Effects of combined antibiotics on nitrification, bacteria and antibiotic resistance genes in activated sludge: Insights from legacy effect of antibiotics

The effect of combined antibiotics exposure on nitrogen removal, microbial community assembly and proliferation of antibiotics resistance genes (ARGs) is a hotspot in activated sludge system. However, it is unclear that how the historical antibiotic stress affects the subsequent responses of microbes and ARGs to combined antibiotics. In this study, the effects of combined sulfamethoxazole (SMX) and trimethoprim (TMP) pollution on activated sludge under legacy of SMX or TMP stress with different doses (0.005-30 mg/L) were investigated to clarify antibiotic legacy effects. Nitrification activity was inhibited under higher level of combined exposure but a high total nitrogen removal (∼70%) occurred. Based on the full-scale classification, the legacy effect of past antibiotic stress had a marked effect on community composition of conditionally abundant taxa (CAT) and conditionally rare or abundant taxa (CRAT). Rare taxa (RT) were the keystone taxa in the microbial network, and the responses of hub genera were also affected by the legacy of antibiotic stress. Nitrifying bacteria and genes were inhibited by the antibiotics and aerobic denitrifying bacteria (Pseudomonas, Thaurea and Hydrogenophaga) were enriched under legacy of high dose, as were the key denitrifying genes (napA, nirK and norB). Furthermore, the occurrences and co-selection relationship of 94 ARGs were affected by legacy effect. While, some shared hosts (eg., Citrobacter) and hub ARGs (eg., mdtD, mdtE and acrD) were identified. Overall, antibiotic legacy could affect responses of activated sludge to combined antibiotic and the legacy effect was stronger at higher exposure levels.