Environmental emergence and dissemination of clinically relevant multidrug-resistant bacteria and resistance genes in sewage and aquatic ecosystems

Objectives: This study characterized multidrug-resistant (MDR) bacteria in sewage, river, and marine ecosystems in South Gujarat, India. Methods: Water samples were collected from 25 hospital drainage, sewage treatment/pumping, Tapi River, and coastal marine sites at multiple time points. From 270 screened colonies, 166 morphologically and biochemically distinct nonduplicate isolates were retained. Antimicrobial susceptibility was assessed, and isolates were classified as MDR, extensively drug-resistant (XDR), or pan-drug-resistant (PDR). Biofilm formation, metabolic activity, extracellular polymeric substance protein, heavy metal tolerance, extended-spectrum β-lactamase production, carbapenemase production, and metallo-β-lactamase activity were assessed phenotypically. Tapi River estuary water was used for taxonomic profiling and antibiotic resistance gene (ARG) detection. Results: The 166 isolates comprised 45 bacterial species, with clinically significant Gram-negative pathogens predominating, including Pseudomonas aeruginosa (n=24, 14.5%), Ochrobactrum intermedium (n=16, 9.6%), Stenotrophomonas maltophilia (n=15, 9.0%), and Escherichia coli (n=12, 7.2%). MDR phenotypes were detected in 80.1% of isolates; 18.1% were XDR, and 1.8% were PDR, with PDR isolates confined to river water samples. Biofilm formation was observed in 86.1% (n=143) of isolates, including 28.3% (n=47) strong, 24.7% (n=41) moderate, and 32.5% (n=54) weak producers. Extended-spectrum β-lactamase production was confirmed in 13.4% of Gram-negative isolates, and carbapenemase activity was detected in 11 isolates. Zinc and copper tolerance were significantly higher in XDR than in MDR isolates (p<0.05). Metagenomics identified efflux pumps as the dominant ARG class (36.2%), followed by target-site mutations (21.3%) and β-lactamases (14.9%), and detected blaCTX-M-15, blaTEM-207, gyrA, and parC. Conclusion: These interconnected aquatic systems represent important reservoirs for community-level antimicrobial resistance transmission.