Molecular epidemiology of methicillin-resistant Staphylococcus aureus through analysis of chromosome DNA profile and typing of Sthaphylococcal Cassete Chromosome mec (SCCmec) in tertiary hospital of Campinas

Orientador: Maria Clara PadoveseDissertação (mestrado) - Universidade Estadual de Campinas. Faculdade de Ciências MédicasResumo: Staphylococcus aureus é reconhecido como um dos principais patógenos humanos capazes de causar uma grande variedade de infecções. As infecções causadas pelo Staphylococcus aureus resistentes a meticilina (MRSA) tiveram grande aumento na última década. MRSA é capaz de produzir uma proteína ligadora de penicilina (PBP) específica, chamada PBP2a ou PBP2' que possui baixa afinidade de ligação aos antibióticos _-lactâmicos. Esta proteína é codificada pelo gene mecA, o qual é carreado por um elemento genético móvel, denominado cassete cromossômico estafilocócico (SCCmec) e está integrado ao cromossomo dos MRSA. Foram descritos cinco tipos de SCCmec, sendo eles tipo I, II, III, IV e V e seus variantes, onde o tipo IIIA é característico do clone endêmico brasileiro. Os objetivos do presente estudo foram identificar o perfil de SCCmec de um conjunto de amostras de MRSA obtidas de pacientes atendidos no Hospital das clínicas da Unicamp (HC-Unicamp), descrever sua distribuição nas diversas unidades do HC e comparar o perfil de SCCmec utilizando-se as técnicas de reação em cadeia da polimerase (PCR-multiplex) com o perfil genômico definido por meio de eletroforese em campo pulsado - Pulsed Field Gel Eletrophoresis (PFGE). Foram estudadas 99 amostras obtidas, por razões clínicas, no período de 1999 a 2004. Todas as amostras foram submetidas ao teste de difusão em disco para confirmação da resistência a oxacilina. Como resultados, foram identificados 26 diferentes perfis de PFGE e seus respectivos perfis relacionados. Os perfis genômicos apresentaram uma distribuição temporal, podendo ser agrupados em conjuntos clonais que foram substituídos ao longo do período, com exceção de uma única cepa, que apresentou ocorrências esporádicas durante o período do estudo. Dentre os 99 isolados, 92% apresentaram SCCmec tipo IIIA, característico do clone endêmico brasileiro e 7% apresentaram SCCmec tipo IV. A revisão de prontuários não indica fatores que possam sugerir aquisição extra-hospitalar das cepas estudadasAbstract: Staphylococcus aureus has been recognized as an important human pathogen capable of causing a wide variety of infections. Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) had greatly increased over the past decade. MRSA is capable of produce a specific penicillin-binding protein (PBP) called PBP2a or PBP2' that hold reduced affinities for binding to _-lactam antibiotics. This protein is encoded by the mecA gene, which is carried by a mobile genetic element, called staphylococcal cassette chromosome mec (SCCmec) and is integrated to the chromosome of MRSA. It has been described five types of SCCmec, which are type I, II, III, IV and V and its variants, where the type IIIA is characteristic of Brazilian endemic clone. The goals of the study were to identify the SCCmec type from a set of MRSA samples obtained from patients seeking assistance at Hospital das Clínicas da Unicamp (HC-Unicamp), to describe its distribution in various units of HC and to compare the SCCmec profile with the genomic profile defined using the polymerase chain reaction (PCR-multiplex) techniques and pulsed-field electrophoresis (PFGE), respectively. It was studied 99 samples collected for clinical reasons during a period that extended from 1999 to 2004. All the samples have been submitted to the disk diffusion test for oxacilina resistance confirmation. It was carried through PFGE for genomic evaluation and PCR-multiplex for SCCmec type detection. It have been found 26 different PFGE profiles and their respective related profiles which have presented a temporal distribution, showing clonal groups of strains that have been substituted throughout the period, except of only one strain that was present sporadically during all the period of the study. Among 99 isolated, 92% have shown SCCmec type IIIA, characteristic of Brazilian endemic clone and 7% have shown SCCmec type IV, in a way that all strains seem to have been acquired in the hospital environmentMestradoCiencias BasicasMestre em Clinica Medic

A observação do céu nos livros didáticos de ciências no Brasil

A atividade de observação do céu remonta da antiguidade e sua presença na educação básica é defendida por distintos pesquisadores. No entanto, as atividades de observação direta do céu requerem tempos (duração e horários das atividades) e espaços (a céu aberto, fora da sala de aula) que fogem do sistema tradicional escolar, além da necessidade de condições climáticas para tal. Para verificar a natureza das atividades propostas, analisaram-se nove coleções de Livros Didáticos de Ciências distribuídos nacionalmente. A análise permitiu perceber a presença de propostas didáticas, no entanto, ainda centradas em curtos períodos de tempo, dificultando a percepção de fenômenos cíclicos e pro¬postas como atividades a serem realizadas fora do espaço escolar dificultando a participação in loco do professor, necessitando para tal um maior detalhamento da proposta e autonomia discente

Genetic and virulence characterization of colistin-resistant and colistin-sensitive A. baumannii clinical isolates.

Treatment of infections caused by A. baumannii is becoming a challenge due to the ability to develop multidrug-resistance, virulence, and high mortality. We described the colistin resistance and virulence genes present in sixA. baumannii clinical isolates using WGS, expression by qPCR, and virulence in the Galleria mellonella model. The colistin-resistant isolates were assigned as ST233 and the colistin-susceptible isolates as ST236 and ST407. The colistin-resistant isolates contained mutations within PmrA/PmrB, and the pmrA showed up-regulation in all of them. Only one colistin-resistant isolate indicating virulence in G. mellonella. This particular isolate belonged to a different clone, and it was the only isolate that presented non-synonymous mutations in pmrB. Colistinresistance in A. baumannii isolates seems to be caused by up-regulation of pmrA gene. Only one isolate appeared to be virulent in the G. mellonella model. This finding indicating low virulence in isolates belonging to emerging clones circulating in our hospital

Determination of antimicrobial combinations effect against Acinetobacter baumannii multidrug-resistant isolates with evaluation of resistance mechanisms

Acinetobacter baumannii é um importante agente de infecções relacionadas à assistência à saúde, principalmente nas unidades de terapia intensiva no Brasil, sua resistência a antimicrobianos vem aumentando nas últimas décadas e as opções para o tratamento são restritas. Em 2011, no HC-FMUSP, ocorreu um surto de infecção por A. baumannii resistente a todos os antibióticos e desde então isolados resistentes a todas as classes de antibióticos vêm sendo identificados no hospital. O estudo investigou o efeito de combinações antimicrobianas contra 20 isolados clínicos de A. baumannii, sendo sete isolados resistentes (2011 a 2012) e treze sensíveis a colistina (2002 a 2004), obtidas do banco de cepas do LIM-54 com diferentes mecanismos de resistência. Foram realizados, concentração inibitória mínima dos antibióticos, avaliação da clonalidade por Pulsedfield gel electrophoresis, detecção de mecanismos de resistência por reação de amplificação em cadeia da polimerase, análise de proteínas da membrana externa e baseado na clonalidade e o sequenciamento total do genoma de quinze isolados. Sinergismo foi investigado usando os métodos de checkerboard e time-kill. Para monitorização da expressão do sistema regulatório PmrCAB e dos genes responsáveis pela biossíntese do lipopolissacarídeo, foi realizada reação em cadeia da polimerase quantitativa. Todos os isolados foram resistentes ao meropenem e a rifampicina. OXA- 23 e OXA-143 foram as carbapenemases mais frequentes. Quatro isolados mostraram perda de uma proteína de membrana externa denominada OMP 43kDa. Os isolados sensíveis a colistina pertenciam a diferentes clones e Multilocus Sequence Types, também apresentaram o maior efeito sinérgico com fosfomicina-amicacina. A resistência a colistina foi associada com a superexpressão do gene pmrA. Seis isolados resistentes a colistina, pertenciam ao Complexo Clonal 113 e o maior efeito sinérgico foi observado com combinações de colistina-rifampicina seguido de colistina-vancomicina. Foram encontrados diferentes genes de virulência envolvidos com formação de biofilme, aderência, produção de enzimas e captação de ferroAcinetobacter baumannii is an important agent of infections related to health care, especially in intensive care units in Brazil, its antimicrobial resistance has increased in recent decades and the options for treatment are restricted. In 2011, in the HC-FMUSP, there was an outbreak of A. baumannii infection resistant to all antibiotics and since then isolates resistant to all classes of antibiotics have been identified in the hospital. The study investigated the effect of antimicrobial combinations against 20 clinical isolates of A. baumannii, seven isolates colistin resistant (2011-2012) and thirteen colistin sensitive (2002-2004) from LIM-54 strains bench with different resistance mechanisms. Minimum inhibitory concentration of antibiotics, clonality evaluation by pulsed-field gel electrophoresis and detection of resistance mechanisms by polymerase chain reaction, outer membrane protein analysis and based on clonality, the whole genome sequencing of fifteen isolates were performed. Synergism was determined using checkerboard and time-kill methods. For expression monitoring of the regulatory system PmrCAB and the genes responsible for the biosynthesis of lipopolysaccharide, it was performed quantitative polymerase chain reaction. All isolates were resistant to meropenem and rifampicin. OXA-23 and OXA-143 were the most frequent carbapenems. Four isolates showed loss of one outer membrane protein called OMP 43kDa. Colistin susceptible isolates belonged to different clones and Multilocus Sequence Types; it also showed the greatest synergistic effect with fosfomycin-amikacin. The colistin resistance was involved in overexpression of the pmrA gene. Six colistin-resistant isolates belonged to Clonal Complex 113 and higher synergistic effect were observed with colistin-rifampicin followed by colistin-vancomycin combinations for these isolates. We found different virulence genes involved in biofilm formation, adhesion, enzyme production and iron uptak

A observação do céu nos livros didáticos de ciências no Brasil

A atividade de observação do céu remonta da antiguidade e sua presença na educação básica é defendida por distintos pesquisadores. No entanto, as atividades de observação direta do céu requerem tempos (duração e horários das atividades) e espaços (a céu aberto, fora da sala de aula) que fogem do sistema tradicional escolar, além da necessidade de condições climáticas para tal. Para verificar a natureza das atividades propostas, analisaram-se nove coleções de Livros Didáticos de Ciências distribuídos nacionalmente. A análise permitiu perceber a presença de propostas didáticas, no entanto, ainda centradas em curtos períodos de tempo, dificultando a percepção de fenômenos cíclicos e pro¬postas como atividades a serem realizadas fora do espaço escolar dificultando a participação in loco do professor, necessitando para tal um maior detalhamento da proposta e autonomia discente

Methicillin-resistant Staphylococcus aureus DNA electrophoretic pattern: temporal changes in an endemic hospital environment Patrón electroforético del ADN de Staphylococcus aureus resistente a la meticilina: cambios temporales en un medio hospitalario endémico

OBJECTIVE: To describe the analysis of geographical and temporal distribution of DNA profiles determined by pulsedfield gel electrophoresis (PFGE) of methicillinresistant Staphylococcus aureus (MRSA) strains isolated from hospitalized patients in a tertiary care university hospital in Brazil. METHODS: Ninetynine samples of MRSA obtained from 89 patients in the period 1999- 2004 were studied. MRSA strains were isolated from central venous catheters (33 isolates) and bloodstream infections (66 strains). PFGE with 20 units of SmaI restriction endonuclease was used for genomic typing. RESULTS: Analysis of DNA PFGE of 99 strains of MRSA revealed 26 profiles and their respective related profiles. The mean time interval for detecting MRSA infection was 26 days from hospital admission. Forty-nine patients (57.6%) had a recent hospitalization. The DNA PFGE MRSA profiles were distributed in three clonal groups-I, II, and III-according to the period of time when the MRSA strains were isolated. DNA PFGE MRSA profiles were spread homogeneously through all hospital wards. CONCLUSIONS: Changes in the distribution of DNA PFGE MRSA profiles were largely temporal, with clonal groups being replaced over time, without predominance in any hospital ward or any specific area of the hospital.<br>OBJETIVO: Analizar la distribución geográfica y temporal de los perfiles de ADN determinados mediante electroforesis en gel de campo pulsado (PFGE) de cepas de Staphylococcus aureus resistente a la meticilina (SARM) aisladas de pacientes internados en un hospital universitario de atención terciaria en el Brasil. MÉTODOS: Se estudiaron 99 muestras de SARM obtenidas 89 de pacientes en el período 1999-2004. Las cepas de SARM se aislaron de infecciones de catéteres venosos centrales (33 aislados) y del torrente sanguíneo (66 cepas). Para la tipificación genómica se empleó PFGE con 20 unidades de endonucleasa de restricción SmaI. RESULTADOS: El análisis del ADN de 99 cepas de SARM mediante PFGE reveló 26 perfiles, con sus respectivos perfiles relacionados. El intervalo medio de detección de la infección por SARM fue de 26 días desde el ingreso al hospital. En 49 pacientes (57,6%) había habido una hospitalización previa reciente. Los perfiles de ADN de SARM determinados mediante PFGE se distribuyeron en tres grupos clonales -I, II y III- según el período en el que se aislaron las cepas de SARM. Estos perfiles de ADN se encontraban distribuidos de manera homogénea en todos los servicios del hospital. CONCLUSIONES: Los cambios en la distribución de los perfiles de ADN de SARM determinados mediante PFGE fueron en gran medida temporales, con reemplazo de los grupos clonales con el transcurso del tiempo, y sin predominio en ningún servicio ni área específica del hospital

Methicillin-resistant Staphylococcus Aureus Dna Electrophoretic Pattern: Temporal Changes In An Endemic Hospital Environment.

To describe the analysis of geographical and temporal distribution of DNA profiles determined by pulsed-field gel electrophoresis (PFGE) of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from hospitalized patients in a tertiary care university hospital in Brazil. Ninetynine samples of MRSA obtained from 89 patients in the period 1999-2004 were studied. MRSA strains were isolated from central venous catheters (33 isolates) and bloodstream infections (66 strains). PFGE with 20 units of SmaI restriction endonuclease was used for genomic typing. Analysis of DNA PFGE of 99 strains of MRSA revealed 26 profiles and their respective related profiles. The mean time interval for detecting MRSA infection was 26 days from hospital admission. Forty-nine patients (57.6%) had a recent hospitalization. The DNA PFGE MRSA profiles were distributed in three clonal groups-I, II, and III-according to the period of time when the MRSA strains were isolated. DNA PFGE MRSA profiles were spread homogeneously through all hospital wards. Changes in the distribution of DNA PFGE MRSA profiles were largely temporal, with clonal groups being replaced over time, without predominance in any hospital ward or any specific area of the hospital.30535-

Pé diabético: apresentação clínica e relação com o atendimento na atenção básica

Estudio transversal, epidemiológico, realizado en un hospital público de Recife, con los siguientes objetivos: evaluar las lesiones de los pies de pacientes con diabetes mellítus por métodos clínicos y verificar la existencia de asociación entre el riesgo de amputación y el uso de la atención primaria de salud. La muestra estuvo conformada por 61 pacientes diabéticos con úlceras en los pies. Se verificó que 41% (25/61) fue clasificado como grado 4 y 80,3% fue caracterizado como de alto riesgo de amputación (49/61). La falta de sensibilidad al monofllamentc (44/51) y diapasón (43/50) y la ausencia de palpación de pulsos distales (44/51 y 41/47) se asociaron con riesgo de amputación. Si no se realiza el examen de los pies, el riesgo de amputación es de 1,9 veces. El estudio demostró la importancia de la evaluación y clasificación del pie diabético para la organización de un plan adecuado de detección y prevención

Antimicrobial Combinations against Pan-Resistant Acinetobacter baumannii Isolates with Different Resistance Mechanisms.

The study investigated the effect of antibiotic combinations against 20 clinical isolates of A. baumannii (seven colistin-resistant and 13 colistin-susceptible) with different resistance mechanisms. Clinical data, treatment, and patient mortality were evaluated. The following methods were used: MIC, PCRs, and outer membrane protein (OMP) analysis. Synergy was investigated using the checkerboard and time-kill methods. Clonality was evaluated by PFGE. Based on clonality, the whole genome sequence of six A. baumannii isolates was analyzed. All isolates were resistant to meropenem, rifampicin, and fosfomycin. OXA-23 and OXA-143 were the most frequent carbapenemases found. Four isolates showed loss of a 43kDa OMP. The colistin-susceptible isolates belonged to different clones and showed the highest synergistic effect with fosfomycin-amikacin. Among colistin-resistant isolates, the highest synergistic effect was observed with the combinations of colistin-rifampicin followed by colistin-vancomycin. All colistin-resistant isolates harbored blaOXA-23-like and belonged to CC113. Clinical and demographic data were available for 18 of 20 patients. Fourteen received treatment and eight patients died during treatment. The most frequent site of infection was the blood in 13 of 14 patients. Seven patients received vancomycin plus an active drug against A. baumannii; however, mortality did not differ in this group. The synergistic effect was similar for colistin-susceptible isolates of distinct clonal origin presenting with the same resistance mechanism. Overall mortality and death during treatment was high, and despite the high synergism in vitro with vancomycin, death did not differ comparing the use or not of vancomycin plus an active drug against A. baumannii

DNA sequences of oligonucleotides used in polymerase chain reactions to detect carbapenemase and outer-membrane proteins of <i>A</i>. <i>baumannii</i> isolates.

<p>DNA sequences of oligonucleotides used in polymerase chain reactions to detect carbapenemase and outer-membrane proteins of <i>A</i>. <i>baumannii</i> isolates.</p