Identification of Enterococci

Enterococci are gram-positive cocci that form chains in culture and that, until recently, were classified in the genus Streptococcus. Because they differ in several characteristics, including the composition of their genetic material, they are now classified in a separate genus, Enterococcus. As the name implies, enterococci are found primarily in the intestinal tract, although they may be found in the upper respiratory tracts of infants and young children. Their primary role in disease is as the agents of urinary tract infection, infective endocarditis (like the viridans group streptococci), and wound infections, especially those contaminated with intestinal contents. In the laboratory, their colonies resemble somewhat those of group B streptococci. They must be differentiated from this organism because their presence at certain body sites has a different meaning. For example, enterococci isolated from a genital tract specimen of a pregnant woman near term may simply represent contamination from the intestinal tract, whereas isolation of group B streptococci from the same specimen represents a potential hazard for the fetus. Enterococcus faecalis is the most common species isolated from persons with enterococcal infections, but another species, Enterococcus faecium, is being isolated more frequently from hospitalized patients with serious infections. This organism is highly resistant to almost all antimicrobial agents including vancomycin, which has been the only drug available for treating some strains of this species. Enterococcal strains resistant to this agent are referred to as vancomycin-resistant enterococci, or VRE. Patients colonized or infected with VRE are treated with special precautions in the hospital to prevent transmission of the organism to others. Treatment of infections caused by VRE is a significant clinical challenge. Although pharmaceutical companies are working to develop new, effective drugs, microbial resistance to them evolves rapidly. Some strains of other enterococcal species, including E. faecalis, are also resistant to vancomycin but not yet to the same extent as E. faecium strains are.

Enterococci previously were known as group D streptococci because they possess a characteristic antigen on their cell wall that reacts in serological tests with group D antibody. Unlike the streptococci, enterococci can grow in a high concentration salt broth (containing 6.5% sodium chloride), are resistant to bile, and hydrolyze a complex carbohydrate, esculin. The last two characteristics are used in a selective and differential medium for enterococci, called bile-esculin agar. The bile inhibits streptococcal but not enterococcal growth. When enterococci hydrolyze the esculin, a black pigment forms in the medium. The pigment results from the reaction of the esculin breakdown products with an iron salt that is also included in the medium (see colorplate 32). This test often becomes positive within 4 hours so that a rapid identification can be made. An even more rapid test that is performed with colonies of enterococci growing on a culture plate is the PYR test. This test detects an enzyme, pyrrolidonylarylamidase, which is produced by enterococci but not most other gram-positive cocci (an important exception is the group A beta-hemolytic Streptococcus, which also produces pyrrolidonylarylamidase and is positive in the PYR test). The substrate for this enzyme is impregnated on disks and its hydrolysis is detected by a simple disk method (see colorplate 33).

Purpose	To identify enterococci in culture
Materials	6.5% sodium chloride broths Plate of bile-esculin agar PYR disks and developer reagent Blood agar plate cultures of Enterococcus faecalis and a group B Streptococcus

Procedures

1. Examine the blood agar plate culture of the Enterococcus. Do the colonies resemble those of the group B Streptococcus? Make a Gram stain of the organisms.
2. Inoculate two sodium chloride broths lightly, one each with a portion of a colony from each plate. After you inoculate them, the broths should not be turbid; otherwise, you will not be able to determine whether the organism grew during incubation.
3. Incubate the broths for 24 hours at 35°C.
4. Mark the bottom of the bile-esculin plate to divide it in half.
5. Streak the Enterococcus across one-half of the bile-esculin agar plate and the group B Streptococcus across the other half. Incubate the plate at 35°C and examine it just before you leave the laboratory (don’t forget to reincubate) and again after 24 hours.
6. With forceps, remove a filter paper disk impregnated with PYR substrate (L-pyrrolidonyl-beta-naphthylamide) from the vial of disks. Place the disk on the surface of a glass microscope slide or in an empty petri dish. Moisten the disk with a small drop of tap or distilled water, taking care not to flood the disk.
7. With your sterilized inoculating loop, pick up several colonies of enterococci and rub them onto the surface of the disk. Be careful not to dig up any blood agar with your inoculum. Resterilize your inoculating loop.
8. After two minutes, add a drop of the developer reagent to the surface of the disk. A red color develops within one minute if the test is positive.
9. Repeat steps 6 through 8 with the culture of group B Streptococcus.
10. After 24 hours examine the salt broths for the presence or absence of growth (turbidity). Compare the inoculated, incubated broths with an uninoculated broth tube.
11. Examine the bile-esculin plate and note the color of the medium in each half.

Results

1. For each organism, record results (+ or −) of the esculin hydrolysis reaction (black pigment formation) at the end of the lab session (less than or equal to [≤] 4 hours) and at 24 hours.
1. Enterococcus faecalis:
• ≤4 hours _____
• 24 hours ______
2. group B Streptococcus:
• ≤4 hours ______
• 24 hours ______
2. Record your observations in the following table.