Monitoring of natural recreational bathing waters relies primarily on fecal indicator bacteria (FIB), particularly Escherichia coli, but bacterial abundance alone provides limited information on contamination sources, virulence potential and antimicrobial resistance (AMR). Here, we investigated the spatial and seasonal dynamics of E. coli in Lake Bled, Slovenia, and its tributaries over a one-year period using an integrated culture-based, phenotypic, phylogenetic and total community DNA approach. E. coli abundance was consistently higher in tributaries than in lake water and increased during periods associated with agricultural fertilization and the bathing season. Phylogenetic analysis revealed a predominance of environmentally adapted phylogroups A and B1. Virulence-associated genes were widespread, with fimH detected in nearly all isolates. Phenotypic AMR was generally low, with the highest resistance observed to ampicillin (19.5%) and tetracycline (13.8%), and significantly more prevalent among tributary and off-season isolates. In contrast, total community DNA analysis demonstrated a diverse resistome across the catchment, with higher ARG diversity and abundance in inflow sites, widespread detection of intI1, and multiple β-lactam, tetracycline, sulfonamide and aminoglycoside resistance genes. Notably, the colistin resistance gene mcr-4 was detected in more than 25% of samples. The findings demonstrate that tributaries represent important entry points for fecal contamination, virulence determinants and AMR into Lake Bled, while agricultural and recreational pressures shape the composition of E. coli populations. Despite excellent bathing water quality based on FIB monitoring, our results reveal a broader and largely undetected reservoir of clinically relevant microbial traits, supporting the need for integrated surveillance of recreational freshwater ecosystems.
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