Aplicação do sequenciamento completo do genoma ba caracterização de amostras de acinetobacter baumannii produtores de carbapenemase
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Data
2018-03-20
Autores
Boettger, Bruno Cruz [UNIFESP]
Orientadores
Pignatari, Antonio Carlos Campos [UNIFESP]
Tipo
Dissertação de mestrado
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Introdução: Infecções por Acinetobacter baumanni apresentam elevadas taxas de morbidade e mortalidade, particularmente quando os isolados são resistentes a antibióticos carbapenêmicos. Surtos de infecções relacionadas à assistência a saúde tem sido detectados com disseminação de clones intra e inter hospitais incluindo a determinação de genes codificadores de oxacilinases, o principal mecanismos de resistência a carbapenêmicos. Técnicas moleculares têm sido utilizadas para tipagem molecular, como Pulsed-Fiel Gel Electrophoresis (PFGE) e MLST por sequenciamento de housekeeping genes e caracterização de genes de resistência aos antimicrobianos por PCR convencional e real time. Recentemente, o sequenciamento de genoma completo tem sido utilizado como um método que permite a tipagem molecular e a caracterização de genes de resistência em diferentes gêneros e espécies bacterianas. Objetivos: Identificar genes codificadores de resistência a antimicrobianos e caracterizar perfis clonais de A. baumannii resistentes a carbapenêmicos por sequenciamento de genoma completo. Métodos: Foram estudadas seis amostras de A. baumannii resistentes a carbapenêmicos isoladas de pacientes internados em hospital geral da cidade de São Paulo no período de 20 de novembro a 03 de dezembro de 2016. O perfil de resistência a antimicrobianos foi realizado por microdiluição em caldo e a tipagem molecular por PFGE. As sequencias de DNA do genoma completo foram obtidas através da plataforma Illumina, anotação de bioinformática pelo software Prokka e análise pelo Center for Genomic Epidemiology. Resultados: Somente a polimixina B, minociclina e tigeciclina apresentaram atividade frente a todos os isolados de A. baumannii resistentes aos carbapenêmicos. Genes de resistência aos aminoglicosídeos, β-lactâmicos, fenicois, macrolídeos, quinolonas, sulfonamidas, tetraciclinas e trimetroprim foram encontrados no genoma dos isolados. Uma coprodução de OXA-23 e OXA-72 foram detectadas em um isolado de A. baumannii pertencente ao ST1/CC1. Dois isolados apresentando mesmo padrão de PFGE pertencente ao ST79/CC79 carreavam os genes blaOXA-23 e blaOXA-72 separadamente localizados no cromossomo e no plasmídeo, respectivamente. O gene qnrD-like foi detectado no isolado de A. baumannii coprodutor de OXA-23 e OXA-72, sendo o primeiro isolado de Acinetobacter spp. reportado a carrear este gene globalmente. Conclusão: O sequenciamento completo de genoma de A. baumanii revelou a presença de genes de resistência a diversos antimicrobianos, incluindo os codificadores de oxacilinases, e contribuiu para a tipagem molecular com finalidades epidemiológicas.
Introduction: Acinetobacter baumannii infections show high rates of morbidity and mortality, particularly for carbapenem-resistant isolates. Outbreaks of nosocomial infections have been observed with clonal dissemination in the hospital and among hospitals including the detection of oxacilinases encoding genes, the main mechanism of resistance to carbapenems in Acinetobacter. Molecular methods have been used for molecular typing, such as Pulsed-Field Tel Electrophoresis (PFGE) and multilocus sequencing typing (MLST), and characterization of antimicrobial resistance genes by conventional or real-time PCR. Recently, the whole genome sequencing has been utilized as a method for molecular typing and resistance genes detection in distinct bacterial species. Objectives: To identify antimicrobial resistance genes and to characterize carbapenem-resistant A. baumannii by whole genome sequencing. Methods: We studied six carbapenem-resistant A. baumannii isolated from patients admitted to a general hospital in the citiy of São Paulo, Brazil, during the period of November 20 to December 03, 2016. The antimicrobial resistance profile was done by broth microdilution method and the molecular typing by PFGE. The whole DNA genome sequence was obtained through the Illumina plataform, bioinformatics annotation by Prokka software, and analysis by the Center for Genomic Epidemiology site. Results: Only polymyxin B, minocycline, and tigecycline showed activity against all isolates. Resistance genes to aminoglycosides, β-lactams, phenicols, macrolides, quinolones, sulphonamides, tetracyclines and trimethoprim were detected in the genomes. A coproduction of OXA-23 and OXA-72 was detected in one isolate belonging to ST1/CC1. Two isolates showing similar PFGE profiles and belonging to ST79/CC79 carried the genes OXA-23 and OXA-72, located in the chromosome and plasmid, respectively. The gene qnrD-like was detected in the isolate coproduce of OXA-23 and OXA-72, the first Acinetobacter spp. reported carrying this gene globally. Conclusion: The A. baumannii whole genome sequencing detected antimicrobial resistance genes, including the oxacilinase encoding genes, with relevant contribution for molecular epidemiology typing.
Introduction: Acinetobacter baumannii infections show high rates of morbidity and mortality, particularly for carbapenem-resistant isolates. Outbreaks of nosocomial infections have been observed with clonal dissemination in the hospital and among hospitals including the detection of oxacilinases encoding genes, the main mechanism of resistance to carbapenems in Acinetobacter. Molecular methods have been used for molecular typing, such as Pulsed-Field Tel Electrophoresis (PFGE) and multilocus sequencing typing (MLST), and characterization of antimicrobial resistance genes by conventional or real-time PCR. Recently, the whole genome sequencing has been utilized as a method for molecular typing and resistance genes detection in distinct bacterial species. Objectives: To identify antimicrobial resistance genes and to characterize carbapenem-resistant A. baumannii by whole genome sequencing. Methods: We studied six carbapenem-resistant A. baumannii isolated from patients admitted to a general hospital in the citiy of São Paulo, Brazil, during the period of November 20 to December 03, 2016. The antimicrobial resistance profile was done by broth microdilution method and the molecular typing by PFGE. The whole DNA genome sequence was obtained through the Illumina plataform, bioinformatics annotation by Prokka software, and analysis by the Center for Genomic Epidemiology site. Results: Only polymyxin B, minocycline, and tigecycline showed activity against all isolates. Resistance genes to aminoglycosides, β-lactams, phenicols, macrolides, quinolones, sulphonamides, tetracyclines and trimethoprim were detected in the genomes. A coproduction of OXA-23 and OXA-72 was detected in one isolate belonging to ST1/CC1. Two isolates showing similar PFGE profiles and belonging to ST79/CC79 carried the genes OXA-23 and OXA-72, located in the chromosome and plasmid, respectively. The gene qnrD-like was detected in the isolate coproduce of OXA-23 and OXA-72, the first Acinetobacter spp. reported carrying this gene globally. Conclusion: The A. baumannii whole genome sequencing detected antimicrobial resistance genes, including the oxacilinase encoding genes, with relevant contribution for molecular epidemiology typing.
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BOETTGER, Bruno Cruz. Aplicação do sequenciamento completo do genoma na caracterização de amostras de acinetobacter baumannii produtores de carbapenemase. 2018. 74 f. Dissertação (Mestrado em Infectologia) - Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo, São Paulo, 2018.