The present study aimed to evaluate bacterial isolates and the microbiome of dairies. The specific aims were: to characterize Staphylococcus spp. isolated from mastitic milk, to evaluate the presence of Lactococcus in mastitic milk as a potential causative agent of mastitis, to evaluate the association between microbiome and milk quality parameters, and to characterize Staphylococcus spp. isolated from production lines of Minas Frescal cheese. The detection of genes encoding virulence factors (enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, selj, selk, sell, selm, seln, selo, selp, seIq, ser, ses, set, selu, selv, and selx), hemolysins (hla, hlb, hld, hlg, and hlgv), exfoliative toxins (eta, etb, and etd), Panton-Valentine leukocidin (pvl), and toxic shock syndrome toxin (tst)), genes encoding antibiotic resistance (resistance to tetracycline (tetK, tetL, and tetM), erythromycin (ermA, ermB, and ermC), methicillin (mecA and mecC), and tobramycin (ant(4\')-Ia)), molecular typing (spa, SCCmec, and agr types), and phenotyping regarding antibiotic resistance were performed in staphylococci isolates from mastitic milk, and from cheese processing plant samples. Staphylococcus aureus was identified in the majority of isolates from both origins. Several virulence factor genes were detected. The distribution of genes encoding staphylococcal enterotoxins (85.0% - 85.7% of isolates were positive for one or more enterotoxin gene) was highlighted and the gene related to H toxin was the most prevalent. Methicillin-resistant Staphylococcus aureus were identified in isolates from mastitic milk (4.1%) and cheese processing (6.0%); the genotyping and phenotyping of these isolates were described. t605 had the highest frequency in the S. aureus population studied. In mastitic milk, Lactococcus was suggested as the causative agent of an outbreak of mastitis in a dairy farm. Using next generation sequencing, the abundance of Lactococcus was observed in microbiome samples. Bacterial isolation and DNA sequencing confirmed the presence of Lactococcus lactis and Lactococcus garvieae. The microbiome of environmental samples and bulk tank milk from the dairy farm showed the Lactococcus genus among the most common bacterial taxa, suggesting other sources of this genus. Regarding milk quality parameters, the microbiome of bulk tank milk from several dairy farms was associated with somatic cell count and bacterial count. The core microbiome was described and many genera of importance were identified. Among the associations performed between microbiome and milk quality parameters, the identification of Streptococcus in samples classified with high somatic cell count and high bacterial count was highlighted. Several bacterial taxa with relative abundance significantly higher in samples classified as high and low cell count and bacterial count were shown. Real-time polymerase chain reaction was also performed associated with bacterial diversity, bacterial taxa, and bacterial count. These findings highlight the need to control and prevent bacterial contamination in the dairy industry, from herd to consumers. / O presente estudo apresentou como objetivo avaliar isolados bacterianos e microbioma de lácteos. Os objetivos específicos foram: caracterizar Staphylococcus spp. isolados de leite de vacas com mastite, avaliar a presença de Lactococcus em leite de vacas com mastite como um potencial agente causador de mastite, avaliar a associação entre microbioma de leite de tanque e parâmetros da qualidade de leite, e caracterizar Staphylococcus spp. isolados de linhas de processamento de queijo Minas frescal. A detecção de genes codificadores de fatores de virulência (enterotoxinas (sea, seb, sec, sed, see, seg, seh, sei, selj, selk, sell, selm, seln, selo, selp, seIq, ser, ses, set, selu, selv, e selx), hemolisinas (hla, hlb, hld, hlg, e hlgv), toxinas exfoliativas (eta, etb e etd), leucocidina de Panton-Valentine (pvl), toxina da síndrome do choque tóxico (tst)), genes codificadores de resistência a antibióticos (resistência a tetraciclina (tetK, tetL e tetM), eritromicina (ermA, ermB e ermC), meticilina (mecA e mecC) e tobramicina (ant(4\')-Ia)), tipagem molecular (spa, SCCmec e agr types), e fenotipagem quanto à resistência a antibióticos foram realizadas em estafilococos isolados de leite de vacas com mastite e de amostras de planta de processamento de queijo. Staphylococcus aureus foi identificado na maioria dos isolados de ambas as origens. Diversos genes de fatores de virulência foram detectados, com destaque para a distribuição de genes codificadores de enterotoxinas estafilocócicas (85,0%-85,7% dos isolados foram positivos para um ou mais genes codificadores de enterotoxinas), sendo o gene relacionado com a toxina H o mais frequente. Staphylococcus aureus meticilina resistente foram identificados em isolados de leite de vacas com mastite (4.1%) e em processamento de queijo (6.0%); o perfil genotípico e fenotípico destes isolados foram descritos. t605 foi o mais freqüente na população de S. aureus estudada. Em leite de vacas com mastite, Lactococcus foi sugerido como o agente causador de um surto de mastite numa fazenda leiteira. Usando sequenciamento de nova geração, a abundância de Lactococcus foi observada no microbioma das amostras. O isolamento e sequenciamento de DNA confirmaram a presença de Lactococcus lactis e Lactococcus garvieae. O microbioma de amostras ambientais e de leite de tanque da fazenda mostrou o gênero Lactococcus entre os mais comuns, sugerindo outras fontes deste gênero. Contemplando parâmetros da qualidade de leite, o microbioma de leite de tanque de várias fazendas leiteiras foi relacionado com contagem de células somáticas e contagem bacteriana. O core microbiome foi descrito e muitos gêneros bacterianos de importância foram identificados. Dentre as análises realizadas associando microbioma com parâmetros da qualidade de leite, foi destacada a identificação de Streptococcus em amostras classificadas com alta contagem de células somáticas e alta contagem bacteriana. Diversos táxons bacterianos com abundância relativa significativamente maior em amostras classificadas com alta e baixa contagem de células somáticas e contagem bacteriana foram mostrados. Reação em cadeia da polimerase em tempo real também foi realizada e associada com diversidade bacteriana, táxons bacterianos e contagem bacteriana. Estes levantamentos confirmam a necessidade de controlar e prevenir a contaminação bacteriana na indústria de lácteos, do rebanho leiteiro até os consumidores.

Infecções invasivas por Trichosporon spp. ocorrem com maior frequência em pacientes neutropênicos, principalmente portadores de doenças hematológicas malignas, e estão associadas a elevados índices de mortalidade devido às dificuldades na identificação do patógeno e à resistência aos fármacos mais empregados na terapêutica antifúngica. A identificação das espécies de Trichosporon é importante tanto para estudos epidemiológicos, como para associar aspectos clínicos com as espécies causadoras das infecções. Além disso, auxilia no tratamento da enfermidade, uma vez que a suscetibilidades aos fármacos antifúngicos pode variar de acordo com a espécie. Além disso, as leveduras do gênero Trichosporon sintetizam a glucuronoxilomanana (GXM) em sua parede celular, que pode estar envolvida no mecanismo de virulência do patógeno. Este estudo teve como objetivo determinar, por identificação fenotípica e molecular, espécies isoladas de pacientes internados em unidades hospitalares, comparando os resultados obtidos por ambos os métodos; avaliar diferenças na distribuição dessas espécies em relação às formas invasivas e não invasivas da infecção; determinar o perfil de suscetibilidade dessas leveduras aos antifúngicos, empregando um método de micro-diluição de referência e um método comercial; e avaliar a presença de GXM na parede celular dos isolados. Foram avaliados 74 isolados obtidos de amostras clínicas de pacientes do Hospital das Clinicas da FMUSP e de outras unidades hospitalares do Estado de São Paulo, no período de 2003 a 2011. Dezenove amostras foram isoladas de sítios estéreis do organismo (infecções invasivas) e 55 foram isoladas de urina e cateter (isolados não invasivos). Para a identificação das espécies, os isolados foram submetidos a análises fenotípicas, que incluíram estudo macro e micromorfológico, provas fisiológicas e avaliação do perfil bioquímico por sistema automatizado VITEK 2. A identificação molecular foi realizada pelo sequenciamento das regiões IGS e D1/D2 do DNA ribossomal. O perfil de suscetibilidade dos 74 isolados foi analisado pelo método de micro-diluição EUCAST (referência) com os fármacos fluconazol (FCZ), itraconazol (ITZ), voriconazol (VCZ), cetoconazol (CTZ), anfotericina B (AMB) e 5-fluocitosina (5FC); e pelo método de micro-diluição comercial Sensititre YeastOne, com os mesmos fármacos empregados no EUCAST, acrescidos do posaconazol (POS) e caspofungina (CAS). Os valores das concentrações inibitórias mínimas (CIM), erros categórico e essencial, bem como outros parâmetros foram comparados entre os dois métodos. A presença de GXM na parede celular dos 74 isolados foi determinada por citometria de fluxo, empregando anticorpo monoclonal anti-GXM. Os resultados dos estudos morfológicos e fisiológicos foram insuficientes para definir as espécies dos 74 isolados. Pela assimilação de carboidratos analisada pelo sistema VITEK 2, verificou-se que 71 isolados foram identificados como T. asahii (17 de infecção invasiva e 54 não invasivos), um isolado como T. mucoides (invasivo), e para dois isolados (um invasivo e um não invasivo), a identificação não foi conclusiva. Para estes últimos foi realizado o auxanograma (método manual), e a identificação permaneceu inconclusiva, pois pelo perfil de assimilação, os isolados poderiam ser identificados como T. asahii ou T. faecale. Pela técnica de sequenciamento, 62 dos 74 isolados foram identificados como T. asahii, demonstrando 82,4% de concordância com o sistema VITEK 2. Onze isolados com identificações discordantes pertenciam às espécies T. inkin (8), T. faecale (2) e T. dermatis (1), como determinado por sequenciamento. Dos dois isolados com identificação inconclusiva pelo VITEK 2, um foi identificado pela técnica molecular como T. asahii, enquanto para o outro isolado não foi possível definir a espécie. Portanto, dos 74 isolados do estudo, 62 foram identificados como T. asahii, 8 como T. inkin, 2 como T. faecale e 1 T. dermatis; dois isolados permaneceram sem identificação conclusiva. Os resultados dos testes de suscetibilidade in vitro mostraram que, em ambos os métodos, VCZ apresentou a melhor atividade antifúngica. Pelo método EUCAST, foram obtidos valores elevados de CIM para AMB, enquanto o mesmo não foi observado no teste comercial. Neste último, foram observados valores elevados de CIM para FCZ, POS e CAS. Em relação à 5FC, os valores de CIM 90% por ambos os testes foram elevados (16mg/L). Diferenças significantes foram observadas entre os valores de CIM obtidas pelos dois métodos, e percentuais relativamente elevados de erros categóricos graves quando o método comercial foi comparado ao de referência. Não houve diferença estatística significante de valores de CIM entre isolados de infecção invasiva e não invasiva, exceto para ITZ e 5FC. Cerca de 30% dos isolados obtidos de casos de infecção invasiva e não invasivos apresentaram resistência cruzada entre os azóis FCZ e VCZ, e uma pequena porcentagem apresentou multirresistência. Para a análise de GXM na parede celular dos 74 isolados do estudo, foi avaliada a intensidade de fluorescência emitida pela citometria de fluxo, não tendo sido observada diferença estatística significante entre isolados invasivos e não invasivos. O estudo permitiu concluir que T. asahii foi a espécie mais isolada das amostras clínicas obtidas de sítios estéreis e não estéreis. A metodologia clássica de identificação fenotípica não foi suficiente para definir as espécies do gênero Trichosporon, e o sistema VITEK 2 apresentou discordância quando comparado à técnica molecular para as espécies não T. asahii. Em relação aos testes de suscetibilidade in vitro, VCZ apresentou-se mais adequado para a inibição das leveduras, enquanto os fármacos AMB, FCZ e POS não foram eficazes para a maior parte dos isolados. As discordâncias encontradas entre o método de referência e o comercial sugerem que, para o segundo, são necessárias mais avaliações para seu emprego em rotina laboratorial para o gênero Trichosporon. A detecção de GXM não resultou em diferenças entre os isolados de ambos os grupos; no entanto, para se determinar o efeito protetor do polissacarídeo contra a ação de macrófagos, ensaios de fagocitose devem ser realizados / Invasive Trichosporon spp. infections occur more frequently in neutropenic patients, especially those with hematologic malignancies, and are associated with high mortality rates due to difficulties in identifying the pathogen and treating patients with drugs most currently employed in antifungal therapy. Trichosporon species identification is important for epidemiological studies and to better define eventual species-specific clinical association. Additionally, antifungal susceptibility may vary according to the species. Furthermore, glucuronoxylomannan (GXM) is a cell wall-associated polysaccharide produced by genus Trichosporon, which may be involved in virulence mechanisms of this pathogen. This study aimed (i) to identify Trichosporon species isolated from hospitalized patients by both phenotypic and molecular methods, comparing results; (ii) to verify the distribution of these species in invasive and non-invasive infection episodes; (iii) to determine the in vitro activities of various antifungals agents against the Trichosporon spp. isolates, employing a reference micro-dilution method and a commercial system; (iv) and to analyze the surface expression of GXM. Seventy-four Trichosporon spp. isolates obtained from clinical specimens of patients admitted to the Hospital das Clínicas-FMUSP and to other hospitals in the state of São Paulo, from 2003 to 2011, were included in the study. Nineteen samples were isolated from sterile deep sites (invasive infections) and 55 were isolated from catheter and urine samples (non-invasive isolates). All isolates were submitted to phenotypic analysis, which consisted in morphological features observation, physiological tests and determination of the biochemical profile by VITEK 2 system. Molecular identification was performed by sequencing of IGS1 and D1/D2 regions from the ribosomal DNA. The susceptibility antifungal profiles of the 74 isolates were analyzed by both the EUCAST micro-dilution method (reference) employing fluconazole (FCZ), itraconazole (ITZ), voriconazole (VCZ), ketoconazole (CTZ), amphotericin B (AMB) and 5 - flucytosine (5FC), and the commercial micro-dilution test Sensititre YeastOne, with the same drugs employed in EUCAST plus posaconazole (POS) and caspofungin (CAS). The minimum inhibitory concentration values (MIC), categorical and essential errors as well as other susceptibility parameters were compared between both methods. The cell wall expression of GXM of all isolates was measured by flow cytometry employing an anti-GXM monoclonal antibody. The morphological and physiological features of the Trichosporon spp. isolates were insufficientto define species. The carbohydrate assimilation analysis, performed by VITEK 2 system, has resulted in 71 isolates identified as T. asahii (17 from invasive infections and 54 non-invasive isolates) and one isolate as T. mucoides (invasive). The species identification for the two remaining isolates (one invasive and one non-invasive) was inconclusive. For this reason, a manual auxanogram was performed with these isolates, resulting again in non-conclusive species identification. By the automated sequencing method, 62 of the 74 isolates were identified as T. asahii, showing 82.4% of agreement with the VITEK 2 identification. Eleven isolates were identified by sequencing as T. inkin (8), T. faecale (2) and T. dermatis (1), showing disagreement identification with the VITEK 2 system. Regarding the two isolates with inconclusive results by the carbohydrate assimilation, the molecular technique identified one as T. asahii, whereas for the other isolate the sequencing was also unable to define species. Therefore, among the 74 studied isolates, 62 were identified as T. asahii, eight as T. inkin, two as T. faecale and one as T. dermatis; and two isolates remained with unconclusive identification. Almost all Trichosporon spp. isolates displayed susceptibility to VCZ with both methods. By the EUCAST method, high values of MIC were observed for AMB, while by the commercial test especially the invasive isolates showed susceptibility to this drug. Additionally, the Sensititre kit provided elevated MIC values for FCZ, POS and CAS. In regards to 5FC, the MIC 90% values were consistently high (16 mg/L) in both methodologies. The MIC values obtained by both EUCAST and commercial methods were compared, resulting in significant differences of MIC values for all tested antifungal drugs; major categorical errors occurred at relatively high percentage with the commercial method. No statistically significant differences in MIC values were verified when invasive and non-invasive isolates were compared. Around 30% of both invasive and non-invasive isolates showed cross-resistance to FCZ and VCZ, while a small number of isolates was multiresistant. The GXM analysis by cytometry demonstrated no significant differences between invasive and non- invasive isolates. This study demonstrated that T. asahii was the most frequently isolated species from both deep and non-sterile sites of the patients. The classical phenotypic methodology was not able to define Trichosporon species, and the VITEK 2 system identification showed disagreement with the sequencing technique for the non-T. asahii species. Regarding the in vitro susceptibility tests, VCZ was the most effective drug against the isolates, whereas most of them appear to be less susceptible to AMB, FCZ and POS. The discrepancies in the Trichosporon spp. susceptibility results between the reference and commercial methods suggest that the latter requires further evaluation tests before it can be used in routine laboratory. Although the GXM expression seemed to be equal in both invasive and non-invasive Trichosporon spp. isolates, phagocytic assays should be performed in order to determine the protective effect of the polysaccharide against phagocytosis

Hintergrund: Die Pathogenese nicht-familiärer kolorektaler Neoplasien ist heute noch nicht bekannt. Die Besonderheiten der Epidemiologie der Erkrankung sprechen für die Beteiligung von Umweltfaktoren, wie sozioökonomischer Faktoren, der Ernährung oder der bakteriellen Flora des Darmes. Eine intrazelluläre, von E.coli dominierte Flora wurde in Kolonbiopsien dieser Patienten beschrieben. Methoden: Es wurden Virulenzfaktoren von Escherichia coli untersucht, die aus gewaschenen koloskopischen Biopsien von 43 Patienten mit kolorektalen Adenomen und Karzinomen isoliert worden waren. 100 Stämme wurden mittels PCR auf Gene für folgende Virulenzfaktoren untersucht: s-Fimbrien (sfa), pyelonephritisassoziierter Pilus (pap), Hämolysin A (hlyA), hitzestabiles und -labiles Toxin (ST, LT, EAST), Intimin (eae), Verotoxin (stx), Invasionsplasmid (ipa), cytolethal distending toxin (cdt) und cytotoxic necrotizing factor 1 (cnf1). Für die Kontrollgruppe wurden E.coli aus Biopsien von 55 Patienten mit chronisch-entzündlichen Darmerkrankungen (CED) und unspezifischer Kolitis, von 16 Patienten mit Colon irritabile (IBS) und aus Stuhlproben von 29 gesunden Probanden isoliert und untersucht. Ergebnisse: Bei 69% der Patienten mit kolorektalen Karzinomen und bei 58% der Patienten mit kolorektalen Adenomen wurde mindestens einer der Virulenzfaktoren gefunden, dagegen nur bei 25 bis 39% der IBS- und CED- Patienten sowie der gesunden Probanden (p / Background: Pathogenesis of non-hereditary colorectal neoplasia is poorly understood. The differences in regional incidence indicate an influence of environmental factors, as socio-economic conditions, nutrition and intestinal flora. An intracellular flora with a predominance of Escherichia coli in colon biopsies has been described in these patients. Methods: We studied virulence factors of Escherichia coli isolated from washed colonoscopic biopsies of 43 patients with colorectal carcinoma and adenoma. 100 strains of E.coli were isolated and used for detection of a broad range of virulence genes by PCR encoding: s-fimbriae (sfa), pyelonephritis-associated pili (pap), hemolysin A (hlyA), heatstable and heatlable toxins (ST, LT, EAST), verotoxin (stx), invasionplasmidantigen (ipaH), intimin (eae), cytolethal distending toxin (cdt) and cytotoxic necrotizing factor 1 (cnf1). E.coli from biopsies of 55 patients with inflammatory bowel disease (IBD) and non-specific colitis, of 16 patients with irritable bowel syndrom (IBS) and from stool samples of 29 healthy individuals were isolated and examined as controls. Results: The prevalence of virulent strains bearing at least one of the tested genes was 69% in colorectal carcinoma and 58% in colorectal adenoma, but only 25 to 39% of IBD and IBS patients and healthy individuals (p

Infecções invasivas por Trichosporon spp. ocorrem com maior frequência em pacientes neutropênicos, principalmente portadores de doenças hematológicas malignas, e estão associadas a elevados índices de mortalidade devido às dificuldades na identificação do patógeno e à resistência aos fármacos mais empregados na terapêutica antifúngica. A identificação das espécies de Trichosporon é importante tanto para estudos epidemiológicos, como para associar aspectos clínicos com as espécies causadoras das infecções. Além disso, auxilia no tratamento da enfermidade, uma vez que a suscetibilidades aos fármacos antifúngicos pode variar de acordo com a espécie. Além disso, as leveduras do gênero Trichosporon sintetizam a glucuronoxilomanana (GXM) em sua parede celular, que pode estar envolvida no mecanismo de virulência do patógeno. Este estudo teve como objetivo determinar, por identificação fenotípica e molecular, espécies isoladas de pacientes internados em unidades hospitalares, comparando os resultados obtidos por ambos os métodos; avaliar diferenças na distribuição dessas espécies em relação às formas invasivas e não invasivas da infecção; determinar o perfil de suscetibilidade dessas leveduras aos antifúngicos, empregando um método de micro-diluição de referência e um método comercial; e avaliar a presença de GXM na parede celular dos isolados. Foram avaliados 74 isolados obtidos de amostras clínicas de pacientes do Hospital das Clinicas da FMUSP e de outras unidades hospitalares do Estado de São Paulo, no período de 2003 a 2011. Dezenove amostras foram isoladas de sítios estéreis do organismo (infecções invasivas) e 55 foram isoladas de urina e cateter (isolados não invasivos). Para a identificação das espécies, os isolados foram submetidos a análises fenotípicas, que incluíram estudo macro e micromorfológico, provas fisiológicas e avaliação do perfil bioquímico por sistema automatizado VITEK 2. A identificação molecular foi realizada pelo sequenciamento das regiões IGS e D1/D2 do DNA ribossomal. O perfil de suscetibilidade dos 74 isolados foi analisado pelo método de micro-diluição EUCAST (referência) com os fármacos fluconazol (FCZ), itraconazol (ITZ), voriconazol (VCZ), cetoconazol (CTZ), anfotericina B (AMB) e 5-fluocitosina (5FC); e pelo método de micro-diluição comercial Sensititre YeastOne, com os mesmos fármacos empregados no EUCAST, acrescidos do posaconazol (POS) e caspofungina (CAS). Os valores das concentrações inibitórias mínimas (CIM), erros categórico e essencial, bem como outros parâmetros foram comparados entre os dois métodos. A presença de GXM na parede celular dos 74 isolados foi determinada por citometria de fluxo, empregando anticorpo monoclonal anti-GXM. Os resultados dos estudos morfológicos e fisiológicos foram insuficientes para definir as espécies dos 74 isolados. Pela assimilação de carboidratos analisada pelo sistema VITEK 2, verificou-se que 71 isolados foram identificados como T. asahii (17 de infecção invasiva e 54 não invasivos), um isolado como T. mucoides (invasivo), e para dois isolados (um invasivo e um não invasivo), a identificação não foi conclusiva. Para estes últimos foi realizado o auxanograma (método manual), e a identificação permaneceu inconclusiva, pois pelo perfil de assimilação, os isolados poderiam ser identificados como T. asahii ou T. faecale. Pela técnica de sequenciamento, 62 dos 74 isolados foram identificados como T. asahii, demonstrando 82,4% de concordância com o sistema VITEK 2. Onze isolados com identificações discordantes pertenciam às espécies T. inkin (8), T. faecale (2) e T. dermatis (1), como determinado por sequenciamento. Dos dois isolados com identificação inconclusiva pelo VITEK 2, um foi identificado pela técnica molecular como T. asahii, enquanto para o outro isolado não foi possível definir a espécie. Portanto, dos 74 isolados do estudo, 62 foram identificados como T. asahii, 8 como T. inkin, 2 como T. faecale e 1 T. dermatis; dois isolados permaneceram sem identificação conclusiva. Os resultados dos testes de suscetibilidade in vitro mostraram que, em ambos os métodos, VCZ apresentou a melhor atividade antifúngica. Pelo método EUCAST, foram obtidos valores elevados de CIM para AMB, enquanto o mesmo não foi observado no teste comercial. Neste último, foram observados valores elevados de CIM para FCZ, POS e CAS. Em relação à 5FC, os valores de CIM 90% por ambos os testes foram elevados (16mg/L). Diferenças significantes foram observadas entre os valores de CIM obtidas pelos dois métodos, e percentuais relativamente elevados de erros categóricos graves quando o método comercial foi comparado ao de referência. Não houve diferença estatística significante de valores de CIM entre isolados de infecção invasiva e não invasiva, exceto para ITZ e 5FC. Cerca de 30% dos isolados obtidos de casos de infecção invasiva e não invasivos apresentaram resistência cruzada entre os azóis FCZ e VCZ, e uma pequena porcentagem apresentou multirresistência. Para a análise de GXM na parede celular dos 74 isolados do estudo, foi avaliada a intensidade de fluorescência emitida pela citometria de fluxo, não tendo sido observada diferença estatística significante entre isolados invasivos e não invasivos. O estudo permitiu concluir que T. asahii foi a espécie mais isolada das amostras clínicas obtidas de sítios estéreis e não estéreis. A metodologia clássica de identificação fenotípica não foi suficiente para definir as espécies do gênero Trichosporon, e o sistema VITEK 2 apresentou discordância quando comparado à técnica molecular para as espécies não T. asahii. Em relação aos testes de suscetibilidade in vitro, VCZ apresentou-se mais adequado para a inibição das leveduras, enquanto os fármacos AMB, FCZ e POS não foram eficazes para a maior parte dos isolados. As discordâncias encontradas entre o método de referência e o comercial sugerem que, para o segundo, são necessárias mais avaliações para seu emprego em rotina laboratorial para o gênero Trichosporon. A detecção de GXM não resultou em diferenças entre os isolados de ambos os grupos; no entanto, para se determinar o efeito protetor do polissacarídeo contra a ação de macrófagos, ensaios de fagocitose devem ser realizados / Invasive Trichosporon spp. infections occur more frequently in neutropenic patients, especially those with hematologic malignancies, and are associated with high mortality rates due to difficulties in identifying the pathogen and treating patients with drugs most currently employed in antifungal therapy. Trichosporon species identification is important for epidemiological studies and to better define eventual species-specific clinical association. Additionally, antifungal susceptibility may vary according to the species. Furthermore, glucuronoxylomannan (GXM) is a cell wall-associated polysaccharide produced by genus Trichosporon, which may be involved in virulence mechanisms of this pathogen. This study aimed (i) to identify Trichosporon species isolated from hospitalized patients by both phenotypic and molecular methods, comparing results; (ii) to verify the distribution of these species in invasive and non-invasive infection episodes; (iii) to determine the in vitro activities of various antifungals agents against the Trichosporon spp. isolates, employing a reference micro-dilution method and a commercial system; (iv) and to analyze the surface expression of GXM. Seventy-four Trichosporon spp. isolates obtained from clinical specimens of patients admitted to the Hospital das Clínicas-FMUSP and to other hospitals in the state of São Paulo, from 2003 to 2011, were included in the study. Nineteen samples were isolated from sterile deep sites (invasive infections) and 55 were isolated from catheter and urine samples (non-invasive isolates). All isolates were submitted to phenotypic analysis, which consisted in morphological features observation, physiological tests and determination of the biochemical profile by VITEK 2 system. Molecular identification was performed by sequencing of IGS1 and D1/D2 regions from the ribosomal DNA. The susceptibility antifungal profiles of the 74 isolates were analyzed by both the EUCAST micro-dilution method (reference) employing fluconazole (FCZ), itraconazole (ITZ), voriconazole (VCZ), ketoconazole (CTZ), amphotericin B (AMB) and 5 - flucytosine (5FC), and the commercial micro-dilution test Sensititre YeastOne, with the same drugs employed in EUCAST plus posaconazole (POS) and caspofungin (CAS). The minimum inhibitory concentration values (MIC), categorical and essential errors as well as other susceptibility parameters were compared between both methods. The cell wall expression of GXM of all isolates was measured by flow cytometry employing an anti-GXM monoclonal antibody. The morphological and physiological features of the Trichosporon spp. isolates were insufficientto define species. The carbohydrate assimilation analysis, performed by VITEK 2 system, has resulted in 71 isolates identified as T. asahii (17 from invasive infections and 54 non-invasive isolates) and one isolate as T. mucoides (invasive). The species identification for the two remaining isolates (one invasive and one non-invasive) was inconclusive. For this reason, a manual auxanogram was performed with these isolates, resulting again in non-conclusive species identification. By the automated sequencing method, 62 of the 74 isolates were identified as T. asahii, showing 82.4% of agreement with the VITEK 2 identification. Eleven isolates were identified by sequencing as T. inkin (8), T. faecale (2) and T. dermatis (1), showing disagreement identification with the VITEK 2 system. Regarding the two isolates with inconclusive results by the carbohydrate assimilation, the molecular technique identified one as T. asahii, whereas for the other isolate the sequencing was also unable to define species. Therefore, among the 74 studied isolates, 62 were identified as T. asahii, eight as T. inkin, two as T. faecale and one as T. dermatis; and two isolates remained with unconclusive identification. Almost all Trichosporon spp. isolates displayed susceptibility to VCZ with both methods. By the EUCAST method, high values of MIC were observed for AMB, while by the commercial test especially the invasive isolates showed susceptibility to this drug. Additionally, the Sensititre kit provided elevated MIC values for FCZ, POS and CAS. In regards to 5FC, the MIC 90% values were consistently high (16 mg/L) in both methodologies. The MIC values obtained by both EUCAST and commercial methods were compared, resulting in significant differences of MIC values for all tested antifungal drugs; major categorical errors occurred at relatively high percentage with the commercial method. No statistically significant differences in MIC values were verified when invasive and non-invasive isolates were compared. Around 30% of both invasive and non-invasive isolates showed cross-resistance to FCZ and VCZ, while a small number of isolates was multiresistant. The GXM analysis by cytometry demonstrated no significant differences between invasive and non- invasive isolates. This study demonstrated that T. asahii was the most frequently isolated species from both deep and non-sterile sites of the patients. The classical phenotypic methodology was not able to define Trichosporon species, and the VITEK 2 system identification showed disagreement with the sequencing technique for the non-T. asahii species. Regarding the in vitro susceptibility tests, VCZ was the most effective drug against the isolates, whereas most of them appear to be less susceptible to AMB, FCZ and POS. The discrepancies in the Trichosporon spp. susceptibility results between the reference and commercial methods suggest that the latter requires further evaluation tests before it can be used in routine laboratory. Although the GXM expression seemed to be equal in both invasive and non-invasive Trichosporon spp. isolates, phagocytic assays should be performed in order to determine the protective effect of the polysaccharide against phagocytosis

Fímbrias do tipo IV estão associadas a diversos fenótipos em bactérias gram-negativas, e o presente estudo consistiu na caracterização da fímbria Pil e investigação de seu papel na adesão bacteriana de isolados de EPEC atípica. Por PCR e RT-PCR foram investigadas a presença e a funcionalidade do operon Pil e os resultados demonstraram que este está sendo transcrito somente nos isolados BA558 e BA956. Os genes pilS e pilV foram clonados em vetor de expressão para obtenção das proteínas Pil recombinantes e produção de anticorpos policlonais. A análise qualitativa dos testes de inibição da adesão utilizando os soros anti-PilS e anti-PilV juntos demonstraram que o isolado BA558 apresentou mudança de fenótipo de adesão. Esses resultados nos permitem concluir que o operon Pil está funcional em BA558 e BA956, e a expressão da fímbria Pil nessas cepas não está relacionada à formação de biofilme e autoagregação, porém a proteína fimbrial PilS juntamente com a adesina PilV parecem exercer uma função acessória importante na interação de BA558 às células HEp-2. / Type IV fimbriae are associated with several phenotypes in gram-negative bacteria. The aim of this study was the characterization of the Pil fimbria and its role in the interaction of atypical EPEC isolates in bacterial adhesion. Using PCR and RT-PCR, we investigated the presence and functionality of the pil operon genes. The results showed that these genes are transcribed only in the BA558 and BA956 isolates. The pilS and pilV genes were cloned into an expression vector for recombinant proteins and polyclonal antibodies production. Qualitative analysis of the adherence inhibition assays using both rabbit sera changed to localized-like the phenotype of BA558 isolate adhesion. Together, these results allow us to conclude that the Pil operon is functional only in the BA558 and BA956 isolates and that the expression of Pil fimbriae in aEPEC is not related to biofilm formation and autoaggregation but, the fimbrial PilS protein together with PilV adhesin seem to play an important accessory function in the interaction between the BA558 and epithelial cells in vitro.

<i>Escherichia coli </i> remains a significant cause of diarrhea worldwide and in recent years a relatively high number of E. coli carrying gene for AIDA-I (adhesin involved in diffuse adherence) has been isolated from cases of neonatal and post-weaning diarr<i>Escherichia coli</i> remains a significant cause of diarrhea worldwide and in recent years a relatively high number of <i>E. coli</i> carrying gene for AIDA-I (adhesin involved in diffuse adherence) has been isolated from cases of neonatal and post-weaning diarrhea in pigs. AIDA-I adhesin and its gene aidA were first identified and characterized in <i>E. coli</i> isolated from a human case of infantile diarrhea. Recent studies have demonstrated a significant degree of homology between the AIDA-I adhesin isolated from porcine neonatal diarrheagenic <i>E. coli</i> isolates and that from a human <i>E. coli</i> isolate; however, the role of AIDA-I adhesin in the pathogenesis of diarrhea and the clinical significance of the AIDA-I <i>E. coli</i> virotype are unknown in humans or in animals. <p>First, in order to evaluate the role of AIDA-I adhesin, colostrum deprived newborn pigs were infected with: i) a wild strain PD20 (AIDA-I+/STb+) <i>E. coli</i>; ii) a mutant strain PD20M (AIDA-I-/STb+), generated by partial deletion of the aidA gene from the wild strain, iii) a complemented strain PD20C (AIDA-I+/STb+), generated by reintroducing the full length aidA gene into PD20M strain, and iv) a nonpathogenic <i>E. coli</i> strain PD71 used as negative control. Pigs infected with wild type (PD20) and complemented (PD20C) strains developed diarrhea between 15-19 h and 27-31 h after oral inoculation, respectively, in contrast to pigs infected with strains PD20M or PD71 that did not developed diarrhea. Intestinal colonization was evaluated by histology, imunohistochemistry (IHC), transmission electron microscopy (TEM), including immunogold electron microscopy (IGEM), and showed heavy bacterial colonization with biofilm formation in the large intestine with AIDA-I+ strains (PD20 and PD20C), but not in pigs infected with AIDA-I- strains (PD20M and PD71). In vitro assays showed marked diffuse adherence to HeLa cells, enhanced bacterial autoaggregation and significant biofilm formation by AIDA-I+ strains, when compared to AIDA-I- strains.<p>Second, 110 F4 negative <i>E. coli</i> isolates from problematic cases of diarrhea in pigs were subjected to multiplex polymerase chain reaction (M-PCR) for detection of the genes encoding the virulence factors F4, F5, F6, F18, F41, AIDA-I, EAE, STa, STb, LT, EAST1 and Stx2e. In this study, the prevalence of aidA gene among the 110 isolates was 8.2%, and the aidA gene was shown to be associated most commonly with EAST1 and STb genes. The genes for the F4, F5, F6 and F41 fimbriae were absent in all the AIDA-I+ <i>E. coli</i> isolates. <p>The clinical significance of the AIDA-I+ <i>E. coli</i> was studied using clinical data available for 35 of the 110 <i>E. coli</i> isolates, originating from 18 cases of diarrhea. Among these 18 diarrhea cases, 3 cases (5 isolates) were found to have AIDA-I+ <i>E. coli</i> and these were significantly associated with diarrhea cases of post-weaning age group. Enterotoxigenic <i>E. coli</i> strains were isolated from the majority (72.5%) of 18 diarrhea cases and a high proportion (23.1%) of these ETEC cases carried AIDA-I+ <i>E. coli</i>. <p>In conclusion, AIDA-I adhesin appears to be a significant virulence factor for intestinal colonization and induction of biofilm formation. Further, experimental studies and clinical data suggest that the AIDA-I/STb virotype may be important in the pathogenesis of pre-weaning and post-weaning diarrhea in pigs. Our results suggest that AIDA-I may play a significant role in the development of diarrhea in pigs. .hea in pigs. AIDA-I adhesin and its gene aidA were first identified and characterized in E. coli isolated from a human case of infantile diarrhea. Recent studies have demonstrated a significant degree of homology between the AIDA-I adhesin isolated from porcine neonatal diarrheagenic E. coli isolates and that from a human E. coli isolate; however, the role of AIDA-I adhesin in the pathogenesis of diarrhea and the clinical significance of the AIDA-I E. coli virotype are unknown in humans or in animals. First, in order to evaluate the role of AIDA-I adhesin, colostrum deprived newborn pigs were infected with: i) a wild strain PD20 (AIDA-I+/STb+) E. coli; ii) a mutant strain PD20M (AIDA-I-/STb+), generated by partial deletion of the aidA gene from the wild strain, iii) a complemented strain PD20C (AIDA-I+/STb+), generated by reintroducing the full length aidA gene into PD20M strain, and iv) a nonpathogenic E. coli strain PD71 used as negative control. Pigs infected with wild type (PD20) and complemented (PD20C) strains developed diarrhea between 15-19 h and 27-31 h after oral inoculation, respectively, in contrast to pigs infected with strains PD20M or PD71 that did not developed diarrhea. Intestinal colonization was evaluated by histology, imunohistochemistry (IHC), transmission electron microscopy (TEM), including immunogold electron microscopy (IGEM), and showed heavy bacterial colonization with biofilm formation in the large intestine with AIDA-I+ strains (PD20 and PD20C), but not in pigs infected with AIDA-I- strains (PD20M and PD71). In vitro assays showed marked diffuse adherence to HeLa cells, enhanced bacterial autoaggregation and significant biofilm formation by AIDA-I+ strains, when compared to AIDA-I- strains. Second, 110 F4 negative E. coli isolates from problematic cases of diarrhea in pigs were subjected to multiplex polymerase chain reaction (M-PCR) for detection of the genes encoding the virulence factors F4, F5, F6, F18, F41, AIDA-I, EAE, STa, STb, LT, EAST1 and Stx2e. In this study, the prevalence of aidA gene among the 110 isolates was 8.2%, and the aidA gene was shown to be associated most commonly with EAST1 and STb genes. The genes for the F4, F5, F6 and F41 fimbriae were absent in all the AIDA-I+ E. coli isolates. The clinical significance of the AIDA-I+ E. coli was studied using clinical data available for 35 of the 110 E. coli isolates, originating from 18 cases of diarrhea. Among these 18 diarrhea cases, 3 cases (5 isolates) were found to have AIDA-I+ E. coli and these were significantly associated with diarrhea cases of post-weaning age group. Enterotoxigenic E. coli strains were isolated from the majority (72.5%) of 18 diarrhea cases and a high proportion (23.1%) of these ETEC cases carried AIDA-I+ E. coli. In conclusion, AIDA-I adhesin appears to be a significant virulence factor for intestinal colonization and induction of biofilm formation. Further, experimental studies and clinical data suggest that the AIDA-I/STb virotype may be important in the pathogenesis of pre-weaning and post-weaning diarrhea in pigs. Our results suggest that AIDA-I may play a significant role in the development of diarrhea in pigs.

<i>Escherichia coli </i> remains a significant cause of diarrhea worldwide and in recent years a relatively high number of E. coli carrying gene for AIDA-I (adhesin involved in diffuse adherence) has been isolated from cases of neonatal and post-weaning diarr<i>Escherichia coli</i> remains a significant cause of diarrhea worldwide and in recent years a relatively high number of <i>E. coli</i> carrying gene for AIDA-I (adhesin involved in diffuse adherence) has been isolated from cases of neonatal and post-weaning diarrhea in pigs. AIDA-I adhesin and its gene aidA were first identified and characterized in <i>E. coli</i> isolated from a human case of infantile diarrhea. Recent studies have demonstrated a significant degree of homology between the AIDA-I adhesin isolated from porcine neonatal diarrheagenic <i>E. coli</i> isolates and that from a human <i>E. coli</i> isolate; however, the role of AIDA-I adhesin in the pathogenesis of diarrhea and the clinical significance of the AIDA-I <i>E. coli</i> virotype are unknown in humans or in animals. <p>First, in order to evaluate the role of AIDA-I adhesin, colostrum deprived newborn pigs were infected with: i) a wild strain PD20 (AIDA-I+/STb+) <i>E. coli</i>; ii) a mutant strain PD20M (AIDA-I-/STb+), generated by partial deletion of the aidA gene from the wild strain, iii) a complemented strain PD20C (AIDA-I+/STb+), generated by reintroducing the full length aidA gene into PD20M strain, and iv) a nonpathogenic <i>E. coli</i> strain PD71 used as negative control. Pigs infected with wild type (PD20) and complemented (PD20C) strains developed diarrhea between 15-19 h and 27-31 h after oral inoculation, respectively, in contrast to pigs infected with strains PD20M or PD71 that did not developed diarrhea. Intestinal colonization was evaluated by histology, imunohistochemistry (IHC), transmission electron microscopy (TEM), including immunogold electron microscopy (IGEM), and showed heavy bacterial colonization with biofilm formation in the large intestine with AIDA-I+ strains (PD20 and PD20C), but not in pigs infected with AIDA-I- strains (PD20M and PD71). In vitro assays showed marked diffuse adherence to HeLa cells, enhanced bacterial autoaggregation and significant biofilm formation by AIDA-I+ strains, when compared to AIDA-I- strains.<p>Second, 110 F4 negative <i>E. coli</i> isolates from problematic cases of diarrhea in pigs were subjected to multiplex polymerase chain reaction (M-PCR) for detection of the genes encoding the virulence factors F4, F5, F6, F18, F41, AIDA-I, EAE, STa, STb, LT, EAST1 and Stx2e. In this study, the prevalence of aidA gene among the 110 isolates was 8.2%, and the aidA gene was shown to be associated most commonly with EAST1 and STb genes. The genes for the F4, F5, F6 and F41 fimbriae were absent in all the AIDA-I+ <i>E. coli</i> isolates. <p>The clinical significance of the AIDA-I+ <i>E. coli</i> was studied using clinical data available for 35 of the 110 <i>E. coli</i> isolates, originating from 18 cases of diarrhea. Among these 18 diarrhea cases, 3 cases (5 isolates) were found to have AIDA-I+ <i>E. coli</i> and these were significantly associated with diarrhea cases of post-weaning age group. Enterotoxigenic <i>E. coli</i> strains were isolated from the majority (72.5%) of 18 diarrhea cases and a high proportion (23.1%) of these ETEC cases carried AIDA-I+ <i>E. coli</i>. <p>In conclusion, AIDA-I adhesin appears to be a significant virulence factor for intestinal colonization and induction of biofilm formation. Further, experimental studies and clinical data suggest that the AIDA-I/STb virotype may be important in the pathogenesis of pre-weaning and post-weaning diarrhea in pigs. Our results suggest that AIDA-I may play a significant role in the development of diarrhea in pigs. .hea in pigs. AIDA-I adhesin and its gene aidA were first identified and characterized in E. coli isolated from a human case of infantile diarrhea. Recent studies have demonstrated a significant degree of homology between the AIDA-I adhesin isolated from porcine neonatal diarrheagenic E. coli isolates and that from a human E. coli isolate; however, the role of AIDA-I adhesin in the pathogenesis of diarrhea and the clinical significance of the AIDA-I E. coli virotype are unknown in humans or in animals. First, in order to evaluate the role of AIDA-I adhesin, colostrum deprived newborn pigs were infected with: i) a wild strain PD20 (AIDA-I+/STb+) E. coli; ii) a mutant strain PD20M (AIDA-I-/STb+), generated by partial deletion of the aidA gene from the wild strain, iii) a complemented strain PD20C (AIDA-I+/STb+), generated by reintroducing the full length aidA gene into PD20M strain, and iv) a nonpathogenic E. coli strain PD71 used as negative control. Pigs infected with wild type (PD20) and complemented (PD20C) strains developed diarrhea between 15-19 h and 27-31 h after oral inoculation, respectively, in contrast to pigs infected with strains PD20M or PD71 that did not developed diarrhea. Intestinal colonization was evaluated by histology, imunohistochemistry (IHC), transmission electron microscopy (TEM), including immunogold electron microscopy (IGEM), and showed heavy bacterial colonization with biofilm formation in the large intestine with AIDA-I+ strains (PD20 and PD20C), but not in pigs infected with AIDA-I- strains (PD20M and PD71). In vitro assays showed marked diffuse adherence to HeLa cells, enhanced bacterial autoaggregation and significant biofilm formation by AIDA-I+ strains, when compared to AIDA-I- strains. Second, 110 F4 negative E. coli isolates from problematic cases of diarrhea in pigs were subjected to multiplex polymerase chain reaction (M-PCR) for detection of the genes encoding the virulence factors F4, F5, F6, F18, F41, AIDA-I, EAE, STa, STb, LT, EAST1 and Stx2e. In this study, the prevalence of aidA gene among the 110 isolates was 8.2%, and the aidA gene was shown to be associated most commonly with EAST1 and STb genes. The genes for the F4, F5, F6 and F41 fimbriae were absent in all the AIDA-I+ E. coli isolates. The clinical significance of the AIDA-I+ E. coli was studied using clinical data available for 35 of the 110 E. coli isolates, originating from 18 cases of diarrhea. Among these 18 diarrhea cases, 3 cases (5 isolates) were found to have AIDA-I+ E. coli and these were significantly associated with diarrhea cases of post-weaning age group. Enterotoxigenic E. coli strains were isolated from the majority (72.5%) of 18 diarrhea cases and a high proportion (23.1%) of these ETEC cases carried AIDA-I+ E. coli. In conclusion, AIDA-I adhesin appears to be a significant virulence factor for intestinal colonization and induction of biofilm formation. Further, experimental studies and clinical data suggest that the AIDA-I/STb virotype may be important in the pathogenesis of pre-weaning and post-weaning diarrhea in pigs. Our results suggest that AIDA-I may play a significant role in the development of diarrhea in pigs.

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Made available in DSpace on 2014-12-17T14:16:35Z (GMT). No. of bitstreams: 1 WalicyranisonPS_DISSERT.pdf: 2780035 bytes, checksum: 20a70f53e533bfb0816b6d9b02b8af32 (MD5) Previous issue date: 2013-06-11 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Candida albicans is a diploid yeast that in some circumstances may cause oral or oropharyngeal infections. The investigation of natural products is mandatory for the discovery of new targets for antifungal drugs development. This study aimed to determine the genotypes of 48 clinical isolates of C. albicans obtained from the oral cavity of kidney transplant patients from two distinct geographic regions of Brazil. In addition, we investigated three virulence factors in vitro: phospholipase activity, morphogenesis and the ability to evade from polymorphonuclear neutrophils. The expression of these virulence factors in vitro was also investigated in the presence of the crude extract of Eugenia uniflora. The genotype A was the most prevalent (30 isolates; 62.5%), followed by genotype C (15 isolates; 31.5%) and genotype B (3 isolates; 6.25%). When microsatellite technique with primer M13 was applied, 80% of the isolates from the South were placed within the same cluster. All Genotype C strains were grouped together within two different clusters. Genotype C was considered more resistant to PMNs attack than genotypes A and B. Strains isolated from the South of Brazil showed higher ability to combat PMNs phagocytosis. We found a high rate of genotype C strains isolated from the oral cavity of this group of patients. The crude extract of E. uniflora inhibited proper hypha formation and phagocytosis by PMNs, but had no significant effect on phospholipase activity. This study characterized oral C. albicans strains isolated from kidney transplant recipients and will contribute for the better understanding of the pathogenesis and alternative therapeutics for oral candidiasis / Candida albicans ? uma levedura dipl?ide que em certas circunst?ncias pode causar infec??es da cavidade oral e da orofaringe. A investiga??o de produtos naturais ? fundamental para a descoberta de novos alvos para o desenvolvimento de drogas antif?ngicas. Este estudo objetivou determinar os gen?tipos de 48 isolados cl?nicos de C. albicans obtidos da cavidade oral de pacientes transplantados renais de duas distintas regi?es geogr?ficas do Brasil. Al?m disso, foram investigados tr?s fatores de virul?ncia in vitro: atividade de fosfolipase, morfog?nese e a capacidade de escapar do ataque de neutr?filos polimorfonucleares. A express?o destes fatores de virul?ncia tamb?m foi investigada na presen?a do extrato bruto de Eugenia uniflora. O gen?tipo A foi o mais prevalente (30 isolados; 62,5%), seguido do gen?tipo C (15 isolados; 31,5%) e do gen?tipo B (3 isolados; 6,25%). Quando a t?cnica do microssat?lite com o primer M13 foi empregada, 80% dos isolados da regi?o Sul foram agrupados no mesmo cluster. Todos os isolados do gen?tipo C foram agrupados juntos em dois diferentes clusters bem definidos. Isolados do gen?tipo C foram considerados mais resistentes ? a??o de PMNs do que os dos gen?tipos A e B. As cepas isoladas do Sul do Brasil demonstraram maior habilidade em combater a fagocitose por PMNs. Encontrou-se uma alta taxa de isolados do gen?tipo C da cavidade oral deste grupo de pacientes. O extrato bruto de E. uniflora inibiu a forma??o de hifa e fagocitose por PMNs, mas n?o apresentou efeito significativo na atividade de fosfolipase. Este estudo caracterizou isolados cl?nicos de C. albicans da cavidade oral de pacientes transplantados renais, contribuindo para um melhor entendimento da patog?nese e terap?utica alternativa para a candid?ase oral