Fates and Characteristics of Antibiotic Resistance Genes Carried by Viruses during a Full-Scale Anaerobic Sludge Digestion.

The transmission of antibiotic resistance genes (ARGs) mediated by viruses in sludge represents an emerging concern for antibiotic resistance dissemination. This study investigated the virus-carried ARGs (vARGs) profiles and potential risks in sludges collected from an entire anaerobic sludge digestion plant. A total of 79 unique vARGs spanning 10 antibiotic resistance classes were identified, with trimethoprim resistance genes being the most diverse. Although most vARGs (94.9%) exhibited low sequence similarity (< 70%) to known ARGs, the representative functional validation showed that virus-carried dfr genes could confer trimethoprim resistance to Escherichia coli. Thermal hydrolysis reduced the levels and diversity of vARGs, whereas subsequent anaerobic digestion led to substantial enrichment. More than 90% of vARGs in sludge viromes were removed after all treatment processes, but a significant number of ARG-carrying viruses (105 - 106 VLPs/g) still remained in the final sludges. In the metagenomes, the levels of vARGs increased approximately 7-fold, suggesting that vARGs were more difficult to be moved than plasmid-associated ARGs and chromosome-associated ARGs. The host prediction suggested associations between some vARG-carrying viruses and potential pathogenic bacteria, which worthing further experimental validation. In summary, the persistence of vARGs in sludge remains a concern, even after the implementation of sludge treatment processes.