INVESTIGATION OF SOME CARBAPENEM RESISTANCE GENES IN ESCHERICHIA COLI AND KLEBSIELLA PNEUMONIAE FROM HOSPITAL WASTE

*Banigo Kalanne Dabota, Confidence Kinikanwo Wachukwu, Easter Godwin Nwokah and Agi Vivian Nkemkanma

ABSTRACT

The hospital waste is a reservoir for multidrug resistant Escherichia coli and Klebsiella pneumoniae and it leads to an increase in the frequency of these resistance genes creating a critical predicament for the society. The aim of this study was to identify some resistance genes found in Escherichia coli and Klebsiella pneumoniae obtained from hospital waste. A total of 100 samples of wastes which were hazardous and non-hazardous were collected from identified waste dumps sites in the Rivers State University hospital into sterile bottles. These were transported to the microbiology laboratory of the Rivers State University using cold chain. The conventional method of culturing, isolating and identifying was used. The samples were cultured on prepared agar plates Cysteine Lactose Electrolyte Deficient (CLED), MacConkey (MAC), Eosin Methylene Blue (EMB), Mueller-Hinton Agar (MHA), Blood Agar (BA) and nutrient agar (NA) and incubated at 37°c for 24hours. The culture plates were examined, the isolates subcultured and used for identification. Gram staining and other biochemical tests were employed for the identification of the isolates. Results showed isolates of Klebsiella pneumoniae constituting a majority of the isolated bacteria forming (27.5%). Escherichia coli, Staphylococcus aureus and Bacillus spp. constituted 25%, 22.5% and 15% respectively while Pseudomonas spp. was 10%. The isolated and identified organisms (E.coli and K. pneumoniae) were subjected to sensitivity tests and the resistant forms were now taken for molecular identification of resistant genes to carbapenem. Molecularly the DNA of the organisms were extracted and quantified using nanodrop 1000 spectrophotometer after which they were amplified using the polymerase chain reaction (PCR) and they were checked using agarose Gel electrophoretic system to be sure they possessed enough DNA and the 16srRNA against the standard known as the ladder. It was then stored frozen in vial form and sent to South Africa where 3510 ABI sequencer was used. The sequences were lodged into the gene bank National Center for Biotechnology Information (NCBI) data base using BLASTN where the organisms were identified molecularly and the genes associated with carbapenems were also identified. The results of the molecular techniques showed the identification of Escherichia coli and Klebsiella pneumoniae and the resistant genes to carbapenems. The New Delhi Metallo-Beta-Lactamase (NDM) resistant gene was detected in three of the isolates of Escherichia coli and Klebsiella pneumoniae as against the other three Klebsiella pneumoniae carbapenemases (KPC), Oxacillinase (OXA) and Verona Integron-encoded Metallo-Beta-Lactamase (VIM) carbapenem genes. This study therefore has identified a gene that is resistant to carbapenem.

Keywords: Carbapenem, Resistance Genes, Hospital waste, organisms, 16srRNA.