Thesis (DClinPsychol)--British Psychological Society. BLDSC no. DX233166.

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Source: Masters Abstracts International, Volume: 47-01, page: 0341. Adviser: Luis Ostrosky. Includes bibliographical references.

Wong, Kai Yi. / Thesis (M.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 101-110). / Abstracts in English and Chinese. / Abstract --- p.4 / 摘要 --- p.7 / Acknowledgements --- p.9 / List of Tables --- p.10 / List of Figures --- p.11 / List of Abbreviations and Symbols --- p.12 / Chapter Chapter 1 --- Introduction --- p.14 / Chapter 1.1 --- Staphylococcus aureus --- p.14 / Chapter 1.2 --- Methicillin-resistant Staphylococcus aureus (MRSA) --- p.14 / Chapter 1.2.1 --- Methicillin resistance of MRSA --- p.15 / Chapter 1.2.2 --- Staphylococcal Chromosomal Cassette mec (SCCmec) --- p.16 / Chapter 1.2.3 --- Hospital-associated MRSA (HA-MRSA) and Community-associated MRSA (CA-MRSA) --- p.22 / Chapter 1.2.3.1 --- Hospital-associated MRSA (HA-MRSA) --- p.23 / Chapter 1.2.3.2 --- Community-associated MRSA (CA-MRSA) --- p.23 / Chapter 1.2.4 --- Pathogenesis of MRSA infection --- p.27 / Chapter 1.2.4.1 --- Possible virulence genes contributing to necrotizing pneumonia --- p.29 / Chapter 1.2.4.1.1 --- Panton-Valentine Leukocidin (PVL) --- p.29 / Chapter 1.2.4.1.2 --- Phenol-soluble modulins (PSMs) --- p.36 / Chapter 1.3 --- Evolution of MRSA --- p.36 / Chapter 1.4 --- Epidemiology of MRSA --- p.38 / Chapter 1.4.1 --- Epidemiology of MRSA worldwide --- p.38 / Chapter 1.4.1.1 --- Epidemiology of HA-MRSA worldwide --- p.38 / Chapter 1.4.1.2 --- Epidemiology of CA-MRSA worldwide --- p.39 / Chapter 1.4.2 --- Epidemiology of MRSA in Hong Kong --- p.40 / Chapter 1.5 --- Clinical significance of MRSA --- p.41 / Chapter 1.6 --- Antibiotics --- p.43 / Chapter 1.6.1 --- Beta-lactams --- p.43 / Chapter 1.6.2 --- Fluoroquinolone --- p.44 / Chapter 1.6.3 --- Linezolid --- p.45 / Chapter 1.6.4 --- Glycopeptides --- p.45 / Chapter 1.6.5 --- Aminoglycosides --- p.46 / Chapter 1.6.6 --- Fusidic acid --- p.46 / Chapter 1.6.7 --- Clindamycin --- p.47 / Chapter 1.7 --- Hypothesis --- p.47 / Chapter Chapter 2 --- Methods and Materials --- p.50 / Chapter 2.1 --- Bacterial isolate --- p.50 / Chapter 2.2 --- Effect of subinhibitory antibiotics on the expression of mRNA in MRSA in vitro --- p.53 / Chapter 2.2.1 --- Collection of bacterial fraction --- p.53 / Chapter 2.2.2 --- RNA extraction and DNA digestion --- p.53 / Chapter 2.2.3 --- Reverse transcription for cDNA synthesis --- p.54 / Chapter 2.2.4 --- Quantitative real-time PCR (qPCR) analysis (pvl and psma\-A expression) --- p.55 / Chapter 2.2.5 --- Preparation of standard controls for quantification of DNA copy number in qPCR reactions... --- p.58 / Chapter 2.3 --- Effect of subinhibitory concentration of antibiotics on MRSA pneumonia in a murine model --- p.60 / Chapter 2.4 --- Statistical Analysis --- p.62 / Chapter Chapter 3 --- Results --- p.63 / Chapter 3.1 --- Effect of subinhibitory antibiotics on the expression ofmRNA in MRSA in vitro --- p.63 / Chapter 3.2 --- Effect of subinhibitory concentration of antibiotics on MRSA pneumonia in a murine model --- p.74 / Chapter Chapter 4 --- Discussion --- p.80 / Chapter 4.1 --- Effect of subinhibitory antibiotics on the expression of mRNA in MRSA in vitro --- p.81 / Chapter 4.2 --- Effect of subinhibitory concentration of antibiotics on MRSA pneumonia in a murine model --- p.87 / Chapter 4.3 --- Correlation of effects of subinhibitory antibiotics on the expression ofmRNA in MRSA in vitro and on MRSA pneumonia in a murine model --- p.91 / Chapter 4.4 --- Limitations of Study --- p.95 / Chapter 4.5 --- Future Work --- p.95 / Chapter Chapter 5 --- Conclusions --- p.97 / References --- p.99 / Chapter Appendix I- --- Materials and Reagents --- p.109 / Chapter Appendix II- --- Average and standard deviation of the copy number ratio (pvl or psmal-4 copy number/JdiS1 copy number) --- p.111 / Chapter Appendix III- --- In-vivo experimental data for infected control group and seven antibiotic groups --- p.116

The gene encoding resistance to β-lactam antibiotics in the staphylococci is found on the chromosome in a genomic island designated Staphylococcal Chromosome Cassette mec or SCCmec. In addition to the resistance gene, mecA, SCCmec also contains site specific recombinase genes, ccrAB, that are capable of catalyzing the chromosomal excision and integration of SCCmec. The increasing prevalence of methicillin-resistant Staphylococcus aureus infections may be due, in part, to the transfer of SCCmec into successful methicillin-sensitive S. aureus lineages. In this work we attempt to better characterize the factors associated with SCCmec transfer, beginning with CcrAB-mediated SCCmec excision in a collection of MRSA containing type IV SCCmec. CcrAB-mediated excision of type IV SCCmec was not demonstrated for all strains tested, as a subset of S. aureus strains with type IV SCCmed did not excise their element. These strains are all highly related and represent a lineage of successful community associated pathogens. In addition, the inability to excise SCCmec in these strains is associated with the presence of a presumptive mobile element containing the gene for staphylococcal enterotoxin H (seh) immediately outside of SCCmec on the chromosome. Staphylococcus aureus strain USA300 contains SCCmec type IV and a genomic island containing an arginine deiminase pathway, known as ACME, inserted adjacent to one another in the SCCmec chromosomal attachment site. Each element was site-specifically excised from the chromosome by CcrAB, resulting in two independent, extra-chromosomal, circularized elements. Therefore the presence of ACME did not disrupt SCCmec excision.Next, we describe three MRSA strains that become resistant to vancomyein during passage on increasing concentrations of the drug. In each case, mecA was lost during passage. Strain 5836VR lost mecA by the site-specific chromosomal excision of SCCmec while the other two strains (3130VR and VP32) deleted portions of their SCCmec elements in a manner that appears to involve IS431. Conversion to vancomycin resistance caused a decrease in growth rate that was partially compensated for by deletion of mecA. In mixed culture competition experiements, vancomycin resistant strains that lacked mecA readily out-competed their mecA-containing counterparts, suggesting that the loss of mecA during conversion to vancomycin resistance was advantageous to the organism.Finally, we examined the genetic structure surrounding the SCCmec attachment site in a diverse collection of methicillin-sensitive S. aureus isolates. This region of the chromosome varies greatly from strain to strain and appaers to be prone to recomination. Open reading frames found in this region were homologous to enterotoxins, restriction-modification enzymes, and transposases. Several open reading frames that have not been previously reported in staphylococci were also present in this region. 28 out of the 42 isolates examined did not contain the attachment site sequence found in S. aureus isolates known to be capable of CcrAB-mediated SCCmec integration or excision. This may indicate that these strains do not contain a functional attachment site and therefore may not have the potential to acquire SCCmec by CcrAB-mediated recombination.

Staphylococcus aureus é um patógeno versátil, capaz de causar uma grande variedade de infecções. Nos últimos anos, ocorreu um aumento da proporção de infecções causadas por S. aureus resistentes a meticilina (MRSA). A resistência a meticilina deve-se à presença do gene mecA, carreado no cassete cromossômico estafilocócico (SCCmec). Pacientes infectados ou colonizados por MRSA são reservatórios e fontes de disseminação deste microorganismo, em instituições de cuidado à saúde, principalmente através de profissionais transitoriamente colonizados. Freqüentemente, a infecção por MRSA é precedida por um período de colonização. Em 2003 foi observado um aumento das taxas de infecções hospitalares por S. aureus na Enfermaria de Dermatologia do Hospital das Clínicas (EDER), em relação aos cinco anos anteriores. Os objetivos do presente estudo foram avaliar a transmissão hospitalar de MRSA, entre os pacientes da EDER e caracterizar os isolados de MRSA, obtidos de pacientes e funcionários colonizados, presentes nessa unidade. Foi realizada a detecção dos pacientes colonizados por MRSA, através de culturas de vigilância das narinas e, quando possível, culturas de vigilância das lesões de pele, num período de seis meses. A identificação fenotípica foi confirmada por reação em cadeia da polimerase (PCR) multiplex para detecção dos genes mecA e coa. Posteriormente, os isolados de MRSA foram submetidos ao teste de sensibilidade aos antimicrobianos pelo método de microdiluição em caldo, PCR multiplex para determinação do tipo de SCCmec e tipagem molecular por eletroforese em campo pulsado (PFGE). Quarenta e cinco por cento dos pacientes eram portadores de MRSA. No início do estudo, 14% dos funcionários eram portadores de MRSA, no fim eram 18%. Foram obtidas 105 amostras de MRSA, sendo que 11 foram isoladas de funcionários da EDER e 94 isoladas de 64 pacientes que eram portadores deste microorganismo.Sessenta e um por cento dos pacientes classificados como portadores de MRSA, eram positivos na primeira coleta realizada e 39% foram identificados durante o seguimento, nas coletas posteriores. O gene da coagulase foi detectado em todas as 105 amostras e o gene mecA em 101. Todos os isolados foram sensíveis a vancomicina e resistentes a oxacilina e penicilina. Trinta e três por cento dos isolados eram multirresistentes, apresentaram SCCmec tipo IIIA e um perfil PFGE predominante. SCCmec tipo IV foi observado em 59% dos isolados. Não foi possível determinar o tipo de SCCmec de quatro isolados. Na PFGE, o perfil B1 foi o mais prevalente, apresentado por isolados de MRSA SCCmec tipo IV, obtidos de nove pacientes e de três funcionários. A transmissão hospitalar foi caracterizada em 39% dos pacientes portadores. Foi possível observar a participação dos funcionários, na transmissão cruzada de MRSA, na EDER. A colonização por MRSA, dos profissionais de cuidado à saúde, foi transitória. Além da transmissão hospitalar de MRSA, foi possível detectar pacientes que eram portadores de MRSA na admissão / Staphylococcus aureus is a versatile pathogen capable of causing a wide variety of infections. The proportion of nosocomial and community-acquired methicillinresistant Staphylococcus aureus (MRSA) infections has increased in the last years. Methicillin resistance is mediated by the mecA gene which is carried on the Staphylococcal Cassette Chromosome mec (SCCmec). In heath care settings, patients who are colonized or infected with MRSA constitute a reservoir and a source of spread of this microorganism, mainly through transiently colonized health care workers (HCWs). MRSA infections are usually preceded by a period of colonization. In 2003, an increase in the rates of MRSA nosocomial infection in the Dermatology ward of Hospital das Clínicas was observed, in comparison with the five previous years. The aims of this study were to evaluate the nosocomial transmission of MRSA in the Dermatology ward and to characterize MRSA isolates obtained from patients and HCWs. Surveillance cultures of the anterior nares and skin lesions were performed to identify patients who were MRSA carriers, during a period of six months. The phenotypic identification was confirmed by multiplex polymerase chain reaction (PCR), to detect mecA and coa genes. Subsequently, MRSA isolates were submitted to antimicrobial susceptibility testing by microdilution method, multiplex PCR for SCCmec typing and molecular typing by pulsed-field gel electrophoresis (PFGE). Forty-five percent of the patients were MRSA carriers. 14% of the HCWs were MRSA carriers at the beginning of the study and 18% at the end. One hundred and five MRSA isolates were obtained, 11 from HCWs and 94 from 64 patients who were MRSA carriers. Sixty-one percent of the patients, classified as MRSA carriers, were positive on the first culture and 39% were identified during the follow up period in the subsequent cultures. The coagulase gene was detected in all 105 isolates and the mecA gene in 101. All MRSA isolates were susceptible to vancomycin and resistant to oxacillin and penicillin. Thirty-three percent of the isolates were multiresistant, presented SCCmec Type IIIA and showed a predominant PFGE type. The SCCmec type IV was found in 59% of the isolates. It was not possible to determine the SCCmec type of four isolates. The B1 PFGE pattern was the most prevalent, presented by MRSA SCCmec type IV isolates, obtained from nine patients and three HCWs. Nosocomial transmission occurred in 39% of the MRSA carriers. It was possible to observe HCWs MRSA cross- transmission in the Dermatology ward. HCWs were transiently colonized. In addition to nosocomial transmission of MRSA, it was possible to detect patients who were MRSA carriers on admission

Os objetivos do presente estudo foram avaliar os perfis de sensibilidade a antimicrobianos de MRSA isolados de sangue, identificar o tipo de SCCmec e analisar o perfil de DNA desses isolados para verificar a existência de linhagens predominantes. Amostras de MRSA isoladas de sangue foram submetidas ao teste de triagem em ágar para detecção de resistência a oxacilina, teste de sensibilidade aos antimicrobianos, PCR multiplex para detecção dos genes mecA e coa e do tipo de SCCmec, tipagem molecular por PFGE. 89% das amostras de MRSA foi de origem hospitalar. Foi observado multirresistência em 69% dos isolados. 83% dos isolados apresentaram SCCmec tipo IIIA e um perfil PFGE predominante. Foram observadas amostras de MRSA sensíveis a diversas classes de antimicrobianos, portando SCCmec tipo IV, associadas a infecção de origem hospitalar e a tipagem molecular permitiu observar o predomínio de um clone, disseminado em todo o complexo HC / The aims of this study were to evaluate the susceptibility profile of MRSA strains isolated from blood, identify the SCCmec types and analyze the DNA profile in order to determine if there were predominant lineages. Consecutive MRSA blood isolates were submitted to oxacillin agar screening test, antimicrobial susceptibility test, multiplex PCR to detect mecA and coa genes, SCCmec typing and molecular typing by PFGE. 89% of the isolates were nosocomial-acquired. 69% of strains were observed to be multiresistant. 83% of the 223 isolates had SCCmec type IIIA and had a predominant DNA pattern by PFGE. Some strains nosocomial-acquired had SCCmec type IV, resistance only to beta-lactams and demonstrated PFGE pattern with one predominant clone

Chio Weng Fan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 131-149). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / Contents --- p.iv / List of tables --- p.ix / List of figures --- p.x / List of abbreviation --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Biology of Staphylococci --- p.1 / Chapter 1.1.1 --- Taxonomy --- p.1 / Chapter 1.1.2 --- Characteristics of S. aureus --- p.2 / Chapter 1.2 --- Methicillin-resistant Staphylococcus aureus --- p.1 / Chapter 1.2.1 --- Description of MRSA --- p.7 / Chapter 1.2.2 --- Antibiotic Resistance of MRSA --- p.7 / Chapter 1.2.2.1 --- Resistance to β-lactam Antibiotics --- p.8 / Chapter 1.2.2.1.1 --- Production of β-lactamase --- p.8 / Chapter 1.2.2.1.2 --- Penicillin-binding Protein PBP2a --- p.8 / Chapter 1.2.2.1.3 --- Borderline Methicillin Resistance --- p.11 / Chapter 1.2.2.2 --- Resistance to Antibiotics other than β-lactams --- p.11 / Chapter 1.2.3 --- Epidemiology of MRSA --- p.15 / Chapter 1.2.3.1 --- Clinical Significance of MRSA --- p.15 / Chapter 1.2.3.1.1 --- Evolution of MRSA --- p.16 / Chapter 1.2.3.1.2 --- Epidemiology of MRSA Worldwide --- p.17 / Chapter 1.2.3.2 --- MRSA in Hong Kong --- p.21 / Chapter 1.3 --- Strain Typing for MRSA --- p.26 / Chapter 1.3.1 --- Phenotypic Methods --- p.27 / Chapter 1.3.1.1 --- Antibiotic Susceptibility Test --- p.27 / Chapter 1.3.1.2 --- Bacteriophage Typing --- p.28 / Chapter 1.3.1.3 --- Multilocus Enzyme Electrophoresis --- p.29 / Chapter 1.3.2 --- Genotypic Methods --- p.30 / Chapter 1.3.2.1 --- Analysis of Chromosomal DNA --- p.30 / Chapter 1.3.2.1.1 --- Pulsed-field Gel Electrophoresis (PFGE) --- p.30 / Chapter 1.3.2.1.2 --- Amplified Fragment Length Polymorphism (AFLP) --- p.31 / Chapter 1.3.2.2 --- Analysis of Gene Polymorphism --- p.32 / Chapter 1.3.2.2.1 --- PCR-Restriction Length Polymorphism (PCR-RFLP) --- p.32 / Chapter 1.3.2.2.2 --- Random Amplification of Polymorphic DNA (RAPD) --- p.33 / Chapter 1.3.2.2.3 --- Nucleotide Sequence Typing --- p.33 / Chapter 1.3.2.2.3.1 --- A Typing --- p.33 / Chapter 1.3.2.2.3.2 --- Multilocus Sequence Typing --- p.33 / Chapter 1.3.2.3 --- Hybridization by Southern Blotting --- p.34 / Chapter 1.3.2.3.1 --- MecA/Tn544 Probe Typing --- p.34 / Chapter 1.3.2.3.2 --- Binary Typing --- p.34 / Chapter 1.3.2.4 --- Analysis of Plasmid DNA --- p.35 / Chapter 1.4 --- Objectives of the Project --- p.37 / Chapter Chapter 2 --- Materials & Methods --- p.38 / Chapter 2.1 --- Bacterial Isolates & Culture Conditions --- p.38 / Chapter 2.1.1 --- Study Period & Sample Sources --- p.38 / Chapter 2.1.2 --- Selection of Bacterial Isolates --- p.38 / Chapter 2.1.3 --- Reference Strains --- p.38 / Chapter 2.1.4 --- Culture & Storage Conditions --- p.39 / Chapter 2.1.5 --- Identification of MRSA --- p.39 / Chapter 2.2 --- Antibiotic Susceptibility Testing --- p.40 / Chapter 2.3 --- PCR for MecA Gene --- p.43 / Chapter 2.3.1 --- DNA Preparation and Primer Design for mecA Gene --- p.43 / Chapter 2.3.2 --- PCR Amplification of mecA Gene --- p.45 / Chapter 2.3.2.1 --- Master Mix Preparation --- p.45 / Chapter 2.3.2.2 --- Polymerase Chain Reaction --- p.45 / Chapter 2.3.2.3 --- Analysis of PCR Products by Agarose Gel Electrophoresis --- p.45 / Chapter 2.3.2.4 --- Precautions to Avoid Cross-contamination --- p.46 / Chapter 2.4 --- Pulsed-field Gel Electrophoresis (PFGE) --- p.47 / Chapter 2.4.1 --- PFGE Protocol --- p.47 / Chapter 2.4.1.1 --- DNA Preparation for PFGE --- p.47 / Chapter 2.4.1.2 --- Restriction Enzyme Digestion --- p.48 / Chapter 2.4.1.3 --- Pulsed-field Gel Electrophoresis --- p.48 / Chapter 2.4.1.4 --- Standards & Markers for PFGE --- p.48 / Chapter 2.4.2 --- Optimization of PFGE --- p.49 / Chapter 2.4.3 --- Computer Analysis of PFGE Patterns --- p.49 / Chapter 2.5 --- Amplified Fragment Length Polymorphism (AFLP) --- p.52 / Chapter 2.5.1 --- Amplified Fragment Length Polymorphism Protocol --- p.52 / Chapter 2.5.1.1 --- DNA Preparation --- p.52 / Chapter 2.5.1.2 --- Enzyme Digestion & Ligation --- p.53 / Chapter 2.5.1.3 --- PCR for AFLP --- p.53 / Chapter 2.5.1.4 --- Gel Electrophoresis for AFLP --- p.54 / Chapter 2.5.1.5 --- Standards & Markers for AFLP --- p.55 / Chapter 2.5.2 --- Computer Analysis for AFLP Patterns --- p.55 / Chapter 2.6 --- Phage Typing --- p.58 / Chapter 2.6.1 --- Phages & Propagating Strains --- p.58 / Chapter 2.6.2 --- Phage Typing Protocol --- p.58 / Chapter 2.6.3 --- Data Analysis and Results Interpretation for Phage Tying --- p.62 / Chapter 2.7 --- Other Typing Methods --- p.63 / Chapter 2.7.1 --- PCR Restriction Fragment Length Polymorphism for the coa gene --- p.63 / Chapter 2.7.1.1 --- Primer Design --- p.63 / Chapter 2.7.1.2 --- DNA Preparation --- p.63 / Chapter 2.7.1.3 --- Optimization of PCR --- p.63 / Chapter 2.7.1.3.1 --- PCR Amplification --- p.64 / Chapter 2.7.1.3.2 --- Restriction Enzyme Digestion --- p.64 / Chapter 2.7.2 --- Multilocus Sequence Typing --- p.64 / Chapter 2.7.2.1 --- Preparation of Chromosomal DNA --- p.65 / Chapter 2.7.2.2 --- PCR for MLST Gene --- p.65 / Chapter 2.7.2.3 --- Purification of DNA for Sequencing --- p.67 / Chapter 2.7.2.4 --- PCR for Sequencing --- p.67 / Chapter 2.7.2.5 --- Sequencing by Automatic DNA Analyzer & Data Analysis --- p.68 / Chapter Chapter 3 --- Results --- p.69 / Chapter 3.1 --- Bacterial Isolates --- p.69 / Chapter 3.2 --- Antibiotic Susceptibility Testing --- p.72 / Chapter 3.3 --- PCR for MecA Gene --- p.78 / Chapter 3.4 --- Pulsed-field Gel Electrophoresis --- p.80 / Chapter 3.4.1 --- Optimization of PFGE --- p.80 / Chapter 3.4.2 --- Analysis of PFGE Profiles --- p.83 / Chapter 3.5 --- Amplified Fragment Length Polymorphism --- p.95 / Chapter 3.6 --- Phage Typing --- p.102 / Chapter 3.7 --- Other Typing Methods --- p.109 / Chapter 3.7.1 --- PCR Restriction Fragment Length Polymorphism for the Coa Gene --- p.109 / Chapter 3.7.1.1 --- Optimization of PCR conditions for the Coa Gene --- p.109 / Chapter 3.7.1.2 --- Analysis of MRSA by PCR-RFLP of the coa Gene --- p.109 / Chapter 3.7.2 --- Multilocus Sequence Typing --- p.115 / Chapter Chapter 4 --- Discussion --- p.116 / Chapter 4.1 --- Evaluation of Typing methods for MRSA --- p.116 / Chapter 4.1.1 --- Antibiotic Susceptibility Testing --- p.116 / Chapter 4.1.2 --- Phage Typing --- p.117 / Chapter 4.1.3 --- Pulsed-field Gel Electrophoresis --- p.118 / Chapter 4.1.4 --- Amplified Fragment Length Polymorphism --- p.119 / Chapter 4.1.5 --- PCR-Restriction Fragment Length Polymorphism for the Coa Gene --- p.120 / Chapter 4.1.6 --- Multilocus Sequence Typing --- p.121 / Chapter 4.1.7 --- Conclusion for Method Evaluation --- p.122 / Chapter 4.2 --- "Analysis of Results of Antibiotic Susceptibility Test, Phage Typing, PFGE, AFLP, PCR-RFLP (Coa) & MLST" --- p.124 / Chapter 4.2.1 --- Correlation between Methods --- p.124 / Chapter 4.2.2 --- Clinical Correlation --- p.125 / Chapter 4.2.3 --- Correlation between MRSA Isolates at PWH and Reference Strains --- p.128 / Chapter 4.3 --- Future Research --- p.129 / Chapter 4.4 --- Conclusion --- p.130 / Reference List --- p.131 / Appendix I Reagent & Material Lists for Methods --- p.150 / Appendix II Buffers & Media --- p.156 / Appendix III Coa Gene Sequences of Isolates from PWH and Reference Strains --- p.158 / Appendix IV Unique antibiotic resistance profiles --- p.165 / "Appendix V Details of MRSA iolates selected for AFLP, Phage typing and MLST" --- p.166

Os objetivos do presente estudo foram avaliar os perfis de sensibilidade a antimicrobianos de MRSA isolados de sangue, identificar o tipo de SCCmec e analisar o perfil de DNA desses isolados para verificar a existência de linhagens predominantes. Amostras de MRSA isoladas de sangue foram submetidas ao teste de triagem em ágar para detecção de resistência a oxacilina, teste de sensibilidade aos antimicrobianos, PCR multiplex para detecção dos genes mecA e coa e do tipo de SCCmec, tipagem molecular por PFGE. 89% das amostras de MRSA foi de origem hospitalar. Foi observado multirresistência em 69% dos isolados. 83% dos isolados apresentaram SCCmec tipo IIIA e um perfil PFGE predominante. Foram observadas amostras de MRSA sensíveis a diversas classes de antimicrobianos, portando SCCmec tipo IV, associadas a infecção de origem hospitalar e a tipagem molecular permitiu observar o predomínio de um clone, disseminado em todo o complexo HC / The aims of this study were to evaluate the susceptibility profile of MRSA strains isolated from blood, identify the SCCmec types and analyze the DNA profile in order to determine if there were predominant lineages. Consecutive MRSA blood isolates were submitted to oxacillin agar screening test, antimicrobial susceptibility test, multiplex PCR to detect mecA and coa genes, SCCmec typing and molecular typing by PFGE. 89% of the isolates were nosocomial-acquired. 69% of strains were observed to be multiresistant. 83% of the 223 isolates had SCCmec type IIIA and had a predominant DNA pattern by PFGE. Some strains nosocomial-acquired had SCCmec type IV, resistance only to beta-lactams and demonstrated PFGE pattern with one predominant clone

Staphylococcus aureus é um patógeno versátil, capaz de causar uma grande variedade de infecções. Nos últimos anos, ocorreu um aumento da proporção de infecções causadas por S. aureus resistentes a meticilina (MRSA). A resistência a meticilina deve-se à presença do gene mecA, carreado no cassete cromossômico estafilocócico (SCCmec). Pacientes infectados ou colonizados por MRSA são reservatórios e fontes de disseminação deste microorganismo, em instituições de cuidado à saúde, principalmente através de profissionais transitoriamente colonizados. Freqüentemente, a infecção por MRSA é precedida por um período de colonização. Em 2003 foi observado um aumento das taxas de infecções hospitalares por S. aureus na Enfermaria de Dermatologia do Hospital das Clínicas (EDER), em relação aos cinco anos anteriores. Os objetivos do presente estudo foram avaliar a transmissão hospitalar de MRSA, entre os pacientes da EDER e caracterizar os isolados de MRSA, obtidos de pacientes e funcionários colonizados, presentes nessa unidade. Foi realizada a detecção dos pacientes colonizados por MRSA, através de culturas de vigilância das narinas e, quando possível, culturas de vigilância das lesões de pele, num período de seis meses. A identificação fenotípica foi confirmada por reação em cadeia da polimerase (PCR) multiplex para detecção dos genes mecA e coa. Posteriormente, os isolados de MRSA foram submetidos ao teste de sensibilidade aos antimicrobianos pelo método de microdiluição em caldo, PCR multiplex para determinação do tipo de SCCmec e tipagem molecular por eletroforese em campo pulsado (PFGE). Quarenta e cinco por cento dos pacientes eram portadores de MRSA. No início do estudo, 14% dos funcionários eram portadores de MRSA, no fim eram 18%. Foram obtidas 105 amostras de MRSA, sendo que 11 foram isoladas de funcionários da EDER e 94 isoladas de 64 pacientes que eram portadores deste microorganismo.Sessenta e um por cento dos pacientes classificados como portadores de MRSA, eram positivos na primeira coleta realizada e 39% foram identificados durante o seguimento, nas coletas posteriores. O gene da coagulase foi detectado em todas as 105 amostras e o gene mecA em 101. Todos os isolados foram sensíveis a vancomicina e resistentes a oxacilina e penicilina. Trinta e três por cento dos isolados eram multirresistentes, apresentaram SCCmec tipo IIIA e um perfil PFGE predominante. SCCmec tipo IV foi observado em 59% dos isolados. Não foi possível determinar o tipo de SCCmec de quatro isolados. Na PFGE, o perfil B1 foi o mais prevalente, apresentado por isolados de MRSA SCCmec tipo IV, obtidos de nove pacientes e de três funcionários. A transmissão hospitalar foi caracterizada em 39% dos pacientes portadores. Foi possível observar a participação dos funcionários, na transmissão cruzada de MRSA, na EDER. A colonização por MRSA, dos profissionais de cuidado à saúde, foi transitória. Além da transmissão hospitalar de MRSA, foi possível detectar pacientes que eram portadores de MRSA na admissão / Staphylococcus aureus is a versatile pathogen capable of causing a wide variety of infections. The proportion of nosocomial and community-acquired methicillinresistant Staphylococcus aureus (MRSA) infections has increased in the last years. Methicillin resistance is mediated by the mecA gene which is carried on the Staphylococcal Cassette Chromosome mec (SCCmec). In heath care settings, patients who are colonized or infected with MRSA constitute a reservoir and a source of spread of this microorganism, mainly through transiently colonized health care workers (HCWs). MRSA infections are usually preceded by a period of colonization. In 2003, an increase in the rates of MRSA nosocomial infection in the Dermatology ward of Hospital das Clínicas was observed, in comparison with the five previous years. The aims of this study were to evaluate the nosocomial transmission of MRSA in the Dermatology ward and to characterize MRSA isolates obtained from patients and HCWs. Surveillance cultures of the anterior nares and skin lesions were performed to identify patients who were MRSA carriers, during a period of six months. The phenotypic identification was confirmed by multiplex polymerase chain reaction (PCR), to detect mecA and coa genes. Subsequently, MRSA isolates were submitted to antimicrobial susceptibility testing by microdilution method, multiplex PCR for SCCmec typing and molecular typing by pulsed-field gel electrophoresis (PFGE). Forty-five percent of the patients were MRSA carriers. 14% of the HCWs were MRSA carriers at the beginning of the study and 18% at the end. One hundred and five MRSA isolates were obtained, 11 from HCWs and 94 from 64 patients who were MRSA carriers. Sixty-one percent of the patients, classified as MRSA carriers, were positive on the first culture and 39% were identified during the follow up period in the subsequent cultures. The coagulase gene was detected in all 105 isolates and the mecA gene in 101. All MRSA isolates were susceptible to vancomycin and resistant to oxacillin and penicillin. Thirty-three percent of the isolates were multiresistant, presented SCCmec Type IIIA and showed a predominant PFGE type. The SCCmec type IV was found in 59% of the isolates. It was not possible to determine the SCCmec type of four isolates. The B1 PFGE pattern was the most prevalent, presented by MRSA SCCmec type IV isolates, obtained from nine patients and three HCWs. Nosocomial transmission occurred in 39% of the MRSA carriers. It was possible to observe HCWs MRSA cross- transmission in the Dermatology ward. HCWs were transiently colonized. In addition to nosocomial transmission of MRSA, it was possible to detect patients who were MRSA carriers on admission