Real-time PCR assays for the detection and quantification of carbapenemase genes (bla KPC, bla NDM, and bla OXA-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 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.

Keywords: Environmental biofilm; Real-time PCR; bla KPC genes; bla NDM genes; bla OXA-48-like genes.