Resistomic features and novel genetic element identified in hospital wastewater with short- and long-read metagenomics.

The global spread of antimicrobial resistance (AMR) poses a serious threat to public health, with hospital wastewater treatment plants (WWTPs) recognized as a key hotspot for resistant pathogens and antibiotic resistance genes (ARGs). This study employed advanced hybrid sequencing platforms to provide a comprehensive resistomic analysis of a Qingdao WWTP in China, revealing previously uncovered AMR transmission risks. We identified 175 ARG subtypes conferring resistance to 38 antimicrobials, including the last-resort antibiotics, highlighting the extensive and concerning resistance reservoir within this environment. Multidrug resistance genes predominated, followed by ARGs targeting aminoglycoside, β-lactam, tetracycline, glycopeptide, and macrolide classes, reflecting clinically relevant resistance patterns. Co-occurrence analysis revealed ARGs were strongly associated with mobile genetic elements, especially for ARGs targeting sulfonamide, glycopeptide, macrolide, tetracycline, aminoglycoside, and β-lactam classes, providing concrete evidence of their high dissemination potential. A striking 85 % of 131 metagenome-assembled genomes (MAGs) carried ARGs, demonstrating prevalent resistance in the wastewater microbiome. Furthermore, the identification of several rarely studied genomic islands (GIs), including those conferring resistance to antibiotics and heavy metals, and notably, the novel variant GIAS409 carrying transposases and heavy metal resistance operons, reveals a significant and previously neglected mechanism for co-selection and dissemination. This study significantly advances our understanding of AMR dynamics in hospital WWTPs, demonstrating that current treatment approaches (42 % ARG removal) have limited efficacy and that WWTP may serve as potential hotspots for multidrug resistance development. Collectively, these findings emphasize the urgent need for improved wastewater management to safeguard public health.