Detection and Real-time PCR Assay for the Quantification of Carbapenemase Gene [bla.sub.NDM-1] in Hospital Effluent

This study aims to isolate gram negative bacteria (GNB) harboring the gene NDM-l from the tertiary care hospital effluents. Also, aims to evaluate the relative copy number of [bla.sub.NDM-1] carried by the positive isolates. The study isolated 215 GNB from 40 effluent samples. The antibiotic susceptibility tests for carbapenems were performed using disc diffusion assay. The isolates resistant to either meropenem or imipenem were checked for the existence of MBL by phenotypic methods. The isolates carrying NDM-l gene were genotypically confirmed by Polymerase chain reaction (PCR). The gene copy number of [bla.sub.NDM-1] were determined by quantative real-time PCR. A total of 22 isolates showed phenotypic resistance to carbapenems and were characterized by biochemical methods. Among them, 12 harbored NDM-l gene by PCR; these bacteria were subjected to qPCR for determining the absolute copy numbers of the NDM-l gene on it. The gene abundance in the strains was in the range of 3.28* [10.sup.5] to 6.05* [10.sup.6] copies/ ng of DNA. Hospital effluents are important pool of antibiotic resistant bacteria harboring the [bla.sub.NDM-1] and infections caused by these bacteria are difficult to treat. Hence, the present study stresses the need for stringent antibiotic use and efficient wastewater treatment policies in these hospital settings, which is paramount in achieving sustainable health. Keywords: wastewater, NDM-l, hospital effluent, carbapenem, qPCR