Extended-spectrum β-lactamases (ESBL)-producing Escherichia coli represents one of the main priorities in the surveillance of antibiotic-resistant bacteria. Its presence is well studied in human and animal reservoirs, whereas it is less explored in environmental reservoirs. The aim of our study was to compare the genotypes of ESBL-producing E. coli from human and environmental sources in Slovenia and to assess their genetic relatedness. Between June 2023 and May 2024, we collected 561 human and 564 environmental isolates from three regions of Slovenia. Environmental samples were obtained from water and sediment of various aquatic sources, including rivers, tributaries, lakes, gravel pits, and inflows to wastewater treatment plants. Whole-genome sequencing was used to perform multilocus sequence typing (MLST) and core genome MLST (cgMLST), to identify ESBL genes, and to analyze plasmid content. In total, 196 sequence types were identified. Environmental isolates showed greater genetic diversity than human isolates, in which sequence type ST131 strongly predominated (45%). A total of 48 sequence types were shared between the two reservoirs, representing 77% of all isolates. Several clonal groups were also identified, comprising isolates from both reservoirs. Among ESBL genes, blaCTX-M-15 predominated in both reservoirs. Greater plasmid diversity was observed in environmental isolates. At the same time, ESBL-encoding plasmids with identical replicon types were identified in both human and environmental reservoirs. The results indicate a significant genetic linkage between human and environmental reservoirs and highlight the importance of including environmental surveillance in strategies for monitoring antibiotic-resistant bacteria.
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