Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves: [89]-98). Also available on the Internet.

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.

Bien que l'initiation de la traduction soit un processus conservé entre les bactéries, nous avons montré que le mécanisme par lequel les ARNm structurés sont reconnus et adaptés sur le ribosome diffère chez Staphylococcus aureus, un micro-organisme avec un bas taux de G+C et chez Escherichia coli. Une particularité du ribosome de S. aureus est l'absence de la protéine ribosomale S1, qui non seulement est plus courte que celle de E. coli mais qui possède également une organisation distincte des domaines. Mes expériences suggèrent que la protéine S1 (SauS1) favorise spécifiquement l'initiation de la traduction de l'opéron α-psm 1-4 en liant son ARNm hautement structuré. En outre, il influence aussi l'expression et la production de facteurs de virulence comme les exotoxines (α-haemolysine, δ-hémolysine et γ- hémolysine) et les exoenzymes (protéases et lipases). En plus de son rôle dans la traduction, SauS1 pourrait être impliquée dans d'autres processus cellulaires tels que le métabolisme de l'ARN et la régulation par des ARN non-codants (ARNnc). Elle forme des complexes in vivo avec plusieurs ARNnc dont la stabilité serait affectée dans la souche délétée du gène rpsA codant S1. SauS1 a donc une activité chaperonne favorisant la cinétique d’appariement entre deux molécules d'ARN et au moins dans un cas, elle stimule la reconnaissance entre un ARNnc et son ARN cible. Ainsi, SauS1 appartient à une nouvelle classe de chaperons d'ARN qui jouent un rôle clé dans la régulation du virulon de S. aureus. / Even if translation initiation is a conserved process among bacteria, we have recently shown that low G+C content Gram-positive, such as Staphylococcus aureus, differ from E. coli on the mechanism by which structured mRNAs are recognized and adapted on the ribosome. One peculiarity of the S. aureus ribosome is the absence of ribosomal protein S1, which is shorter than E. coli S1 and has different domains organization. My work could demonstrate that S. aureus S1 (SauS1) specifically promotes translation initiation of the α-psm 1-4 operon by binding its highly structured mRNA. Moreover, it influences the expression and production of other exotoxins (α-haemolysin, δ-haemolysin and γ-haemolysins) and exoenzymes (proteases and lipases). Besides its role in translation, SauS1 could be implicated in other cellular processes such as RNA maturation/degradation and sRNA-mediated regulation. It forms in vivo complexes with several sRNAs whose level is affected in a strain deleted of rpsA gene, coding for S1. Preliminary results show that SauS1 has a chaperone activity promoting the kinetic of annealing of two model RNA molecules and at least in one case, we could demonstrate that it stimulates the recognition between a sRNA and its target RNA. Taken together, SauS1 belongs to a new class of RNA chaperones that play key roles in the regulation of S.aureus virulon.

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

Digitized by Kansas State University Libraries

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

No description available.

No description available.