Detection and Real-time PCR Assay for the Quantification of Carbapenemase Gene blablaNDM-1 in Hospital Effluent

M Divyashree,Shama Prakash K,Madhu K Mani

doi:10.22207/jpam.15.4.65

Abstract

This study aims to isolate gram-negative bacteria (GNB) harboring the gene NDM-1 from the tertiary care hospital effluents. Also, aims to evaluate the relative copy number of blaNDM-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-1 gene were genotypically confirmed by Polymerase chain reaction (PCR). The gene copy number of blaNDM- 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-1 gene by PCR; these bacteria were subjected to qPCR for determining the absolute copy numbers of the NDM-1 gene on it. The gene abundance in the strains was in the range of 3.28× 105 to 6.05× 106 copies/ ng of DNA. Hospital effluents are important pool of antibiotic-resistant bacteria harboring the blaNDM-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.

Highlights

Hospitals are a selective atmosphere for antibiotic resistant bacteria, disseminating them to the surrounding environment primarily from discharges of hospital effluents.[1,2] It is quite natural where antibiotics are heavily used for the existence of resistance seems to be high
This study aims to isolate gram negative bacteria (GNB) harboring the gene New Delhi Metallobeta-lactamases-1 (NDM-1) from the tertiary care hospital effluents
NDM-1 gene was detected only among the isolates from untreated effluent, those positive strains only used for the real time quantification studies

Summary

Introduction

Hospitals are a selective atmosphere for antibiotic resistant bacteria, disseminating them to the surrounding environment primarily from discharges of hospital effluents.[1,2] It is quite natural where antibiotics are heavily used for the existence of resistance seems to be high. A new class of New Delhi Metallobeta-lactamases-1 (NDM-1) has been reported worldwide since 2008. These NDM-1 producers show resistance to other classes of antibiotics.[7] the spread of these bacteria carrying the NDM-1 gene is a major global health threat.[8,9] The blaNDM-1 is located on large plasmids with various sizes.[10] Uncontrollable spread and infection caused by these NDM-1 producing bacteria leaves a limited treatment option the term "superbugs" is used.[7] it is important to study the bacteria containing NDM-1 gene in the hospital effluents. Current study interested in detecting these NDM-1 carrying isolates from the tertiary care hospital effluents and to carry out real- time PCR assay as a powerful tool for the quantification of blaNDM-1 in hospital effluent

Objectives

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Results

Conclusion