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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

Antimicrobial resistance in Enterobacteriaceae is an emerging global public health problem. Numerous studies have reported community-acquired AmpC beta-lactamase and extended spectrum beta-lactamase (ESBL) producing Enterobacteriaceae in Nepal. However, there are limited data on community-acquired Metallo-beta-lactamase (MBL) producing Enterobacteriaceae. A hospital-based descriptive cross-sectional study was conducted using 294 Enterobacteriaceae isolates from a total of 2,345 different clinical specimens collected from patients attending a tertiary care hospital in Nepal. Bacteria were isolated using standard microbiological growth media and identified using biochemical tests. For antimicrobial susceptibility testing, Kirby-Bauer disc diffusion technique was used. AmpC, ESBL, and MBL productions were detected by using combined disc method. AmpC, ESBL, and MBL productions were detected in 19.4%, 29.6%, and 8.5% of total Enterobacteriaceae isolates respectively. Higher rates of beta-lactamases production were seen among the isolates from in-patients in comparison with those from out-patients. However, 11.6%, 25%, and 3.7% of the total isolates from out-patients were AmpC, ESBL, and MBL producers respectively. The co-production of the beta-lactamases was also detected, with two Klebsiella pneumoniae isolates producing all three beta-lactamases. One MBL producing Proteus vulgaris isolate that was pan-resistant with no remaining treatment options was also isolated. Prevalence of drug resistant Enterobacteriaceae in our study was very high. Detection of AmpC, ESBL, and MBL positive isolates from out-patients, who did not have recent history of hospital visit, indicated the community dissemination of the drug resistant bacteria. This is a matter of great concern and an immediate attention to formulate strategies to prevent further development and spread of antibiotic resistance is required., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Bhandari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: 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 (( bla NDM-1, bla NDM-1 and bla NDM-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 bla NDM-1 , bla NDM-2 and bla NDM-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., Competing Interests: The authors report no conflicts of interest in relation to this work., (© 2022 Thapa et al.)

Swine nasal samples [n = 282] were collected from 12 randomly selected farms around Kathmandu, Nepal, from healthy animals. In addition, wild monkey (Macaca mulatta) saliva samples [n = 59] were collected near temples areas in Kathmandu using a non-invasive sampling technique. All samples were processed for MRSA using standardized selective media and conventional biochemical tests. MRSA verification was done and isolates characterized by SCCmec, multilocus sequence typing, whole genome sequencing [WGS] and antibiotic susceptibilities. Six (2.1%) swine MRSA were isolated from five of the different swine herds tested, five were ST22 type IV and one ST88 type V. Four (6.8%) macaques MRSA were isolated, with three ST22 SCCmec type IV and one ST239 type III. WGS sequencing showed that the eight ciprofloxacin resistant ST22 isolates carried gyrA mutation [S84L]. Six isolates carried the erm(C) genes, five isolates carried aacC-aphD genes and four isolates carried blaZ genes. The swine linezolid resistant ST22 did not carry any known acquired linezolid resistance genes but had a mutation in ribosomal protein L22 [A29V] and an insertion in L4 [68KG69], both previously associated with linezolid resistance. Multiple virulence factors were also identified. This is the first time MRSA ST22 SCCmec IV has been isolated from livestock or primates.