Antimicrobial resistance characteristics of enterococcus and analysis of intra-farm transmission associations in large-scale laying hen production.

The emergence and transmission of antimicrobial resistance (AMR) in layer production has drawn considerable attention. Enterococcus spp. are recognized as indicator bacteria for AMR, and systematic investigation of these organisms can help elucidate the dynamics of AMR risks. In this study, a total of 1,016 samples were collected at multiple time points and from various sites across the production chain on a large-scale layer farm. Enterococci were isolated and cultured, and selected strains were subjected to antimicrobial susceptibility testing and whole-genome sequencing. The overall isolation rate of Enterococcus was 80.0%, with the highest rate observed in floor samples (92.9%). The predominant resistance phenotypes were to macrolides, tetracyclines, and sulfonamides. Genome sequencing revealed that all sequenced E. faecalis strains harbored tet(L), lsa(A), and optrA, whereas all E. faecium strains carried aac(6')-Ii and msr(C). Mobile genetic elements were present in 97.2% of the isolates, and the transposon Tn554 was identified in 13 strains across all production stages. Multilocus sequence typing (MLST) identified 12 sequence types (STs) among E. faecalis, with ST1024 predominating, and 7 STs among E. faecium, with ST92, ST1091, and ST160 being dominant. Single nucleotide polymorphism (SNP) analysis revealed high genetic homology between ST1024 strains isolated from farm workers and layer cloacal samples, between ST1091 strains from water and floor samples, and between ST160 strains from air and eggshell surfaces. These findings demonstrate genetic relatedness across different niches, suggesting potential within-farm transmission and pointing to cross-transmission risks of resistant Enterococcus between humans and layers, between feed/water and the environment, and between the environment and eggs. Furthermore, high homology detected between ST92 strains isolated from flies and workers five months apart indicates persistent transmission and colonization via biological vectors. Resistance phenotypes for enrofloxacin and sulfisoxazole were strongly associated with environmental samples, highlighting the farm environment as a critical potential control point for AMR risk mitigation. Leveraging whole-genome sequencing, this study explores the transmission risk of antimicrobial-resistant Enterococcus in layer production and provides insights for preventing AMR risks originating from layer farms.