Unveiling the Hidden Resistome: A Comprehensive Risk Assessment of Latent Antibiotic Resistance Genes in China's...

Wastewater systems are important reservoirs of antibiotic resistance genes (ARGs), but the ecological and health risks of numerous latent ARGs (LARGs) remain unclear. In this study, we analysed 636 wastewater metagenomic samples from China and constructed a database containing 1587 LARGs. Across all environments, LARGs encoding serine-β-lactamases were the most abundant and prevalent. A comprehensive risk assessment, integrating host pathogenicity, gene mobility and environmental prevalence, was performed on 561 LARGs identified in metagenome-assembled genomes. Most LARGs exhibited low levels across all three dimensions, suggesting limited transmission risk. Nevertheless, 37 high-risk LARGs were identified, indicating non-negligible threats. Functional validation showed that the top three extremely high-risk LARGs significantly enhanced host resistance to ampicillin and ciprofloxacin when expressed in Escherichia coli, while AlphaFold3 revealed typical resistance protein folding, further supporting their functional activity. Horizontal gene transfer analysis indicated that these high-risk genes have disseminated from wastewater to natural water bodies such as rivers via plasmid-mediated mechanisms. Collectively, wastewater acts not only as an 'accumulation pool' for LARGs but also as a potential source releasing 'super-risky' resistance gene into the environment. Therefore, urgent efforts are needed to monitor and control these high-risk LARGs and their mobile genetic elements to block their environmental spread.