Enterococcus faecalis is a Gram-positive, facultative anaerobe that inhabits the gastrointestinal tract of humans and animals as part of the commensal flora. Despite its natural presence, E. faecalis has emerged as one of the leading opportunistic pathogens in healthcare settings. It is responsible for a substantial percentage of hospital-acquired (nosocomial) infections, including urinary tract infections, endocarditis, and bacteremia. The ability of this bacterium to tolerate harsh conditions and its rising resistance to multiple antibiotics make its accurate and timely identification a high priority for clinical laboratories.
Macroscopic Colony Appearance
Laboratory identification begins by observing the macroscopic characteristics of colonies grown on culture media. On standard agar, E. faecalis colonies are small, typically measuring one to two millimeters in diameter after 24 hours of incubation. They appear circular, with a slightly raised or convex elevation, and have a smooth, moist, or creamy texture.
On Blood Agar, the colonies usually present as grayish-white or occasionally light pink. The organism often exhibits gamma-hemolysis (no visible change in the surrounding red blood cells). However, some strains may display alpha-hemolysis, resulting in a greenish discoloration around the colony due to partial breakdown of blood cells.
Growth on selective and differential media provides further visual clues. E. faecalis grows well on Bile Esculin Agar, a medium containing bile salts that inhibit many other organisms. The colonies on this agar are surrounded by a distinct black precipitate, a visual marker of the organism’s biochemical activity. Furthermore, on chromogenic media, E. faecalis typically produces colonies with a specific color, such as turquoise blue or green, which aids in presumptive identification.
Initial Biochemical Screening Assays
Once macroscopic characteristics suggest an Enterococcus species, biochemical tests are performed for presumptive identification. The Bile Esculin test is a differential test assessing the organism’s ability to grow in bile and hydrolyze esculin. The enzyme esculinase breaks down esculin, and the resulting product reacts with ferric ions in the medium to form a visible black complex, confirming a positive result.
The Pyrrolidonyl Arylamidase (PYR) hydrolysis assay is also used. E. faecalis possesses the enzyme L-pyrrolidonyl arylamidase, which hydrolyzes the substrate L-pyrrolidonyl-β-naphthylamide. The subsequent addition of a color developer results in a bright pink or cherry-red color, which is a positive reaction for all Enterococcus species.
The Catalase test distinguishes Enterococcus from Staphylococcus species, which are catalase-positive. E. faecalis is typically catalase-negative, meaning it does not produce effervescence when exposed to hydrogen peroxide.
A critical screening step for confirming the genus Enterococcus is the 6.5% Sodium Chloride (NaCl) tolerance test. The organism is inoculated into a broth containing a high concentration of salt. Visible turbidity (cloudiness) in the broth, often within 24 hours, confirms the organism’s high salt tolerance and distinguishes it from non-enterococcal Group D streptococci.
Advanced and Differential Identification
While initial screening confirms the genus Enterococcus, advanced methods are necessary for definitive species-level identification and differentiation, particularly between E. faecalis and the closely related Enterococcus faecium. Traditional differential carbohydrate fermentation tests leverage metabolic differences between the species. For instance, E. faecalis is characterized as a sorbitol and pyruvate fermenter, but it is typically unable to ferment arabinose.
These physiological tests are increasingly complemented or replaced by automated identification systems, such as Vitek or Phoenix instruments, which utilize panels of biochemical reactions for rapid species confirmation. For high-throughput clinical and epidemiological studies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) provides a fast and accurate protein-based identification.
Molecular techniques offer the highest specificity for definitive identification. Polymerase Chain Reaction (PCR) assays targeting species-specific genes, such as groESL or Ef0027, confirm the presence of E. faecalis. Furthermore, techniques like Peptide Nucleic Acid Fluorescent In Situ Hybridization (PNA FISH) allow for rapid identification of E. faecalis directly from positive blood cultures, providing results faster than traditional methods. 16S ribosomal RNA gene sequencing and PCR-Restriction Fragment Length Polymorphism (RFLP) analysis also offer reliable methods for confirming species identity and for detailed strain typing.