Long-read metagenomic sequencing reveals that high-copy small plasmids shape the highly prevalent antibiotic resistance genes in animal fecal microbiome

The emergence and prevalence of animal-derived antibiotic resistance genes (ARGs) pose a great threat to public health globally. Long-read metagenomic sequencing is increasingly being used to decipher the fate of environmental ARGs. However, the investigations of the distribution, co-occurrence patterns, and host information of animal-derived environmental ARGs with long-read metagenomic sequencing have received little attention. To cover the gap, we employed a novel QitanTech nanopore long-read metagenomic sequencing method to perform a comprehensive and systematic investigation of the microbial communities and antibiotic resistance profiles, as well as to analyze the host information and genetic structures of ARGs in the feces of laying hens. Our results showed that highly abundant and diverse ARGs were detected in the feces of different ages of laying hens, indicating that feeding animal feces was an important reservoir for the enrichment and maintenance of ARGs. The distribution pattern of chromosomal ARGs was more strongly associated with fecal microbial communities than plasmid-mediated ARGs. Further long-read host tracking analysis revealed that ARGs from Proteobacteria are commonly located on plasmids, whereas in Firmicutes, they are usually carried by chromosomes. Co-occurrence analysis displayed that co-selection phenomena of different ARGs were common occurrences and highly active insertion sequences (ISs) could result in the serious prevalence of many ARGs. Notably, small high-copy plasmids played a significant role in the dissemination of several ARGs, such as floR and tet(L), which could disturb the compositions of fecal ARGs. Overall, our findings significantly expand our knowledge of the comprehensive landscape of feeding animal feces resistome, which is important for the prevention and management of multi-drug resistant bacteria in laying hens.