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• 30 mg/L of combined SMX and TMP increased TN removal (∼70%) and achieved SND. • Abundant genera were more affected by legacy of antibiotics than rare genera. • Rare taxa were keystone taxa in network based on the full-scale classification. • Shared hosts of ARGs and hub ARGs were observed under legacy of antibiotics stress. • Antibiotics legacy effect was more obvious at higher levels of antibiotic stress. 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. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • Compare with AZM, nitrification was more inhibited by QSI. • AZM and QSI had more inhibitions on bacteria and archaea, respectively. • Functions differ little in bacteria/archaea and AZM had more impact on metabolism. • AZM had main impact on nitrifying bacteria and specific DNB were enriched. • Bacteria were main hosts for most ARGs and archaea carried multiple ARGs. Effects of antibiotics (azithromycin, AZM, 1–40 mg/L) and quorum sensing inhibitor (QSI, 2(5H)-furanone, 1–40 mg/L) combined pollution with environmental concentration of copper on bacterial/archaeal community and antibiotic resistance genes (ARGs) in activated sludge system were explored. QSI inhibited nitrification more obviously than AZM. AZM and QSI were synergistic inhibitions on bacterial diversity, and AZM inhibited bacterial compositions more than QSI. While, QSI had more impacts on archaeal diversity/compositions. Less interactions among bacteria and archaea communities with Aquimonas as keystone genus. Functional differences in bacteria/archaea communities were little, and AZM had more effects on metabolism. AZM mainly affected nitrifying bacteria (Candidatus Nitrospira nitrificans and Nitrosomonas). Specific denitrifying bacteria were enriched by AZM (Brevundimonas , 1.76–31.69%) and QSI (Comamonas , 0.61–9.61%), respectively. AZM enriched ARGs more easily than QSI and they were antagonistic to proliferation of ARGs. Bacteria were main hosts of ARGs (macrolide-lincosamide-streptogramin B, other/efflux, etc.) and archaea (Methanosphaerula , Methanolobus) carried multiple ARGs. [ABSTRACT FROM AUTHOR]