Caracterização de plasmídeos de isolados clínicos de acinetobacter spp. produtores de carbapenemases

Detalhes bibliográficos
Autor(a) principal: Silva, Adriana Pereira de Matos Marques [UNIFESP]
Data de Publicação: 2017
Tipo de documento: Tese
Idioma: por
Título da fonte: Repositório Institucional da UNIFESP
Texto Completo: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=5255798
http://repositorio.unifesp.br/handle/11600/50666
Resumo: ABSTRACT Acinetobacter spp. is a nosocomial pathogen frequently isolated in ICU patients. High resistance rates have been detected among Acinetobacter spp.. Carbapenem Hydrolyzing Class D β-lactamases (CHDL) production is the most frequent and the most important resistance mechanism. This species is able to acquire plasmids and mobile genetic elements (MGEs) that mobilize resistance genes. This study aimed to describe plasmids of carbapenemase producing Acinetobacter spp. clinical isolates. Six carbapenem resistant Acinetobacter spp. strains were selected as OXA-58, OXA-143, OXA-231 and OXA-253 producing. Strain identification was performed on MALDI-TOF MS and confirmed by rpoB sequencing. Carbapenemase detection was confirmed by PCR and susceptibility testing was performed by broth microdilution. MLST was performed to evaluate the ancestrality. The pool of plasmids was extracted using Mini Prep Extraction Kit® and quantified in Qubit® 3.0. Libraries were constructed using Illumina® TruSeq Nano DNA LT Library Preparation Kit – SetA generating ~550 bp and sequencing was performed in Illumina® MiSeq 2x300 bp in paired-end mode. Sequences were assembled using softwares Newbler 3.0 and Ray 2.3.1. Automatic annotation was performed using SABIA/LNCC platform and manual validation was performed using NCBI Blast, UniProt, ISFinder e ResFinder 2.1 softwares. The illustration of the circularized plasmids and in silico analysis of replicase gene group (AbGR) was obtained of Snap Gene® program. Promoter sequences were suggested by BPROM tool. Five of six strains were identified as A. baumanni and one was identified as A. seifertii. High MIC rates were observed to the β-lactams tested. Only polymyxin B and minocyclin presented good activity to the tested strains, except A. seifertii that showed resistance to polymyxin B (MIC: 4μg/mL). OXA-231 producing strain was included on ST107, OXA-253 on ST25 and OXA-58 producing A. seifertii and A. baumannii were included on ST551 and ST15, respectively. A total of 23 plasmids were obtained from the six analyzed strains. Of strain A-58.015 plasmids pAb58015_a (118.738 bp/173 ORFs; blaOXA-143 carrying), pAb58015_b (55.356 bp/62 ORFs), pAb58015_c (6.520 bp/9 ORFs) and pAb58015_d (2.368 bp/3 ORFs) were obtained; on strain 81048, three identic plasmids pAb58015_b, pAb58015_c and pAb58015_d were observed and also plasmids pAb81048_a (113.646 bp/177 ORFs), pAb81048_b (87.915 bp/101 ORFs), pAb81048_c (13.910 bp/21 ORFs), pAb81048_d (9.304 bp/15 ORFs), pAb81048_e (7.763 bp/6 ORFs; blaOXA-143 carrying), pAb81048_f (5.300 bp/6 ORFs) and pAb81048_g (2.845 bp/5 ORFs) were obtained; on strain A-41.652, plasmids pAb41652_a (101.946 bp/137 ORFs), pAb41652_b (11.844 bp/12 ORFs) and pAb41652_c (3.955 bp/3 ORFs; blaOXA-231 carrying) were observed; on strain 86, plasmids pAb86_a (234.383 bp/256 ORFs) and pAb86_b (13.453 bp/14 ORFs; blaOXA-253 carrying) were sequenced; on strain 1069, plasmids pAs1069_a (24.672 bp/44 ORFs; blaOXA-58 carrying) and pAs1069_b (13.129 bp/14 ORFs); and from strain A-45.063, plasmids pAb45063_a (183.767 bp/209 ORFs) and pAb45063_b (19.808 bp/24 ORFs; blaOXA-58 carrying) were verified. Plasmids were classified in six different AbGR groups (GR2, GR3, GR4, GR6, GR8 and GR19). Resistance genes of distinct antimicrobial classes were found in approximately half of the sequenced plasmids. Nine plasmids had two or three virulence genes. A new genetic environment in OXA-143 and variants OXA-231 and OXA-253 was described in our isolates. The presence of an ISAba825 upstream blaOXA-58 in A. seifertii was the main difference on the genetic environment of blaOXA-58 inserted in plasmid pAb45063_b in A. baumannii. The number of MGEs found in plasmids evaluated in this study suggests a high mobilization capacity of those structures and consequently horizontal transfer of resistance.
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spelling Caracterização de plasmídeos de isolados clínicos de acinetobacter spp. produtores de carbapenemasesCharacterization of plasmids of carbapenemase producing acinetobacter spp. clinical isolatesAcinetobacter SppResistência aos carbapenêmicosOxacilinasesPlasmídeos em acinetobacter SppFarmacorresistência bacterianaABSTRACT Acinetobacter spp. is a nosocomial pathogen frequently isolated in ICU patients. High resistance rates have been detected among Acinetobacter spp.. Carbapenem Hydrolyzing Class D β-lactamases (CHDL) production is the most frequent and the most important resistance mechanism. This species is able to acquire plasmids and mobile genetic elements (MGEs) that mobilize resistance genes. This study aimed to describe plasmids of carbapenemase producing Acinetobacter spp. clinical isolates. Six carbapenem resistant Acinetobacter spp. strains were selected as OXA-58, OXA-143, OXA-231 and OXA-253 producing. Strain identification was performed on MALDI-TOF MS and confirmed by rpoB sequencing. Carbapenemase detection was confirmed by PCR and susceptibility testing was performed by broth microdilution. MLST was performed to evaluate the ancestrality. The pool of plasmids was extracted using Mini Prep Extraction Kit® and quantified in Qubit® 3.0. Libraries were constructed using Illumina® TruSeq Nano DNA LT Library Preparation Kit – SetA generating ~550 bp and sequencing was performed in Illumina® MiSeq 2x300 bp in paired-end mode. Sequences were assembled using softwares Newbler 3.0 and Ray 2.3.1. Automatic annotation was performed using SABIA/LNCC platform and manual validation was performed using NCBI Blast, UniProt, ISFinder e ResFinder 2.1 softwares. The illustration of the circularized plasmids and in silico analysis of replicase gene group (AbGR) was obtained of Snap Gene® program. Promoter sequences were suggested by BPROM tool. Five of six strains were identified as A. baumanni and one was identified as A. seifertii. High MIC rates were observed to the β-lactams tested. Only polymyxin B and minocyclin presented good activity to the tested strains, except A. seifertii that showed resistance to polymyxin B (MIC: 4μg/mL). OXA-231 producing strain was included on ST107, OXA-253 on ST25 and OXA-58 producing A. seifertii and A. baumannii were included on ST551 and ST15, respectively. A total of 23 plasmids were obtained from the six analyzed strains. Of strain A-58.015 plasmids pAb58015_a (118.738 bp/173 ORFs; blaOXA-143 carrying), pAb58015_b (55.356 bp/62 ORFs), pAb58015_c (6.520 bp/9 ORFs) and pAb58015_d (2.368 bp/3 ORFs) were obtained; on strain 81048, three identic plasmids pAb58015_b, pAb58015_c and pAb58015_d were observed and also plasmids pAb81048_a (113.646 bp/177 ORFs), pAb81048_b (87.915 bp/101 ORFs), pAb81048_c (13.910 bp/21 ORFs), pAb81048_d (9.304 bp/15 ORFs), pAb81048_e (7.763 bp/6 ORFs; blaOXA-143 carrying), pAb81048_f (5.300 bp/6 ORFs) and pAb81048_g (2.845 bp/5 ORFs) were obtained; on strain A-41.652, plasmids pAb41652_a (101.946 bp/137 ORFs), pAb41652_b (11.844 bp/12 ORFs) and pAb41652_c (3.955 bp/3 ORFs; blaOXA-231 carrying) were observed; on strain 86, plasmids pAb86_a (234.383 bp/256 ORFs) and pAb86_b (13.453 bp/14 ORFs; blaOXA-253 carrying) were sequenced; on strain 1069, plasmids pAs1069_a (24.672 bp/44 ORFs; blaOXA-58 carrying) and pAs1069_b (13.129 bp/14 ORFs); and from strain A-45.063, plasmids pAb45063_a (183.767 bp/209 ORFs) and pAb45063_b (19.808 bp/24 ORFs; blaOXA-58 carrying) were verified. Plasmids were classified in six different AbGR groups (GR2, GR3, GR4, GR6, GR8 and GR19). Resistance genes of distinct antimicrobial classes were found in approximately half of the sequenced plasmids. Nine plasmids had two or three virulence genes. A new genetic environment in OXA-143 and variants OXA-231 and OXA-253 was described in our isolates. The presence of an ISAba825 upstream blaOXA-58 in A. seifertii was the main difference on the genetic environment of blaOXA-58 inserted in plasmid pAb45063_b in A. baumannii. The number of MGEs found in plasmids evaluated in this study suggests a high mobilization capacity of those structures and consequently horizontal transfer of resistance.RESUMO Acinetobacter spp. é um patógeno frequentemente isolado em infecções nosocomiais em pacientes de UTI. Altas taxas de resistência aos carbapenêmicos têm sido observadas entre estes isolados. A produção de enzimas da classe D que hidrolisam carbapenêmicos (CHDLs) é o mecanismo de resistência mais frequente e mais importante. Estas espécies possuem habilidade em adquirir plasmídeos e elementos genéticos móveis (EGMs) que mobilizam genes de resistência. Este estudo teve como objetivo descrever os plasmídeos de isolados clínicos de Acinetobacter spp. carreadores de genes codificadores de carbapenemases. Foram selecionadas seis cepas de Acinetobacter spp. resistentes aos carbapenêmicos produtoras de OXA-58, OXA-143, OXA-231 e OXA-253. A identificação foi realizada pelo MALDI-TOF MS e confirmada pelo sequenciamento do rpoB. A detecção de carbapenemases foi confirmada por PCR/sequenciamento e o teste de sensibilidade foi realizado por microdiluição em caldo. O MLST foi realizado para análise da ancestralidade. O pool de plasmídeos foi extraído utilizando o Mini Prep Extraction Kit® e quantificado no fluorômetro Qubit® 3.0. As bibliotecas foram construídas utilizando Illumina® TruSeq Nano DNA LT Library Preparation Kit - SetA gerando fragmentos de ~550 pb e o sequenciamento foi realizado na plataforma Illumina® MiSeq 2x300 bp no modo paired-end. As sequências foram submetidas à montagem utilizando os softwares Newbler 3.0 e Ray 2.3.1. Para anotação automática foi utilizada a plataforma SABIA/LNCC e para validação manual foram utilizados os softwares NCBI Blast, UniProt, ISFinder e ResFinder 2.1. A figura circularizada dos plasmídeos e a análise in silico dos grupos de replicase (AbGR) foram obtidas do programa Snap Gene®. As sequências promotoras foram sugeridas pela ferramenta BPROM. Cinco das seis cepas foram identificadas como A. baumannii e uma foi identificada como A. seifertii. Altas CIMs foram observadas para os β-lactâmicos testados. Somente polimixina B e minociclina apresentaram boa atividade frente às cepas testadas, exceto para A. seifertii que foi resistente à polimixina B (CIM: 4μg/mL). A cepa produtora de OXA-231 foi incluída no ST107, a cepa produtora de OXA-253 no ST25 e as cepas de A. seifertii e A. baumannii produtoras de OXA-58 no ST551 e ST15, respectivamente. Foram obtidos um total de 23 plasmídeos das seis cepas analisadas. Na cepa A-58.015 foram obtidos os plasmídeos pAb58015_a (118.738 pb/173 ORFs; carreador do gene blaOXA-143), pAb58015_b (55.356 pb/62 ORFs), pAb58015_c (6.520 pb/9 ORFs) e pAb58015_d (2.368 pb/3 ORFs); na cepa 81048, três plasmídeos idênticos aos pAb58015_b, pAb58015_c e pAb58015_d e os plasmídeos pAb81048_a (113.646 pb/177 ORFs), pAb81048_b (87.915 pb/101 ORFs), pAb81048_c (13.910 pb/21 ORFs), pAb81048_d (9.304 pb/15 ORFs), pAb81048_e (7.763 pb/6 ORFs; carreador do gene blaOXA-143), pAb81048_f (5.300 pb/6 ORFs) e pAb81048_g (2.845 pb/5 ORFs); na cepa A-41.652 os plasmídeos pAb41652_a (101.946 pb/137 ORFs), pAb41652_b (11.844 pb/12 ORFs) e pAb41652_c (3.955 pb/3 ORFs; carreador do gene blaOXA-231); na cepa 86 os plasmídeos pAb86_a (234.383 pb/256 ORFs) e pAb86_b (13.453 pb/14 ORFs; carreador do gene blaOXA-253); na cepa 1069 os plasmídeos pAs1069_a (24.672 pb/44 ORFs; carreador do gene blaOXA-58) e pAs1069_b (13.129 pb/14 ORFs); e na cepa A-45.063 os plasmídeos pAb45063_a (183.767 pb/209 ORFs) e pAb45063_b (19.808 pb/24 ORFs; carreador do gene blaOXA-58). Seis grupos AbGR foram encontrados (GR2, GR3, GR4, GR6, GR8 e GR19). Genes de resistência a diversas classes de antimicrobianos foram encontrados em cerca de metade dos plasmídeos sequenciados. Nove plasmídeos possuíam de dois a três genes de virulência. Um novo contexto genético nos isolados produtores de OXA-143 e suas variantes OXA-231 e OXA-253 foi descrito nestes isolados. A presença de uma ISAba825 à montante do gene blaOXA-58 no plasmídeo pAs1069_a em A. seifertii é a principal diferença no contexto genético deste gene quando comparado ao plasmídeo pAb45063_b carreado por A. baumannii. O grande número de EGMs encontrados nos plasmídeos avaliados neste estudo, sugere que estas estruturas possuem alta capacidade de mobilização e, consequentemente transmissão horizontal.Dados abertos - Sucupira - Teses e dissertações (2017)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo (UNIFESP)Gales, Ana Cristina [UNIFESP]Silva, Rodrigo Cayô da [UNIFESP]http://lattes.cnpq.br/5699739668358897http://lattes.cnpq.br/8402272715765172http://lattes.cnpq.br/1117846722738557Universidade Federal de São Paulo (UNIFESP)Silva, Adriana Pereira de Matos Marques [UNIFESP]2019-06-19T14:58:14Z2019-06-19T14:58:14Z2017-12-05info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/publishedVersion186 f.application/pdfhttps://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=5255798http://repositorio.unifesp.br/handle/11600/50666porSão Pauloinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UNIFESPinstname:Universidade Federal de São Paulo (UNIFESP)instacron:UNIFESP2024-08-10T15:29:57Zoai:repositorio.unifesp.br/:11600/50666Repositório InstitucionalPUBhttp://www.repositorio.unifesp.br/oai/requestbiblioteca.csp@unifesp.bropendoar:34652024-08-10T15:29:57Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)false
dc.title.none.fl_str_mv Caracterização de plasmídeos de isolados clínicos de acinetobacter spp. produtores de carbapenemases
Characterization of plasmids of carbapenemase producing acinetobacter spp. clinical isolates
title Caracterização de plasmídeos de isolados clínicos de acinetobacter spp. produtores de carbapenemases
spellingShingle Caracterização de plasmídeos de isolados clínicos de acinetobacter spp. produtores de carbapenemases
Silva, Adriana Pereira de Matos Marques [UNIFESP]
Acinetobacter Spp
Resistência aos carbapenêmicos
Oxacilinases
Plasmídeos em acinetobacter Spp
Farmacorresistência bacteriana
title_short Caracterização de plasmídeos de isolados clínicos de acinetobacter spp. produtores de carbapenemases
title_full Caracterização de plasmídeos de isolados clínicos de acinetobacter spp. produtores de carbapenemases
title_fullStr Caracterização de plasmídeos de isolados clínicos de acinetobacter spp. produtores de carbapenemases
title_full_unstemmed Caracterização de plasmídeos de isolados clínicos de acinetobacter spp. produtores de carbapenemases
title_sort Caracterização de plasmídeos de isolados clínicos de acinetobacter spp. produtores de carbapenemases
author Silva, Adriana Pereira de Matos Marques [UNIFESP]
author_facet Silva, Adriana Pereira de Matos Marques [UNIFESP]
author_role author
dc.contributor.none.fl_str_mv Gales, Ana Cristina [UNIFESP]
Silva, Rodrigo Cayô da [UNIFESP]
http://lattes.cnpq.br/5699739668358897
http://lattes.cnpq.br/8402272715765172
http://lattes.cnpq.br/1117846722738557
Universidade Federal de São Paulo (UNIFESP)
dc.contributor.author.fl_str_mv Silva, Adriana Pereira de Matos Marques [UNIFESP]
dc.subject.por.fl_str_mv Acinetobacter Spp
Resistência aos carbapenêmicos
Oxacilinases
Plasmídeos em acinetobacter Spp
Farmacorresistência bacteriana
topic Acinetobacter Spp
Resistência aos carbapenêmicos
Oxacilinases
Plasmídeos em acinetobacter Spp
Farmacorresistência bacteriana
description ABSTRACT Acinetobacter spp. is a nosocomial pathogen frequently isolated in ICU patients. High resistance rates have been detected among Acinetobacter spp.. Carbapenem Hydrolyzing Class D β-lactamases (CHDL) production is the most frequent and the most important resistance mechanism. This species is able to acquire plasmids and mobile genetic elements (MGEs) that mobilize resistance genes. This study aimed to describe plasmids of carbapenemase producing Acinetobacter spp. clinical isolates. Six carbapenem resistant Acinetobacter spp. strains were selected as OXA-58, OXA-143, OXA-231 and OXA-253 producing. Strain identification was performed on MALDI-TOF MS and confirmed by rpoB sequencing. Carbapenemase detection was confirmed by PCR and susceptibility testing was performed by broth microdilution. MLST was performed to evaluate the ancestrality. The pool of plasmids was extracted using Mini Prep Extraction Kit® and quantified in Qubit® 3.0. Libraries were constructed using Illumina® TruSeq Nano DNA LT Library Preparation Kit – SetA generating ~550 bp and sequencing was performed in Illumina® MiSeq 2x300 bp in paired-end mode. Sequences were assembled using softwares Newbler 3.0 and Ray 2.3.1. Automatic annotation was performed using SABIA/LNCC platform and manual validation was performed using NCBI Blast, UniProt, ISFinder e ResFinder 2.1 softwares. The illustration of the circularized plasmids and in silico analysis of replicase gene group (AbGR) was obtained of Snap Gene® program. Promoter sequences were suggested by BPROM tool. Five of six strains were identified as A. baumanni and one was identified as A. seifertii. High MIC rates were observed to the β-lactams tested. Only polymyxin B and minocyclin presented good activity to the tested strains, except A. seifertii that showed resistance to polymyxin B (MIC: 4μg/mL). OXA-231 producing strain was included on ST107, OXA-253 on ST25 and OXA-58 producing A. seifertii and A. baumannii were included on ST551 and ST15, respectively. A total of 23 plasmids were obtained from the six analyzed strains. Of strain A-58.015 plasmids pAb58015_a (118.738 bp/173 ORFs; blaOXA-143 carrying), pAb58015_b (55.356 bp/62 ORFs), pAb58015_c (6.520 bp/9 ORFs) and pAb58015_d (2.368 bp/3 ORFs) were obtained; on strain 81048, three identic plasmids pAb58015_b, pAb58015_c and pAb58015_d were observed and also plasmids pAb81048_a (113.646 bp/177 ORFs), pAb81048_b (87.915 bp/101 ORFs), pAb81048_c (13.910 bp/21 ORFs), pAb81048_d (9.304 bp/15 ORFs), pAb81048_e (7.763 bp/6 ORFs; blaOXA-143 carrying), pAb81048_f (5.300 bp/6 ORFs) and pAb81048_g (2.845 bp/5 ORFs) were obtained; on strain A-41.652, plasmids pAb41652_a (101.946 bp/137 ORFs), pAb41652_b (11.844 bp/12 ORFs) and pAb41652_c (3.955 bp/3 ORFs; blaOXA-231 carrying) were observed; on strain 86, plasmids pAb86_a (234.383 bp/256 ORFs) and pAb86_b (13.453 bp/14 ORFs; blaOXA-253 carrying) were sequenced; on strain 1069, plasmids pAs1069_a (24.672 bp/44 ORFs; blaOXA-58 carrying) and pAs1069_b (13.129 bp/14 ORFs); and from strain A-45.063, plasmids pAb45063_a (183.767 bp/209 ORFs) and pAb45063_b (19.808 bp/24 ORFs; blaOXA-58 carrying) were verified. Plasmids were classified in six different AbGR groups (GR2, GR3, GR4, GR6, GR8 and GR19). Resistance genes of distinct antimicrobial classes were found in approximately half of the sequenced plasmids. Nine plasmids had two or three virulence genes. A new genetic environment in OXA-143 and variants OXA-231 and OXA-253 was described in our isolates. The presence of an ISAba825 upstream blaOXA-58 in A. seifertii was the main difference on the genetic environment of blaOXA-58 inserted in plasmid pAb45063_b in A. baumannii. The number of MGEs found in plasmids evaluated in this study suggests a high mobilization capacity of those structures and consequently horizontal transfer of resistance.
publishDate 2017
dc.date.none.fl_str_mv 2017-12-05
2019-06-19T14:58:14Z
2019-06-19T14:58:14Z
dc.type.driver.fl_str_mv info:eu-repo/semantics/doctoralThesis
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
format doctoralThesis
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http://repositorio.unifesp.br/handle/11600/50666
url https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=5255798
http://repositorio.unifesp.br/handle/11600/50666
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dc.coverage.none.fl_str_mv São Paulo
dc.publisher.none.fl_str_mv Universidade Federal de São Paulo (UNIFESP)
publisher.none.fl_str_mv Universidade Federal de São Paulo (UNIFESP)
dc.source.none.fl_str_mv reponame:Repositório Institucional da UNIFESP
instname:Universidade Federal de São Paulo (UNIFESP)
instacron:UNIFESP
instname_str Universidade Federal de São Paulo (UNIFESP)
instacron_str UNIFESP
institution UNIFESP
reponame_str Repositório Institucional da UNIFESP
collection Repositório Institucional da UNIFESP
repository.name.fl_str_mv Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)
repository.mail.fl_str_mv biblioteca.csp@unifesp.br
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