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Front. Microbiol. | doi: 10.3389/fmicb.2019.00025

Development of a novel loop-mediated isothermal amplification method to detect Guiana extended-spectrum (GES) β-lactamase genes in Pseudomonas aeruginosa

 Chika Takano1,  Mitsuko Seki1, 2*,  Dong Wook Kim3*,  Humphrey Gardner4, Robert E. McLaughlin5,  Paul E. Kilgore6, Kazunari Kumasaka7 and  Satoshi Hayakawa1
  • 1Nihon University, Japan
  • 2Meikai University, Japan
  • 3Hanyang University, South Korea
  • 4Evelo Biosciences, United States
  • 5Institute for Life Science Entrepreneurship, United States of America
  • 6Wayne State University, United States
  • 7Ageo Central General Hospital, Japan

Infections caused by multidrug-resistant Pseudomonas aeruginosa in hospitalised patients are often fatal, and nosocomial infections caused by Guiana extended-spectrum (GES) β-lactamase-producing strains are of growing concern. Several genotypes of the GES β-lactamase gene (blaGES) 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 blaGES and another LAMP method to discriminate carbapenemase genotypes of blaGES. We evaluated the two assays using clinical P. aeruginosa strains.
Two primer sets targeting blaGES (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 blaGES 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 blaGES with high sensitivity in all DNA-spiked samples; PCR did not detect blaGES in blood samples. The GES-LAMP method correctly detected the 5 isolates containing blaGES among the 14 isolates tested. Using these isolates, we confirmed that our Carba-GES-LAMP method of detecting point mutations correctly identified the two blaGES positive organisms with carbapenemase activity.
To the best of our knowledge, this is the first report of the GES β-lactamase gene detection assay using the LAMP method. Our new assays effectively detect blaGES and critical unique mutations.

Keywords: blaGES, β-lactamase, Point Mutation, carbapenemase, Loop-mediated isothermal amplification, Pseudomonas aeruginosa

Received: 08 Sep 2018; Accepted: 09 Jan 2019.

Edited by:

Leonard Peruski, Centers for Disease Control and Prevention (CDC), United States

Reviewed by:

Mariagrazia Perilli, University of L'Aquila, Italy
Amro A. Amara, City of Scientific Research and Technological Applications, Egypt  

Copyright: © 2019 Takano, Seki, Kim, Gardner, McLaughlin, Kilgore, Kumasaka and Hayakawa. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
Dr. Mitsuko Seki, Nihon University, Chiyoda, Tokyo, 102-0074, Tōkyō, Japan, seki.mitsuko@nihon-u.ac.jp
Prof. Dong Wook Kim, Hanyang University, Seoul, 133-791, South Korea, dongwook@hanyang.ac.kr