Abstract
<jats:p>Enterococcus faecalis and Enterococcus faecium are commensal bacteria of poultry but also important opportunistic pathogens and key indicators for antimicrobial resistance (AMR) surveillance within a One Health framework. However, data on the presence and resistance of enterococci in breeding hens remain limited despite their critical role in the poultry production pyramid. This study evaluated the prevalence, antimicrobial resistance profiles, and resistance genes of E. faecalis and E. faecium isolated from two commercial breeding hen farms in eastern Spain. A total of 330 isolates were obtained from different production stages and sample types. Species identification was performed by multiplex PCR, antimicrobial susceptibility was assessed using disk diffusion according to CLSI guidelines, and resistance genes (erm and tet) were detected by PCR. A significant age-related shift from E. faecalis in early stages to E. faecium in adult hens was observed. High resistance rates were detected for tetracycline (up to 95.2%) and erythromycin, whereas resistance to critically important antimicrobials such as vancomycin, aminoglycosides, and ampicillin was rare or absent. Significant differences between farms were identified, with higher resistance levels and multidrug resistance for both farms. The most prevalent resistance genes were ermB, tetL, and tetM, frequently co-occurring. This study shows that breeding hens constitute a relevant reservoir of AMR within poultry production systems. Species dynamics and resistance patterns appear to be strongly influenced by age and farm-related factors. These findings highlight the need for continuous AMR surveillance integrating phenotypic and genotypic approaches under a One Health perspective.</jats:p>