Molecular analysis of B-lactamase gene in multidrug resistant clinical isolates of pseudomonas aeruginosa

Abstract

The multi-drug resistant Pseudomonas aeruginosa which is one of the most prevalent opportunistic nosocomial pathogen is on the rise. Its major defense against β-lactam antibiotics is production of metallo-β-lactamases (MBLs) which degrade this group of antibiotics including carbapenems. Carbapenems are the drugs of choice for the treatment of P. aeruginosa infection but carbapenemases (MBLs) have emerged and have spread from this bacterium to Enterobacteriaceae. The finding of carbapenem resistance is a menacing development that challenges this “last resort antibiotic” and organisms harboring the enzyme may lead to therapeutic dead ends. The aim of the study is to determine the β-lactamase gene responsible for causing antimicrobial resistance and its prevalence in clinical isolates of P. aeruginosa in the context of Nepal.

The clinical isolates of P. aeruginosa were collected from different hospitals of Kathmandu valley. The isolates were subjected to the biochemical test for confirmation and then to the antibiotic susceptibility test. ESBL production was tested by double disk synergy test. For MBL production, the carbapenem resistant isolates were screened out and then tested for the presence of MBL enzyme and its detection was done by EDTA combined disk test. Molecular identification of the responsible gene blaIMP, blaVIM and blaNDM causing MDR in those MBL producers was done by Polymerase Chain Reaction (PCR) using gene specific primers. Sequencing was then carried out. Of total 67 consecutive isolates of P. aeruginosa, 52 (77.61%) were MDR P. aeruginosa, none of them were ESBL producers and 30 (75%) of total 40 carbapenem resistant isolates showed positive result for MBL phenotypically. Additionally, the results of PCR method showed that 15 strains (37.5%) of carbapenem resistant isolates contained blaNDM (New Delhi Metallo-β-lactamase) gene. No other gene was found in the examined samples. Further sequence analysis showed the presence of blaNDM-1 in majority of sequenced isolates and 3 novel variants of this gene was also detected. The prevalence of the MBLs has been increasing worldwide, particularly among P. aeruginosa, leading to severe limitations in the therapeutic options. Thus, antimicrobial stewardship should be implemented to minimize the emergence of this β-lactamase producing pathogens. Molecular surveillance on a regular basis and proper resistance screening measures needs to be adopted to prevent spreading of these superbugs.

Keywords: blaNDM, Carbapenem, Metallo-β-lactamases, Pseudomonas aeruginosa, Sequencing.