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The widespread use of antibiotics has created an antibiotic resistance genes (ARGs)-enriched environment, which causes high risks on human and animal health. Although antibiotics can be partially adsorbed and degraded in wastewater treatment processes, striving for a complete understanding of the microbial adaptive mechanism to antibiotic stress remains urgent. Combined with metagenomics and metabolomics, this study revealed that anammox consortia could adapt to lincomycin by spontaneously changing the preference for metabolite utilization and establishing interactions with eukaryotes, such as Ascomycota and Basidiomycota. Specifically, quorum sensing (QS) based microbial regulation and the ARGs transfer mediated by clustered regularly interspaced short palindromic repeats (CRISPR) system and global regulatory genes were the principal adaptive strategies. Western blotting results validated that Cas9 and TrfA were mainly responsible for the alteration of ARGs transfer pathway. These findings highlight the potential adaptative mechanism of microbes to antibiotic stress and fill gaps in horizontal gene transfer pathways in the anammox process, further facilitating the ARGs control through molecular and synthetic biology techniques., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The anaerobic ammonium oxidation (anammox) process is regarded as a promising approach to treat antibiotic-containing wastewater. Therefore, it is urgent to elucidate the effects of various antibiotics on the anammox process. Moreover, the mechanism of extracellular polymeric substance (EPS) as protective barriers to relieve antibiotic stress remain unclear. Therefore, the single and combined effects of erythromycin (ETC) and sulfamethoxazole (SMZ), and interactions between EPS and antibiotics were investigated in this study. Based on a 228-day continuous flow experiment, high concentrations of ETC and SMZ had significant inhibitory effects on the nitrogen removal performance of the anammox process, with the abundances of corresponding antibiotic resistance genes (ARGs) increasing. In addition, the combined inhibitory effect of the two antibiotics on the anammox process was more significant and longer-lasting than that of the single. However, the anammox process was able to quickly recover from deterioration. The tolerance of anammox granules to the stress of low-concentration antibiotics was probably attributed to the increase in ARGs and secretion of EPS. Molecular docking simulation results showed that proteins in EPS could directly bind with SMZ and ETC at the sites of GLU-307, HYS-191, ASP-318 and THR-32, respectively. These findings improved our understanding of various antibiotic effects on the anammox process and the interaction mechanism between antibiotics and proteins in EPS., (Copyright © 2021. Published by Elsevier Ltd.)