Metagenomic assembly reveals the circadian oscillations of the microbiome and antibiotic resistance genes in a model of laying hens.

The increasing risks of antibiotic resistance genes (ARGs) in livestock feces have attracted global attention. However, how the rhythmic activity of ARGs changes in fecal microbiota remains largely unclear. In our study, we collected 52 fresh fecal samples every 6 h over 72 h from laying hens and characterized circadian oscillations of bacteria and ARGs using an approach based on assembled metagenome shotgun sequencing. We found that 14% of commensal bacterial taxonomic units fluctuated over 24 h. A total of 33 out of 281 ARGs and 17 of 574 mobile genetic elements (MGEs) featured rhythmic patterns in feces. lnuC and ANT(6)H-lb were the two most abundant ARGs with circadian oscillation identified from feces, and they increased during the day and decreased at night. Acetate, butyrate, propionate, and 78 out of 392 MetaCyc pathways relating to short-chain fatty acid (SCFA) metabolism featured circadian oscillations. Assessment of the above ARG-other element connections revealed that 17 ARGs presented strong correlations with 7 MGEs, and 2 SCFAs (acetate and propanoate) and bacterial species in feces. Structural equation model (SEM) analysis showed that ARGs were directly affected by microbial β-diversity and MGEs. These results showed a comprehensive map of ARGs over 24 h and revealed circadian oscillations of ARGs, which are influenced by key bacterial species, MGEs, and metabolites. Together, our findings advance comprehension of circadian oscillations of ARGs in the fecal microbiota and provide a reference for ARGs control and management.