Caracterização microbiológica e epidemiologia molecular de Enterococcus spp. isolados de infecções da corrente sanguínea de pacientes do Projeto SCOPE Brasil
Data
2015-05-25
Autores
Chagas Neto, Thomas Cardoso das 
Orientadores
Pignatari, Antonio Carlos Campos
Tipo
Tese de doutorado
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ISSN da Revista
Título de Volume
Resumo
Introdução: Infecções de corrente sanguínea (ICS) por Enterococcus spp., especialmente quando relacionadas com isolados resistentes à vancomicina, apresentam altas taxas de morbidade e terapia antimicrobiana limitada. Objetivos: Caracterizar o perfil epidemiológico e molecular de Enterococcus spp. isolados de hemoculturas de pacientes com ICS participantes do programa SCOPE Brasil. Material e Métodos: Entre junho de 2007 e dezembro de 2011, 144 cepas de Enterococcus spp. foram isoladas a partir do primeiro episódio de ICS hospitalar de pacientes de 12 centros médicos de quatro regiões brasileiras. Todas as 144 amostras foram re-identificadas pelo sistema automatizado Phoenix® e a identificação final das espécies foi confirmada por espectrometria de massa por ionização e dessorção a laser - assistida por matriz - matrix associated laser desorption-ionization - time of flight (MALDI-TOF MS) (Bruker Microflex, Vitek MS® BMX) e reação da polimerase em cadeia (PCR) (oligonucleotídeos, ddlE.faecalis, ddlE.faecium). Além disso, os isolados foram submetidos a testes de sensibilidade aos antimicrobianos pelo sistema Phoenix® e para aqueles que apresentaram resistência à vancomicina, foram submetidos ao Etest® para confirmação das CIMs de daptomicina, linezolida, teicoplanina e vancomicina. O mecanismo de resistência a vancomicina foi confirmado por PCR para os genes vanA e vanB e a tipagem por gel de eletroforese em campo pulsado - pulsed field gel electrophoresis (PFGE) foram feitas para os isolados resistentes à vancomicina. A tipagem pela técnica de sequenciamento de múltiplos loci - multilocus sequence typing MLST foi realizada para 22 isolados selecionados com base nos perfis de PFGE. Avaliou-se a aplicabilidade da técnica de MALDI-TOF MS para tipagem de Enterococcuss spp. comparando os dendrogramas do tipo UHCA e UPGMA com o algoritimo de Ward. Resultados: A espécie mais prevalente foi o E. faecalis com 110 isolados, seguidos por E. faecium com 33 isolados e apenas um E. durans. Todos os 35 isolados resistentes à vancomicina apresentaram o gene vanA, dos quais 15 da espécies E. faecalis (EFSRV) e 20 da E. faecium (EFMRV). De acordo com a PFGE foram encontrados dois padrões (B e C), que prevalecem entre EFSRV entre EFMRV foi observado um maior polimorfismo e três grupos, sendo padrão A, o predominante. MLST evidenciou em E. faecalis os STs 9; 103; 525 e 526. Enquanto em E. faecium STs encontrados foram 18, 412, 478 e 896. Conclusões: E. faecalis foi a espécie mais prevalente. Neoplasias foram as doenças de base mais encontradas. ST526 foi o mais encontrado entre os EFSRV e o ST412 o mais prevalente entre os EFMRV. A tipagem com os espectros produzidos no Microflex LT® e analisados no BioNumerics 7.5 apresentou uma boa concordância com o MLST para E. faecalis, quando utilizado o algoritmo de Ward desconsiderando a intensidade dos espectros. Mesmo utilizando Ward para E. faecium não houve boa correlação com o MLST.
Introduction: Bloodstream infection (BSI) caused by Enterococcus spp. particulally related to vancomycin-resistant isolates, present high rates of morbi-mortality and limited antimicrobial therapy. Objective: To characterize the epidemiological and molecular profile of Enterococcus spp. isolates from patients of SCOPE Brazilian program followed with BSI from June 2007 to December 2011. Methods: we enrolled 144 strains of Enterococcus spp. isolated from the first hospital episode of BSI of patients admited to 12 hospital from four Brazillian regions . All these 144 isolates were reviewed by the automated system BD PhoenixTM and their final species identification was confirmed by Matrix Associated Laser Desorption-Ionization ? Time of Flight (MALDI-TOF) (Bruker Microflex, VitekMS BMX) and PCR (ddlE.faecalis, ddlE.faecium). In addition, the isolates were submited to antimicrobial susceptibility testing by PhoenixBD. Those isolates resistant to vancomycin were also examined using EtestTM to confirm the minimum inhibitory concentration (MIC) for daptomycin, linezolid, vancomycin and teicoplanin. The mechanism of resistance to vancomycin was confirmed by using vanA and vanB gene PCR and pulsed-field gel electrophoresis (PFGE) was used to typing. We performed multilocus sequence typing (MLST) in 22 isolates that were selected based on PFGE profiles. We further evaluated the applicability of Matrix-Assisted Laser Desorption Ionization Time-of- Flight (MALDI-TOF) technology for Enterococcus spp. typing comparing the dendrograms obtained by UHCA, UPGMA and Ward algoritm. Results: The most prevalent species was E. faecalis (110 isolates), followed by E. faecium (33 isolates) and only one E. durans. All 35 vancomycin resistant isolates (15 E. faecalis, EFSRV and 20 E. faecium, EFMRV) carried vanA gene. According with PFGE analysis, we found the patterns B and C as more prevalent in EFSRV. Among the EFMRV isolates we observed a greater polymorphism (patterns A, B, and C) among all three groups, being predominant the pattern A. We evidenced by MLST the sequence types (STs) 9; 103; 525, and 526 in E. faecalis, whereas 18, 412, 478 and 896 in E. faecium. Conclusions: E. faecalis was the most prevalent specie. Cancer was the most frequent underlying disease on the course of BSI. ST526 and ST412 were the most frequent among EFSRV and EFMRV isolates respectively. EFSRV typing by using Microflex LTTM and BioNumerics 7.5 program correlated with MLST results when Ward's algorithm was applied not taking into account the intensity of the spectra. However, we do not observe the same correlation among EFMRV isolates.
Introduction: Bloodstream infection (BSI) caused by Enterococcus spp. particulally related to vancomycin-resistant isolates, present high rates of morbi-mortality and limited antimicrobial therapy. Objective: To characterize the epidemiological and molecular profile of Enterococcus spp. isolates from patients of SCOPE Brazilian program followed with BSI from June 2007 to December 2011. Methods: we enrolled 144 strains of Enterococcus spp. isolated from the first hospital episode of BSI of patients admited to 12 hospital from four Brazillian regions . All these 144 isolates were reviewed by the automated system BD PhoenixTM and their final species identification was confirmed by Matrix Associated Laser Desorption-Ionization ? Time of Flight (MALDI-TOF) (Bruker Microflex, VitekMS BMX) and PCR (ddlE.faecalis, ddlE.faecium). In addition, the isolates were submited to antimicrobial susceptibility testing by PhoenixBD. Those isolates resistant to vancomycin were also examined using EtestTM to confirm the minimum inhibitory concentration (MIC) for daptomycin, linezolid, vancomycin and teicoplanin. The mechanism of resistance to vancomycin was confirmed by using vanA and vanB gene PCR and pulsed-field gel electrophoresis (PFGE) was used to typing. We performed multilocus sequence typing (MLST) in 22 isolates that were selected based on PFGE profiles. We further evaluated the applicability of Matrix-Assisted Laser Desorption Ionization Time-of- Flight (MALDI-TOF) technology for Enterococcus spp. typing comparing the dendrograms obtained by UHCA, UPGMA and Ward algoritm. Results: The most prevalent species was E. faecalis (110 isolates), followed by E. faecium (33 isolates) and only one E. durans. All 35 vancomycin resistant isolates (15 E. faecalis, EFSRV and 20 E. faecium, EFMRV) carried vanA gene. According with PFGE analysis, we found the patterns B and C as more prevalent in EFSRV. Among the EFMRV isolates we observed a greater polymorphism (patterns A, B, and C) among all three groups, being predominant the pattern A. We evidenced by MLST the sequence types (STs) 9; 103; 525, and 526 in E. faecalis, whereas 18, 412, 478 and 896 in E. faecium. Conclusions: E. faecalis was the most prevalent specie. Cancer was the most frequent underlying disease on the course of BSI. ST526 and ST412 were the most frequent among EFSRV and EFMRV isolates respectively. EFSRV typing by using Microflex LTTM and BioNumerics 7.5 program correlated with MLST results when Ward's algorithm was applied not taking into account the intensity of the spectra. However, we do not observe the same correlation among EFMRV isolates.
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Citação
CHAGAS NETO, Thomas Cardoso das. Caracterização microbiológica e epidemiologia molecular de Enterococcus spp. isolados de infecções da corrente sanguínea de pacientes do Projeto SCOPE Brasil. 2015. 153 f. Tese (Doutorado em Infectologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2015.