O surgimento de cepas bacterianas resistentes a múltiplos fármacos impulsiona a busca por agentes antimicrobianos que possuem novos mecanismos de ação, incluindo compostos antivirulência. Apesar da ampla variedade de moléculas derivadas de química combinatória produzidas pela indústria farmacêutica, produtos naturais continuam a desempenhar um papel chave no desenvolvimento de fármacos. A seleção de plantas como fonte de compostos antimicrobianos é adequada do ponto de vista ecológico, uma vez que elas naturalmente produzem uma grande variedade de metabólitos secundários que atuam como defesa química contra micro-organismos no ambiente. Neste estudo, nós relatamos que miricetina (Myr), um flavonoide comum derivado de vegetais, frutas, nozes, frutas e chá, pode diminuir a produção de vários fatores de virulência de Staphylococcus aureus utilizando diferentes ensaios fenotípicos. Para explorar o mecanismo pelo qual Myr inibe a virulência de S. aureus, enquanto a sua forma glicosilada não, verificamos os níveis de expressão de genes relacionados à virulência e empregamos simulações de dinâmica molecular com enzimas cruciais no processo de patogênese. Além disso, Myr conferiu um grau significativo de proteção contra a infecção estafilocócica em modelo in vivo de Galleria mellonella. Outro foco deste estudo e com base em dados anteriores, o extrato de Harpochilus neesianus foi selecionado para o fracionamento bioguiado, uma vez que não há estudos fitoquímicos e de atividade biológica relatados na literatura para esta espécie. Utilizando o ensaio de proteinase e análises por MALDI-TOF, peptídeos foram identificados como os compostos bioativos, sendo então isolados por cromatografia em Sephadex G-50 e RPC18. Este estudo revela compostos derivados de plantas com um elevado potencial como protótipos antivirulência contra agentes bacterianos patogênicos e uma possível aplicação destes agentes na concepção de superfícies biomédicas anti-infectivas. / The emergence of drug-resistant bacterial strains drives the search for antimicrobials possessing new modes of action, including antivirulence compounds. Despite the wide variety of molecules derived from combinatorial chemistry by the pharmaceutical industry, natural products still play a key role in the development of pharmaceuticals. The selection of plants as source of antimicrobial compounds is appropriate from the ecological standpoint, since they naturally produce a wide range of secondary metabolites that act as a chemical defense against microorganisms in the environment. In this study, we report that myricetin (Myr), a common flavonol derived from vegetables, fruits, nuts, berries and tea, can remarkably decrease the production of several Staphylococcus aureus virulence factors using different phenotypic assays. To explore the mechanism by which Myr inhibits S. aureus virulence, while its glycosylated form does not, we verified the relative expression levels of virulence related genes and employed molecular dynamics simulations with pivotal enzymes in pathogenesis process. Furthermore, Myr conferred a significant degree of protection against staphylococcal infection in Galleria mellonella in vivo model. In addition to this study and based on previous data, Harpochilus neesianus extract was selected for the bioguided fractionation, since no phytochemical studies and biological activity is reported in the literature for this species. By using proteinase assay and MALDI-TOF analyses, peptides were identified as bioactive compounds which were isolated by Sephadex G-50 and RP-C18. This study reveals plant-derived compounds with high potential as antivirulence prototypes against bacterial pathogens and a possible application of these agents in the design of anti-infective biomedical surfaces.

A specific method has been developed for the extraction and measurement of staphylococcal nuclease in cheese in which Staphylococcus aureus has grown. Ten grams of cheese sample were homogenized with ninety milliliters of pH ten buffer for three minutes. Ammonium sulfate fractionation was used and a forty to eighty percent fraction was collected and concentrated using ultrafilters. The nuclease activity was determined using a toluidine blue deoxyribonucleic acid agar slide method and a spectophotometric method. The DNA agar slide method was used to compare staphylococcal growth with nuclease production in cheese under varying conditions. When Staphylococcus aureus plate counts indicated populations of three to four thousand per milliliter, it was possible to detect nuclease in the cheese sample. A method has also been developed to detect Staphylococcus aureus colonies using DNase agar and toluidine blue, utilizing the heat stability of Staphylococcus aureus nuclease.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-12-13T12:38:59Z No. of bitstreams: 1 marne-chagas.pdf: 1100888 bytes, checksum: 7a199f01e03cd65e615b7c57af7901f0 (MD5) / Made available in DSpace on 2012-12-13T12:38:59Z (GMT). No. of bitstreams: 1 marne-chagas.pdf: 1100888 bytes, checksum: 7a199f01e03cd65e615b7c57af7901f0 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil. / O surgimento de resistência a antimicrobianos em bactérias está se agravando ano após ano, constituindo-se um problema de saúde pública em hospitais e centros de tratamento ao redor do mundo. No Brasil, a resistência a meticilina em Staphylococcus aureus(MRSA) chega a 29 % dos casos detectados anualmente. Atualmente, a metodologia convencional para a identificação de MRSA pode levar 48 horas, portanto, este trabalho tem por objetivo o desenvolvimento de um teste para diagnóstico rápido baseado na tecnologia de imunocromatografia de fluxo lateral usando um anticorpo específico contra a proteína PBP2a, a qual confere resistência ao Staphylococcus aureus, permitindo a identificação em minutos, a partir de colônias isoladas. O anticorpo monoclonalanti-PBP2a foi purificado e empregado em um processo de conjugação a microesferas de látex coloridas para a implementação do teste rápido. Os resultados iniciais demonstraram a capacidade do anticorpo de reconhecer a PBP2a em amostras de MRSA, porém foram observados problemas de inespecificidade que deverão ser solucionados para dar confiabilidade ao teste proposto. / The emergence of antibiotic resistance in bacteria is getting worse year after year, becoming a public health problem in hospitals and treatment centers around the world. In Brazil, methicillin resistance in Staphylococcus aureus(MRSA) reaches 29% of cases detected annually. Currently, the conventional methodology for identification of MRSA may take 48 hours of lengthyphenotypical tests, so this work aims at developing a rapid diagnostic test based onthe lateral flow immunochromatography protocol using a specific antibody against the protein PBP2a, which confers the methicillin-resistance phenotype to Staphylococcus aureus, allowing a prompt identification from isolated colonies. The monoclonal antibody anti-PBP2a was purified and used in a process of conjugation to colored latex microspheres for the implementation of the rapid test. Initial results demonstrated the ability of the antibody to recognize the PBP2a in samples of MRSA, but inespecificity issues will have to be solved to improve reliability of the test proposed.

Orientador: Maria de Lourdes Ribeiro de Souza da Cunha / Resumo: A epidemiologia das infecções estafilocócicas tem sofrido importante modificação nas últimas décadas. A emergência de linhagens de Staphylococcus aureus resistentes à meticilina associados à comunidade (CA-MRSA) representa risco especial para populações reconhecidamente vulneráveis como os idosos. Neste âmbito, duas situações distintas são de especial interesse: os indivíduos institucionalizados, vivendo em casas de repouso, que representam um espaço intermediário entre a comunidade e o hospital; e os dependentes (acamados) cuidados em domicílio, expostos de forma intermitente aos serviços de saúde. Nosso objetivo foi identificar a prevalência e fatores associados ao carreamento nasal, oral e retal de S. aureus e MRSA em indivíduos acamados ou residindo em instituições de longa permanência para idosos (ILPIs) no município de Botucatu, SP, bem como a identificação de clones importantes de S. aureus e MRSA nessa população. Estudo com delineamento transversal, em que swabs da nasofaringe, orofaringe e reto de 226 indivíduos (150 residentes em nove ILPIs e 76 acamados em domicílio) foram coletados juntamente com um questionário que, através de entrevista com o próprio indivíduo ou responsável legal, levantou informações como dados demográficos (gênero e idade), tempo de institucionalização ou restrição ao leito, dados clínicos (comorbidades), dispositivos invasivos, internações recentes, doenças infeccionas e uso de antimicrobianos, para identificação dos fatores de risco. O isol... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The epidemiology of staphylococcal infections has undergone important changes in recent decades. The emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains represents a special risk to populations recognized as vulnerable, such as the elderly. Within this context, two different situations are of special interest: institutionalized individuals living in nursing homes, which represent an intermediary space between the community and the hospital, and dependent (bedridden) patients cared for at home, who are intermittently exposed to health services. Our objective was to determine the prevalence and factors associated with nasal, oral and rectal carriage of S. aureus and MRSA in bedridden patients and residents of long-term care facilities (LTCF) for the elderly in the city of Botucatu, SP, and to identify important S. aureus and MRSA clones in this population. In a cross-sectional study, nasopharyngeal, oropharyngeal and rectal swab samples were collected from 226 individuals (150 individuals from nine LTCF and 76 bedridden patients living at home). In addition, a questionnaire was applied by interview with the subject himself or the legal representative for the collection of demographic data (gender and age), length of institutionalization or bedridden period, clinical data (comorbidities), invasive devices, recent hospitalizations, infectious diseases, and antimicrobial use in order to identify risk factors. Staphylococcus aureus was is... (Complete abstract click electronic access below) / Mestre

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Digitized by Kansas State University Libraries

LD2668 .T4 1962 K34

LD2668 .T4 1962 O33

Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen associated with significant morbidity and mortality. Previous colonisation with this pathogen is a risk factor for the development of subsequent infection. Tolllike receptors (TLRs) are a family of transmembrane receptors of the innate immune system that recognize pathogen-associated molecular patterns. The role of nasal colonisation of S. aureus has started to receive more attention. In spite of this, there are not enough studies looking at its effects on human primary nasal epithelial cells and their response to TLR ligands. The respiratory tract itself seems to pose a contradiction given by the clinical observation that its upper portion (nasal compartment) allows the growth of bacteria, acting like a reservoir, whereas the lower portion (lung compartment) reacts with an exuberant inflammatory response to the same organisms, as noted during pneumonia. The mechanism related with this phenomenon remains to be elucidated. A negative regulator of the TLR signalling cascade called toll-interacting protein (tollip) has been demonstrated to induce hyporesponsiveness in the gastrointestinal tract in the presence of bacteria. So far, tollip has not been demonstrated in the respiratory tract. Aims: To compare the responses of the upper and lower respiratory tract to TLR ligands, to characterise the role of tollip in the respiratory tract and its effects in the induction of tolerance, and to determine the cellular response to nasal carriage of S. aureus. Materials and Methods: The cell line RPMI 2650 (representative of nasal epithelium) and the cell line A549 (representative of type II alveolar epithelium) were used to establish the cytokine response to stimulation with TLR ligands and to demonstrate the presence of tollip protein by immunocytochemistry and enzymelinked immunosorbent assay (ELISA). Primary human nasal epithelial and type II alveolar epithelial cells were isolated and cultured from consented subjects. The cytokine response to stimulation was measured using cytokine bead array and the presence of tollip was determined by immunofluorescence and quantitative polymerase chain reaction. The presence of TLRs was assessed by immunocytochemistry in primary nasal and type II alveolar epithelial cells and the response to stimulation with the TLR9 agonist CpG-C ODN was assessed in these cells as well as in primary human type II alveolar epithelial cells. Subjects were also assessed for nasal carriage of S. aureus and their associated cytokine responses. Results: The RPMI 2650 cell line, despite retaining phenotypic characteristics of the nasal epithelium, appears unresponsive to stimulation with TLR ligands. In contrast, the A549 cell line responded significantly to stimulation with TLR ligands. Primary human nasal epithelial cells responded by secreting higher amounts of interleukin (IL)-8 and IL-6 in response to stimulation with S. aureus peptidoglycan (PGN) and tumour necrosis factor alpha (TNF-α) with a strong trend toward statistical significance. These cells did not respond to stimulation with Pseudomonas aeruginosa LPS. Primary type II alveolar epithelial cells responded significantly to stimulation with S. aureus PGN by increasing the secretion of IL-8, IL-6, IL-1β, TNF-α and IL-10 into cultured supernatant. Cells from the upper respiratory tract displayed a more tolerant phenotype given by the lower levels in cytokine production in response to stimulation with S. aureus PGN, in contrast to alveolar epithelial cells. TLRs were identified in primary nasal epithelial cells. The negative regulator tollip was identified in cell lines as well as primary cells of the respiratory tract in its three segments: nasal, bronchial and type II alveolar. It was not possible to demonstrate an up-regulation of tollip after stimulation with TLR ligands in any of the cell types studied, although, it was possible to observe a significantly higher constitutive level in tollip mRNA transcripts from primary nasal epithelial cells in comparison to type II alveolar epithelial cells. TLR9 was identified in human primary nasal epithelial cells, although it was not possible to observe an increase in cytokine production after stimulation with a TLR9 agonist. TLR9 was expressed strongly in primary type II alveolar epithelial cells which responded by significantly increasing IL-8 production after stimulation with CpG-C ODN. Primary nasal epithelial cells from individuals who carry S. aureus exhibit a proinflammatory profile, as evidenced by higher basal levels of IL-8 and IL-6 in comparison to non-colonised controls. Conclusion: The upper respiratory tract epithelium displays a tolerant phenotype in response to stimulation with TLR ligands in comparison to the lower respiratory epithelium, potentially favouring nasal colonisation by S. aureus. Tollip m-RNA transcripts appear to be up-regulated constitutively in the nasal epithelium which might favour this response. Staphylococcus aureus colonisation is however associated with a local pro-inflammatory state in the nasal epithelium of carrier individuals.

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy