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

In this study, we have developed real-time PCR assays using SYBRGreen 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 blaKPC, blaNDM, 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.