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Anisha Thapa,1,* Milan Kumar Upreti,1,* Nabin Kishor Bimali,1 Basudha Shrestha,2 Anil Kumar Sah,3 Krishus Nepal,1 Binod Dhungel,4 Sanjib Adhikari,4 Nabaraj Adhikari,4 Binod Lekhak,1,4 Komal Raj Rijal4 1Department of Microbiology, Golden Gate International College, Kathmandu, Nepal; 2Kathmandu Model Hospital, Kathmandu, Nepal; 3Annapurna Neurological Institute and Allied Sciences, Kathmandu, Nepal; 4Central Department of Microbiology, Tribhuvan University, Kathmandu, Nepal*These authors contributed equally to this workCorrespondence: Komal Raj Rijal, Central Department of Microbiology, Tribhuvan University, Kirtipur, Kathmandu, Nepal, Email rijalkomal@gmail.comBackground: Increasing burden of carbapenem resistance among Enterobacterales is attributable to their ability to produce carbapenemase enzymes like metallo-beta-lactamase (MBL), Klebsiella pneumoniae carbapenemase (KPC), and OXA-type. This study aimed to determine the prevalence of carbapenemases and MBL genes ((blaNDM-1, blaNDM-1 and blaNDM-3) among E. coli and K. pneumoniae isolates.Methods: A total of 2474 urine samples collected during the study period (July–December 2017) were processed at the microbiology laboratory of Kathmandu Model Hospital, Kathmandu. Isolates of E. coli and K. pneumoniae were processed for antimicrobial susceptibility testing (AST) by disc diffusion method. Carbapenem-resistant isolates were subjected to Modified Hodge Test (MHT) for phenotypic confirmation, and inhibitor-based combined disc tests for the differentiation of carbapenemase (MBL and KPC). MBL-producing isolates were screened for NDM genes by polymerase chain reaction (PCR).Results: Of the total urine samples processed, 19.5% (483/2474) showed the bacterial growth. E. coli (72.6%; 351/483) was the predominant isolate followed by K. pneumoniae (12.6%; 61/483). In AST, 4.4% (18/412) isolates of E. coli (15/351) and K. pneumonia (3/61) showed resistance towards carbapenems, while 1.7% (7/412) of the isolates was confirmed as carbapenem-resistant in MHT. In this study, all (3/3) the isolates of K. pneumoniae were KPC-producers, whereas 66.7% (10/15), 20% (3/15) and 13.3% (2/15) of the E. coli isolates were MBL, KPC and MBL/KPC (both)-producers, respectively. In PCR assay, 80% (8/10), 90% (9/10) and 100% (10/10) of the isolates were positive for blaNDM-1, blaNDM-2 and blaNDM-3, respectively.Conclusion: Presence of NDM genes among carbapenemase-producing isolates is indicative of potential spread of drug-resistant variants. This study recommends the implementation of molecular diagnostic facilities in clinical settings for proper infection control, which can optimize the treatment therapies, and curb the emergence and spread of drug-resistant pathogens.Keywords: E. coli, K. pneumoniae, MBL, KPC, NDM variants