Prevalence of Biofilm-Forming, ESβLs and Metallo-β-lactamase Producing Gram-Negative MDR Bacteria in the Domestic and Hospital Wastewater of Aligarh City.

Gram-negative pathogenic bacteria are a major contributor to antibiotic-resistant infections in hospitals and communities. The emergence of multidrug resistance (MDR) and biofilm formation complicates chemotherapy. This study aimed to assess the prevalence of multidrug-resistant (MDR) biofilm-forming, extended-spectrum beta-lactamase (ESβL) and metallo-beta-lactamase (MβL) producers in wastewater, which pose a public health threat. During 2022-2023, 117 enteric/Gram-negative isolates were isolated using selective culture techniques. Antimicrobial resistance was assessed via disc diffusion assay. ESβL and MβL production was confirmed through phenotypic and PCR-based methods. Biofilm formation was determined using a microtiter plate assay. Biofilms developed on glass coverslips were visualized by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Isolates with bla-CTX-M, bla-SHV, and bla-NDM-1 genes were identified by partial 16S rRNA gene sequencing. A total of 93.16% of isolates were resistant to multiple drug classes (≥ 4), with the increased resistance to ampicillin (100%) and the lowest to gentamicin (12.82%). Biofilm assays indicated that 32.48% of MDR strains formed strong biofilms, 31.62% moderate, and 35.90% weak. β-lactamase activity appeared in 58.97% of isolates, with 56.41% confirmed as ESβL producers. PCR detection in ESβL-positive strains showed 84.61% carried CTX-M, 46.15% SHV, and 53.84% NDM-1 genes. 16S rRNA gene sequencing identified selected MDR strains as Escherichia coli (5), Klebsiella pneumoniae (1), Pseudomonas aeruginosa (1), Salmonella sp. (1), Enterobacter sp. (1), Citrobacter sp. (1), and Proteus sp. (1). The findings reveal the prevalence of biofilm-forming, ESβL, and MβL-producing Gram-negative pathogens in Aligarh's wastewater, underscoring the need for effective treatment to reduce public health risks from MDR bacteria and AMR genes.