Hidden antibiotic resistance risks and key drivers during tertiary wastewater treatment deciphered by an integrated metagenomic framework

Potential high-risk antibiotic resistance genes (ARGs) were considered as higher public health threats in wastewater treatment systems. While tertiary wastewater treatment processes (TWTPs) effectively remove conventional and emerging pollutants, their impact on ARGs with potential higher risk remains unclear. In this study, metagenomic assembly and binning were applied to profile potential-risk ARGs and identify key factors shaping their distribution during TWTPs. Results showed that potential-risk ARGs accounted for 34.32 ± 1.98% to 59.71 ± 1.55% of total ARGs, indicating their widespread persistence. Notably, DB significantly increased the relative abundance of potential-risk ARGs, particularly those conferring resistance to multidrug, bacitracin, and aminoglycoside. In parallel, DB treatment elevated the abundance of mobile genetic elements (MGEs), primarily transposase-related. DB treatment facilitated the co-occurrence of potential-risk ARGs and MGEs, especially multidrug and transposase/recombinase. Key bacterial hosts carrying potential-risk ARGs, such as Pseudomonas and Acinetobacter, were highly enriched after DB treatment, contributing substantially to the proliferation of these ARGs. In contrast, UV disinfection and CW treatment continuously reduced the abundance and risk levels of potential-risk ARGs, highlighting their complementary roles in mitigating antibiotic resistance risks during TWTPs. Variation partitioning analysis showed that bacterial community composition explained 36.15% of the variation in potential-risk ARG profiles, underscoring its primary role in ARG dynamics. Overall, this study provides genome-resolved insights into the hidden risks of ARGs and key drivers during TWTPs, highlights the necessity of optimizing operational parameters to mitigate antibiotic resistance dissemination.