Antimicrobial resistance, conjugative plasmids and pathogenicity in wastewater and freshwater Escherichia spp. in Stockholm, Sweden

Abstract To investigate antibiotic resistance genes (ARGs), conjugative plasmids, and virulence genes in Swedish waterborne Escherichia spp., water samples were collected from urban freshwater and Baltic Sea beaches and a primary wastewater treatment plant (WWTP) in Stockholm, Sweden. During the summer of 2022, 68 isolates were recovered using ESBL-selective and non-selective agar, including 40 from wastewater and 28 from fresh or brackish water. Isolates were characterised by phenotypic antibiotic susceptibility testing, conjugation assays, and whole-genome sequencing. Antibiotic residues were quantified, with higher concentrations detected at WWTP inlets and outlets than in natural water sources. Overall, 28 isolates (41.17%) were phenotypically multidrug-resistant (MDR), and 18 (26.47%) carried ≥3 ARGs. WWTP-derived isolates showed a significantly higher prevalence of extended-spectrum β-lactamase (ESBL) genes than freshwater isolates (p < 0.0001). Isolates represented diverse multilocus sequence types (MLST), and most harboured ≥1 plasmid. Sixteen strains transferred conjugative plasmids encoding resistance to cefotaxime, tetracycline, streptomycin, and trimethoprim. ESBL genes, including bla CTX-M-15 , were located on IncF, IncN, IncB/O/K/Z, and IncI plasmids, with IncF plasmids showing lower transfer frequencies than IncN and IncI1 conjugative plasmids. These findings identify WWTPs as a major source of MDR and pathogenic E. coli , highlighting their role in environmental dissemination.