AUTHOR=Takano Chika , Seki Mitsuko , Kim Dong Wook , Gardner Humphrey , McLaughlin Robert E. , Kilgore Paul E. , Kumasaka Kazunari , Hayakawa Satoshi 

TITLE=Development of a Novel Loop-Mediated Isothermal Amplification Method to Detect Guiana Extended-Spectrum (GES) β-Lactamase Genes in Pseudomonas aeruginosa

JOURNAL=Frontiers in Microbiology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2019.00025

DOI=10.3389/fmicb.2019.00025

ISSN=1664-302X

ABSTRACT=<p>Infections caused by multidrug-resistant <italic>Pseudomonas aeruginosa</italic> in hospitalized 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 (<italic>bla</italic><sub>GES</sub>) 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 <italic>bla</italic><sub>GES</sub> and another LAMP method to discriminate carbapenemase genotypes of <italic>bla</italic><sub>GES</sub>. We evaluated the two assays using clinical <italic>P. aeruginosa</italic> strains. Two primer sets targeting <italic>bla</italic><sub>GES</sub> (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) <italic>P. aeruginosa</italic> clinical isolates, collected from diverse geographical locations between 2003 and 2012. The novel LAMP assay targeting <italic>bla</italic><sub>GES</sub> 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 <italic>bla</italic><sub>GES</sub> with high sensitivity in all DNA-spiked samples; PCR did not detect <italic>bla</italic><sub>GES</sub> in blood samples. The GES-LAMP method correctly detected the 5 isolates containing <italic>bla</italic><sub>GES</sub> among the 14 isolates tested. Using these isolates, we confirmed that our Carba-GES-LAMP method of detecting point mutations correctly identified the two <italic>bla</italic><sub>GES</sub> 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 <italic>bla</italic><sub>GES</sub> and critical unique mutations.</p>