title: Real-time PCR assays for the detection and quantification of carbapenemase genes (blaKPC, blaNDM, and blaOXA-48) in environmental samples.
abstract: In this study, we have developed real-time PCR assays using SYBR Green chemistry to detect all known alleles of blaKPC, blaNDM, and blaOXA-48-like carbapenemase genes in water, sediment, and biofilm samples collected from hospital and wastewater treatment plant (WWTP) effluents and rivers receiving chronic WWTP discharges. The amplification of blaKPC, blaNDM, and blaOXA-48DNA was linear over 7 log dilutions (R2between 0.995 and 0.997) and showing efficiencies ranging from 92.6% to 100.3%. The analytical sensitivity indicated that the reaction for blaKPC, blaNDM, and blaOXA-48-like genes was able to detect 35, 16, and 19 copy numbers per assay, respectively. The three carbapenemase genes were detected in hospital effluents, whereas only the blaKPCand blaNDMgenes were detected in biofilm and sediment samples collected from wastewater-impacted rivers. The detection of blaKPC, blaNDM, and blaOXA-48-like genes in different matrices suggests that carbapenem-resistant bacteria occur in both planktonic and benthic habitats thus expanding the range of resistance reservoirs for last-resort antibiotics. We believe that these real-time PCR assays would be a powerful tool for the rapid detection and quantification of blaKPC, blaNDM, and blaOXA-48-like genes in complex environmental samples.
authors.affiliation: Catalan Institute for Water Research (ICRA), Scientific and Technological Park of the University of Girona, Girona, Spain.
authors.forename: Jèssica
authors.initials: J
authors.lastname: Subirats
authors.affiliation: Group of Molecular Microbial Ecology, Institute of Aquatic Ecology, University of Girona, Girona, Spain.
authors.forename: Elena
authors.initials: E
authors.lastname: Royo
authors.affiliation: Catalan Institute for Water Research (ICRA), Scientific and Technological Park of the University of Girona, Girona, Spain. jlbalcazar@icra.cat.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Balcázar
authors.affiliation: Catalan Institute for Water Research (ICRA), Scientific and Technological Park of the University of Girona, Girona, Spain.|Group of Molecular Microbial Ecology, Institute of Aquatic Ecology, University of Girona, Girona, Spain.
authors.forename: Carles M
authors.initials: CM
authors.lastname: Borrego
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D052160:Benzothiazoles; D018441:Biofilms; D003959:Diamines; D004783:Environmental Microbiology; D005456:Fluorescent Dyes / Q000737:chemistry; D005798:Genes, Bacterial; D057230:Limit of Detection; D009930:Organic Chemicals / Q000737:chemistry; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction; D062065:Wastewater / Q000382:microbiology; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D062065:Wastewater; D001618:beta-Lactamases; C098022:SYBR Green I; C063912:carbapenemase
keywords: Environmental biofilm; Real-time PCR; bla KPC genes; bla NDM genes; bla OXA-48-like genes