The human gastrointestinal system, vaginal tracts, and oral cavity are home to the
common gram-positive, facultative anaerobic bacteria known as Enterococcus species. When
recovered from clinical specimens of hospitalized patients, the most prevalent species of the
broad genus Enterococcus bacteria—which contains over 50 species—are E. faecalis and E.
faecium. This study aim to isolation and identifaction of E.faecalis depended on phenotype and
molecular method, the phenotypic patterns using traditional, and then diagnosed it based on the
genotypes and using specialized primers for 16srRNA and D-Ala:D-Ala ligase genes using
polymerase chain reaction as well as molecular detection of SprE and PAI virulence genes and
carrying out antibiotic susceptibility test . Between June and September 2024 , one hundred and
forty five urine samples were collected from patients of different ages (from 26 to 60 years old)
and of both sexes (males number 81 and female number 64) that suffering from UTIs that avoid
from taking antibiotics for at least a week, Mid-stream urine samples were collected in sterilized
screw-cap containers. To prevent any potential contamination, the specimens were gathered in the
correct manner. Three Babylonian hospitals—Al-Hilla Teaching Hospital, Marjan Hospital, and
Imam Al-Sadq Hospital—were the sites of sample collection, The result showed presence
(16srRNA and ddl) genes in all isolation bacteria at 100% and sprE, PAI genes were performed
by Conventional PCR amplification was 35.71 % , 21.42 % respectively . Antibiotic sensitivity
tests were performed using Vancomycin, linezolid, ampicillin, chloramphenicol, nitrofurantoin
and ciprofloxacin ,the result of antibiotic sensitivity tests showed very high resistant (96.42%) to
ampicillin, whereas a few isolates exhibited strong ciprofloxacin resistance (85.71%) and
vancomycin (82.14%), while some isolates showed moderate resistance (46.42%) to
nitrofurantoin, and (57.14%) to chloramphenicol, and show low resistance to linezolid (10.71%).