Made available in DSpace on 2012-05-07T14:44:01Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 000068.pdf: 816656 bytes, checksum: c8740fc970374a5b1d5b8e0e1ab000ba (MD5) Previous issue date: 2007 / A cólera é uma doença bacteriana historicamente conhecida por seu potencial de provocar epidemias, tendo levado milhares de indivíduos à morte. É causada pelo bacilo Gramnegativo Vibrio cholerae O1 toxigênico. No Brasil, apesar de grandes avanços no que se refere a prevenção, a infecção ainda persiste e, embora não seja causa de alta mortalidade, sua presença é motivo de preocupação para a rede de Saúde Pública local. A infecção colérica é endêmica no nordeste brasileiro e está relacionada, principalmente, a condições sanitárias precárias. O diagnóstico clássico da infecção é a cultura bacteriana, complementada pela detecção da toxina colérica, o que retarda o resultado do exame. O objetivo principal deste trabalho foi avaliar a técnica de nested PCR em tubo único com dois genes alvos (MSTNPCR) na detecção de V. cholerae O1 diretamente do meio de cultura. Utilizando DNA, a técnica foi capaz de amplificar até 1 pg de V. cholerae O1, além de ter possibilitado a detecção do vibrião diretamente do meio de cultura líquido, sem prévia extração de DNA, apresentando limite de detecção de três Unidades Formadoras de Colônia. Além disso, a MSTNPCR mostrou-se específica para V. cholerae O1 quando testada com vários microrganismos diferentes. Um kit diagnóstico foi montado e estocado a -20 ºC, permanecendo estável durante os quatro meses em que foi testado. A MSTNPCR descrita neste trabalho pode ser útil no diagnóstico da infecção colérica e em investigações epidemiológicas, complementando os resultados obtidos com a cultura bacteriana

Made available in DSpace on 2014-06-12T18:03:27Z (GMT). No. of bitstreams: 2 arquivo3705_1.pdf: 1739643 bytes, checksum: d5c033b4b7bcbc4ee46e7a8e20bbe0df (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A emergência do Vibrio cholerae sorogrupo O139 como um segundo agente etiológico da cólera serviu de alerta para o surgimento de outros clones epidêmicos que possam passar despercebidos pelos métodos tradicionais de diagnóstico da cólera, geralmente baseados no uso de antissoro contra o sorogrupo O1 tradicional. Em estudos prévios, a partir da análise de 179 cepas de V. cholerae não-O1/não-O139 isoladas de casos clínicos de cólera no Brasil, foram selecionadas sete cepas de V. cholerae O26, e outra cepa de sorogrupo não tipável (17155), que possuíam genes de virulência associados ao desenvolvimento desta enfermidade. Destas, duas cepas (4756 e 17155) possuíam o gene rfb, específico do sorogrupo O1, sugerindo serem genotipicamente deste sorogrupo, e também expressaram a toxina colérica (CT) em cultura. Este trabalho buscou uma análise genética mais detalhada destas oito cepas comparando a classificação sorológica com outros marcadores moleculares. Neste sentido foi realizada a amplificação, clonagem e seqüenciamento da região espaçadora ribossomal 16S-23S (ISR) de diferentes operons de V. cholerae. A partir da análise da seqüência de cinco grupos de operons distintos (de um total de 210 clones seqüenciados), foram construídas três árvores filogenéticas em que as cepas 4756 e 17155 ficaram agrupadas no mesmo clado com cepas O1 controle, e as demais cepas O26 foram agrupadas separadamente. Conclui-se, desta forma, que as cepas 4756 e 17155 são filogeneticamente do sorogrupo O1 e a diferença nos resultados de sorologia pode ser uma conseqüência de soroconversão provocada por mudanças em genes de biossíntese do antígeno O

Vibrio cholerae y Shigella dysenteriae, entre otras bacterias enteropatógenas, causan enfermedades gastrointestinales, la segunda causa de muerte en niños menores de cinco años en el mundo. Los sucesos de gastroenteritis que desencadenan dichos microorganismos son mediados por la acción molecular de toxinas secretadas de tipo AB, CTX y STX, respectivamente para V, cholerae y S, dysenteriae. Dichas toxinas causan desbalances iónicos y lisis celular, generando deshidratación, pérdida de electrolitos y nutrientes. El correcto funcionamiento de ambas toxinas requiere una relación estricta de cinco subunidades de la proteína B por cada subunidad A, sin embargo, los mecanismos moleculares que regulan la expresión y mantienen la relación 5:1 entre las subunidades son poco entendidos. El objetivo de este estudio es analizar la regulación post transcripcional de las toxinas CTX y STX mediante aproximaciones experimentales in vitro y usando reacciones libres de células, en un entorno altamente puro y controlado. Nuestros resultados in vitro indican que CTX se expresa de manera más eficiente que STX. En el caso de CTX, la subunidad B tuvo mayor expresión que A, con una relación cercana a 5:1 como visto en estudios previos realizados in vivo. Por el contrario, STX fue sintetizada con baja eficiencia independientemente de la presencia o ausencia de la región codificantes para STXA, desviándose notablemente de una relación activa de 5 B por cada subunidad A. La adición de ppGpp, molécula implicada en la respuesta al estrés en bacterias, resultó en una ligera disminución de la expresión de ambas toxinas. En conclusión, nuestros resultados indican que la relación de 5 a 1 entre B y A de las toxinas CTX de V. cholerae es regulada durante la síntesis de proteínas. Por el contrario, la ausencia de regulación a nivel transcripcional o traduccional de STX del presente estudio in vitro, fue también observada en estudios in vivo, indicando que en la célula existen otros factores que podrían modular la estequiometría de síntesis. Si bien ambas toxinas, CTX y STX, permanecen a la misma familia de exotoxinas, los resultados del presente estudio indican que la regulación de su expresión difiere ampliamente entre ellas. Los resultados aquí reportados brindan nuevas perspectivas para el desarrollo de moléculas inhibidoras, con especial énfasis en perturbar el ribosoma de V, cholerae, para un desbalance en la síntesis de la toxina CTX y así reducir la patogenicidad de la bacteria. / Vibrio cholerae and Shigella dysenteriae, among other enteropathogenic bacteria, cause gastrointestinal diseases, the second cause of death in children under five years of age in the world. The events of gastroenteritis that trigger these microorganisms are mediated by the molecular action of secreted toxins of type AB, CTX and STX, respectively for V. cholerae and S. dysenteriae. These toxins cause ionic imbalances and cell lysis, generating dehydration, loss of electrolytes and nutrients. The correct functioning of both toxins requires a strict relationship of five subunits of protein B for each subunit A, however, the molecular mechanisms that regulate expression and maintain the 5: 1 ratio between the subunits are poorly understood. The objective of this study is to analyze the post transcriptional regulation of CTX and STX toxins by in vitro experimental approaches and using cell-free reactions, in a highly pure and controlled environment. Our in vitro results indicate that CTX is expressed more efficiently than STX. In the case of CTX, subunit B had greater expression than A, with a ratio close to 5: 1 as seen in previous studies conducted in vivo. In contrast, STX was synthesized with low efficiency regardless of the presence or absence of the region coding for STXA, deviating markedly from an active ratio of 5 B for each subunit A. The addition of ppGpp, a molecule involved in the stress response in bacteria, resulted in a slight decrease in the expression of both toxins. In conclusion, our results indicate that the ratio of 5 to 1 between B and A of CTX toxins of V. cholerae is regulated during protein synthesis. On the contrary, the absence of transcriptional or transcriptional STX regulation of the present in vitro study was also observed in in vivo studies, indicating that the cell may harbor other factors that could modulate the synthesis stoichiometry. Although both toxins, CTX and STX, belong to the same family of exotoxins, the results of the present study indicate that the regulation of their expression differs widely. The results reported here provide new perspectives for the development of inhibitory molecules, with special emphasis on disturbing the ribosome of V. cholerae, to provoke an imbalance in the synthesis of CTX toxin and thus to reduce the pathogenicity of the bacteria. / Tesis

Please read the abstract in the section, 00front, of this document / Dissertation (MSc)--University of Pretoria, 2001. / Microbiology and Plant Pathology / MSc / unrestricted

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No description available.

Distributional ecology is a branch of ecology which aims to reconstruct and predict the geographic range of free-living and symbiotic organisms in terrestrial and aquatic ecosystems. More recently, distributional ecology has been used to map disease transmission risk. The implementation of distributional ecology for disease transmission has, however, been erroneous in many cases. The inaccurate representation of disease distribution is detrimental to effective control and prevention. Furthermore, ecological niche modeling experiments are generally developed and tested using data from terrestrial organisms, neglecting aquatic organisms in case studies. Both disease and aquatic systems are often data limited, and current modeling methods are often insufficient. There is, therefore, a need to develop data-driven models that perform accurately even when only limited amounts of data are available or when there is little to no knowledge of the species' natural history to be modeled. Here, I propose a data-driven ecological niche modeling method that requires presence-only data (i.e., absence, pseudoabsence, or background records are not needed for model calibration). My method is expected to reconstruct environmental conditions where data-limited aquatic organisms are more likely to be present, based on a density-based clustering algorithm as a proxy of the realized niche (i.e., abiotic, and biotic environmental conditions occupied by the organism). Supported by ecological theories and methods, my central hypothesis is that because density-based clustering machine-learning modeling prevents extrapolation and interpolation, it can robustly reconstruct the realized niche of a data-limited aquatic organism. First, I assembled a comprehensive dataset of abiotic (temperature) and biotic (phytoplankton) environmental conditions and presence reports using Vibrio cholerae, a well-understood aquatic bacterium species in coastal waters globally (Chapter 2). Second, using V. cholerae as a model system, I developed detailed parameterizations of density-based clustering models to determine the parameter values with the best capacities to reconstruct and predict the species' distribution in global seawaters (Chapter 3). Finally, I compared the performance of density-based clustering modeling against traditional, correlative machine-learning ecological niche modeling methods (Chapter 4). Density-based clustering models, when assessed based on model fit and prediction, had comparable performance to traditional 'data-hungry' machine-learning correlative methods used in modern applications of ecological niche modeling. Modeling the environmental and geographic ranges of V. cholerae, an aquatic organism of free-living and parasitic ecologies, is a novel approach itself in distributional ecology. Ecological niche modeling applications to pathogens, such as V. cholerae, provide an opportunity to further the knowledge of directly-transmitted emerging diseases for which only limited data are available. Density-based clustering ecological niche modeling is termed here as Marble, honoring a previous, experimental version of this analytical approach, and is expected to provide new opportunities to understand how an ecological niche modeling method influences estimates of the distribution of data-limited organisms of complex ecology. These are lessons applicable to novel, rare, and cryptic aquatic organisms, such as emerging diseases, endangered fishes, and elusive aquatic species. / Master of Science / Distributional ecology is a branch of ecology which aims to reconstruct and predict the geographic distribution of land and water organisms. In the case of diseases, a correct representation of their geographic distributions is key for successful management. Previous studies highlight the need to develop new models that perform accurately even when limited amounts of data are available and there is little to no knowledge of the organisms' ecology. This thesis proposes a data-driven method, originally termed Marble. Marble is expected to help reconstruct environmental conditions where data-limited aquatic organisms are more likely to be found. Supported by ecological theories and methods, my hypothesis is that because Marble prevents under- and over-fitting, this method will produce results which better fit the data. Using V. cholerae, an aquatic organism, as a model system, I compared the performance of Marble against other traditional modeling algorithms. I found that Marble, in terms of model fit, performed similarly to traditional methods used in distributional ecology. Modeling the ecology of V. cholerae is a new approach in and of itself in ecological modeling. Furthermore, modeling pathogens provides an opportunity to further the knowledge of directly transmitted diseases, and Marble is expected to provide opportunities to understand how algorithm selection can reconstruct (or not) the distribution of data-limited aquatic organisms of diverse ecologies.

Vibrio cholerae é uma bactéria autóctone em ecossistemas aquáticos, os fatores responsáveis pela virulência podem contribuir com a patogenicidade, influenciados por fatores genéticos e ambientais. Considerando a importância de conhecer e monitorar o V. cholerae, o estudo pretende caracterizar isolados da especie e padronizar uma metodologia para detecção em amostras de água. Os isolados foram avaliados por metodologias clássicas e moleculares, para confirmar espécie. Também, foi avaliada a presença de genes de virulência, susceptibilidade aos antibióticos e resposta em modelo invertebrado. Tres marcadores moleculares foram avaliados por PCR quantitativa. Observou-se que setenta dos isolados pertenciam a espécie V. cholerae e mostraram variação na prevalência dos genes de virulência e ao perfil de suscetibilidade ao antibióticos. Mostrou uma influencia da temperatura e concentração do inoculo no modelo invertebrado. Os marcadores moleculares selecionados mostraram a viabilidade da metodologia proposta neste estudo pela alta especificidade e sensibilidade. / Vibrio cholerae is an autochthonous bacterium in aquatic ecosystems, factors responsible for virulence may contribute to pathogenicity, influenced by genetic and environmental factors. Considering the importance of knowing and monitoring V. cholerae, the study pretend to characterize selected isolates and to standardize a methodology for detection in water samples. The isolates were evaluated by classical and molecular methodologies to confirm species. Also, the presence of factors associated with virulence, antibiotics susceptibility and response in invertebrate model were evaluated. Three molecular markers were evaluated by quantitative PCR. It was observed that seventy of the isolates belonged to the V. cholerae species and showed a variation in the prevalence of the virulence genes and the antibiotic susceptibility profile. Also, showed an influence of the inoculum temperature and concentration on the invertebrate model. The selected molecular markers showed the viability of the methodology proposed in this study for the high specificity and sensitivity.

Vibrio cholerae é uma bactéria autóctone em ecossistemas aquáticos, os fatores responsáveis pela virulência podem contribuir com a patogenicidade, influenciados por fatores genéticos e ambientais. Considerando a importância de conhecer e monitorar o V. cholerae, o estudo pretende caracterizar isolados da especie e padronizar uma metodologia para detecção em amostras de água. Os isolados foram avaliados por metodologias clássicas e moleculares, para confirmar espécie. Também, foi avaliada a presença de genes de virulência, susceptibilidade aos antibióticos e resposta em modelo invertebrado. Tres marcadores moleculares foram avaliados por PCR quantitativa. Observou-se que setenta dos isolados pertenciam a espécie V. cholerae e mostraram variação na prevalência dos genes de virulência e ao perfil de suscetibilidade ao antibióticos. Mostrou uma influencia da temperatura e concentração do inoculo no modelo invertebrado. Os marcadores moleculares selecionados mostraram a viabilidade da metodologia proposta neste estudo pela alta especificidade e sensibilidade. / Vibrio cholerae is an autochthonous bacterium in aquatic ecosystems, factors responsible for virulence may contribute to pathogenicity, influenced by genetic and environmental factors. Considering the importance of knowing and monitoring V. cholerae, the study pretend to characterize selected isolates and to standardize a methodology for detection in water samples. The isolates were evaluated by classical and molecular methodologies to confirm species. Also, the presence of factors associated with virulence, antibiotics susceptibility and response in invertebrate model were evaluated. Three molecular markers were evaluated by quantitative PCR. It was observed that seventy of the isolates belonged to the V. cholerae species and showed a variation in the prevalence of the virulence genes and the antibiotic susceptibility profile. Also, showed an influence of the inoculum temperature and concentration on the invertebrate model. The selected molecular markers showed the viability of the methodology proposed in this study for the high specificity and sensitivity.

MSc (Microbiology) / Department of Microbiology / See the attached abstract below