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Doctoral Thesis
DOI
10.11606/T.76.2014.tde-13032014-085110
Document
Author
Full name
Andrei Nicoli Gebieluca Dabul
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Carlos, 2014
Supervisor
Committee
Camargo, Ilana Lopes Baratella da Cunha (President)
Darini, Ana Lucia da Costa
Mamizuka, Elsa Masae
Minarini, Luciene Andrade da Rocha
Negrini, Bento Vidal de Moura
Title in Portuguese
Estudo epidemiológico molecular de Staphylococcus aureus resistentes à meticilina (MRSA) isolados no Brasil e estudo da proteína reguladora de resposta GraR
Keywords in Portuguese
Staphylococcus
Epidemiologia
Genômica
Microbiologia médica
Proteínas recombinantes
Abstract in Portuguese
S. aureus é um patógeno que sempre surpreende equipes médicas quanto à sua capacidade de resistir em curto espaço de tempo às mais novas drogas lançadas no mercado. Algumas alterações genéticas que poderiam causar a emergência de VISA foram previamente identificadas, dentre elas uma mutação no sistema de dois componentes GraRS. As proteínas GraR e GraR*, esta última com a mutação que levaria ao fenótipo de VISA, foram estudadas com o intuito de resolver a estrutura tridimensional de ambas e determinar seu papel no processo de resistência à vancomicina. Ensaios de clonagem, expressão, purificação das proteínas e dicroísmo circular foram realizados, porém não houve sucesso na cristalização. No estudo epidemiológico, PFGE dividiu os 36 isolados de MRSA (25 de infecção e 11 de colonização) de um hospital mineiro em 8 pulsotipos. Análise do MLST e SCCmec do pulsotipo A (58,3% das amostras) mostrou que os isolados pertenciam ao CC5 (ST5 e ST105) e continham SCCmecII. Todos os 3 isolados ST239 continham SCCmecIII, sendo relacionados ao BEC. A maioria dos isolados ST5 continha SCCmecII como o Clone NY/J. Observou-se 24% de resistência à tigeciclina nos isolados de sítios de infecção. Dois isolados foram sensíveis à daptomicina depois de 24 horas de incubação mas resistentes após 48 horas. Os resistentes à tigeciclina eram todos ST105-SCCmecII, e de 5 pacientes diferentes. Os resistentes à daptomicina eram ST5-SCCmecII, de pacientes diferentes, e apresentaram algumas mutações no gene rpoB. Uma mudança na linhagem prevalente nos hospitais brasileiros tem sido reportada e, de fato, BEC não era prevalente neste hospital em 2009. ST5-SCCmecII e ST105-SCCmecII foram prevalentes, e ainda o último apresenta o agravante da resistência à tigeciclina quando esta droga ainda não era utilizada no hospital. Não houve disseminação de apenas um pulsotipo, sugerindo que essas linhagens sejam endêmicas ao hospital. O conhecimento do perfil das linhagens locais auxilia na adequação do tratamento empírico dado aos pacientes, além de demonstrar que é preciso ter cuidado ao se administrar novas drogas indiscriminadamente para evitar seleção de clones mais resistentes.
Title in English
Molecular epidemiological study of methicillin-resistant Staphylococcus aureus (MRSA) isolated in Brazil and study of the response regulator protein GraR
Keywords in English
Staphylococcus
Epidemiology
Genomics
Medical microbiology
Recombinant proteins
Abstract in English
S. aureus is a pathogen that always surprises the medical staff regarding its capability to resist to the newest drugs marketed, in a short period of time. Some genetic changes which might cause the emergence of VISA were previously identified, among them a mutation on the twocomponent system GraRS. The proteins GraR and GraR*, the last one with the mutation that would lead to the VISA phenotype, were studied aiming to solve the tridimensional structure of both and determine their role on the process of vancomycin resistance. Cloning, expression, protein purification and circular dichroism experiments were performed, however, the crystallization was not successful. In the epidemiological study PFGE distributed the 36 isolates (25 from infection sites and 11 from colonization) from a hospital in Minas Gerais in 8 pulsotypes. Analysis of MLST and SCCmec of pulsotype A (58.3% of all samples) showed that the isolates belonged to CC5 (ST5 and ST105) and harbored SCCmecII. All three ST239 harbored SCCmecIII, being related to Brazilian Endemic Clone (BEC). Most ST5 isolates harbored SCCmecII as the NY/J clone. It was observed 24% of resistance to tigecycline on the infection sites isolates. On microdilution, 2 isolates were susceptible to daptomycin after 24 hours of incubation, but resistant after 48 hours. Tigecycline-resistants were all ST105-SCCmecII and were isolated from 5 different patients. Daptomycin-resistants were ST5-SCCmecII, from different patients, and they presented some mutations on the gene rpoB. A change in the prevalent lineage of the Brazilian hospitals has been reported and, in fact, the clone BEC was not prevalent in this hospital in 2009. ST5-SCCmecII and ST105-SCCmecII were prevalent, and also, the last one presents the aggravating factor of tigecycline resistance when this drug was not used in the hospital. However, there was no dissemination of only one pulsotype, suggesting these lineages to be endemic to the hospital. Knowledge of the profile of the local lineages helps on the adequation of empiric treatment given to the patients, besides demonstrating that care is needed on administering new drugs indiscriminately to avoid selection of the more resistant clones.
 
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Publishing Date
2014-03-18
 
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