Plasmid-borne multidrug resistance genes in environmental isolates from hospital effluent: Implications for resistance dissemination.

Antibiotic resistance (AR) in environmental bacteria poses a critical challenge to public health, driven largely by the dissemination of antibiotic resistance genes (ARGs) via mobile genetic elements such as plasmids. In our previously reported work, environmental bacterial isolates were obtained from diverse aquatic sources contaminated by hospital effluents, and their antibiotic susceptibility profiles were determined. Species-level identification of isolates as Aeromonas spp. was achieved through 16S rRNA sequencing, revealing a diverse microbiota harbouring multidrug resistance (MDR) traits. In the present study, plasmids sequence was reconstructed using the plasmidSPAdes assembler. The assembled plasmid sequences were interrogated against the Comprehensive Antibiotic Resistance Database (CARD), enabling the identification of ARGs and associated accessory genes. Comparative analysis of the five reconstructed plasmid sequences was conducted through network analysis, which allowed the segregation of ARGs shared across all plasmids from those uniquely present. Representative ARGs identified as common were selected for experimental validation. PCR amplification and sequencing detected these target ARGs within the plasmids of different isolates. Subsequently, their abundance was quantified using absolute qPCR, providing precise copy number/μL. This integrated approach from plasmid reconstruction to ARG network mapping and quantitative validation offers a robust framework for understanding the distribution and prevalence of plasmid borne ARGs in environmental bacteria. The findings underscore the pivotal role of plasmids in ARG dissemination and highlight the importance of coupling in silico analysis with experimental confirmation for comprehensive resistance surveillance.