Copper in different forms and tetracycline affect behavior and risk of antibiotic resistome in thermophilic anaerobic digestion of cattle manure

The metagenomics-based behavior and risk of antibiotic resistance genes (ARGs) were investigated during cattle manure thermophilic anaerobic digestion with tetracycline and copper, namely, bulk-copper oxide, nano-copper oxide, and copper sulfate, which are common feed additives. Although bulk-copper oxide reduced ARGs’ diversity, it enriched high-risk ARGs the most than the other two copper species, while copper sulfate could strongly mitigate the ARG risk by decreasing their abundances. Compared to corresponding individual effects, copper and tetracycline combinations may decrease ARGs’ co-occurrence potential by 22.0%, and particularly, tetracycline combined separately with copper sulfate and nano-copper oxide reduces the ARGs’ risk in abundance (by 7.2%) and human health (by 4.0%). These were mainly driven by bioavailable copper, volatile fatty acids, and pH, as well as the main potential hosts in phyla Firmicutes, Coprothermobacterota, and Euryarchaeota. Notably, the twin risks of pathogenicity and ARGs should be emphasized due to the ARGs’ positive correlation with human pathogens of Clostridioides difficile and Arcobacter peruensis . These findings are important for understanding the potential ARGs’ risk in treatments of livestock wastes containing feed additives of different sizes and speciation.