Co-occurrence of triple ESBL-associated genes (blaCTX−M, blaTEM and blaSHV) in Gram-negative bacteria from clinical specimens at a provincial hospital in Far-Western Nepal

Antimicrobial resistance (AMR) among Gram-negative bacteria, particularly those producing extended-spectrum β-lactamase (ESBLs), represents a significant global healthcare challenge. CTX-M enzymes have achieved global dominance, often disseminated through plasmid-mediated horizontal gene transfer, complicating treatment in resource-limited settings. This study aimed to determine prevalence, antimicrobial resistance patterns and ESBL-associated gene profiles in clinical Gram-negative isolates. A hospital-based cross-sectional study was conducted at Mahakali Provincial Hospital from November 2024 to February 2026. Using a consecutive sampling technique, 1485 clinical specimens (including urine, pus, blood, wound swabs, throat swabs and tissues) were processed for bacterial culture, identification, antimicrobial susceptibility testing and ESBL detection following CLSI 2024 guidelines. ESBL-associated genes ( bla CTX−M , bla TEM and bla SHV ) were detected by PCR. Of 1485 specimens, 478 (32.3%) were culture-positive, yielding 411 (86.0%) Gram-negative bacteria. Urine samples accounted for 88.3% of isolates. E. coli (71.8%) and K. pneumoniae (15.6%) were the most common pathogens. Overall, 54.7% of isolates were multidrug-resistant (MDR). Among ESBL-screened Enterobacterales ( n = 385), 108 (28.1%) were confirmed ESBL producers. Molecular analysis of 108 ESBL- producing MDR isolates revealed bla CTX−M (56.5%) as the most prevalent gene followed by bla TEM (44.4%) and bla SHV (32.4%). Co-occurrence of multiple genes was observed in 56 (51.9%) isolates, with 6 (5.6%) harboring all three genes. To the best of our knowledge, this is the first report from Far-Western Nepal documenting the co-carriage of triple ESBL-associated genes in clinical Enterobacterales. These findings highlight a high regional burden of community-level resistance and emphasize the urgent need for continuous molecular surveillance, targeted antimicrobial stewardship and region-specific treatment guidelines.