Submitted by Aline Batista (alinehb.ufpel@gmail.com) on 2018-05-16T13:41:30Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Aline_de_Oliveira_Rodrigues.pdf: 787004 bytes, checksum: 42043083bf6f4a2ee9080a2164a23e88 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-05-16T20:21:01Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Aline_de_Oliveira_Rodrigues.pdf: 787004 bytes, checksum: 42043083bf6f4a2ee9080a2164a23e88 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-05-16T20:21:17Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Aline_de_Oliveira_Rodrigues.pdf: 787004 bytes, checksum: 42043083bf6f4a2ee9080a2164a23e88 (MD5) / Made available in DSpace on 2018-05-16T20:21:17Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Aline_de_Oliveira_Rodrigues.pdf: 787004 bytes, checksum: 42043083bf6f4a2ee9080a2164a23e88 (MD5) Previous issue date: 2015-04-30 / Sem bolsa / O setor hoteleiro vem se expandindo mundialmente a cada ano e dentre os serviços oferecidos, a gastronomia é um dos mais requisitados, através da qual busca-se a satisfação do cliente, oferecendo produtos seguros e de qualidade. Paralelamente ao incremento do turismo, a incidência de doenças de origem alimentar está crescendo em todo mundo. Acredita-se que o consumo de alimentos contaminados seja a principal causa de doenças em turistas. Neste contexto o objetivo deste estudo foi avaliar a aplicação das boas práticas em restaurantes hoteleiros da cidade de Pelotas/RS e Rio Grande/RS e relacioná-las com a qualidade microbiológica dos alimentos oferecidos, além de identificar fontes de contaminação. O setor de alimentos e bebidas (A&B) de quatro hotéis foi avaliado quanto às condições higiênicossanitárias em duas visitas, com intervalo mínimo de 30 dias, através de aplicação de check list fundamentado na norma da RDC 216/2004, e de análises microbiológicas de amostras coletadas no local. Foram realizadas coletas de amostras de superfícies de utensílios, equipamentos e mãos de manipuladores para contagem de Staphylococcus coagulase positiva e de coliformes termotolerantes. Isolados obtidos das culturas dessas contagens foram comparados através de perfis de genéticos obtidos por rep-PCR. Considerando as não conformidades observadas no check list, na primeira visita apenas um hotel foi classificado como ótimo, dois foram classificados como bons e um recebeu a classificação ruim. Entretanto, na segunda visita o hotel classificado como ruim passou para bom, devido à realização de melhorias no restaurante. Alguns resultados das análises microbiológicas apresentaram limite acima do permitido para a contagem dos micro-organismos analisados, identificando falha na aplicação das boas práticas. Através da técnica de rep-PCR, foi identificada uma mesma cepa em amostras de queijo e mãos, manteiga e tábua de corte, queijo e tábua de corte e salada de frutas e mãos, evidenciando contaminação cruzada devido falhas no processo de higiene e manipulação dos alimentos. / The hotel industry has been expanding every year and among the services offered, the cuisine is one of the most requested, which seeks customer satisfaction by offering safe and quality products. Parallel to the tourism development, the incidence of foodborne diseases is growing worldwide. It is believed that the consumption of contaminated food is the major cause of disease in tourists. This study goal was to evaluate the application of best practices in hotel restaurants in the cities of Pelotas/RS and Rio Grande/RS and relate it to the microbiological quality of the food offered, besides to identify sources of contamination. The sector of food and beverages (F&B) of four hotels was evaluated in two visits with an interval of 30 days regarding hygiene-sanitary conditions, by application of check list and microbiological analyzes of samples collected on site. Surface, utensils, equipment and hands handlers samples have been collected for counting coagulase positive Staphylococcus and thermotolerant coliform. Isolates obtained from these counting cultures were compared by bands profiles obtained by rep-PCR. According to the non-conformity observed in the check list on the first visit, only one hotel was rated as great, two were classified as good and one received a bad rating. However, on the second visit, the one considered bad got a good rate due to improvements in its unit. Some microbiological analysis showed a limit above the allowed for the count of analyzed microorganisms, identifying failure in the hygiene-sanitary process. By rep-PCR, was identified the same strain in samples of cheese and hands, butter and cutting board, cutting board and cheese and fruit salad and hands, showing cross-contamination due to flaws in the process of care and handling of food.

Made available in DSpace on 2014-08-20T13:42:05Z (GMT). No. of bitstreams: 1 Dissertacao_ Caroline_Peixoto_ Bastos.pdf: 482424 bytes, checksum: 0f1a1fb787f50f0f7f89d2501df4b4db (MD5) Previous issue date: 2008-07-02 / Staphylococcus aureus, Staphylococcus hyicus e Staphylococcus intermedius are species of genus Staphylococcus capable of producing enterotoxins, as well as the enzymes termonuclease and coagulase, and are called Staphylococcus Coagulase Positive (SCP). They show very similar biochemical and morphological characteristics, this fact, makes their differentiation and they identification in food by phenotypic techniques very difficult. In this way, the aim of this work was the development of a Multiplex PCR (mPCR), targetting the nuc gene (encoding for thermonuclease) as target, for the identification of S. aureus, S. intermedius, and S. hyicus. Initially, the mPCR was standartizeted using reference strains of S. aureus, S. warneri, S. lugdunensis and Listeria monocytogenes and isolates of S. hyicus and S. intermedius, affer words it was tested with 16 isolates of the three species of SCP. The primers annealing to each of the three species, the conditions for mPCR, the suitability of the Internal Amplification Control (IAC) for the genus Staphylococcus, and the sequences obtained through the amplification by PCR were evaluated. After this, the results were compared with those achieved by biochemical tests for differentiation of SCP. Primers NUC1-NUC2 (for sequences of the S. aureus nuc gene), NUC5-NUC6 (for sequences of the S. intermedius nuc gene), NUC7-NUC8 (for sequences of the S. hyicus nuc gene) and, as IAC, the primers 16S1 16S2 (for sequences of the 16S of rRNA gene) were used. The mPCR proposed enable the identification of S. aureus, S. hyicus e S. intermedius, and the IAC was suitable for the genus. The sequences obtained in PCR showed a high degree of similarity with the sequences of the thermonuclease gene deposited in GenBank. The mPCR showed greater discriminatory power that the biochemical tests for identification the vii S. aureus, S. hyicus e S. intermedius, as well as reduced the time required for the identification of ECP (reduction of 24h) or identification of specie (reduction of 72h). / Staphylococcus aureus, Staphylococcus hyicus e Staphylococcus intermedius são espécies do gênero Staphylococcus com capacidade de produzir enterotoxinas, bem como as enzimas termonuclease e coagulase, sendo denominadas de Estafilococos Coagulase Positiva (ECP). Apresentam características morfológicas e bioquímicas extremamente semelhantes, o que torna difícil sua diferenciação assim como sua identificação em alimentos através de técnicas fenotípicas de análise microbiológica. Dessa forma, este trabalho teve como objetivo o desenvolvimento de um Multiplex PCR (mPCR), tendo-se como alvo o gene nuc (codifica para a termonuclease), para a identificação de S. aureus, S. intermedius e S. hyicus. Inicialmente, o mPCR foi padronizado utilizando-se cepas padrão de S. aureus, S. warneri, S. lugdunensis e Listeria monocytogenes e isolados de S. hyicus e S. intermedius, e, posteriormente, foi testado em 16 isolados das três espécies de ECP. Foram avaliadas a sensibilidade dos primers para cada uma das três espécies, as condições da mPCR, a sensibilidade do Controle Interno de Amplificação (IAC) para o gênero Estafilococos, e as seqüências obtidas através da amplificação por PCR. Posteriormente, os resultados foram comparados aos obtidos com testes bioquímicos de diferenciação de ECP. Foram utilizados os primers NUC1-NUC2 (para seqüências do gene nuc de S. aureus), NUC5-NUC6 (para seqüências do gene nuc de S. intermedius), NUC7-NUC8 (para seqüências do gene nuc de S. hyicus) e, para amplificação do IAC, os primers 16S1 16S2 (para seqüências do gene 16S do rRNA). O mPCR proposto possibilitou a identificação das espécies S. aureus, S. hyicus e S. intermedius, bem como o IAC utilizado apresentou sensibilidade para o gênero. As seqüências obtidas na PCR apresentaram elevado v grau de similaridade com as seqüências do gene da termonuclease depositadas no GenBank. A mPCR apresentou maior poder discriminatório que os testes bioquímicos para identificação de S. aureus, S. hyicus e S. intermedius, bem como reduziu significativamente o tempo necessário para a identificação de ECP (redução de 24h) ou de identificação em nível de espécie (redução de 72h).

The mozzarella cheese is a product of high consumption, but very susceptible to microbiological contamination. Therefore, this study aimed to evaluate the microbiological and physical-chemistry of sliced mozzarella cheese in bulk and vacuum-packed, sold in supermarkets in the Northwest region of Rio Grande do Sul State. We evaluated five marks (50 samples) of vacuum-packed mozzarella cheese (A, B, C, D and E) and a mark (25 samples) sold in bulk (F). Microbiological tests were performed according to Normative Instruction 62, August 26th, 2003: fecal coliform counts (45ºC), Salmonella sp. and coagulase positive Staphylococcus in two different periods during the validity period (period I) and after the expiration of the samples (period II). Were also conducted analysis of pH, fat and moisture, according to the Normative Instruction 68, December 12th, 2006. The physicochemical parameters classified the samples as fat cheese and medium humidity and all attended the legal parameters established by the Brazilian legislation. The microbiological results showed that all samples were within the standards for fecal coliform and coagulase positive Staphylococcus in both periods of analysis. However, in period I detected the presence of Salmonella sp. in one of the marks (16.67 %), being improper for human consumption. / O queijo Mussarela é um produto de elevado consumo, porém muito susceptível à contaminação microbiológica. Portanto, este trabalho teve como objetivo avaliar a qualidade microbiológica e físico-química do queijo Mussarela fatiado à granel e embalado à vácuo, comercializado em supermercados da região Noroeste do estado do Rio Grande do Sul. Foram avaliadas 5 marcas de queijo Mussarela embalado à vácuo (A, B, C, D e E), totalizando 50 amostras, e uma marca comercializada à granel (F), totalizando 25 amostras. As análises microbiológicas realizadas foram: contagem de coliformes termotolerantes (45°C), Salmonella sp. e Staphylococcus coagulase positiva, em dois diferentes períodos, durante o prazo de validade (período I) e após o vencimento das amostras (período II). Também foram realizadas as análises de pH, teor de gordura e umidade. Os parâmetros físico-químicos classificaram as amostras como sendo queijos gordos e de média umidade e atenderam os padrões exigidos pela legislação brasileira. Os resultados microbiológicos mostraram que todas as amostras apresentaram-se dentro dos padrões para coliformes termotolerantes e Staphylococcus coagulase positiva, nos dois períodos de análise, porém, no período I, foi detectada a presença de Salmonella sp. em uma das marcas (16,67 %), encontrando-se imprópria para o consumo humano.

Orientador: Pasqual Barretti / Resumo: A peritoniteé complicação grave e responsável pela maioria dos casos de falência da técnica de diálise peritoneal (DP). Os cocos Gram-positivos são o grupo etiológico principal, sendo os estafilococos coagulase-negativa (ECN) os germes mais comuns e o Staphylococcus aureus(S.aureus), associado a episódios mais graves e commenor frequência de resolução. O conhecimento dos fatores de risco e dospreditoresda sua evolução podem contribuir para a melhoria das estratégias de prevenção e de tratamento. Objetivo:Os objetivos do presente estudo foram avaliar os fatores de risco para o primeiro episódio de peritonite por estafilococos e os fatores associados à resolução e falência da técnica após resolução do episódio de peritonite, em uma grande coorte brasileira de pacientes em DP (BRAZPD). Métodos:De uma coorte de 5707 pacientes incidentes adultos, com mais de 90 dias de tratamento por DP, foram incluídos, entre dezembro de 2004 e novembro de 2011, aqueles que apresentaram um primeiro episódio de peritonite por S. aureus ou ECN. As covariáveis, potencialmente associadas aos desfechos, foram testadas em análise univariada e aquelas com p ≤ 0,10 incluídas no modelo multivariado. Resultados:Durante o seguimento,389pacientes apresentaram um primeiro episódio de peritonite estafilocócica. Destes, 234 foram causados por S. aureus e 155 por ECN. Os grupos que apresentaram peritonite por S. aureus ou por ECN foram semelhantes para a maior parte das características basais. Entre os pacientes... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Peritonitis is a serious complication of peritoneal dialysis (PD) and the main cause of technique failure. Gram-positive cocci are the most frequent etiological group, coagulase-negative staphylococci (CNS) are the most common germs, and Staphylococcus aureus (S. aureus) is associated with more severe episodes and lower resolution rate. Objective: The objectives of this study were to evaluate the risk factors for the first episode of staphylococcal peritonitis, and the factors associated with resolution and technique failure after peritonitis episode resolution, in a large Brazilian cohort of PD patients (BRAZPD). Methods: From a cohort of 5707 adult incident patients with more than 90 days of PD treatment, between December 2004 and November 2011, those who had a first episode of S. aureus or CNS peritonitis were included. The covariates potentially associated with the outcomes were tested in univariate analysis and those with p ≤ 0.10 included in the multivariate model. Results: During follow-up, 389 patients had a first episode of staphylococcal peritonitis. Of these, 234 were caused by S. aureus and 155 by CNS. The groups of patients with S. aureus or CNS peritonitis were similar for most baseline characteristics. Among S. aureus peritonitis, there was resolution in 190 (81.2%); technique failure in 41 (21.6%), and episode-related death in 18 (7.7%). In the episodes by CNS, there were 127 resolutions (82.6%); 33 technique failures (25.8%), and 12 episode-related deaths (7.... (Complete abstract click electronic access below) / Doutor

Orientador: Maria de Lourdes Ribeiro de Souza da Cunha / Banca: Elisabeth Loschagin Pizzolitto / Banca: Eduardo Bagagli / Resumo: Os estafilococos coagulase-negativa (ECN) estão entre os microrganismos mais envolvidos em infecções nosocomiais, principalmente em recém-nascidos (RN) prematuros e de baixo peso. Entre os fatores de risco para infecções por esses agentes, a produção de um polissacarídeo extracelular e a conseqüente formação do biofilme, permitindo aderência às superfícies plásticas e lisas de cateteres e outros dispositivos médicos desempenham um papel importante. Uma coleção de 100 amostras de ECN, sendo 50 isoladas de pontas de cateteres e 30 de hemoculturas obtidas de RN da Unidade Neonatal do Hospital das Clínicas da Faculdade de Medicina de Botucatu, UNESP e 20 obtidas de fossas nasais de portadores saudáveis foi analisada quanto à produção de biofilme pelos métodos do tubo (TM), ágar vermelho congo (CRA) e através do método quantitativo de aderência a placa de poliestireno (TCP). Esses métodos foram comparados com a pesquisa dos genes icaA, icaD e icaC envolvidos na produção de biofilme em ECN. Das 100 amostras de ECN estudadas, 82% foram positivas pela técnica de PCR, 82% pelo método do tubo, 81% pelo método da placa de poliestireno e 76% pelo CRA. O método de aderência ao tubo (TM) foi o teste que mostrou resultados mais confiáveis, com uma sensibilidade e especificidade de 100% e boa concordância quando comparado a técnica de PCR. Com o objetivo de determinar o papel do biofilme como fator de risco na ocorrência de infecções em RN, 130 amostras de estafilococos coagulasenegativa (ECN) isoladas de recém-nascidos (RN) internados na Unidade Neonatal do Hospital das Clínicas da Faculdade de Medicina de Botucatu foram identificadas e classificadas em significativas e contaminantes, baseado em dados clínicos e laboratoriais obtidos dos prontuários dos RN. Foram pesquisados fatores perinatais de risco para infecção, evolução clínica dos RN e antibiotico... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Coagulase-negative staphylococci (CNS) are most often associated with nosocomial infections, especially in premature and under weight newborns. The most important pathogenic factor of these microorganisms is the production of extracellular polysaccharide and the consequent formation of biofilm which facilitates their adherence to the surfaces of catheters and other medical devices. A total of 100 clinical isolates of coagulase-negative staphylococci (CNS) isolated from infected medical devices received from the Neonatal Unit, University Hospital, Botucatu Medical Schooll, including 50 isolated from catheter tips, 30 from blood cultures, and 20 collected from the nasal cavity of healthy subjects were investigated in order to evaluate the efficiency of three phenotypic methods of detection of biofilm formation, and also analyze the icaA, icaD and icaC genes, using the PCR method. The clinical isolates were screened by tissue culture plate (TCP), Tube method (TM), and Congo Red Agar (CRA) method. Of the 100 tested isolates, 82% were positive in the PCR method; in the TM, 82%; in the TCP assay, 81%; and 76% in CRA method. The method of adherence to the test tube was shown that more reliable results, with a sensitivity and specificity of 100% and good agreement when compared with the PCR technique. In order to determine the role of biofilm as a risk factor in the occurrence of infections in newborns 130 clinical isolates of CNS were identified and classified as significant and contaminant, based on clinical data obtained from the patients' reports. Perinatal risk factors for infection, clinical evolution and antibiotic therapy for the newborns were studied, as well as the detection of the genes responsible for the production of biofilm in CNS.Of the 130 clinical isolates of CNS, 66 (50.8%) were classified as clinically significant and 64 (49.2%) as contaminant... (Complete abstract click electronic access below) / Mestre

Les toxi-infections alimentaires collectives staphylococciques représentent ces dernières années en France la seconde cause de toxi-infections alimentaires bactériennes. Ces dernières sont dues à l'ingestion de toxines préformées dans les aliments : les entérotoxines. Actuellement, une vingtaine d'entérotoxines (SEA àSElV) ont été décrites dans la littérature ; parmi celles-ci, toutes possédent une activité superantigéniques mais seules quelques unes possèdent une activité émétique prouvée et présentent donc un risque sanitaire pour le consommateur. Deux problèmes majeurs se posent pour confirmer le diagnostic des épisodes toxiques à staphylocoques. D'une part, les staphylocoques sont sensibles aux traitements thermiques appliqués aux aliments alors que les entérotoxines ne le sont pas, et d'autre part les méthodes de détection des entérotoxines staphylococciques actuellement disponibles ne couvrent pas l'ensemble des entérotoxines décrites à ce jour. Ainsi, et afin de proposer une alternative innovante aux tests immuno-enzymatiques utilisés pour la recherche des entérotoxines staphylococciques dans les matrices alimentaires, nous avons mis en place une démarche intégrée « du gène à la protéine » utilisant des outils de biologie moléculaire, d'immunochimie et de physisco-chimie des protéines pour caractériser, comprendre et élucider les toxi-infections alimentaires collectives à staphylocoques.

Σκοπός της παρούσας ερευνητικής εργασίας ήταν η επιδημιολογική μελέτη των σταφυλοκοκκικών λοιμώξεων από πηκτάση–αρνητικούς σταφυλοκόκκους (CNS) σε ασθενείς της Μονάδας Εντατικής Θεραπείας των πρόωρων νεογνών του Πανεπιστημιακoύ Γενικού Νοσοκομείου Πατρών (ΠΓΝΠ) μεταξύ 2002-2004. Κατά τη διάρκεια της μελέτης, συλλέχθηκαν συνολικά 287 στελέχη CNS. Τα στελέχη ελέγχθηκαν για την παραγωγή της πρωτεΐνης PBP2α και την ύπαρξη του γονιδίου mecA για τον προσδιορισμό των ανθεκτικών στη methicillin στελεχών CNS. Από το σύνολο των 287 CNS, τα 132 (46%) χαρακτηρίσθηκαν ως MRCNS. Τα MRCNS αποτελούν σοβαρό πρόβλημα για τα νοσοκομεία, γιατί αυξάνουν τον χρόνο παραμονής των ασθενών στο νοσοκομείο, άρα και το κόστος νοσηλείας και θεραπείας. Έγινε μελέτη του φαινοτύπου αντοχής των στελεχών σε αντιβιοτικά, της κλωνικής τους διασποράς και της ικανότητάς παραγωγής βιομεμβράνης σε σύγκριση με την παρουσία των γονιδίων του οπερονίου ica. Για την επιδημιολογική μελέτη των λοιμώξεων που οφείλονται σε MRCNS, μέθοδος αναφοράς είναι η PFGE, κυρίως σε συνδυασμό και με υβριδισμό του χρωμοσωμικού DNA με ειδικούς ανιχνευτές. Όλα τα στελέχη MRCNS ήταν πολυανθεκτικά και η πλειονότητά τους παρήγαγε βιομεμβράνη (89%). Μεταξύ των 115 στελεχών S. epidermidis αναδείχθηκε ένας κυριάρχος κλώνος (z) (77 στελέχη) και ακολούθησε ο κλώνος (g). Τα δέκα από τα δεκαέξι στελέχη του είδους S. haemolyticus ανήκαν στον κλώνο (y). Το οπερόνιο ica συμβάλλει στην παραγωγή της εξωκυττάριας ουσίας polysaccharide-intercellular-adhesin (ΡΙΑ) που είναι το κύριο συστατικό του biofilm. Η έκφραση των γονιδίων του οπερονίου ica επάγεται από την παρουσία του ρυθμιστικού γονιδίου sarA, ενώ καταστέλλεται από το γονίδιο icaR. Τα περισσότερα στελέχη που ήταν biofilm-θετικά (80%) έφεραν όλα τα γονίδια του οπερονίου ica και το τρανσποζόνιο IS256, ενώ τα υπόλοιπα 23 είχαν ποικίλους συνδυασμούς των γονιδίων. Η παρουσία όλων των γονιδίων του οπερονίου ica ανιχνεύθηκε σε τρία από τα δεκαπέντε biofilm-αρνητικά στελέχη. Αναδείχθηκαν ένας κύριος και δύο δευτερεύοντες biofilm-θετικοί, πολυανθεκτικοί MRCNS κλώνοι, που αποίκισαν και προξένησαν λοιμώξεις στα πρόωρα νεογνά. Μέρος της παρούσας ερευνητικής εργασίας ήταν η διερεύνηση της επίδρασης του χρόνου επώασης στη γονιδιακή έκφραση σε δεκατέσσερα αντιπροσωπευτικά στελέχη. Αυτό έγινε μέσω της ποσοτικοποίησης με αλυσιδωτή αντίδραση πολυμεράσης πραγματικού χρόνου (RT-PCR) των γονιδίων icaA και icaD τα οποία κυρίως σχετίζονται με την παραγωγή biofilm, καθώς και δύο ρυθμιστικών γονιδίων icaR και sarA σε σύγκριση με ένα συντηρημένο γονίδιο αναφοράς (23S rDNA). Ο έλεγχος έγινε σε στελέχη S. epidermidis που ήταν ανθεκτικά στη methicillin, κατατάσσονταν σε διαφορετικούς κλώνους, εμφάνιζαν διαφορετικό φαινότυπο σύνθεσης βιομεμβράνης και έφεραν ποικίλους συνδυασμούς γονίδιων. Οι αντιδράσεις απόλυτης ποσοτικοποίησης έκφρασης των γονιδίων είχαν υψηλή απόδοση (1,86 και 1,84 για την επώαση στις τέσσερις ώρες και δύο ώρες, αντίστοιχα). Η μαθηματική ανάλυση δεν έδειξε ιδιαίτερα μεγάλες διαφορές μεταξύ των αποτελεσμάτων για τα τέσσερα γονίδια και τους δύο χρόνους επώασης. Αυτό που παρατηρήθηκε ήταν οτι η έκφραση ήταν υψηλότερη στα biofilm-θετικά στελέχη. Προκειμένου να μελετηθεί η επίδραση της βακτηριακής προσκόλλησης στις διάφορες επιφάνειες τροποποιημένου και μη τροποποιημένου γυαλιού, πραγματοποιήθηκαν δυναμικά πειράματα, κάτω από δύο διαφορετικές συνθήκες ροής, σε δύο χρόνους επώασης σε τέσσερα στελέχη S. epidermidis. Η βακτηριακή προσκόλληση, η παραγωγή biofilm και ο σχηματισμός βιομεμβράνης στις τροποποιημένες επιφάνειες ήταν αυξημένα σε σχέση με τις μη τροποποιημένες. Αυτό ήταν σε συμφωνία με τα αποτελέσματα της γονιδιακής έκφρασης των γονιδίων icaA και icaD, μετά από ανάλυση της έκφρασης των γονιδίων icaA και icaD με RT-PCR, που έδειξε αυξημένες τιμές στις τροποποιημένες επιφάνειες, κυρίως στην υψηλή ροή. Επομένως, ο συνδυασμός της δράσης της χημείας της επιφάνειας και της ροής, επηρεάζουν την προσκόλληση, τον φαινότυπο και το γονότυπο των βακτηριακών στελεχών, όπως και την προσαρμογή τους στις αλλαγές του περιβάλλοντος. Η μελέτη της γονιδιακής έκφρασης μπορεί να βοηθήσει στην κατανόηση των αλληλεπιδράσεων της βακτηριακής προσκόλλησης με το βιοϋλικό. / A total of 132 methicillin-resistant coagulase-negative staphylococci (MRCNS) isolated from preterm neonates were studied for antibiotic resistance patterns, clonal distribution and biofilm formation in relation to the presence of ica operon. All MR-CNS were multi-resistant while the majority (89%) produced biofilm. Among 115 Staphylococcus epidermidis, a major clone (z) was identified (77 strains), followed by clone g. Ten out of 16 S. haemolyticus belonged to a common clone (y). Most of the biofilm-positive strains (80%) possessed all tested genes, while the remaining 23 had a variety of gene combinations. Presence of the entire ica cluster was detected in three out of 15 biofilm-negative strains. One major and two minor biofilm-positive, multi-resistant MRCNS clones were disseminated, colonizing and infecting hospitalized preterm neonates. Fourteen representative MR- S. epidermidis strains were selected and investigated for the expression of icaA and icaD genes, and two regulatory genes, icaR (repressor) and sarA (inducer), towards a housekeeping gene (23SrDNA), by Real-Time PCR (RT-PCR). These strains belonged to various clones, had different combinations of ica genes and biofilm-forming phenotypes. The experiments were performed in two incubation times, after two and four hours. Νο differences were observed at the gene expression level at the two tested times. In general, biofilm-positive strains had higher expression rates for ica operon and sarA genes and lower for the repressor of ica cluster gene (icaR). In order to investigate the interaction of bacteria with the surfaces after attachment, dynamic experiments were performed under two flow conditions. Two different surfaces were used, a modified and a non-modified glass surface. Bacterial adhesion, as well as biofilm production and biofilm formation, was much higher on the modified surface than on the non-modified one for four S. epidermidis strains. This was in agreement with icaA and icaD gene expression that showed increased expression by RT-PCR, for the bacteria adhered at the modified substrate, especially under the high shear rate. Therefore, the combined effect of surface chemistry and shear significantly influence the adhesion of interacting bacterial cells. This indicates that analysis of gene expression not only can greatly refine our knowledge of bacterial-material interactions, but also yield novel biomarkers for potential use in biocompatibility assessment.

Orientador: Luiz Augusto do Amaral / Banca: Naiá Carla Marchi de Rezende Lago / Banca: Oswaldo Durival Rossi Junior / Resumo: O objetivo deste trabalho foi avaliar os riscos para a saúde pública apresentado por tilápias, produzidas em cativeiro, comercializadas em supermercados na região nordeste de São Paulo. Para tal, análises microbiológicas foram realizadas, em 40 amostras de tilápias in natura e em 50 filés de tilápias. No peixe in natura foi analisado a água de enxaguadura da pele e o trato gastrintestinal. Os números mais prováveis de coliformes totais e termotolerantes foram determinados assim como a contagem Staphylococcus coagulase positivo e a presença de Salmonella spp. de acordo com a American Public Health Association (APHA). Os resultados obtidos foram analisados utilizando valores logarítmicos e as médias comparadas pelo teste de Tukey, com nível de 5% de probabilidade. A pele foi o tecido com maior nível de contaminação em relação ao trato gastrintestinal e músculo de tilápia in natura. O número de amostras com presença de coliformes totais e termotolerantes na água de enxaguadura da pele foi, respectivamente, 72,5% e 60%, no trato gastrintestinal 25% e 12,5% e no músculo 17,5% e 5%. A carga bacteriana foi significativamente maior no filé em relação ao músculo. Em duas amostras de filé foram verificados Staphylococcus coagulase positivo, uma acima dos parâmetros microbiológicos estabelecidos pela Resolução RDC nº 12/2001 da ANVISA. Salmonella spp. não foi detectada em nenhuma amostra analisada. As amostras de peixes estavam em boas condições para consumo, com exceção de duas amostras de filé. Os filés apresentaram maior contaminação que o músculo da tilápia in natura. Indicadores de poluição fecal demonstraram que a pele foi o órgão com maior contaminação / Abstract: The objective of this work was to evaluate the risks to public health of consumption of tilápia fish growed in captivity and commercialised in supermarkets in the Northwest region of São Paulo state. In order to accomplish with the objective the researcher undertook analysis in 40 samples of fresh tilápias and in 50 fillets of the fish. In the fresh fish it was also analysed the washing water of the skin and of the gastrointestinal tract. It were determined, in accordance to the American Public Health Association (APHA), the number of total and thermotolerant fecal coliforms as well as the number of coagulase-positive Staphylococcus and presence of the bacteria Salmonella spp. The results were analyzed using logarithmic values and means compared by Tukey test with 5% of probability. The skin was the tissue with higher levels of contamination comparing with the gastrointestinal tract and muscle of the fresh tilapia. The percentage of samples with total and thermotolerant fecal coliforms in the skin washing water was 72.5% and 60% respectively; 25% and 12.5% in the gastrointestinal tract and 17.5% and 5% in the muscles. Bacterial presence was significantly higher in fillets comparing to muscles. Moreover, it was noticed presence of coagulase-positive Staphylococcus in two samples of fillet; one of them showed levels above of the microbiological parameters established by the RDC resolution no. 12/2001 ANVISA. Additionally, it was not identified presence of Salmonella spp in any of the samples analyzed. Generally, the studied samples were in good condition for human consumption, except for two samples of fillets. Fillets presented higher levels of contamination comparing with muscles of the fresh tilapia. Fecal pollution indicators have proved that the skin was the organ with higher levels of contamination / Mestre

Vancomicina é o antimicrobiano de escolha no tratamento de bacteremias causadas por estafilococos resistentes à meticilina. No entanto, estudos recentes têm reportado que a vancomicina apresenta atividade reduzida contra Staphylococcus aureus resistentes à meticilina com concentrações inibitórias mínimas (CIMs) próximas do limite do ponto de corte de suscetibilidade, conforme critérios do CLSI, indicando falha terapêutica. Entretanto, existem poucos estudos à respeito dos Staphylococcus sp. coagulase negativa resistentes à meticilina (SCoNRM). Ademais, para determinação da CIM, deveria ser utilizado o método de referência microdiluição em caldo (MDC), mas a maioria dos laboratórios clínicos utiliza a técnica de Etest ou sistemas automatizados. Alguns estudos com S. aureus demonstraram discrepâncias entre MDC e Etest, contudo não existem dados referentes aos SCoN. Os objetivos deste estudo foram avaliar a correlação entre as CIMs de vancomicina determinadas pelas técnicas de MDC e Etest em 130 SCoNRM isolados de hemocultura, bem como verificar a relação entre valores de CIM e falha terapêutica entre pacientes com bacteremia por SCoNRM tratados com este antimicrobiano. A maioria dos resultados de CIM por MDC (98,5%) foram ≤1,0 mg/mL, enquanto o Etest apresentou 72,3% de CIM ≥ 1,5 mg/mL. As CIMs de vancomicina obtidas por Etest foram, em geral, uma a duas diluições maiores do que as CIMs obtidas por MDC. Os resultados indicam que a técnica de Etest gera valores de CIM consistentemente maiores do que os obtidos por MDC nos SCoNRM. Apenas 37 (28,5%) dos 130 pacientes com hemocultura positiva para SCoNRM apresentaram dados clínicos compatíveis com bacteremia. A maioria dos pacientes com bacteremia comprovada (n=24) apresentaram CIMs de vancomicina ≥1,5 mg/mL, sendo que 13 pacientes (35,1%) obtiveram CIM < 1,5 mg/mL. Este estudo não observou relação estatisticamente significativa entre valores de CIM de vancomicina que pudessem ser associados com falha terapêutica em pacientes com bacteremia por SCoNRM. / Vancomycin is the first-line therapy for methicillin-resistant staphylococci bacteremia. However, recent studies have reported that vancomycin demonstrates reduced activity against methicillin-resistant Staphylococcus aureus bacteremia, with vancomycin MICs at the high end of the CLSI susceptibility range indicating treatment failure. There is, however, little data considering methicillin-resistant coagulase-negative staphylococci (MRCoNS) bacteremia. Besides, the reference method that should be used for MIC determination is the broth microdilution (BMD), but many clinical laboratories use the commercial Etest technique or automated systems. Some reports have showed a growing number of vancomycin MIC discrepancies between BMD and Etest method for S. aureus, but there are no studies about CoNS. The aims of this study were to evaluated the correlation between the vancomycin MIC determined by the Etest and the BMD method for a total of 130 MRCoNS bloodstream isolates as well as to examine the relationship between vancomycin MICs and failure among patients with MRCoNS bacteremia treated with vancomycin. The vast majority (98.5%) of MIC results by BMD were ≤1.0 mg/mL in contrast to MIC by Etest which majority (72.3%) was ≥1.5 mg/mL. The vancomycin MICs obtained by the Etest for the same isolates were, in general, one to twofold higher than those obtained by the BMD method. The results indicate that the Etest provides vancomycin MIC values consistently higher than those obtained by BMD method for MRCoNS. Only 37 (28.5%) out of the 130 patients with a positive MRCoNS bloodstream culture met the eligibility criteria to be considered bacteremic. The majority of these patients (n = 24, 64.9%) presented vancomycin MIC ≥ 1.5 mg/mL, in opposite to 13 patients (35.1%) with MIC < 1.5 mg/mL. This study did not observe any statistical significative relationship between vancomycin MIC and treatment failure.

Made available in DSpace on 2015-12-10T14:23:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-05-17. Added 1 bitstream(s) on 2015-12-10T14:29:41Z : No. of bitstreams: 1 000850005.pdf: 1236403 bytes, checksum: b1877b4be5ffb104086b39681ebd86a7 (MD5) / As hortaliças são itens indispensáveis na dieta dos seres humanos, atuando como adjuvantes na prevenção e tratamento de doenças. Porém, destaca-se o aumento de surtos de Doenças Transmitidas por Alimentos (DTA) associados ao consumo de produtos hortícolas. O objetivo desse trabalho foi verificar o perfil microbiológico de diferentes tipos de saladas de cozinhas hospitalares. No período de 2010 a 2014, o Serviço de Orientação a Alimentação Pública (SOAP) recebeu 641 amostras de saladas provenientes de hospitais da região de Bauru e Botucatu, onde foram submetidas às análises microbiológicas para a determinação do número mais provável de coliformes a 35°C e 45°C, pesquisa de Salmonella spp e contagem de estafilococos coagulase positiva. Os resultados revelaram que em 30,56% das amostras a contagem de coliformes a 35°C foi maior que 1.100 NMP/g e 12,17% apresentaram coliformes a 45°C acima de 100 NMP/g, limite máximo estabelecido pela legislação brasileira. A prevalência de amostras contaminadas sem tratamento térmico foi de 97,44% e de 2,56% para amostras com tratamento térmico, cozidas ou refogadas. Todas as amostras foram negativas para presença de Salmonella spp e apresentaram contagem de estafilococos coagulase positiva < 1,0X102 UFC/g. Apesar das amostras de saladas não oferecerem risco microbiológico associado à patógenos, deve-se atentar para os micro-organismos indicadores, pois as refeições são servidas a indivíduos hospitalizados / Vegetables crops are indispensable items in human diet, acting as adjuvants in the prevention and treatment of diseases. However, the number of Foodborne Diseases outbreaks associated with their consumption increases. The aim of this study was determine the microbiological profile of different salads from hospital kitchens. In the period between 2010 and 2014 the Public feeding orientation service (SOAP) received 641 samples of salads from hospital kitchens in the region of Botucatu and Bauru. They, where subjected to microbiological analysis to determine the most probable number of coliforms at 35 ° C and 45 ° C, besides Salmonella spp presence and coagulase-positive staphylococci counts. The results reveal that 30.56% of the samples had counts for coliform at 35°C greater than 1.100 MPN/g and 12.17% had MPN/g of coliforms at 45°C above 100 MPN/g, the maximum limit established by Brazilian legislation. The prevalence of samples without heat treatment was 2.56% and 97.44% with heat treatment, cooked or braised. All samples were negative for Salmonella spp count and coagulase positive staphylococci < 1,0x102 UFC/g. Although most salads had an adequate microbiological quality, attention should be paid to the safety indicators as the meals are served to hospitalized individuals