RESUMO
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.
RESUMO
Metallo-ß-lactamases (MBLs) hydrolyze all classes of ß-lactams except monobactams and are not inhibited by classic serine ß-lactamase inhibitors. Gram-negative pathogens isolated from patient infections were collected from 202 medical centers in 40 countries as part of a global surveillance study from 2012 to 2014. Carbapenem-nonsusceptible Enterobacteriaceae and Pseudomonas aeruginosa were characterized for bla genes encoding VIM, IMP, NDM, SPM, and GIM variants using PCR and sequencing. A total of 471 MBL-positive isolates included the following species (numbers of isolates are in parentheses): P. aeruginosa (308), Klebsiella spp. (85), Enterobacter spp. (39), Proteeae (16), Citrobacter freundii (12), Escherichia coli (6), and Serratia marcescens (5) and were submitted by sites from 34 countries. Of these, 69.6% were collected in 9 countries (numbers of isolates are in parentheses): Russia (72), Greece (61), Philippines (54), Venezuela (29), and Kuwait, Nigeria, Romania, South Africa, and Thailand (20 to 25 isolates each). Thirty-two different MBL variants were detected (14 VIM, 14 IMP, and 4 NDM enzymes). Seven novel MBL variants were encountered in the study, each differing from a previously reported variant by one amino acid substitution: VIM-42 (VIM-1 [V223I]), VIM-43 (VIM-4 [A24V]), VIM-44 (VIM-2 [K257N]), VIM-45 (VIM-2 [T35I]), IMP-48 (IMP-14 [I69T]), IMP-49 (IMP-18 [V49F]), and NDM-16 (NDM-1 [R264H]). The in vitro activities of all tested antibiotics against MBL-positive Enterobacteriaceae were significantly reduced with the exception of that of aztreonam-avibactam (MIC90, 0.5 to 1 µg/ml), whereas colistin was the most effective agent against MBL-positive P. aeruginosa isolates (>97% susceptible). Although the global percentage of isolates encoding MBLs remains relatively low, their detection in 12 species, 34 countries, and all regions participating in this surveillance study is concerning.