Extended spectrum β-lactamase producing Escherichia coli (ESBL E. coli) is a primary concern for hospital and community healthcare settings, often linked to an increased incidence of nosocomial infections. This study investigated the characteristics of ESBL E. coli isolated from hospital environments and clinical samples. In total, 117 ESBL E. coli isolates were obtained. The isolates were subjected to molecular analysis for the presence of resistance and virulence genes, antibiotic susceptibility testing, quantitative adherence assay, ERIC-PCR for phylogenetic analysis and whole genome sequencing of four highly drug resistant isolates. Out of the 117 isolates, 68.4% were positive for bla_CTX-M, 39.3% for bla_TEM, 30.8% for bla_NDM-1, 13.7% for bla_OXA and 1.7% for bla_SHV gene. Upon screening for diarrheagenic genes, no isolates were found to harbour any of the tested genes. In the case of extraintestinal pathogenic E. coli (ExPEC) virulence factors, 7.6%, 11%, 5.9%, 4.3% and 21.2% of isolates harbored the focG, kpsMII, sfaS, afa and iutA genes, respectively. At a temperature of 25°C, 14.5% of isolates exhibited strong biofilm formation with 21.4% and 28.2% exhibiting moderate and weak biofilm formation respectively, whereas 35.9% were non-biofilm formers. On the other hand at 37°C, 2.6% of isolates showed strong biofilm formation with 3.4% and 31.6% showing moderate and weak biofilm formation respectively, whereas, 62.4% were non-biofilm formers. Regarding antibiotic susceptibility testing, all isolates were found to be multidrug-resistant (MDR), with 30 isolates being highly drug resistant. ERIC-PCR resulted in 12 clusters, with cluster E-10 containing the maximum number of isolates. Hierarchical clustering and correlation analysis revealed associations between environmental and clinical isolates, indicating likely transmission and dissemination from the hospital environment to the patients. The whole genome sequencing of four highly drug resistant ExPEC isolates showed the presence of various antimicrobial resistance genes, virulence factors and mobile genetic elements, with isolates harbouring the plasmid incompatibility group IncF (FII, FIB, FIA). The sequenced isolates were identified as human pathogens with a 93.3% average score. This study suggests that ESBL producing E. coli are prevalent in the healthcare settings of Bangladesh, acting as a potential reservoir for AMR bacteria. This information may have a profound effect on treatment, and improvements in public healthcare policies are a necessity to combat the increased incidences of hospital-acquired infections in the country.

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