Dynamic changes in the plasmidome and resistome in the gastrointestinal tract of chickens.

The expansion of intensive poultry farming has led to a substantial increase in antibiotic use, which, in turn, has promoted the accumulation of antibiotic resistance genes (ARGs). The chicken gut serves as a reservoir for these genes and provides favorable conditions for their horizontal transfer via mobile genetic elements, such as plasmids. Through this process, commensal bacteria can transfer ARGs to pathogens, facilitating their spread and increasing the risk of transmission to humans. In this study, long-read sequencing was used to characterize the plasmidome and resistome in 12 fecal samples from 3 houses of a commercial broiler chicken farm. All chickens received enrofloxacin in the first days of life, with one house additionally treated with sulfamethoxazole/trimethoprim combination. For comparison, metagenomic analysis using short-read sequencing was performed on the same samples. This study revealed the presence of various ARGs associated with resistance to 25 antibiotic classes. A strong genetic association between MOBP-type plasmids and fluoroquinolone resistance was observed within broiler chicken farms. Temporal trends indicated progressive mobilization of these ARGs, suggesting an increasing potential for horizontal gene transfer. While fluoroquinolone resistance expanded over time, diaminopyrimidine resistance remained stable despite the antibiotic treatment. Most ARGs were carried on small plasmids, and complete plasmid reconstructions ranged from 2.6 to 47.6 kb. Our findings demonstrate that plasmidome sequencing enables high-resolution detection of resistance-associated plasmids that may be overlooked by conventional metagenomic approaches. The observed patterns are consistent with an association between fluoroquinolone use in poultry farms and the presence of plasmid-mediated resistance genes with potential for horizontal dissemination.IMPORTANCEDespite the crucial role of plasmids in antimicrobial resistance (AMR) dissemination, studies focusing on plasmidomes, defined as the complete set of plasmids, remain limited. This study is the evidence that chicken farms, where fluoroquinolone treatment is a standard practice, act as an important reservoir of plasmid-mediated antibiotic resistance which may not be revealed by commonly used approaches. Combining a metagenomic approach with a focus on plasmids enhances our ability to understand the genetic context and mechanisms underlying AMR transmission. The findings emphasize the importance of targeted plasmid analysis to improve surveillance and risk assessment of AMR transmission in microbial ecosystems.