Fig. 1: Experimental design and microbial consortium responses to combined antibiotic pollution.

a Workflow for domesticating aquatic microbial communities from the same water sample via exposure to tetracycline (TC), oxytetracycline (OTC), and their combination (TCs). Functional microbiomes were analyzed by amplicon sequencingand metabolic modeling of the top 50 abundant strains. Potential keystone species were thus identified by integrating abundance shifts and strain isolation. Untargeted metabolomics was applied to single-, co-, and tri-strain cocultures under three treatments: antibiotic-free control (CK), exposure to a single antibiotic (TC or OTC), and exposure to the combined TCs treatment. Since metabolomic analysis was only performed on co-cultures with up to three strains, the microbial community modeling framework SuperCC was applied to construct multi-strain metabolic models with a larger number of strains, aiming to predict strain-specific biomass and identify key interspecies metabolites. Model predictions were subsequently validated through experimental analyses. Based on these conclusions, we proposed a synthetic microbiome construction strategy: the Degrader–Helper–Potentiator Consortium (DHP-Com) paradigm, and provided the definition of potentiator. b Enhanced degradation efficiency of TC and OTC by acclimated microbial consortia. The degradation efficiency was determined by measuring residual antibiotic concentrations in degradation medium (DM) supplemented with 50 mg/L TC or 25 mg/L OTC at 0, 3, 7, and 10 days after inoculation with the consortia acclimated over 10 rounds. Data are presented as mean values ± SD (n = 3 biological independent replicates). c The Chao1 index was used to evaluate microbiome α-diversity within each consortium. Statistical significance of observed differences was determined by one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, n = 3 biological independent replicates). Data are presented as mean values ± SD. d β-diversity was assessed through Principal Coordinates Analysis (PCoA) using Bray-Curtis dissimilarity metrics. e Gene profiles related to aromatic compound degradation. The relative abundance of key aromatic degradation genes at the KEGG Orthology (KO) level across treatments was visualized by heatmap, with functional predictions generated using PICRUSt2. Source data for this figure is available in the Source data file.