraconazole in C. parapsilosis induces clinical azole resistance by CDR overexpression too.

Resumo

Pythium insidiosum é um oomiceto aquático e agente etiológico da pitiose, uma doença infecciosa, não contagiosa, de difícil tratamento e prognóstico desfavorável, que acomete mamíferos que habitam áreas pantanosas e alagadas. O presente estudo teve como objetivos: a) avaliar e padronizar um inóculo a partir de cultivo micelial de P. insidiosum; b) avaliar a suscetibilidade in vitro de P. insidiosum aos óleos essenciais de Origanum vulgare, Origanum majorana, Mentha piperita e Rosmarinus officinalis; c) investigar a ação antimicrobiana dos óleos essenciais de O. vulgare e M. piperita sozinhos, associados e em combinação com imunoterapia no tratamento da pitiose experimental. O inóculo foi confeccionado a partir de cultura micelial de P. insidiosum e testado frente a antifúngicos azólicos. Os resultados foram comparados aos testes de suscetibilidade empregando o inóculo padrão de zoósporos frente aos mesmos antifúngicos. O inóculo proposto mostrou padrões de suscetibilidade comparáveis ao inoculo padrão, indicando, portanto, que pode ser um método adequado para avaliar-se a suscetibilidade deste oomiceto, particularmente quando não é possível obter-se o inóculo padrão. Os óleos foram analisados por cromatografia gasosa acoplada a espectrometria de massa. Os componentes majoritários dos óleos essenciais foram os seguintes: O. vulgare: carvacrol (93,10%) e beta; O. majorana: 1,4-terpineol (34,34%); M. piperita: mentona (57,53%); R. officinalis: 1,8-cineol (64,53%). A atividade antimicrobiana foi determinada pelo método de microdiluição em caldo frente a 22 isolados de P. insidiosum. Os óleos foram submetidos a uma série de diluições, obtendo-se concentrações de 14 a 0,025mg/mL. As concentrações inibitórias mínimas para O. majorana, M. piperita e R. officinallis variaram de 0,11 a 3,5mg/mL e para O. vulgare de 0,05 a 1,75mg/mL. Os resultados evidenciaram que os óleos essenciais avaliados apresentaram ação antimicrobiana sobre P. insidiosum, ressaltando-se a melhor atividade do óleo essencial de O. vulgare. A partir destes dados delineou-se o experimento in vivo, no qual foram utilizados 18 coelhos com pitiose experimental, divididos em seis grupos de três animais sendo: grupo 1 controle; grupo 2 tratados com óleo essencial de M. piperita; grupo 3 tratados com óleo essencial de O. vulgare; grupo 4 tratados com imunoterápico Pitium Vac®; grupo 5 tratados com associação dos óleos de M. piperita e O. vulgare e grupo 6 tratados com associação dos óleos plus imunoterápico. Os óleos foram formulados em creme tópico e as lesões foram tratadas diariamente por 45 dias; os animais dos grupos 4 e 6 receberam uma dose do imunoterápico a cada 14 dias. Os resultados revelaram que a evolução das lesões dos grupos 5 e 6, não diferiram entre si, porém diferiram dos demais grupos. Evidenciou-se que as lesões do grupo 5 aumentaram 3.16 vezes a cada dia, enquanto àquelas do grupo 6, aumentaram 1.83 vezes, indicando o menor crescimento das lesões quando o tratamento empregou a combinação das terapias. Este estudo é pioneiro no tratamento da pitiose experimental empregando óleos essenciais de plantas e combinação de terapias com óleos em pitiose. Demonstrou que o emprego de óleos essenciais pode se constituir numa alternativa viável de tratamento da pitiose cutânea, particularmente quando utilizados em combinação ou em associação com imunoterapia.

Pythium insidiosum is an aquatic oomycete and etiological agent of Pythiosis, an infectious, non-contagious, difficult-to-treat disease. It affects mammals that inhabit marshy and flooded areas. This study aimed to: a) evaluate and standardize an inoculum from P. insidiosum mycelial culture; b) evaluate P. insidiosum in vitro susceptibility to Origanum vulgare, Origanum majorana, Mentha piperita and Rosmarinus officinalis essential oils; c) investigate the antimicrobial activity of O. vulgare and M. piperita essential oils separately, associated and in combination with immunotherapy in the treatment of experimental Pythiosis. The inoculum was prepared from P. insidiosum mycelial culture and tested against azole antifungals. The results were compared to susceptibility tests by using zoospore standard inoculum against the same antifungals. The proposed inoculum showed susceptibility patterns comparable to standard inoculum, thus indicating that it can be a suitable method to evaluate oomycete susceptibility, particularly when it is not possible to obtain standard inoculum. The oils were analyzed by gas chromatography mass spectrometry. Essential oils main components were as follows: O. vulgare: carvacrol (93.10%); O. majorana: 4-terpineol (34.34%); M. piperita: menthone (57.53%); A. officinalis: 1.8cineole (64.53%). Antimicrobial activity was determined by the broth microdilution method against 22 P. insidiosum isolates. The oils were subjected to serial dilutions, and 14-0.025mg/mL concentrations were obtained. O. marjorana, M. piperita and R. officinalis minimum inhibitory concentrations ranged from 0.11 to 3.5 mg/mL, and those for O. vulgare, from 0.05 to 1.75 mg/mL. The results showed that the tested essential oils had antimicrobial effect on P. insidiosum, with O. vulgare essential oil being the most active. The in vivo experiment was outlined from these results. Eighteen rabbits with experimental Pythiosis, divided into six groups of three animals, were used: group 1 control; group 2 was treated with Mentha piperita essential oil; group 3 was treated with Origanum vulgare essential oil; group 4 was treated with commercial immunotherapic; group 5 was treated with a combination of Mentha piperita and Origanum vulgare oils, and group 6 was treated with a combination of immunotherapy plus oils. The oils were formulated as topical cream and lesions were treated daily for 45 days; animals in groups 4 and 6 received an immunotherapy dose every 14 days. The results revealed that the evolution of lesions in groups 5 and 6 did not differ between each other, but differed from the other groups. Group 5 lesions increased 3.16 fold every day, whereas those in group 6 increased 1.83 fold, thus showing that the smallest lesion growth occurred when an combination of therapies was employed. This is the first study to use essential oils from plants and a combined oil therapy in theexperimental treatment of Pythiosis. It was demonstrated that the use of essential oils can be a viable alternative for skin Pythiosis treatment, particularly when used in combination with or associated to immunotherapy.

Resumo

The Candida genus is composed by yeasts that live as commmensal on human and animals' microbiota. In general, they do not cause any damage to their hosts. However, due instability on chemical, physical and immunological defenses, these microorganisms can become pathogens. Candida spp. infections are rare on Veterinary Medicine. However, on the last years, a considerable raise of illness caused by these yeasts has been related on varied animal species. Several species of this genus has been mentioned as responsible for infectious diseases in animals, being C. albicans the main of them, followed by C. tropicalis and C. parapsilosis. Considering the pathogenic role of the genus Candida, allied to the emerging of resistant strains to the azole derivatives, in vitro, the present research proposed to perform a detailed review, approaching clinic-laboratorial, etiologic and therapeutic aspects of the candidosis on Veterinary medicine.

O gênero Candida é composto por leveduras que vivem como comensais na microbiota de homens e animais. Em geral, não causam nenhum dano aos seus hospedeiros, entretanto, em virtude de desequilíbrios nas defesas química, física e imunológica, esses microrganismos podem se tornar patogênicos. Infecções por Candida spp. são pouco frequentes na Medicina Veterinária no entanto, nos últimos anos, tem sido observado aumento considerável de relatos de enfermidades causadas por essas leveduras, acometendo diferentes animais. Várias espécies do gênero são implicadas em quadros infecciosos, sendo a C. albicans a principal delas, seguida por C. tropicalis e C. parapsilosis. Considerando-se o potencial patogênico do gênero Candida, aliado ao surgimento de cepas resistentes a derivados azólicos, in vitro, o presente trabalho se propôs a realizar detalhada revisão de literatura, abordando os aspectos clínico-laboratoriais, etiológicos e terapêuticos da candidose na Medicina Veterinária.

Resumo

Ten clinical isolates of Candida albicans, five strains belonging to each of fluconazole resistant and susceptible groups isolated from diabetic patients, were studied for the membrane fluidity and lipid composition. Compared to fluconazole susceptible strains, fluconazole resistant ones exhibited enhanced membrane fluidity as measured by fluorescence polarization technique. The increased membrane fluidity was reflected in the decreased p-values exhibited by the resistant strains. On the other hand, susceptible isolates contained higher amount of ergosterol, almost twice as compared to resistant isolates which might have contributed to their lower membrane fluidity. However, no significant alteration was observed in the phospholipid and fatty acid composition of these isolates. Labeling experiments with fluorescamine dye revealed that the percentage of the exposed aminophospholipid, phosphatidylethanolamine was highest in the resistant strains as compared to the susceptible strains, indicating a possible overexpression of CDR1 and CDR2 genes in resistant strains. The results presented here suggest that the changes in the ergosterol content and overexpression of ABC transporter genes CDR1 and CDR2 could contributeto fluconazole resistance in C. albicans isolated from diabetic patients.

Dez isolados clínicos, sendo cinco resistentes e cinco sensíveis ao fluconazol, obtidos de pacientes diabéticos, foram estudados quanto à fluidez e composição química da membrana. Quando comparados aos isolados sensíveis ao fluconazol, os isolados resistentes apresentaram fluidez de membrana aumentada, conforme mensurado pela técnica de polarização fluorescente. A fluidez de membrana aumentada refletiu-se pelos valores mais baixos de p. Por outro lado, os isolados sensíveis continham quantidades mais elevadas de ergosterol, quase o dobro dos isolados resistentes, o que pode ter contribuído para a fluidez de membrana mais baixa. Entretanto, não se observou alteração significativa na composição fosfolipídica e de ácidos graxos nesses isolados. Experimentos de marcação com corante fluorescamina indicaram que a porcentagem de aminofosfolípides e fosfatidiletanolamina expostos foi mais elevada nos isolados resistentes do que nos sensíveis, indicando uma possível superexpressão dos genes CDR1 e CDR2 nos isolados resistentes. Os resultados aqui apresentados sugerem que alterações no teor de ergosterol e superexpressão dos genes ABC transportadores CDR1 e CDR2 podem contribuir na resistência ao fluconazol em isolados de C. albicans de pacientes diabéticos.