Genomic insights into the extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolated from hospital wastewater in Nepal

• Whole genome sequencing of two ESBL-producing isolates from hospital wastewater was analyzed. • Multiple ARGs and MGEs were detected in both E. coli and K. pneumoniae subsp. pneumoniae . • Phylogenetic analysis of the bla CTX-M-15 contigs revealed global dissemination of ESBL genes. • Plasmids in bacteria carry conserved resistance regions, indicating shared global evolutionary lineages. • Enhanced wastewater management and genomic surveillance are critical to curb AMR in Nepal. Hospital wastewater (HWW) serves as a significant reservoir for cephalosporin-resistant Gram-negative pathogens, particularly those harboring extended-spectrum β-lactamase (ESBL) genes. However, comprehensive genomic understanding of bla CTX-M and bla TEM -carrying Enterobacteriaceae in HWW in low- and middle-income countries (LMICs) like Nepal remain limited. To fill this gap, the genome of Escherichia coli and Klebsiella pneumoniae subsp. pneumoniae were characterized, and comparative genome analysis with a global database was further conducted. The E. coli was identified as ST3873, phylotype B1 and serotype O115:H10, while K. pneumoniae subsp. pneumoniae belonged to ST15, capsule type KL24, and antigen type O1/O2v1. In E. coli, a bla CTX-M-15 -positive putative plasmid contig exhibited phylogenetic similarity to 36 sequences from E. coli and Shigella strains reported in 13 countries, mainly from human, animal, and environmental sources. Notably, a plasmid from Japan shared the highest similarity with the tnpA-bla CTX-M-15 - parA1-impB-impC region, while its plasmid backbone segment, sul2-trbC-trbB-trbA , aligned with strains from Germany and UK. In K. pneumoniae , a putative plasmid contig carrying bla CTX-M-15 and bla TEM showed phylogenetic similarity with 15 contigs from K. pneumoniae across five countries, mainly from human clinical samples. A segment containing Tn As3-bla TEM - tnpR- Tn 2-bla CTX-M-15 -Tn 2 shared a strong phylogenetic relationship with a plasmid from the USA. Additionally, a multidrug arrangement ( mphA-Mrx- IS 6100-tnpA-sul1-qacE∆1-aadA2-dfrA12-int ) found in the K. pneumoniae isolate was widely distributed globally. This whole genome sequencing data expands the inventory of ARG epidemiology in Nepal, confirming the global dissemination of antibiotic resistance in diverse countries. Genomic surveillance of hospital wastewater is essential to monitor antibiotic resistance in Nepal and more LMICs.