Carbon sources shape activated sludge resistome through direct carbon-assimilating bacteria

Carbon sources significantly influence pollutant removal efficiency at wastewater treatment plants (WWTPs), primarily by shaping the structure and function of microbial communities. Nevertheless, how carbon sources drive the emergence and evolution of antibiotic resistance remains to be elucidated. In this study, we compared the effects of glucose and acetate on the evolution of antibiotic resistance at both community and species levels. Under ciprofloxacin exposure, activated sludge fed with glucose developed resistance levels about 12.5-18.3 times higher than those fed with acetate, accompanied by a 21.05% increase in antibiotic resistance gene (ARG) abundance. DNA stable isotope probing demonstrated that direct glucose assimilators (e.g., Giesbergeria) carried more ARGs, and exhibited a stronger linkage between community composition and resistome structure. Representative strains isolated from the activated sludge (e.g., Enterobacter kobei, Pseudomonas aeruginosa, and Escherichia coli) showed 1.56-10.2 fold higher resistance levels when grown on glucose than on acetate with ciprofloxacin. Glucose utilization provides a robust energy supply that facilitates resistance evolution by mitigating the metabolic burdens associated with mutations at the species level. The results highlight that carbon source management strategies at WWTPs should simultaneously address pollutant removal performance and the mitigation of antibiotic resistance.