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Real-time PCR assays for the detection and quantification of carbapenemase genes (bla KPC, bla NDM, and bla OXA-48) in environmental samples

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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.

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This work was supported by the Spanish Ministry of Economy and Competitiveness through the project TRACE (JPIW2013-129), the European Union through the European Regional Development Fund (FEDER), and the Generalitat de Catalunya (2014 SGR 291) and the Spanish Government through the Ramón y Cajal program (RYC-2011-08154). We thank Alexandre Sànchez-Melsió for his technical assistance and Jordi René, Dr. Vicenç Acuña, and Dr. Gianluigi Buttiglieri for their assistance during field work.

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Correspondence to José Luis Balcázar.

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Responsible editor: Diane Purchase

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Supplementary data. This file contains the alignments of all known alleles of bla KPC, bla NDM, and bla OXA-48-like genes (PDF 172 kb)


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Subirats, J., Royo, E., Balcázar, J.L. et al. Real-time PCR assays for the detection and quantification of carbapenemase genes (bla KPC, bla NDM, and bla OXA-48) in environmental samples. Environ Sci Pollut Res 24, 6710–6714 (2017).

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