Biofilm Formation, Virulence Traits, and Antimicrobial Resistance Profiles of <i>Enterococcus faecalis</i> in Layer Parent Stock in Bangladesh

<i>Enterococcus faecalis</i> is an opportunistic pathogen of growing concern in both human and veterinary medicine due to its virulence traits, biofilm-forming ability, and resistance to multiple antibiotics. This study was aimed at investigating the occurrence, virulence factors, biofilm formation, and antimicrobial resistance (AMR) of <i>E. faecalis</i> in layer parent stock birds in Bangladesh. Samples (<i>n</i> = 80) were collected from healthy (cloacal swabs, <i>n</i> = 60) and dead (liver tissues, <i>n</i> = 20) birds. PCR was used for <i>E. faecalis</i> confirmation and detection of virulence genes. Biofilm formation was assessed using Congo red agar, and antimicrobial susceptibility was determined by disc diffusion. <i>E. faecalis</i> was detected in 76.3% of samples, with higher detection in live birds (80%) than in dead birds (65%). Biofilm production was found in 75.4% of isolates, with a higher rate in dead birds (84.6%) than live birds (72.9%). Strong and intermediate biofilm-forming capacities were more prevalent in isolates from dead birds. All eight tested virulence genes were commonly distributed, particularly <i>pil</i> (95.8%), <i>ace</i> (93.4%), and <i>agg</i> (91.8%), with no significant differences between live and dead bird isolates. High resistance was observed against ampicillin (93.4%), ciprofloxacin (80.3%), erythromycin (78.7%), and tetracycline (72.1%). Multidrug resistance (MDR) was found in 79.2% of isolates from live birds and 69.2% from dead birds, with multiple antibiotic resistance indices ranging from 0.27 to 0.72. To the best of our knowledge, this is the first study in Bangladesh determining MDR and virulence determinants in <i>E. faecalis</i> isolates from layer parent stock. These findings highlight <i>E. faecalis</i> as a prevalent, multidrug-resistant, and virulent bacterium in breeder flocks, emphasizing the need for routine AMR monitoring in parent stock farms.