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Background  The prevalence of antimicrobial resistance (AMR) among Gram-negative bacteria is alarmingly high. Reintroduction of colistin as last resort treatment in the infections caused by drug-resistant Gram-negative bacteria has led to the emergence and spread of colistin resistance. This study was designed to determine the prevalence of drug-resistance among beta-lactamase-producing strains of  Escherichia coli  and  Klebsiella pneumoniae,  isolated from the clinical specimens received at a tertiary care centre of Kathmandu, Nepal during the period of March to August, 2019.    Methods  A total of 3216 different clinical samples were processed in the Microbiology laboratory of Kathmandu Model Hospital. Gram-negative isolates ( E. coli and K. pneumoniae ) were processed for antimicrobial susceptibility test (AST) by using modified Kirby-Bauer disc diffusion method. Drug-resistant isolates were further screened for extended-spectrum beta-lactamase (ESBL), metallo-beta-lactamase (MBL), carbapenemase and  K. pneumoniae  carbapenemase (KPC) production tests. All the suspected enzyme producers were processed for phenotypic confirmatory tests. Colistin resistance was determined by minimum inhibitory concentration (MIC) using agar dilution method. Colistin resistant strains were further screened for plasmid-mediated  mcr-1  gene using conventional polymerase chain reaction (PCR).    Results  Among the total samples processed, 16.4% (529/3216) samples had bacterial growth. A total of 583 bacterial isolates were recovered from 529 clinical samples. Among the total isolates, 78.0% (455/583) isolates were Gram-negative bacteria. The most predominant isolate among Gram-negatives was  E. coli  (66.4%; 302/455) and  K. pneumoniae  isolates were 9% (41/455). In AST, colistin, polymyxin B and tigecycline were the most effective antibiotics. The overall prevalence of multidrug-resistance (MDR) among both of the isolates was 58.0% (199/343). In the ESBL testing, 41.1% (n = 141) isolates were confirmed as ESBL-producers. The prevalence of ESBL-producing  E. coli  was 43% (130/302) whereas that of  K .  pneumoniae  was 26.8% (11/41). Similarly, 12.5% (43/343) of the total isolates, 10.9% (33/302) of  E. coli  and 24.3% of (10/41)  K. pneumoniae  were resistant to carbapenem. Among 43 carbapenem resistant isolates, 30.2% (13/43) and 60.5% (26/43) were KPC and MBL-producers respectively. KPC-producers isolates of  E. coli  and  K. pneumoniae  were 33.3% (11/33) and 20% (2/10) respectively. Similarly, 63.6% (21/33) of the  E. coli  and 50% (5/10) of the  K. pneumoniae  were MBL-producers. In MIC assay, 2.2% (4/179) of  E. coli  and 10% (2/20) of  K. pneumoniae  isolates were confirmed as colistin resistant (MIC ≥ 4 µg/ml). Overall, the prevalence of colistin resistance was 3.1% (6/199) and acquisition of mcr-1 was 16.6% (3/18) among the  E. coli  isolates.    Conclusion  High prevalence of drug-resistance in our study is indicative of a deteriorating situation of AMR. Moreover, significant prevalence of resistant enzymes in our study reinforces their roles in the emergence of drug resistance. Resistance to last resort drug (colistin) and the isolation of  mcr-1  indicate further urgency in infection management. Therefore, extensive surveillance, formulation and implementation of effective policies, augmentation of diagnostic facilities and incorporation of antibiotic stewardship programs can be some remedies to cope with this global crisis.    Supplementary Information  The online version contains supplementary material available at 10.1186/s13099-021-00441-5.

gut pathogens

Antibiotic resistance and detection of plasmid mediated colistin resistance mcr-1 gene among Escherichia coli and Klebsiella pneumoniae isolated from clinical samples