Occurrence and characterization of pathogenic bacteria producing β-lactamase in biomedical waste water from hospitals in Dhaka city

Abstract

Antimicrobial resistance (AMR), particularly among Gram-negative pathogens, presents a serious risk to environmental safety and public health worldwide. Hospitals are recognized as primary contributors to this crisis due to their release of high loads of antibiotics and resistant pathogens into waste water systems. This study focused on the detection, characterization, resistance pattern, and genomic analysis of extended-spectrum β-lactamase (ESBL) and carbapenemase-producing Escherichia. coli and Pseudomonas aeruginosa obtained from biomedical waste water collected from several major hospitals in Dhaka City, Bangladesh. A total of 300 waste water samples were collected from hospital effluents and analyzed through standard microbiological, phenotypic, and molecular techniques. Out of 184 isolates, 42.7% (n=38) were E. coli, 25.9% (n=23) were Acinetobacter baumannii, 25.9% (n=23) were P. aeruginosa and 5.6% (n=5) were Enterobacter cloacae. Antibiotic susceptibility testing revealed that E. coli and P. aeruginosa exhibited multidrug resistance (MDR), with notably high resistance rates against ampicillin, cefuroxime and ceftriaxone. Alarming resistance to meropenem, a last-line antibiotic, was also observed. Phenotypic confirmation of ESBL production using double-disc synergy method showed that 48.4% (n = 89) isolates were ESBL-positive. Carbapenemase activity was confirmed in isolates resistant to carbapenems. Multiplex polymerase chain reaction (PCR) indicated the existence of several key resistance genes: blaCTX-M (60%), blaTEM (48%), blaSHV (45%), and carbapenemase genes including blaNDM-1 (26%), blaOXA (22%), and blaKPC (18%). Plasmid profiling confirmed that many of these genes were plasmid-borne, indicating the likelihood of horizontal gene transfer across bacterial species in the waste water environment. In addition to phenotypic and molecular characterization, in silico docking analysis was employed to assess the binding affinities of clinically important β-lactam antibiotics (cefuroxime, ceftriaxone, and meropenem) against selected β-lactamase enzymes. Results indicated moderate binding affinities, with cefuroxime and ceftriaxone showing docking scores of –7.1 kcal/mol and –8.2 kcal/mol, respectively, against CTX-M-type enzymes. Modified derivatives with specific functional groups demonstrated enhanced binding, with docking energies improving up to –8.9 kcal/mol, suggesting that structural modification could potentially improve drug efficacy against resistant strains. Whole genome sequencing (WGS) of selected MDR isolates revealed comprehensive resistomes containing not only ESBL and carbapenemase genes but also multiple virulence Abstract 2 factors and transferable genetic materials, such as integrons and transposons. Several isolates contained co-localized resistance determinants, including blaCTX-M-15, blaNDM-1, and sul1 on the same plasmid structures, reinforcing the threat of horizontal gene transfer. A comparative analysis between isolates from hospital effluents and other urban water sources revealed significant genomic similarity, confirming the environmental spread of antimicrobial resistance genes (ARGs). The present study provides molecular insights into the structure-function relationship of antibiotics and β-lactamases, contributing to the larger field of drug redesign through computational approaches. Additionally, the findings emphasize the critical role that hospital waste water plays in the spread of multidrug-resistant bacteria and the pressing need for better waste management practices in densely populated urban centers. This integrated study—combining environmental microbiology, molecular diagnostics, bioinformatics, and structural biology—advances our understanding of the AMR burden in biomedical waste and presents a multidimensional strategy for surveillance, mitigation, and future therapeutic development. The outcomes emphasize the importance of adopting One Health frameworks and implementing robust environmental monitoring systems to address the escalating AMR threat at the human–environment interface.