Wastewaters are plausible arenas for antibiotic resistance evolution and transmission, yet selection for resistance by municipal wastewater has rarely been empirically demonstrated. Here, we experimentally investigate the potential of untreated municipal wastewater from 47 countries to select for antibiotic resistance and explore possible drivers. Using a functional selection assay with 340 mixed Escherichia coli strains, we find that sterile-filtered samples from 14 countries select significantly for resistance to at least one antibiotic class compared to baseline, while the majority select significantly against resistance. Similar results were generated using natural wastewater communities. Additionally, we report a comprehensive characterization of antibiotics and antibacterial biocides in the wastewaters. None of the 22 analyzed antibiotics could be assigned as key drivers for selection in E. coli, whereas e.g. folate pathway antagonists and macrolides often exceed predicted non-selective concentrations for other bacteria by >10-fold. Despite detecting 13 (out of 20 analyzed) organic antibacterial biocides, their potential for co-selection remains unclear. Measured chemical constituents correlate only weakly with observed selection, suggesting complex mixture effects and/or selection by unmeasured compounds. The clear deselection of resistance by most samples indicates that many resistant strains have impaired fitness in wastewaters with limited antibiotic selection pressure.

Centre for Antibiotic Resistance Research at the University of Gothenburg Gothenburg Sweden

Department of Chemistry Umea University Umeå Sweden

Department of Infectious Diseases Institute of Biomedicine The Sahlgrenska Academy at the University of Gothenburg Gothenburg Sweden

Research Group for Genomic Epidemiology National Food Institute Technical University of Denmark Kgs Lyngby Denmark

Research Institute of Fish Culture and Hydrobiology South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses Faculty of Fisheries and Protection of Waters University of South Bohemia in České Budějovice Vodňany Czech Republic

The Sahlgrenska University Hospital Gothenburg Sweden

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