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ESTUDO DA DIVERSIDADE GENÉTICA, CARACTERIZAÇÃO FENOTÍPICA E MOLECULAR DE MECANISMOS DE RESISTÊNCIA A ANTIMICROBIANOS E VIRULÊNCIA EM ACINETOBACTER BAUMANNII ISOLADOS EM HOSPITAIS DO RIO DE JANEIRO
Carvalho, Karyne Rangel | Date Issued: 2013
Alternative titleStudy of genetic diversity, phenotypic and molecular mechanisms of antimicrobial resistance and virulence in acinetobacter baumannii isolated in hospitals of Rio de Janeiro
Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Rio de Janeiro, RJ, Brasil.
Abstract in Portuguese
Acinetobacter baumannii é um patógeno oportunista com crescente importância em infecções relacionadas a assistência em saúde. No Brasil é particularmente problemático devido à sua alta prevalência e multirresistência, com as carbapenemases tipo OXA representando o principal mecanismo responsável por esta resistência. Esse trabalho teve como objetivo determinar a diversidade genética, caracterizar fenotípica e molecularmente a resistência a antimicrobianos e avaliar fatores de virulência em Acinetobacter baumannii isolados de 10 hospitais públicos e privados no Rio de Janeiro no período de 2005 a 2007. Durante este período foram estudados 141 isolados de A. baumannii coletados de pacientes internados em UTIs ou enfermarias cirúrgicas. A identificação da espécie foi confirmada através da detecção do gene intrínseco blaOXA-51.Os 110 isolados de A. baumannii resistentes ao imipenem apresentaram perfil de multirresistência com taxas superiores a 98,2% para 8 dos 10 antimicrobianos testados e 98(87,3%) produziram a carbapenemase OXA-23. Não houve produto de amplificação para os genes blaOXA-24, blaOXA-58, blaIMP, blaVIM e Int1 pela técnica de PCR. As drogas com atividade in vitro foram a polimixina B e tigeciclina. Observou-se uma diversidade clonal, com a presença de 5 genótipos tipados por PFGE, com prevalência dos genótipos A (71,8%) e B(22,7%), presente em 7 e 5 hospitais, respectivamente. Dentre os 96 isolados produtores de OXA-23, fori selecionada uma cepa representativa de cada genótipo que foram submetidos a métodos de tipagem baseados em sequenciamento...
Acinetobacter baumannii is an opportunistic pathogen with increasing importance in hospital infections. In Brazil, it is particularly problematic because of its high prevalence and multidrug resistance, with the OXA-type carbapenemases representing the main mechanism responsible for such resistance. This study aimed to determine the genetic diversity, as well as phenotypic and molecular characterization of antimicrobial resistance and evaluation of virulence factors in Acinetobacter baumannii isolated from 10 public and private hospitals in Rio de Janeiro from 2005 to 2007. During this period 141 isolates of A. baumannii collected from patients admitted to ICUs or surgical wards have been studied. Species identification has been confirmed by detection of gene intrinsic blaOXA-51. The 110 isolates of A. baumannii resistant to imipenem showed multidrug resistance profile with rates above 98.2% for 8 of the 10 antibiotics tested and 98 (87.3%) produced the carbapenemase OXA-23. There was no amplification product for genes blaOXA-24, blaOXA-58, blaIMP, blaVIM and Int1 by PCR. The drugs with in vitro activity were polymyxin B and tigecycline. There was a clonal diversity, the presence of five genotypes typed by PFGE, with a prevalence of genotypes A (71.8%) and B (22.7%), present in 7 and 5 hospitals, respectively. Among the 96 strains producing OXA-23, one representative for each genotype that were subjected to typing methods based on sequencing was selected. By MLST (related to the database Oxford) four new STs were detected: ST131, ST132, ST133 and ST134 and when using the MLST-IP (developed by Institut Pasteur) the ST79, ST15 and two new allelic profiles were detected. Four SGs (SG1, SG4 and two new profiles) have been identified, allowing the association of 70% of isolates with the European Clone II. Sequencing of the blaOXA-51-like gene revealed the presence of blaOXA-66, blaOXA-95 and blaOXA-132. The blaOXA-23 gene was consistently found associated with the transposon Tn2006 and was chromosomally encoded in all isolates. Of 31 isolates susceptible to imipenem, 5 showed the blaOXA-23 gene and the insertion sequence ISAba1. However, the association of this sequence to the gene blaOXA-23 was detected only in isolates resistant to imipenem used as controls. The ISAba4 insertion sequence was not found in any isolated. These isolates were susceptible to imipenem and meropenem, with MICs ε 4 mg / mL for both antimicrobials. These isolates producing blaOXA-23 have been grouped into four distinct genotypes (B, C, G and I). Most isolates included in this study had plasmid (83.7%), with profiles ranging from 1 to 4. In search of biofilm production by colorimetric assay using crystal violet it was possible to observe the production of biofilm in 96.5% of isolates. Two representative isolates of the prevalent genotypes were evaluated for the degree of association at monolayer of A-549 cells by flow cytometry, and fluorescence optical microscopy. The adhesion was observed in all methodologies, with low rates of association for genotype A (11.9%) and genotype B (9.7%). The search for quorum-sensing carried out with representative isolates of the same prevalent genotypes were negative for the production of AHL inducer using Agrobacterium tumefaciens strain NT1. While the extract of the supernatant of the positive control of P. aeruginosa PAO1 and the reference standard AHL-C8 and AHL -C12 were positive. Data from this study are relevant to public health because they allow the knowledge of the molecular epidemiology of this species, as well as some of the virulence factors, which have been rarely described in Brazil, thus enhancing the monitoring and implementing control measures.