Antimicrobial effects, and selection for AMR by non-antibiotic drugs in a wastewater bacterial community

Antimicrobial resistance (AMR) is a major threat to human, animal, and crop health. AMR can be directly selected for by antibiotics, and indirectly co-selected for by biocides and metals, at environmentally relevant concentrations. Some evidence suggests that non-antibiotic drugs (NADs) can co-select for AMR, but previous work focused on exposing single model bacterial species to predominately high concentrations of NADs. There is a significant knowledge gap in understanding a range of NAD concentrations, (including lower µg/L concentrations found in the environment) on mixed bacterial communities containing a diverse mobile resistome. Here, we determined the antimicrobial effect and selective potential of diclofenac, metformin, and 17-β-estradiol, NADs that are commonly found environmental pollutants, in a complex bacterial community using a combination of culture based, metagenome, and metratranscriptome approaches. We found that diclofenac, metformin, and 17-β-estradiol at 50 µg/L, 26 µg/L, and 24 µg/L respectively, significantly reduced growth of a bacterial community although only 17-β-estradiol selected for an AMR marker using qPCR (from 7 µg/L to 5400 µg/L). Whole metagenome sequencing indicated that there was no clear selection by NADs for antibiotic resistance genes, or effects on community composition. Additionally, increases in relative abundance of some specific metal resistance genes (such as arsB) were observed after exposure to diclofenac, metformin, and 17-β-estradiol. These results indicate that environmentally relevant concentrations of NADs are likely to affect community growth, function, and potentially selection for specific metal resistance genes.