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Introduction: Enterococcus spp. has become recognized as a significant cause of hospital-acquired infections. Two main virulence factors namely gelatinase and hemolysin of Enterococci have been proved to cause severe infections. In addition, biofilm formation is causing infection by enhancing the persistence of Enterococci in medical indwelling devices. Therefore, the study aimed to evaluate the presence of hemolysin, gelatinase, and biofilm formation in Enterococcus spp. and the impact of biofilm on antimicrobial susceptibility patterns. Materials & Methods: Total 104 Enterococcal isolates obtained from different clinical samples were included in the study for expression of virulence factors. All isolates were evaluated for biofilm formation by the tissue culture plate method. Hemolysin production was checked by using 5% sheep blood agar and gelatinase production by peptone yeast extract agar containing 3% gelatin. Antimicrobial susceptibility testing was done by the Vitek2 compact automated system. Results: Out of 104 isolates, 1(1%) were strong biofilm producers while 4(3.85% %) and 54(52%) were moderate and weak biofilm producers respectively. Hemolysin production was observed in 19(18%) isolates and gelatinase production was universal. Conclusion: Biofilm-producing strains showed higher resistance to beta-lactam drugs and high-level aminoglycosides. Hence, amongst three virulence factors, studying biofilm formation can be an important tool to develop a hospital’s antibiotic policy and other virulence factors can be helpful to understand the pathogenesis of infection caused by Enterococcus spp. as well as for antimicrobial usage strategies.

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