Objective: Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae represent a significant global health concern with elevated prevalence in sub-Saharan Africa. Comprehensive molecular characterization data from Nigerian isolates remain limited.

Patients and Methods: A prospective laboratory surveillance study with retrospective clinical data extraction was conducted at Prince Abubakar Audu University Teaching Hospital from January to December 2023. Clinical isolates of Enterobacteriaceae were screened for ESBL production using phenotypic and molecular methods. Antimicrobial susceptibility testing was performed using disk diffusion and broth microdilution. Multiplex Polymerase Chain Reaction (PCR) detected bla_CTX-M, bla_TEM, and bla_SHV genes. Whole genome sequencing (WGS) was performed on 45 selected multidrug-resistant isolates to identify sequence types and resistance mechanisms.

Results: Of 1,247 Enterobacteriaceae isolates, 542 (43.5%) were confirmed ESBL producers. Escherichia coli (43.2%) and Klebsiella pneumoniae (50.0%) predominated. The bla_CTX-M-15 gene was detected in 55.0% of ESBL-positive isolates, while bla_TEM-1 and bla_SHV-11 were found in 36.5% and 16.4%, respectively. Carbapenem resistance occurred in 23.4% of ESBL-producing isolates, with bla_NDM-1 genes detected in 65.4% of carbapenem-resistant isolates. WGS revealed five novel sequence types and diverse plasmid-mediated resistance mechanisms.

Conclusions: This study documents ESBL production rates of 43.5% among clinical Enterobacteriaceae isolates in a Nigerian tertiary healthcare facility. The predominance of bla_CTX-M-15 and emergence of carbapenem-resistant strains carrying bla_NDM-1 indicate established transmission of internationally disseminated resistance mechanisms. Identified risk factors support targeted implementation of antimicrobial stewardship and enhanced infection control measures.