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 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-48 DNA was linear over 7 log dilutions (R2 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 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 blaKPC and blaNDM genes 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.