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Development of a Novel Loop-Mediated Isothermal Amplification Method to Detect Guiana Extended-Spectrum (GES) ß-Lactamase Genes in Pseudomonas aeruginosa.
Takano, Chika; Seki, Mitsuko; Kim, Dong Wook; Gardner, Humphrey; McLaughlin, Robert E; Kilgore, Paul E; Kumasaka, Kazunari; Hayakawa, Satoshi.
Afiliação
  • Takano C; Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, Tokyo, Japan.
  • Seki M; Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, Tokyo, Japan.
  • Kim DW; Division of Pediatric Dentistry, Meikai University School of Dentistry, Sakado, Japan.
  • Gardner H; Department of Pharmacy, College of Pharmacy, Hanyang University, Ansan, South Korea.
  • McLaughlin RE; Institute of Pharmacological Research, Hanyang University, Ansan, South Korea.
  • Kilgore PE; Evelo Biosciences, Cambridge, MA, United States.
  • Kumasaka K; Institute for Life Science Entrepreneurship, Union, NJ, United States.
  • Hayakawa S; Department of Pharmacy Practice, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI, United States.
Front Microbiol ; 10: 25, 2019.
Article em En | MEDLINE | ID: mdl-30778337
Infections caused by multidrug-resistant Pseudomonas aeruginosa in hospitalized patients are often fatal, and nosocomial infections caused by Guiana extended-spectrum (GES) ß-lactamase-producing strains are of growing concern. Several genotypes of the GES ß-lactamase gene (bla GES) include a single missense mutation, a change from G to A at nucleotide position 493 (G493A) that changes glycine to serine; the mutant enzyme exhibits carbapenemase activity. Rapid and reliable identification of drug-resistance is important in clinical settings; however, culture methods remain the gold standard. Conventional and real-time PCR cannot identify carbapenemase-producing genotypes, and direct DNA sequencing is essential. We established a novel loop-mediated isothermal amplification (LAMP) method to detect various genotypes of bla GES and another LAMP method to discriminate carbapenemase genotypes of bla GES. We evaluated the two assays using clinical P. aeruginosa strains. Two primer sets targeting bla GES (GES-LAMP) and the point mutation (Carba-GES-LAMP) were designed and evaluated for specificity and sensitivity. The detection limit of the GES-LAMP method was assessed using purified DNA and DNA-spiked clinical samples (urine, sputum, and blood). To determine the clinical usefulness of the methods, we used different (genotypically and phenotypically) P. aeruginosa clinical isolates, collected from diverse geographical locations between 2003 and 2012. The novel LAMP assay targeting bla GES was highly specific. The detection limit was 10 DNA copies per reaction; the assay was 10-fold more sensitive than conventional PCR. The LAMP assay detected bla GES with high sensitivity in all DNA-spiked samples; PCR did not detect bla GES in blood samples. The GES-LAMP method correctly detected the 5 isolates containing bla GES among the 14 isolates tested. Using these isolates, we confirmed that our Carba-GES-LAMP method of detecting point mutations correctly identified the two bla GES positive organisms with carbapenemase activity. To the best of our knowledge, this is the first report of the GES ß-lactamase gene detection assay using the LAMP method. Our new assays effectively detect bla GES and critical unique mutations.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE País/Região como assunto: America do sul / Caribe ingles / Guyana Idioma: En Revista: Front Microbiol Ano de publicação: 2019 Tipo de documento: Article País de afiliação: Japão País de publicação: Suíça

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE País/Região como assunto: America do sul / Caribe ingles / Guyana Idioma: En Revista: Front Microbiol Ano de publicação: 2019 Tipo de documento: Article País de afiliação: Japão País de publicação: Suíça