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

• Ano de defesa: 2017
• Autor(a) principal: Silva, Adriana Pereira de Matos Marques [UNIFESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Acinetobacter Spp; Resistência aos carbapenêmicos; Oxacilinases; Plasmídeos em acinetobacter Spp; Farmacorresistência bacteriana
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=5255798; http://repositorio.unifesp.br/handle/11600/50666

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.