Deciphering extracellular antibiotic resistance genes (eARGs) in activated sludge by metagenome

Antibiotic resistance genes (ARGs) including extracellular ARGs (eARGs) and intracellular ARGs (iARGs), are recognized as emerging environmental contaminants. Despite extensive efforts to profile ARGs in their "hotspots" wastewater treatment plants (WWTPs), the contribution of eARGs to antibiotic resistance spread remains unclear. Here, we applied metagenomic sequencing to investigate the distribution, mobility and microbial hosts of eARGs in activated sludge from five WWTPs. The total relative abundance eARGs ranged from 9.5 × 10^-6 to 1.3 × 10^-4, mainly encompassing elfamycin, dual drug, and aminoglycoside resistance genes. Multiple eARGs (e.g., EF-Tu, ropB, and rpsL mutants) were shared among the five WWTPs, and some clustered in the same genetic element (e.g., EF-Tu-rpsJ). eARGs were found to frequently co-localize with the eMGEs (e.g., sul1-3'CS-TnAs3, sul2-intI1-ISVsa3, and tetX-p63039), which may facilitate the mobilization of eARGs. Most eARGs likely originated from the genera Mycobacterium (6.7%), Nitrosomonas (5.3%), Steroidobacter (5.3%), Nitrospira (5.2%) and Pseudomonas (5.1%). No significant difference in the diversity, abundance, and mobility was observed between eARGs and iARGs. The host composition of eARGs and iARGs from municipal WWTPs are consistently dominated by Nitrosomonas, Steroidobacter, Nitrospira and Pseudomonas, while some differentially enriched genera (especially Nitrospira) in the hosts of iARGs compared with those of eARGs from the swine WWTP were identified. Our findings corroborate the mobile eARGs reservoir in WWTPs, thereby laying foundation for mitigating widespread antibiotic resistance.