Comprehensive assessment of antibiotic impacts and risk thresholds on aquatic microbiomes and resistomes

Understanding the impacts of environmentally relevant low-level antibiotics on aquatic microbiomes and resistomes is crucial for risk assessment of anthropogenic antibiotic contamination. Here, we investigated the effects of seven subinhibitory concentrations of trimethoprim and lincomycin (10 ng/L to 10 mg/L), individually and in combination, on surface water microcosms over 1 and 7 days, using unspiked samples as controls. Metagenomic sequencing revealed a decrease in bacterial community α-diversity and an increase in resistome α-diversity with rising antibiotic concentrations upon 7 days of exposure. Notably, the β-diversity of both bacterial communities and resistomes exhibited a biphasic response, decreasing and then increasing with breakpoint concentrations of 2.73 µg/L and 0.68 µg/L, respectively. We also observed concentration-dependent increases in certain metagenome-assembled antibiotic-resistant bacteria (MAARB) and antibiotic resistance genes (ARGs), with minimum selective concentrations (MSCs) of 2.28 µg/L for trimethoprim targeting OXA-21 and 32.4 µg/L for lincomycin targeting erm(F). Among various metrics for identifying risk thresholds that induce significant changes in microbial taxa, resistomes, individual ARGs, and MAARB, the breakpoint concentration derived from resistome β-diversity was the most conservative. We propose integrating this metric into environmental risk assessment frameworks for antibiotics. Our study provides a systematic evaluation of antibiotic impacts on aquatic microbiomes and resistomes, offering key insights for refining risk assessments of antibiotic contamination in aquatic environments.