Insights into antibiotic resistomes from gut meta-genome-assembled genomes of the free-range chickens.

Antibiotic resistance is a growing global threat, and the chicken gut microbiome is a significant reservoir of antibiotic resistance genes (ARGs). To investigate the presence of ARGs in free-range chickens, which are in closer contact with humans and live in closer proximity to human settlements. In this study, we used metagenomic sequencing to characterize the resistome of the chicken gut. We collected 120 fecal samples from free-range chickens across four Chinese provinces and constructed both metagenome-assembled genomes (MAGs) and comprehensive gene catalogs to investigate microbial community structures and ARG distributions. A total of 2,146 MAGs were reconstructed, encompassing 660 species from 26 phyla, forming a comprehensive genomic catalog of the chicken gut microbiota. We identified 254,316 ARGs representing 159 unique resistance genes across 35 antibiotic classes, with multidrug, tetracycline, glycopeptide, and peptide resistance being most prevalent. Notably, ARG distribution showed strong regional variation, influenced by environmental factors and local farming practices. Mobile genetic elements (MGEs), averaging 33.8 per MAG, were positively correlated with ARG abundance (R = 0.77), underscoring their role in facilitating resistance gene dissemination. Specific MAGs-including strains of Escherichia coli and Klebsiella pneumoniae-harbored hundreds of ARGs and virulence factors, highlighting potential high-risk vectors for resistance spread. Our findings reveal a diverse and regionally dynamic antibiotic resistome in the chicken gut, shaped by microbial composition, environment, and host factors. This study provides a valuable genomic resource and emphasizes the need for targeted interventions and surveillance strategies to mitigate antibiotic resistance in poultry production systems.