Antimicrobial Resistance and Genomic Insights into <i>Enterococcus faecalis</i> Isolates from Broilers and Their Handlers in Punjab, India

<i>Enterococcus faecalis</i>, a common constituent of human and animal gut microbiota, has emerged as a significant pathogen due to its antimicrobial resistance (AMR) capabilities. This study investigates AMR profiles, virulence factors, and genetic diversity of <i>E. faecalis</i> isolates from broiler farms in Ludhiana, Punjab, India, to explore transmission dynamics between poultry and handlers. A total of 240 samples were collected from 20 farms, comprising 200 broiler droppings, 34 hand swabs from poultry handlers, and 6 human stool samples. Selective media and standard microbiological techniques were employed to isolate and identify <i>E. faecalis</i>. Whole genome sequencing (WGS) was performed on representative isolates to uncover antimicrobial resistance genes (ARGs) and virulence factors. <i>E. faecalis</i> was isolated from 47% of broiler droppings and 26.47% of handler hand swabs, whereas no isolates were recovered from stool samples. High resistance was observed for erythromycin (96.11%), tetracycline (78.64%), ciprofloxacin (76.69%), streptomycin (76.69%), and linezolid (65.04%), with 83.49% of isolates exhibiting multidrug resistance (MDR). Vancomycin resistance genes (<i>vanC1</i>-34.95% and <i>vanC2/C3</i>-9.70%) were also detected. WGS analysis of four isolates identified ARGs such as <i>dfrE</i>, <i>efrA</i>, <i>vanT</i> gene in <i>vanG</i> cluster, <i>vanY</i> gene in <i>vanB</i> cluster, <i>ermB</i>, <i>tet45</i>, <i>tetM</i>, <i>tetA</i>, <i>aac(6')-Ie-aph(2″)-Ia</i>, <i>ant(4')-Ib</i>, <i>aph(3')-Ia</i>, <i>sul1</i>, <i>sul3</i>, <i>mphA</i>, <i>qnrS1</i>, and <i>catA8</i>, as well as virulence factors associated with biofilm formation, adherence, capsule formation, and protease production. Sequence typing identified was ST-1866, ST-7317, and ST-403, with ST-7317 common between broiler droppings and handler swab isolates, indicating potential transmission. While these findings highlight poultry environments as reservoirs for MDR <i>E. faecalis</i>, the directionality of transmission (zoonotic vs. reverse zoonotic) remains unclear. This underscores the need for expanded surveillance and molecular studies to better understand transmission dynamics and mitigate risks to farm workers and public health.