Real-time PCR assays for the detection and quantification of carbapenemase genes (blaKPC, blaNDM, and blaOXA-48) in environmental samples

• Published in: Environmental science and pollution research international Vol. 24; no. 7; pp. 6710 - 6714
• Main Authors: Subirats, Jèssica, Royo, Elena, Balcázar, José Luis, Borrego, Carles M.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.03.2017; Springer Nature B.V
• Subjects: Antibiotics; Aquatic Pollution; Atmospheric Protection/Air Quality Control/Air Pollution; Bacteria; Bacterial Proteins - genetics; beta-Lactamases - genetics; Bioassays; Biofilms; Cloning; Design; Drug resistance; Earth and Environmental Science; Ecotoxicology; Effluents; Environment; Environmental Chemistry; Environmental Health; Environmental Microbiology; Environmental science; Enzymes; Fluorescent Dyes - chemistry; Genes; Genes, Bacterial; Green chemistry; Hospital wastes; Limit of Detection; Medical wastes; Organic Chemicals - chemistry; Plasmids; Real-Time Polymerase Chain Reaction; Research Article; Rivers; Sediment samplers; Sediments; Software; Studies; Waste Water - microbiology; Waste Water Technology; Wastewater treatment plants; Water Management; Water Pollution Control; Water treatment; Water treatment plants; like genes; Environmental biofilm; Real-time PCR; genes; bla KPC genes; bla NDM genes; bla OXA-48-like genes
• ISSN: 0944-1344; 1614-7499
• DOI: 10.1007/s11356-017-8426-6

In this study, we have developed real-time PCR assays using SYBR Green chemistry to detect all known alleles of bla KPC , bla NDM , and bla OXA-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 bla KPC , bla NDM , and bla OXA-48 DNA was linear over 7 log dilutions ( R 2 between 0.995 and 0.997) and showing efficiencies ranging from 92.6% to 100.3%. The analytical sensitivity indicated that the reaction for bla KPC , bla NDM , and bla OXA-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 bla KPC and bla NDM genes were detected in biofilm and sediment samples collected from wastewater-impacted rivers. The detection of bla KPC , bla NDM , and bla OXA-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 bla KPC , bla NDM , and bla OXA-48 -like genes in complex environmental samples.