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.

Bakgrund: Antibiotikaresistenta bakterier är idag ett stort hot mot folkhälsan. Det här arbetet fokuserar på MRSA och lyfter fram problematiken som dess spridning innebär för patienten, samhället och vårdpersonal. MRSA-bärarskap kan ge både fysiskt och psykiskt lidande och en infektion orsakad av MRSA kan i värsta fall leda till döden för patienten. Sjuksköterskan har en viktig roll i att upprätthålla sin kompetens och arbeta preventivt för att förhindra spridning av MRSA. Syfte: Syftet var att identifiera och beskriva faktorer som leder till spridning av MRSA vid vårdarbetet av patienter på somatisk vårdavdelning. Metod: En litteraturstudie där 11 kvantitativa och en kvalitativ artikel analyserades utifrån Graneheim och Lundmans (2004) metod för innehållsanalys. Artiklarna kvalitetsgranskades med hjälp av Forsberg och Wengströms (2016) granskningsmallar. Resultat: Resultatet delades in i fem huvudkategorier med nio underkategorier som var och en visar på kompetensbrist hos sjuksköterskor. Litteraturstudien påvisar att faktorerna som leder till spridning av MRSA kan vara flera och att hela vårdteamet bär ett ansvar för att förhindra smittspridning. Sjuksköterskans roll i vårdteamet är att undervisa patienter och närstående, följa gällande rutiner och att upprätthålla sin egen kompetens för att arbeta evidensbaserat. Slutsats: Förhindra spridning av antibiotikaresistenta bakterier är en av sjuksköterskans viktigaste arbetsuppgifter i sin roll att lindra lidande hos patienten. Det är många faktorer som kan göra att MRSA sprids, sjuksköterskor måste reflektera och vara självkritiska i sitt arbete på vårdavdelningar. Evidensbaserat arbete var enligt Nightingale sjuksköterskans möjlighet att förhindra lidande för patienten och ligger till grund för den sjuksköterskeutbildning som finns idag. / Background: Antibiotic resistant bacteria are today a major threat to the public health. This work focuses on methicillin resistant staphylococcus aureus, highlighting the problem that it involves for the patient, society and healthcare professionals. MRSA carriership can cause both physical and mental suffering and an infection caused by MRSA can in worst case lead to the patient’s death. Nurses play an important role in maintaining their work skills to prevent MRSA from spreading in the somatic care. Aim: The aim of this study was to identify and describe factors that lead to the spread of MRSA to patients in somatic care. Method: A literature study where 11 quantitative and one qualitative article were analyzed based on Graneheim and Lundman's (2004) method. The articles quality were reviewed using Forsberg and Wengström’s (2016) checklists. Results: The results were divided into five main categories with nine subcategories; each showing that nurses had a lack of competence. The study shows that the factors that lead to the spread of MRSA may be several, and that the entire healthcare team has a responsibility to prevent infection. The nurse's role in the team is to teach patients and close relatives, follow current procedures and maintain their own skills to work evidence based. Conclusion: Preventing the spread of antibiotic resistant bacteria is one of the most important tasks in role of nursing and in alleviating patient suffering. There are many factors that can cause MRSA to spread. Nurses must reflect and be self-critical in their work in health care departments. Evidence based work was, according to Nightingale, the nurse's ability to prevent suffering for the patient and is the baseline in nursing education currently available today.

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

Introdução: a colonização de indivíduos com HIV/aids por microrganismos patogênicos tem sido associada a maior risco de morbidade e mortalidade, principalmente quando esse microrganismo é o Staphylococcus aureus. Identificar precocemente esta condição permite implementar medidas preventivas do adoecimento a ele relacionado, em nível individual e coletivo. Objetivo: avaliar a prevalência de colonização por Staphylococcus aureus em indivíduos com HIV/aids internados em um hospital escola. Metodologia: estudo de corte transversal, tendo como sujeito pessoas vivendo com HIV/aids, internadas em duas unidades especializadas em HIV/aids de um Hospital Escola do município de Ribeirão Preto- SP. Todos os preceitos éticos foram criteriosamente respeitados. No período de Agosto/2011 a Julho/2012, todos os indivíduos internados foram abordados e para aqueles que aceitaram participar, procedeu-se a coleta de amostra de saliva e secreção nasal, além da coleta de dados sociodemográficos, clínicos e imunológicos, obtidos por meio do prontuário e entrevista individual. As amostras foram encaminhadas e processadas pelo Laboratório de Microbiologia e Sorologia da instituição em estudo. Foram semeadas em meios de cultura ágar sangue e manitol, e após, transferidas para o sistema automatizado Vitek® 2 (BioMérieux(TM)), por meio dos cartões GP Test Kit Vitek® 2, para bactérias gram-positivas. Foram empregados cartões AST-P585 para avaliar a sensibilidade dos Staphylococcus aureus meticilina resistente (MRSA) aos antibióticos. Os dados foram armazenados em planilhas do Microsoft Office Excel 2011 for Mac e organizados por meio do software Statistical Package for the Social Sciences (SPSS), versão 17.0 for Windows. Resultados: De 229 indivíduos com HIV/aids internados nas unidades, 169 constituíram os sujeitos desta pesquisa, dos quais 57,4% eram do sexo masculino, 39,6% apresentaram idade de 40 a 49 anos e 45% tinham o primeiro grau completo. Foram obtidas 338 amostras (169 de secreção nasal e 169 de saliva). A prevalência de colonização por Staphylococcus aureus foi identificada em 20,4% das amostras, com 21,7% de resistência à oxacilina, sendo em secreção nasal 66,7% e em saliva 33,3%. Apresentaram contagem de linfócitos T CD4 abaixo de 200 células/mm3 60,0% dos indivíduos com MRSA nasal e 80,0% estavam em uso de antimicrobianos. Em 40,0% dos indivíduos com MRSA na saliva carga viral foi igual ou superior a 500.001 cópias/mL, e 80,0% destes também usavam antimicrobianos, MRSA nasal e saliva foi identificado em 60,0% dos indivíduos que não estavam em uso de antirretroviral. Conclusão: a prevalência de colonização por Staphylococcus aureus em indivíduos com HIV/aids foi predominante em secreção nasal, com baixa contagem de linfócitos T CD4, com história de internação prévia, uso de antimicrobiano e ausência do uso de antirretroviral, podendo representar importante fonte de infecção. / Introduction: colonization by pathogenic microorganisms in individuals with HIV/AIDS has been associated with increased risk of morbidity and mortality, especially when that organism is Staphylococcus aureus. Early identification of this condition allows implementing preventive measures of illness related to it, both individually and collectively. Objective: to evaluate the prevalence of Staphylococcus aureus colonization in individuals with HIV/AIDS in a teaching hospital. Method: cross-sectional study; the subjects were people living with HIV/AIDS and hospitalized in two specialized HIV/AIDS care units of a Teaching Hospital in the city of Ribeirão Preto. All ethical principles were carefully observed. In the period from August 2011 to July 2012, all subjects hospitalized were approached and, for those who agreed to participate, the collection of saliva and nasal discharge sample was performed, in addition to collecting sociodemographic, clinical and immunological data, obtained through medical record and individual interviews. The samples were forwarded and processed by the Laboratory of Microbiology and Sorology of the institution. They were seeded in blood agar and mannitol-salt-agar culture medium, and thereafter, transferred to the automated system Vitek® 2 (BioMérieux(TM)) through Vitek® 2 Test Cards for Gram-positive bacteria. AST-P585 cards were used to assess the sensitivity of methicillin-resistant Staphylococcus aureus (MRSA) to the antibiotic. Data were stored in spreadsheets of Microsoft Office Excel 2011 for Mac and organized by the Statistical Package for the Social Sciences (SPSS) version 17.0 for Windows. Results: of the 229 individuals with HIV/AIDS hospitalized in the units, 169 were the subjects in this study, of whom 57.4% were male, 39.6% were aged from 40 to 49 years, and 45% had completed elementary school. 338 samples were collected (169 of nasal discharge and 169 of saliva). The prevalence of Staphylococcus aureus colonization was identified in 20.4% of samples, with 21.7% of oxacillin resistance, being 66.7% in nasal discharge and 33.3% in saliva. 60.0% of individuals with MRSA in nasal had lymphocytes T CD4 count below 200 cells/mm3 , and 80.0% were taking antimicrobials. In 40.0% of the individuals with MRSA in saliva, the viral load was equal or higher than 500.001 copies/mL, and 80.0% of these also used antimicrobials; MRSA in nasal and in saliva were detected in 60.0% of individuals who were not taking antiretroviral. Conclusion: the prevalence of Staphylococcus aureus colonization in individuals with HIV/AIDS was prevalent in nasal discharge, had lymphocytes T CD4 low count, with a history of previous hospitalization, antimicrobial use and the absence of antiretroviral use, and it may represent an important source of infection.

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

Introdução: Staphylococcus aureus é um importante patógeno responsável por diversas infecções no ambiente hospitalar com elevada morbi-mortalidade. Os profissionais de saúde, sobretudo os de enfermagem, apresentam elevado risco de colonização por meio do contato direto com indivíduos suscetíveis ou pelo contato com fômites em suas atividades laborais. Com isso, esses profissionais podem disseminar esses microrganismos tanto no ambiente hospitalar como na comunidade. Objetivo: Avaliar a colonização por Staphylococcus aureus em profissionais de enfermagem e a adesão às precauções-padrão. Metodologia: Trata-se de um estudo transversal realizado em clínica médica e em unidades especializadas em prestação de cuidados a pessoas com HIV/aids de um hospital escola do município de Ribeirão Preto. A população foi composta por 100 profissionais de enfermagem que prestam cuidados direto aos pacientes dessas unidades. Foram coletadas amostras de saliva, de secreção nasal e um swab do telefone celular dos profissionais. A coleta ocorreu no período de abril de 2014 a fevereiro de 2015, em três momentos, nos meses zero, quatro e oito. A obtenção dos dados demográficos, profissionais e individuais foi feita por meio de um questionário estruturado. Para avaliar a adesão dos profissionais às precauções-padrão, foram aplicadas dez escalas psicométricas do tipo Likert, já traduzidas e validadas para o português. As amostras coletadas foram encaminhadas e processadas pelo Laboratório de Microbiologia e Sorologia do referido hospital. Resultados: Dos 100 profissionais de enfermagem, 43,0% estavam colonizados por Staphylococcus aureus nas amostras de saliva e/ou de secreção nasal; 36,0% eram Staphylococcus aureus sensível à oxacilina e 7,0% resistente à oxacilina. A prevalência foi de 32,0% na secreção nasal, 1,0%, na saliva e 11,0%, nas amostras de saliva e de secreção nasal. Observou-se que 93,0% dos Staphylococcus aureus apresentaram resistência à penicilina, 43,0%, à eritromicina e 39,5%, à clindamicina. Nenhuma das amostras coletadas da base do telefone celular dos profissionais apresentou Staphylococcus aureus. Os profissionais apresentaram escores médios altos para a Escala de Adesão às Precauções-padrão e Escala de Personalidade de Risco. Não houve diferença significante ao comparar a média dos escores das escalas entre o grupo de colonizados e não colonizados. Armazenamento da escova dental em compartimento fechado/protegido (RP=2,07; IC95%=1,07-3,80) foi um fator de risco para a colonização. Enquanto que, o conhecimento sobre as PP (RP=0,53; IC95%=0,44-0,64) e participação em treinamento sobre as PP (RP=0,52; IC95%=0,43-0,64) apresentaram-se como um fator de proteção para a não colonização. Conclusão: A cavidade nasal foi um importante sítio de colonização quando comparada à cavidade oral, sendo um sítio relevante e indicado para a coleta em estudos que investigam a prevalência de colonização por Staphylococcus aureus. O conhecimento sobre precauções-padrão e a participação em treinamentos foram fatores associados à proteção para a não colonização. No entanto, um dos fatores determinantes para a adesão às precauções-padrão é a percepção de suscetibilidade do profissional de adquirir e disseminar esses microrganismos / Introduction: Staphylococcus aureus is an important pathogen responsible for several infections in hospitals with high morbidity and mortality rates. Health professionals, especially nurses, are at increased risk of colonization through direct contact with susceptible individuals or by contact with fomites in their work activities. As a consequence, these professionals can disseminate these microorganisms both in the hospital and in the community. Objective: To assess colonization by Staphylococcus aureus in nursing professionals and the adherence to standard precautions. Methods: This cross-sectional study was carried out in an outpatient clinic and in specialized units that provide care to people with HIV/Aids, in a teaching hospital of Ribeirão Preto. The population consisted of 100 nursing professionals who provide direct care to patients of these units. Samples of saliva, nasal secretions and a swab from the mobile phone of professionals were collected. The collection took place from April 2014 to February 2015 on three occasions, in months zero, four and eight. A structured questionnaire was used to obtain demographic, occupational and personal data. To assess the adherence of professionals to standard precautions, ten Likert-type psychometric scales, translated and validated for Portuguese, were applied. The collected samples were forwarded to and processed by the Microbiology and Serology Laboratory of the hospital. Results: Of the 100 nursing professionals, 43% were colonized with Staphylococcus aureus in saliva samples and/or nasal secretions; 36% were oxacillin-sensitive Staphylococcus aureus and 7.0% oxacillin-resistant. The prevalence was 32% in nasal secretion, 1% in saliva and 11% in saliva samples and nasal discharge. It was observed that 93% of Staphylococcus aureus were penicillin-resistant strains, 43% resistant to erythromycin and 39.5% to clindamycin. None of the samples collected from the base of the mobile phone of the professionals presented Staphylococcus aureus. Professionals had high mean scores for the Compliance with Standard Precautions Scale and the Risk Personality Scale. There was no significant difference when comparing the average scores of scales between the colonized and non-colonized groups. Storing the toothbrush in a closed/protected space (PR=2.07; CI95%=1.07-3.80) was a risk factor for colonization. Knowledge of the SP (PR=0.53, CI95%=0.44-0.64) and participation in training on SP (PR=0.52, CI95%=0.43-0.64) were a protective factor for non- colonization. Conclusion: The nasal cavity was an important colonization site compared to the oral cavity, the nasal site is relevant and recommended for collection in studies investigating the prevalence of colonization for Staphylococcus aureus. Knowledge of the standard precautions and participation in training on standard precautions were protective factors for non-colonization. However, one of the determining factors for adherence to standard precautions is the perceived susceptibility of professional to acquire and disseminate these microorganisms

Introdução: Staphylococcus aureus é um importante patógeno responsável por diversas infecções no ambiente hospitalar com elevada morbi-mortalidade. Os profissionais de saúde, sobretudo os de enfermagem, apresentam elevado risco de colonização por meio do contato direto com indivíduos suscetíveis ou pelo contato com fômites em suas atividades laborais. Com isso, esses profissionais podem disseminar esses microrganismos tanto no ambiente hospitalar como na comunidade. Objetivo: Avaliar a colonização por Staphylococcus aureus em profissionais de enfermagem e a adesão às precauções-padrão. Metodologia: Trata-se de um estudo transversal realizado em clínica médica e em unidades especializadas em prestação de cuidados a pessoas com HIV/aids de um hospital escola do município de Ribeirão Preto. A população foi composta por 100 profissionais de enfermagem que prestam cuidados direto aos pacientes dessas unidades. Foram coletadas amostras de saliva, de secreção nasal e um swab do telefone celular dos profissionais. A coleta ocorreu no período de abril de 2014 a fevereiro de 2015, em três momentos, nos meses zero, quatro e oito. A obtenção dos dados demográficos, profissionais e individuais foi feita por meio de um questionário estruturado. Para avaliar a adesão dos profissionais às precauções-padrão, foram aplicadas dez escalas psicométricas do tipo Likert, já traduzidas e validadas para o português. As amostras coletadas foram encaminhadas e processadas pelo Laboratório de Microbiologia e Sorologia do referido hospital. Resultados: Dos 100 profissionais de enfermagem, 43,0% estavam colonizados por Staphylococcus aureus nas amostras de saliva e/ou de secreção nasal; 36,0% eram Staphylococcus aureus sensível à oxacilina e 7,0% resistente à oxacilina. A prevalência foi de 32,0% na secreção nasal, 1,0%, na saliva e 11,0%, nas amostras de saliva e de secreção nasal. Observou-se que 93,0% dos Staphylococcus aureus apresentaram resistência à penicilina, 43,0%, à eritromicina e 39,5%, à clindamicina. Nenhuma das amostras coletadas da base do telefone celular dos profissionais apresentou Staphylococcus aureus. Os profissionais apresentaram escores médios altos para a Escala de Adesão às Precauções-padrão e Escala de Personalidade de Risco. Não houve diferença significante ao comparar a média dos escores das escalas entre o grupo de colonizados e não colonizados. Armazenamento da escova dental em compartimento fechado/protegido (RP=2,07; IC95%=1,07-3,80) foi um fator de risco para a colonização. Enquanto que, o conhecimento sobre as PP (RP=0,53; IC95%=0,44-0,64) e participação em treinamento sobre as PP (RP=0,52; IC95%=0,43-0,64) apresentaram-se como um fator de proteção para a não colonização. Conclusão: A cavidade nasal foi um importante sítio de colonização quando comparada à cavidade oral, sendo um sítio relevante e indicado para a coleta em estudos que investigam a prevalência de colonização por Staphylococcus aureus. O conhecimento sobre precauções-padrão e a participação em treinamentos foram fatores associados à proteção para a não colonização. No entanto, um dos fatores determinantes para a adesão às precauções-padrão é a percepção de suscetibilidade do profissional de adquirir e disseminar esses microrganismos / Introduction: Staphylococcus aureus is an important pathogen responsible for several infections in hospitals with high morbidity and mortality rates. Health professionals, especially nurses, are at increased risk of colonization through direct contact with susceptible individuals or by contact with fomites in their work activities. As a consequence, these professionals can disseminate these microorganisms both in the hospital and in the community. Objective: To assess colonization by Staphylococcus aureus in nursing professionals and the adherence to standard precautions. Methods: This cross-sectional study was carried out in an outpatient clinic and in specialized units that provide care to people with HIV/Aids, in a teaching hospital of Ribeirão Preto. The population consisted of 100 nursing professionals who provide direct care to patients of these units. Samples of saliva, nasal secretions and a swab from the mobile phone of professionals were collected. The collection took place from April 2014 to February 2015 on three occasions, in months zero, four and eight. A structured questionnaire was used to obtain demographic, occupational and personal data. To assess the adherence of professionals to standard precautions, ten Likert-type psychometric scales, translated and validated for Portuguese, were applied. The collected samples were forwarded to and processed by the Microbiology and Serology Laboratory of the hospital. Results: Of the 100 nursing professionals, 43% were colonized with Staphylococcus aureus in saliva samples and/or nasal secretions; 36% were oxacillin-sensitive Staphylococcus aureus and 7.0% oxacillin-resistant. The prevalence was 32% in nasal secretion, 1% in saliva and 11% in saliva samples and nasal discharge. It was observed that 93% of Staphylococcus aureus were penicillin-resistant strains, 43% resistant to erythromycin and 39.5% to clindamycin. None of the samples collected from the base of the mobile phone of the professionals presented Staphylococcus aureus. Professionals had high mean scores for the Compliance with Standard Precautions Scale and the Risk Personality Scale. There was no significant difference when comparing the average scores of scales between the colonized and non-colonized groups. Storing the toothbrush in a closed/protected space (PR=2.07; CI95%=1.07-3.80) was a risk factor for colonization. Knowledge of the SP (PR=0.53, CI95%=0.44-0.64) and participation in training on SP (PR=0.52, CI95%=0.43-0.64) were a protective factor for non- colonization. Conclusion: The nasal cavity was an important colonization site compared to the oral cavity, the nasal site is relevant and recommended for collection in studies investigating the prevalence of colonization for Staphylococcus aureus. Knowledge of the standard precautions and participation in training on standard precautions were protective factors for non-colonization. However, one of the determining factors for adherence to standard precautions is the perceived susceptibility of professional to acquire and disseminate these microorganisms