Multivariate risk assessment of the taxonomic and virulence landscapes of culturable antibiotic-resistant bacteria in an Indian Ramsar site

Introduction Antibiotic resistance is spreading throughout aquatic ecosystems, especially wetlands that receive urban and industrial effluents. These environments are large reservoirs under anthropogenic selection pressure, favouring the persistence and transmission of multidrug-resistant (MDR) diseases. This study investigated the biofilm-forming capacity and antimicrobial resistance profiles of 32 environmental bacterial isolates recovered in 2024, assessing the microbiological water quality of a sewage-fed aquaculture unit within the East Kolkata Wetlands (EKW), an Indian Ramsar site. Methods To characterise persistence-associated biofilm formation, phenotypic antimicrobial resistance, and hemolytic activity, isolates were evaluated using quantitative biofilm assays, antimicrobial susceptibility testing, and blood-agar hemolysis profiling, respectively. We constructed a composite phenotypic risk score using a risk-quadrant approach to classify isolates based on the convergence of multidrug resistance, strong biofilm formation, and hemolytic activity. Results The findings showed a positive correlation between biofilm development and the multiple-antibiotic-resistance (MAR) index, indicating a close relationship between sessile microbial lifestyles and enhanced survival under antibiotic stress. Pseudomonas and Citrobacter , two genera known to flourish in moist environments, were well represented in the taxonomic profile, which was dominated by the genus Enterobacter . Phylogenetic characterisation using 16S rDNA sequencing revealed a diverse community, with distinct groups of Enterobacter , Pseudomonas , and Citrobacter . BLASTn identity scores ranged from 99.43 to 100%, supporting robust taxonomic classification. Alpha hemolysis predominated, suggesting a significant presence of potential opportunistic pathogens, as supported by the identification of high-risk isolates. Discussion These evolutionary linkages underscore the wetland’s significance in conserving therapeutically relevant bacterial lineages amid sustained anthropogenic assault. The clustering of these variables suggests the potential for environmental transmission of high-MAR strains via aquatic routes. These findings emphasise the need to integrate ecological monitoring into broader biosafety frameworks to mitigate the risks posed by persistent antimicrobial resistance in wetland reservoirs.