Overlooked Roles of Pharmaceutical Metabolic Products in Stimulating Microbial Metabolism and Antibiotic Resistance Gene Dissemination of Anaerobic Sludge

The roles of non-antibiotic pharmaceuticals in shaping the dissemination behaviours of antibiotic resistance genes (ARGs) in wastewater treatment systems remain poorly understood, and the influences of their transformation products have been overlooked. Here, we unveil more profound impacts of the metformin (MET) biotransformation product than the parent pollutant on the microbial community structure and ARG propagation of wastewater anaerobic sludge. The exposure to MET and its metabolic products guanylurea (GUA) at environmentally relevant concentrations both raised the methane production and resulted in up to 52.5% higher sludge ARGs abundance relative to the unexposed control. Especially, the GUA group showed up to 188-fold upregulation in several ARGs including bcrA, PmrF, acrB and mexF, enabled 3218-fold enrichment of plasmids from several bacteria. The underlying mechanisms were elucidated by integrated metagenomics, molecular dynamics simulations, and metabolic profiling analyses. MET and GUA were found to trigger coordinated cellular responses including disrupted glycerophospholipid metabolism, increased membrane permeability and broad metabolic reprogramming, which collectively boosted the ARGs dissemination. Overall, this work establishes a mechanistic link between micropollutant-induced microbial stress and ARGs propagation in anaerobic sludge, and advocates for re-evaluating the environmental risks of non-antibiotic pharmaceuticals and integrating resistance control into wastewater management framework.