This study describes the biochemical activity and antimicrobial susceptibility patterns (antibiograms) of 104 gram-negative bacteria, represented by six species of bacilli, isolated from ten macaws of the genus Ara. Bacterial samples were acquired from fecal matter of six different species of macaws, ages one to three years, housed in a variety of locations. Bacteria from these samples were cultured onto selective media for classification. Identification and biochemical characterization were accomplished with the API 20E system. Escherichia (E.) coli accounted for 78% (81/1 04) of the total number of isolates. Antimicrobial susceptibility testing was done using the Kirby-Bauer method. Biotype and antimicrobial susceptibility data gathered in this study correspond with data of studies done on pigeons and llamas. These data show specific E. coli biotypes as members of the microflora of macaws.

No description available.

<P> Type II Secretion (T2S) and type IV pilus (T4P) systems in Gram-negative bacteria share many features that suggest a common ancestral origin. This study examined the role of the minor pilins FimU, PilV, PilW, PilX and PilE, as well as the putative adhesin PilYl in both the T4P and T2S systems, and elucidated the role of these proteins in pilus assembly. Genetic analysis of the major pilin cluster and the minor pilin operon revealed that the major pilin alleles are associated with a specific set of minor pilins, and that unrelated strains of the same major pilin type have identical minor pilin genes, suggesting that the two gene clusters were horizontally acquired as a 'pilin island'. We observed that the minor pilins required a specific stoichiometric ratio for proper assembly, as overexpression either completely abolished, or significantly reduced twitching motility in mutant backgrounds. We demonstrated that the minor pilins were incorporated into the pilus fibre, and that they were dependent on PilA for surface localization. The T4P minor pilins were also shown to play a role in the secretion of effectors through the T2S system, as elastase and haemolytic phospholipase C secretion was reduced in minor pilin mutants, while overexpression of FimU or PilX significantly increased secretion of T2S exoproteins. Therefore, the minor pilins may participate in T2S substrate recognition. We found that PilYl was not essential for assembly in the absence of retraction, but that its absence caused changes in the levels of other T4P biogenesis proteins, namely FimU, PilW, PilF and PilQ secretin multimers. Finally we show that the minor pilin, PilX functions as a strain-specific factor, potentially through specific interactions with non-conserved residues of PilQ that are necessary to induce opening of the secretin. </p> / Thesis / Doctor of Philosophy (PhD)

No description available.

Orientador: Marcos Rogério André / Banca: Ana Patrícia Yatsuda Natsui / Banca: Rosangela Zacarias Machado / Resumo: Embora espécies de Anaplasma spp. sejam agentes Rickettsiales altamente difundidos em ruminantes domésticos e selvagens, com ampla distribuição mundial, poucos estudos foram realizados até momento com o intuito de detectar e/ou investigar a diversidade de Anaplasma spp. circulantes em bovinos em Moçambique. No presente estudo, ensaios sorológicos e moleculares foram utilizados para investigar a ocorrência de Anaplasma spp. em 219 bovinos amostrados nos distritos de Boane, Magude, Matutuíne, Moamba e Namaacha em Maputo, Moçambique. No teste iELISA para detecção de anticorpos IgG anti- A. marginale, 86,3% (189/219) das amostras mostraram-se soropositivas. Em ensaios de qPCR para os genes msp1β de A. marginale e msp2 para A. phagocytophilum, 97,3% (213/219) e 2,74% (6/219) dos animais mostraram-se positivos, respectivamente. A combinação de dois protocolos diferentes de cPCR baseados no gene 16S rRNA evidenciou 100% de amostras positivas para Anaplasma spp. Sequências de 16S rRNA filogeneticamente relacionadas a A. platys, A phagocytophilum, 'Candidatus Anaplasma boleense', A. centrale, A. marginale e A. ovis foram detectadas neste estudo. Inferências filogenéticas baseadas nos genes msp4 e msp5 posicionaram as sequências obtidas no clado de A. marginale, com a evidência de circulação de 1 e 2 haplótipos diferentes para cada gene, respectivamente. Anaplasma sp. filogeneticamente associado a A. platys foi evidenciado nas análises filogenéticas baseadas nos genes 16S rRNA e groEL.... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Although species of Anaplasma spp. are widespread Rickettsiales agents in domestic and wild ruminants, showing a worldwide distribution, few studies have been conducted in order to detect and/or investigate the diversity of Anaplasma spp. circulating in cattle in Mozambique. In the present study, serological and molecular tests were used to investigate the occurrence of Anaplasma spp. in 219 bovine sampled in the districts of Boane, Magude, Matutuíne, Moamba and Namaacha in Maputo, Mozambique. In the iELISA test for detection of IgG antibodies to A. marginale, 86.3% (189/219) of the samples were positive. In quantitative Real-time PCR tests targeting the msp1β genes of A. marginale and msp2 for A. phagocytophilum, 97.3% (213/219) and 2.74% (6/219) of the animals were positive, respectively. The combination of two different protocols of conventional PCR targeting the 16S rRNA gene evidenced 100% of positive samples for Anaplasma spp. Sequences of 16S rRNA phylogenetically related to A. platys, A. phagocytophilum, 'Candidatus Anaplasma boleense', A. centrale, A. marginale and A. ovis were detected in this study. Phylogenetic analysis based on msp4 and msp5 genes positioned the obtained sequences in the clade of A. marginale, with the evidence of circulation of 1 and 2 different haplotypes for each gene, respectively. Anaplasma sp. phylogenetically related to A. platys was evidenced in the phylogenetic analyses based on the 16S rRNA and groEL genes. In conclusion,a high diversit... (Complete abstract click electronic access below) / Mestre

Este trabalho utiliza a Análise de Fluxos Metabólicos para estudar o aumento da eficiência da linhagem Burkholderia sacchari (LFM101) na produção de PHB. Foram avaliadas as eficiências de conversão de açúcares em PHA de LFM101. Esta também foi cultivada em batelada alimentada em reator, apresentando o máximo teórico durante um estado pseudo-estacionário sob oferta de glicose. Estes dados, submetidos ao software Metatool, resultaram em mapa metabólico contendo os fluxos das reações centrais ede PHA ocorrido no experimento. Através do cultivo de LFM101 e C. necator sob oferta de glicose marcada com 13C, determinou-se que estas utilizam as mesmas vias para produção de PHA, não justificando a baixa eficiência de LFM101. Em um projeto paralelo, estudou-se a eficiência da produção de PHB utilizando melaço de cana, glicerol cru e acetato, em produtores de hidrogênio e PHA, onde verificou-se não apenas o aumento de PHA em mutantes nifh- de R. capsulatus mas a interação dos parâmetros luz e nitrogênio a partir das metodologias DOE e RSM. / This work applies Metabolic Flux Analysis to discuss the eficiency of Burkholderia sacchari (LFM101) in PHA production from sugars . Conversion yields of LFM101 and Cupriavidus necator from carbohydrates to PHA were assessed. LFM101 was also grown in reactor fed-batch cultivations, and presented the theoretical maximum in a pseudo-steady stage while grown on glucose. These data were submitted to Metatool and resulted in a metabolic network containing the experimental fluxes of central and PHA metabolism. Cultivation of LFM101 and C. necator under 13C- labeled glucose showed that both species use the same metabolic pathways for the biodegradable polymer synthesis. On a parallel project, the efficiency of biopolymer production from molasses, raw glycerol and acetate in strains producing hydrogen and PHA was tested. Results showed that there was not only an increase in PHA production by R. capsulatus nifH- mutants but also the interaction of light and nitrogen effects were studied by DOE and RSM.

Introdução: Bactérias intestinais influenciam a resposta imune e conhecendo-se as ações imunomoduladoras da vitamina D passou-se a investigar sua relação com a microbiota. O status adequado desta vitamina está associado a uma composição mais saudável da microbiota, enquanto sua deficiência pode acarretar disbiose intestinal, endotoxemia, inflamação e resistência à insulina. O conhecimento de interações do status de vitamina D com a microbiota pode melhorar a compreensão da gênese de doenças crônicas mediadas pela inflamação. Objetivo: Examinou-se a associação da ingestão e concentração de vitamina D com a composição da microbiota fecal, marcadores inflamatórios e perfil bioquímico de participantes do Nutritionists Health Study. Métodos: Nesta análise transversal, 150 adultos jovens foram estratificados em tercis de consumo e de concentração de 25(OH)D e comparados quanto ao perfil clínico e inflamatório. A associação de 25(OH)D com a microbiota (sequenciamento do 16S rRNA, região V4, Illumina® MiSeq) foi testada por regressão linear múltipla. Resultados: A ingestão de vitamina D se associou aos níveis séricos (p < 0,05). Não foram observadas diferenças significantes de variáveis clínicas e inflamatórias entre os tercis de ingestão, exceto tendência de aumento do LPS com a redução da 25(OH)D (p-trend < 0,05). Prevotella foi mais abundante (log2FC 1,67; p < 0,01), e Haemophilus and Veillonella menos abundantes (log2FC -2,92 e -1,46; p < 0,01, respectivamente) no subgrupo com maior ingestão de vitamina D (referência) comparado aos outros grupos (primeiro e segundo tercis). PCR (r = -0,170; p = 0,039), selectina-E (r = -0,220; p = 0,007) e abundância de Coprococcus (r = -0,215; p = 0,008) e de Bifdobacterium (r = -0,269; p = 0,001) foram inversamente correlacionados com 25(OH)D. Após ajustes por idade, sexo, estação do ano e IMC, a 25(OH)D manteve associação inversa com Coprococcus ( = -9,414; p = 0,045) e Bifdobacterium ( = -1,881; p = 0,051), mas a significância desapareceu com a adição de marcadores inflamatórios aos modelos. Conclusão: Associações de ingestão e concentração de vitamina D com abundância de certos gêneros da microbiota sugerem que sua ação imunomoduladora poderia influenciar a composição bacteriana. Abundância relativamente maior de gram-negativos (Haemophilus e Veillonella) pode ter sido facilitada pela baixa ingestão e/ou concentração da vitamina. Menor proporção de bactérias benéficas (Coprococcus e Bifidobacterium) poderia estimular a resposta imune e inflamação. Concluímos que a participação da vitamina D na manutenção da homeostase imune deve ocorrer em parte pelas interações com a microbiota intestinal, embora o delineamento transversal impeça assegurar relações tipo causa-efeito. / Introduction: Gut bacteria influence the immune response and due the immunomodulatory actions of vitamin D, it has been investigated its relationship with the microbiota. Adequate status of this vitamin is associated with adequate composition of the microbiota, while its deficiency can cause gut dysbiosis, endotoxemia, inflammation and insulin resistance. The knowledge of interactions of vitamin D status with the microbiota may improve the understanding of the genesis of inflammation-mediated chronic diseases. Objective: We examined the association of vitamin D intake and concentration with the composition of fecal microbiota, inflammatory markers and biochemical profile of participants from the Nutritionists\' Health Study. Methods: In this cross-sectional analysis, 150 healthy young adults were stratified into tertiles of intake and concentrations of vitamin D and their clinical and inflammatory profiles were compared. The association between 25(OH)D and fecal microbiota (16S rRNA sequencing, V4 region, Illumina® MiSeq) was tested by multiple linear regression.) Results: Vitamin D intake was associated with its concentration (p<0.05). There were no significant differences in clinical and inflammatory variables across tertiles of intake, except for a trend of LPS increases with reduction of 25(OH)D (p-trend <0.05). Prevotella was more abundant (log2FC 1.67; p <0.01), and Haemophilus and Veillonella less abundant (log2FC -2,92 e -1.46; p <0.01, respectively) in subset with the highest vitamin D intake (reference) than that observed in the other subset (first plus second tertiles). CRP (r=-0.170, p=0.039), E-selectin (r=-0.220, p=0.007) and abundances of Coprococcus (r=-0.215, p=0.008) and Bifdobacterium (r=-0.269, p=0.001) were inversely correlated with 25(OH)D. After adjusting for age, sex, season and BMI, the 25(OH)D maintained inversely associated with Coprococcus (=-9.414, p=0.045) and Bifdobacterium (=-1.881, p=0.051), but significance disappeared following the addition of inflammatory markers in the regression models. Conclusion: Association of vitamin D intake and concentration with abundance of certain genera of microbiota suggests that its immunomodulatory action could influence the bacterial composition. Relatively higher abundance of gram-negative (Haemophilus and Veillonella) may have been facilitated by the low intake and/or concentration of the vitamin. Lower proportion of beneficial bacteria (Coprococcus and Bifidobacterium) could stimulate the immune response and inflammation. We conclude that the role of vitamin D in maintaining immune homeostasis should occur in part by interactions with the gut microbiota, although the cross-sectional design does not allow ensuring cause-effect relationships.

Orientador: Carlos Magno Castelo Branco Fortaleza / Coorientador: Lenice do Rosário de Souza / Banca: Anna Sara S. Levin / Banca: Elisei Alves Waldman / Banca: Maria de Lourdes Ribeiro de Souza da Cunha / Banca: Adriano Dias / Resumo: Estudos recentes identificaram comportamento sazonal de alguns microrganismos implicados na etiologia das Infecções Hospitalares ou Relacionadas à Assistência em Saúde (IH/IRAS). A maior parte desses estudos foi conduzida em países de clima temperado. Para identificar a sazonalidade de patógenos hospitalares em uma região tropical, nós conduzimos um estudo ecológico e um caso-controle, envolvendo hemoculturas positivas de presumível origem hospitalar (colhidas após o terceiro dia de internação) do Hospital das Clínicas da Faculdade de Medicina de Botucatu no período de 2005 a 2010. O estudo foi realizado em duas fases. No estudo ecológico, foram realizadas: (a) comparações de incidência de bacteremias por microrganimos ou grupos específicos em meses "quentes/úmidos" (outubro-março) e "frios/secos" (abril a setembro); (b) análise de regressão para identificar relação entre temperatura e umidade médias e incidência mensal de bacteremias; (c) análise de séries temporais para identificação de sazonalidade.No "casocontrole", foram abordados fatores climáticos (temperatura e umidade médias dos sete dias que precederam a coleta de cada hemocultura) para identificar preditores do isolamento de microrganismos e grupos específicos, em modelo de regressão logística. Os resultados demonstraram consistência entre as diversas abordagens, identificando para três grupos (Gram-negativos como um todo, Acinetobacter baumannii e Enterobacter spp) um aumento de incidência nos meses "quentes/úmidos", relação significante entre temperatura média mensal e incidência e sazonalidade em modelos estocásticos. Neste último teste, também se detectou sazonalidade para estafilococos coagulase-negativa, aparentemente não relacionada a fatores climáticos. No estudo de base individual ("caso-controle") identificamos correlação entre temperaturas elevadas na semana anterior ... / Abstract: Recent studies identified seasonal behavior of some microorganisms implicated in the etiology of Healthcare-associated Infections (HAIs). Most of these studies were conducted in developed countries with temperate climate. In order to identify the seasonality of nosocomial pathogens in a tropical region, we conducted an ecological study involving positive blood cultures of suspected nosocomial origin (collected after the third day of hospitalization) in the Hospital das Clinicas from Faculdade de Medicina de Botucatu in the period 2005-2010. The study was conducted in two phases. In the first ("ecologic study") we performed: (a) comparisons of the incidence of bacteremia caused by specific organisms or groups during "warm" (October to March) and "cold" (April to September) months; (b) regression analysis, aimed at identifying the association between temperature and humidity and the average monthly incidence of bacteremia, (c) time series analysis. In the second phase, ("casecontrol"), weather factors (temperature and humidity averages of the seven days preceding the collection of each blood cultures) were addressed in order to identify predictors of isolation of specific microorganisms and groups in the logistic regression model. The results showed consistency between the various approaches. Three groups (Gram-negative as a whole, Acinetobacter baumannii and Enterobacter spp) presented increased incidence in "warm" months, significant relation between mean temperature and monthly incidence and seasonality in stochastic models. Those models also detected seasonality for coagulase-negative staphylococci, apparently not related to climatic factors. In individualbased analysis ("case-control") we identified correlation between high temperatures in the past week and recovery of Gram-negatives in general, and among these, A. baumannii. The findings are consistent with the literature, showing ... / Mestre

Orientador: José Paes de Almeida nogueira Pinto / Banca: Luiz Carlos de Souza / Banca: Andréa Pereira Pinto / Banca: Rogério Salvador / Banca: Thais Heleno Constantino Patelli / Resumo: O Brasil lidera o ranking de maior exportador de carne bovina no mundo desde 2008. Garantir a segurança microbiológica desses alimentos tem sido um dos principais focos da indústria processadora de carnes. A aplicação de análises nas diversas etapas do processo industrial tem sido vital para implantar e manter programas de autocontrole como o APPCC. Entre as enfermidades mais frequentes causadas pela ingestão de alimentos contaminados, especialmente os de origem animal, destacam-se as salmoneloses, sendo que sua transmissão se dá primariamente pela via fecal oral. Diante disso, esse trabalho teve como objetivo pesquisar a presença de Salmonella spp. em amostras de conteúdo ruminal coletadas durante o abate de bovinos em uma planta frigorífica localizada na região metropolitana de Curitiba - PR. Duzentos e dois animais distribuídos em oito lotes foram avaliados entre os meses de agosto a dezembro de 2013. Ao final do experimento, 37,5% dos lotes mostraram-se positivos para o agente sendo que 2,97% (6/202) das amostras de conteúdo ruminal isolaram o micro-organismo. A totalidade dos animais positivos recebia como alimento apenas pasto de azevém e aveia. Não foi observada a influência de fatores como o estresse do transporte ou temperatura ambiental sobre o isolamento do patógeno. Entretanto a detecção do mesmo sorovar, Salmonella Schwarzengrund na totalidade dos isolamentos nos permite levantar a hipótese de que a contaminação dos animais tenha se dado na própria indústria, a partir das estruturas físicas, responsáveis por conter os bovinos ou conduzi-los ao box de insensibilização / Abstract: Brazil leads the world ranking of bovine meat exportation since 2008. To ensure the microbiological safety of these food products has been one of the primary focuses of the meat processing industry. The application of analyses on diverse stages of the industrial process has been vital for deploy and keeping of self-control programs like APPCC. Beyond the most frequent diseases caused by the ingestion of contaminated food, specially animal origin ones, the salmonellosis stand out being transmitted primarily via fecal-oral route. In light of this, the work had the objective to research the presence of Salmonella spp. in rumen fluid samples collected during cattle slaughter at a slaughtering plant localized in Curitiba's metropolitan area. Two hundred animals distributed over eight lots was evaluated between august and december, 2013. At the end of the experiment, 37.5% of the lots were positive for the agent of which 2.97% (6/202) of samples of rumen contents isolated the micro-organism.The total of the positive animals had been feed with ryegrass pastures and oat. Not the influence of factors like transportation stress or environment temperature over pathogen isolation was observed. However, the detection of the same serovar, Salmonella Schwarzengrund on overall insulation allow us to raise the hypothesis that all animal contamination have been given inside the industry itself, from the infrastructure responsible of holding the cattle or conducting it to the stunning box / Doutor

Antibacterial activities in the salivary glands of female mosquitoes Aedes aegypti were investigated in this study. The mean salivary bacteriolytic activity, during a period of 14-day of female mosquitoes exposed to five different concentrations of Gram-positive bacteria Micrococcus lysodeikticus in the sucrose meal, was detected by a lysoplate method. A logarithmic regression (R²=0.73) fits the different levels of bacteriolytic activity during the entire period. As the concentration of bacteria in the sugar meal increases, the level of mean salivary bacteriolytic activity increases. A maximum level of bacteriolytic factor may exist in the salivary gland when the concentration of M. lysodeikticus in the sucrose meal exceeds 0.6 g/100 ml. One way analysis of variance and multiple range analysis for the different levels of bacteriolytic activity further validate this finding. The mortality of mosquitoes in the different treatments was not affected by the quanti ties of this nonpathogenic bacteria in the sugar meal. Salivary bacteriostatic activity of female mosquitoes against Gram-negative bacteria Escherichia coli was not detectable in our study, despite positive preliminary results. / Graduation date: 1993