Orientador: Antonio Carlos Pizzolitto / Banca: Eliana Aparecida Varanda / Banca: Taís Maria Bauab / Banca: Ioshie Ibara Tanaka / Banca: Antonio Fluminhan Júnior / Resumo : Staphylococcus aureus é um dos principais agentes de infecção humana, especialmente em indivíduos hospitalizados. Cepas MRSA (Methicillin Resistant Staphylococcus aureus) constituem um grave problema em hospitais de todo o mundo, aumentando a morbidade e mortalidade de indivíduos infectados. A vancomicina é uma das únicas opções terapêuticas. No entanto, o uso excessivo desse antibiótico pode selecionar cepas resistentes, agravando ainda mais o problema. Trabalhadores hospitalares podem carrear S. aureus nas narinas anteriores e pele e, assim, constituem um importante elo na epidemiologia das infecções nosocomiais. Nesse trabalho foram coletadas amostras das mãos e narinas de 100 trabalhadores do Hospital Universitário “Dr. Domingos Leonardo Cerávolo” da Universidade do Oeste Paulista - UNOESTE, em Presidente Prudente (SP). Desse total, 68% não eram portadores de S. aureus, 27% carreavam S. aureus sensível a meticilina, 4% carreavam MRSA e 1% carreava BORSA (Borderline Oxacillin Resistant Staphylococcus aureus). No mesmo período (julho a dezembro de 2002), 54,3% das infecções estafilocócicas em pacientes internados nessa Instituição tinham como agente MRSA. A técnica da PCR (Polymerase Chain Reaction) Multiplex para a detecção do gene femA (gene espécie-específico), mecA (resistência à meticilina) e ileS-2 (resistência à mupirocina) foi comparada com o método da difusão com discos e provas convencionais de identificação. Os resultados da PCR Multiplex apresentaram completa concordância com os resultados obtidos com os testes fenotípicos convencionais. Para verificar a relação genética entre as 30 cepas MRSA, 5 isoladas de trabalhadores e 25 de pacientes, foram realizadas a antibiotipagem e tipagem molecular através da técnica RAPD (Random Amplified Polymorphic DNA). Pela antibiotipagem as cepas MRSA foram... (Resumo completo, clicar acesso eletrônico abaixo). / Abstract: Staphylococcus aureus is considered as a major infeccious disease agent in human, especially in hospitalized people. Methicillin-resistant strains (MRSA) constitute a serious problem in hospitals around the world, increasing the morbity and mortality rate of infected people. Usually, vancomycin is the only therapeutic choice for the treatment of MRSA infections. However, excessive use of this antibiotic can select resistant strains, aggravating the problem. Health workers can carry S. aureus in the anterior nares and skin, constituting an important link for the epidemiology of nosocomial infections. In this study, samples of the hands and nares of one hundred workers from the University Hospital “Dr. Domingos Leonardo Cerávolo”, at Presidente Prudente, SP, Brazil, were assessed. Among the one hundred workers, 68% did not carry S. aureus, methicillin-susceptible S. aureus was found in 27/100 (27%) of those people; MRSA in 4/100 (4%) and BORSA (Borderline Resistant Staphylococcus aureus) was found in 1 (1%) health worker. In the same period (July to December of 2002), 54,3% of the staphylococcal infections in hospitalized patients were caused by MRSA. Multiplex PCR assay for the detection of femA (species-specific gene), mecA (methicillin resistance gene) and ileS-2 (mupirocin resistance gene) was compared to the disk diffusion susceptibility test and conventional identification test methods. The Multiplex PCR technic results were in complete agreement with the results obtained from conventional methods. Genetic relationship among the 30 MRSA strains, five isolated from workers and twenty five from patients, was established by antibiotyping and molecular typing by RAPD. On the basis of the antibiotyping, the 30 strains were grouped into four clusters (A to D), of a which 87% were grouped into antibiotype A. According to the profile of RAPD, eight clusters... (Complete abstract, click electronic address below). / Doutor

M.Tech.

Orientador : Fumio Yokoya / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos e Agricola / Made available in DSpace on 2018-07-17T16:24:25Z (GMT). No. of bitstreams: 1 Machado_RubemAbreu_M.pdf: 13249981 bytes, checksum: a57183ef3191e80424b84620cdf3a80a (MD5) Previous issue date: 1979 / Resumo: Foram realizados ensaios para a determinação do numero mais provável de 43 linhagens de Staphylococcus aureus. Após uma série de ensaios preliminares, usando diferentes meios de cultura e modificando-lhes em alguns componentes ou mesmo acrescentando outros, foi finalmente escolhido o meio BHI como meio basal para os estudos. As alterações efetivadas nesse meio proporcionaram u ma recuperação altamente satisfatória para os estafilococos ensaiados. Basicamente essas alterações constaram do acréscimo de NaCl a 10% e vermelho neutro a 0,01 e 0,001 % . Além das linhagens caracterizadas como coagulase e DNA'se positivas, foram também utilizadas, nos ensaios com o meio proposto para a determinação do NMP, linhagens negativas para esses testes, bem como linhagens reconhecidamente entero-toxigênicas. Foi testado também com esse meio, uma linhagem de Micrococcus (Micrococcus glutamicus) para ser determinado o pó der inibidor do corante em relação ao crescimento desse microorganismo. Esse procedimento foi adotado tendo em vista que o Micrococcus sp. apresenta condições de desenvolvimento nos substratos contendo alta percentagem de NaCl. Pelo menos para a linhagem testada, não houve crescimento algum; nem após 72 horas de incubação. Uma das principais vantagens desse meio é que poderão ser feitos, após a leitura dos tubos para o número mais provável, os testes respectivos de coagulase e DNA'se. Esses testes mostraram ser bastante efetivos, tendo em vista que logo nas primeiras horas de incubação já foram evidenciados os resultados positivos. Para a verificação de falsos resultados positivos, foram usadas duas linhagens coagulase e DNA'se negativa se os resultados não foram diferentes / Abstract: The most probable number (MPN) technique is frequently used in food microbiology. However, many individuals are not able to understand the real meant of the method, because the statistics involved on it. To overcome these difficulties, a review about it was introduced in this work. Certain theoretical and mathematical considerations in regard to the dilution method for estimating bacterial densities were presented. About 43 Staphylococcus aureus strains were used , some of them are enterotoxins producer (food-poisoning staphy-lococci); the last were obtained from the "Food Research Institute", University of Wisconsin, Madison. The basal BHI broth was used as proposed medium, added of 10% NaCl and neutral red at 0,01 and/or 0,001% final concentration. This modified medium has presented a good per- formance to Staphylococcus aureus recovering. About 70% of strains presented more then 100% of recovering, compared to control medium (BHI agar). Furthermore, it was possbile to perform the DNA'se and coagulase tests using the MPN proposed medium. Thus, becoming easier and more practical the enumeration and characterization of Staphylococcus aureus / Mestrado / Mestre em Ciência de Alimentos

Thesis (M.S.)--Youngstown State University, 2008. / Includes bibliographical references (leaves 75-78). Also available via the World Wide Web in PDF format.

Thesis (M.S.)--Youngstown State University, 2008. / Includes bibliographical references (leaves 32-39). Also available via the World Wide Web in PDF format.

Hung Ming Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 146-169). / Abstracts in English and Chinese. / Abstract --- p.I / Abstract (Chinese version) --- p.III / Acknowledgments --- p.V / Contents --- p.VI / List of Tables --- p.XII / List of Figures --- p.XIV / List of Abbreviations & Symbols --- p.XVII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Methicillin-resistant Staphylococcus aureus --- p.1 / Chapter 1.1.1 --- Definition and Identification --- p.1 / Chapter 1.1.2 --- Antibiotic resistance --- p.2 / Chapter 1.1.2.1 --- Methicillin/oxacillin resistance --- p.2 / Chapter 1.1.2.2 --- Other antibiotic resistance --- p.2 / Chapter 1.2 --- Evolution of MRSA --- p.3 / Chapter 1.2.1 --- Staphylococcal Chromosome Cassette mec (SCCmec) --- p.3 / Chapter 1.2.2 --- The origin of mec element --- p.7 / Chapter 1.2.3 --- The acquisition of SCCmec: from MSSA to MRSA --- p.7 / Chapter 1.2.4 --- Five pandemic clones --- p.8 / Chapter 1.3 --- Epidemiology of MRSA --- p.10 / Chapter 1.3.1 --- MRSA Infections --- p.12 / Chapter 1.3.2 --- Community-acquired MRSA --- p.13 / Chapter 1.4 --- Staphylococcal Gene Regulators --- p.14 / Chapter 1.4.1 --- Accessory gene regulator (agr) --- p.14 / Chapter 1.4.1.1 --- Structure and functions of accessory gene regulator (agr) --- p.15 / Chapter 1.4.1.2 --- Autoinducing Peptide (AIP) --- p.16 / Chapter 1.4.1.3 --- Implication of agr I-IV --- p.18 / Chapter 1.4.2 --- Staphylococcal accessory regulator (sar) --- p.19 / Chapter 1.4.2.1 --- Structure and function of sar --- p.19 / Chapter 1.4.3 --- Difference in Functions of sar and agr --- p.20 / Chapter 1.4.4 --- "Other sar A homologues, SarS, R，T, U, and other Gene Regulators" --- p.22 / Chapter 1.5 --- Adhesin and toxin genes --- p.25 / Chapter 1.5.1 --- Adhesins --- p.25 / Chapter 1.5.2 --- Exotoxins --- p.26 / Chapter 1.6 --- Typing Methods --- p.29 / Chapter 1.6.1 --- Phenotypic typing method --- p.31 / Chapter 1.6.1.1 --- Antibiotic-susceptibility Test --- p.31 / Chapter 1.6.1.2 --- Bacteriophage Typing --- p.31 / Chapter 1.6.1.3 --- Serotyping --- p.32 / Chapter 1.6.1.4 --- Electrophoretic Protein Typing and Immunoblotting --- p.33 / Chapter 1.6.1.5 --- Multilocus Enzyme Electrophoresis --- p.34 / Chapter 1.6.1.6 --- Limitations of phenotyping --- p.34 / Chapter 1.6.2 --- Genotyping methods --- p.35 / Chapter 1.6.2.1 --- PCR Assays --- p.35 / Chapter 1.6.2.2 --- Pulsed-Field Gel Electrophoresis (PFGE) --- p.36 / Chapter 1.6.2.3 --- Multi-Locus Sequence Typing (MLST) --- p.37 / Chapter 1.6.2.4 --- Amplified Fragment Length Polymorphism (AFLP) --- p.38 / Chapter 1.6.2.5 --- Ribotyping --- p.38 / Chapter 1.6.2.6 --- Plasmid Typing --- p.39 / Chapter 1.6.2.7 --- Restriction Fragment Length Polymorphism --- p.39 / Chapter 1.6.2.8 --- Nucleic Acid Hybridization --- p.40 / Chapter 1.7 --- Objectives of the project --- p.41 / Chapter Chapter 2 --- Materials and Methods --- p.42 / Chapter 2.1 --- Bacterial Isolates & Culture conditions --- p.42 / Chapter 2.1.1 --- Bacterial Isolates --- p.43 / Chapter 2.1.2 --- Reference Strains --- p.43 / Chapter 2.1.3 --- Identification of MRSA --- p.44 / Chapter 2.2 --- Antibiotic Susceptibility Test --- p.45 / Chapter 2.3 --- Pulsed-Field Gel Electrophoresis (PFGE) --- p.47 / Chapter 2.3.1 --- DNA preparation --- p.47 / Chapter 2.3.2 --- Restriction digestion --- p.48 / Chapter 2.3.3 --- PFGE --- p.48 / Chapter 2.3.4 --- Analysis of band patterns --- p.48 / Chapter 2.4 --- Phage Typing --- p.50 / Chapter 2.4.1 --- Source of Phages & Propagating Strains --- p.50 / Chapter 2.4.2 --- Procedures of phage typing --- p.50 / Chapter 2.4.3 --- Routine check of the lytic strength of phages --- p.51 / Chapter 2.4.4 --- Evaluation of the reproducibility of phage typing --- p.51 / Chapter 2.5 --- Detection of SCCmec and mec Genes by Polymerase Chain Reaction (PCR) --- p.55 / Chapter 2.5.1 --- DNA preparation for PCR --- p.55 / Chapter 2.5.2 --- Master mix preparation and PCR conditions --- p.55 / Chapter 2.5.3 --- Electrophoresis of DNA Amplicors (PCR products) --- p.55 / Chapter 2.5.4 --- Detection of mecA gene by PCR --- p.56 / Chapter 2.5.5 --- Detection of SCCmec I-IV by PCR --- p.58 / Chapter 2.6 --- Multiplex Polymerase Chain Reaction --- p.60 / Chapter 2.6.1 --- Detection of agr I-IV --- p.60 / Chapter 2.6.2 --- Detection of Pyrogenic Toxin Genes --- p.62 / Chapter 2.6.2.1 --- Primer Mix Preparation for Multiplex PCR --- p.66 / Chapter 2.6.2.1.1 --- Sea-see Multiplex PCR --- p.66 / Chapter 2.6.2.1.2 --- Seg-sej Multiplex PCR --- p.66 / Chapter 2.6.2.1.3 --- "Eta, etb, tsst-1 for multiplex PCR" --- p.66 / Chapter 2.6.2.2 --- Multiplex Master Mix Preparation --- p.67 / Chapter 2.6.2.3 --- "PCR controls for pyrogenic toxins (sea-sej, eta-b, tsst-1)" --- p.67 / Chapter 2.6.2.4 --- Sequencing of PCR products --- p.68 / Chapter 2.6.3 --- PCR for adhesin and toxin genes --- p.69 / Chapter 2.6.3.1 --- "Multiplex Master mix preparation for lukE, fib, cna, icaD" --- p.71 / Chapter 2.6.3.2 --- "Multiplex Master mix preparation for fnbA,fnbB, hla, hlb, icaA" --- p.71 / Chapter 2.6.3.3 --- PCR controls for adhesin and toxin genes --- p.71 / Chapter 2.6.3.4 --- Size of PCR products resolved by gel electrophoresis --- p.72 / Chapter 2.6.3.5 --- Sequencing of PCR products --- p.72 / Chapter 2.7 --- Multi-locus sequence typing --- p.73 / Chapter 2.7.1 --- Bacterial Isolates --- p.73 / Chapter 2.7.2 --- Primer design --- p.73 / Chapter 2.7.3 --- PCR Master mix preparation --- p.73 / Chapter 2.7.4 --- PCR for MLST gene --- p.75 / Chapter 2.7.5 --- Sequencing of PCR products --- p.75 / Chapter 2.7.6 --- Data Analysis --- p.75 / Chapter 2.8 --- Sequence typing of Coa gene --- p.76 / Chapter 2.8.1 --- Bacterial Isolates --- p.76 / Chapter 2.8.2 --- Amplification of Coa Gene by PCR --- p.76 / Chapter 2.8.3 --- coa sequencing --- p.77 / Chapter 2.8.4 --- Sequence Analysis --- p.77 / Chapter Chapter 3 --- Results --- p.79 / Chapter 3.1 --- Bacterial Isolates --- p.79 / Chapter 3.2 --- PCR for mecA gene --- p.80 / Chapter 3.3 --- Antibiotics Susceptibility Test --- p.81 / Chapter 3.3.1 --- Antibiotic Resistance Profiles of MRSA --- p.82 / Chapter 3.4 --- Pulsed-field Gel Electrophoresis --- p.85 / Chapter 3.4.1 --- Optimization of PFGE and Inter-gel Variation --- p.85 / Chapter 3.4.2 --- Analysis of PFGE profiles --- p.87 / Chapter 3.4.3 --- PFGE patterns and antibiotic profiles --- p.93 / Chapter 3.5 --- Phage Typing --- p.96 / Chapter 3.6 --- SCCmec Typing --- p.98 / Chapter 3.6.1 --- PCR Optimization --- p.98 / Chapter 3.6.2 --- PCR and distribution of SCCmec types --- p.101 / Chapter 3.6.3 --- PFGE patterns and SCCmec types --- p.103 / Chapter 3.7 --- agr Typing --- p.104 / Chapter 3.7.1 --- Optimization of PCR --- p.104 / Chapter 3.7.2 --- PCR and Distribution of agr groups --- p.106 / Chapter 3.7.3 --- PFGE patterns and agr groups --- p.108 / Chapter 3.7.4 --- SCCmec types and agr groups --- p.108 / Chapter 3.8 --- "PCR for adhesin, pyrogenie and other toxin genes" --- p.110 / Chapter 3.8.1 --- Optimization of PCR --- p.110 / Chapter 3.8.2 --- "Distribution of adhesin, pyrogenic and other toxin genes" --- p.120 / Chapter 3.9 --- Multi-Locus Sequence Typing --- p.124 / Chapter 3.10 --- coa Sequence Typing --- p.126 / Chapter Chapter 4 --- Discussion --- p.128 / Chapter 4.1 --- Evaluation of the Typing methods for MRSA --- p.128 / Chapter 4.1.1 --- Antibiotic Susceptibility Test --- p.128 / Chapter 4.1.2 --- PFGE --- p.128 / Chapter 4.1.3 --- Phage Typing --- p.130 / Chapter 4.1.4 --- "PFGE, SCCmec typing and agr typing" --- p.131 / Chapter 4.1.5 --- MLST --- p.132 / Chapter 4.1.6 --- Sequencing typing of coa gene --- p.133 / Chapter 4.2 --- Characteristics of MRSA --- p.134 / Chapter 4.2.1 --- PCR for adhesin and toxin genes --- p.134 / Chapter 4.3 --- Key Characteristics of Hong Kong isolates --- p.137 / Chapter 4.4 --- Conclusion --- p.144 / Chapter 4 5 --- Future Research --- p.145 / Reference List --- p.146 / Appendix I - Materials/Reagents for Methods --- p.170 / Appendix II - Reagents Formula --- p.178 / Appendix III - coa sequence alignment --- p.180 / Appendix IV - Typing patterns and Characteristics of reference isolates --- p.193 / Appendix V - Miscellaneous --- p.195 / Appendix VI - Typing Patterns and Characteristics of PFGE type isolates --- p.204

Thesis (M.S.)--Youngstown State University, 2008. / Includes bibliographical references (leaves 73-74). Also available via the World Wide Web in PDF format.

Thesis (M.S.)--Youngstown State University, 2008. / Includes bibliographical references (leaves 80-83). Also available via the World Wide Web.

Thesis (Ph. D.)--University of Pennsylvania, 1934. / Bibliography: p. 40-41.

Thesis (doctoral)--Swedish University of Agricultural Sciences, 2000. / Includes bibliographical references.