ENTEROCOCCUS

SOIL MICROBIOLOGY

• Gram positive
• Cocci shape
• Nonmotile
• Occur in pairs or short chains
• Cells are one micrometer in diameter
• Predominately inhabit human intestines
• Faculative anaerobes (prefer anaerobic)
• Complex and variable nutritional requirements
• Resistant to many Gram positive antibiotics
• Perform simple fermentation
• Mechanism of pathogenicity unknown
• Used as indicators of fecal pollution in the purification of water and dried and frozen foods
• Members of genus streptoccous
• Belong to Lancefield's serologic group D Streptococcus
• Catalase negative
• Can grow in 6.5% NaCl
• Can grow at a pH range of 9.6 to 4.6
• Can grow at temperatures ranging from 10 to 45°C
• Optimunm growth at 37°C
• Sensitive to chlorination

The above picture shows a Streptococcus. Cells that are oval in appearance are in the process of actively dividing.

Picture at left shows chains of enterococci at 10,000X magnification.

Selected differential physiological characteristics for species of the enterococci.

Ethyl violet azide (EVA) broth can be used as a confirmation. Fecal enterococci from water can be isolated, cultivated, and enumerated in this broth. Growth of fecal enterococci in EVA results in turbidity and a purple sediment in the bottom of liquid cultures. There is also a tyrosine decarboxylase activity procedure and a mentagan test that works well.

Enterococci are able to grow in the presence of bile and hydrolyze the esculin; the liberated diphydroxycourmarin complexes with ferric citrate present in the media to form a dark brown/black soluble compound. The picture on the left shows the differential reaction that identifies the enterococci on bile esculin agar.

Enterococci occur naturally in soil and can be readily isolated from most plant roots as well. They are also found routinely in frozen seafood, cheese, dried whole egg powder, raw and pasteurized milk, frozen fruits, fruit juices, and vegetables. Occasionally they are used as starter cultures for making hard cheese. Some strains produce high levels of the amines tyramine and histamine. Tyramine may be involved in causing migraines. They are capable of producing extracellular proteinases and peptidases to hydrolyse large peptides and transport them into the cell to convert them to amino acids. Due to diet, E. faecalis dominates the guts of humans in the United States and England. In India and Japan, E. faecalis and E. faecium are equally found in the intestines. They get into food through vegetation, processing equipment, processing environments, or fecal contamination. Symptoms are similar to B. cereus and C. perfringens. Symptoms include nausea, vomiting, and diarrhea, but are milder than those caused by other food borne illnesses. The picture at left shows hemolysis on blood agar by S. pyogenes, a group A streptococcus. Blood agar is often used as a diagnostic test for the enterocococci, especially when isolations are made from food or clinical samples. Two of the five enterococcal species (faecalis and durans) will usually produce hemolysis on blood agar (see above table).

For the presumptive test procedure of the multiple-tube technique, a series of azide dextrose broth tubes are inoculated and incubated. If not turbid, tubes are reincubated. Tubes showing turbidity are streaked onto Pfizer selective enterococcus (PSE) agar. Brownish-black colonies with brown halos confirm the presence of fecal streptococci. These colonies are transferred to a tube of brain-heart infusion broth containing 6.5% NaCl. Growth indicates colonies of the enterococcus group.

In the membrane filter technique, the sample is filtered, the filter containing the colonies are transferred to an agar medium which is incubated. The filter is transferred to EIA medium containing esculin and ferric acid as selective agents. Pink to red enterococci colonies develop a black or reddish-brown precipitate. A well isolated colony from brain-heart infusion agar is then transferred onto a brain-heart infusion broth tube and incubated. After growth, a sample of the culture is transferred to bile esculin agar, brain-heart infusion broth, and brain-heart infusion broth with 6.5% NaCl. Growth at 45°C in 6.5% NaCl indicates presence of enterococcus group.

For clinical or food samples, additional tests that may be conducted include bile solubility (above left picture; the tube on the far left is positive), and antibiotic sensitivity (the above right picture shows the antibiotic disk assay for bacitracin).

Rose, A.H., ed. 1983. Economic Microbiology: Food Microbiology. Academic Press. New York. Volume 8. 207 pp.

Defigueiredo, Mario and Don Splittstoesser, ed. 1976. Food Microbiology: Public Health and Spoilage Aspects. AVI Publishing Company. Connecticut. 276-281 pp.

Ray, Bibek. 1996. Fundamental Food Microbiology. CRC Press. New York. 363-364 pp.

Banwart, George. 1979. Basic Food Microbiology. AVI Publishing Company. Connecticut. 400-404 pp.

5. LINKS TO SITES ON THE ENTEROCOCCI

Gram Positive Facultative_Cocci

Detection of Enterococci in Recreational Waters

Cancer Control Journal

Taxonomy - Enterococcus

The NCGR Microbial Genome Site