ENTEROCOCCUS

SOIL MICROBIOLOGY

BIOL/CSES 4684



This webpage was created by Daniel Crossan


1. IDENTIFYING CHARACTERISTICS

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


2. TAXONOMIC DESCRIPTION
The enterococcus group is a subgroup of the fecal streptococci that includes at least five species: S. faecalis, S. faecium, S. durans, S. gallinarum, and S. avium. The enterococci are differentiated from other streptococci by their ability to grow at high pH (9.6 at 10), high temperature (45°C) and in high salt concentrations (6.5% sodium chloride). The enterococcus are generally resistant to many Gram positive antibiotics such as the tetracyclines, aminoglycosides, sulfonamides, some penicillins, and lincosamides. E. faecalis and E. faecium are the most frequent species found in humans. E. faecalis is the only enterococcus species that has been genetically characterized. Its genome is 3 mb in length. The two genetic mechanisms first discovered in the enterococci were conjugative transposons and sex pheromone plasmids. Some strains require vitamin B and amino acids for growth.

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







Selected differential physiological characteristics for species of the enterococci.

E.faecalis

E. faecium

E. durans

E. bovis

E.equinus

Hemolysis

-/+

-

+/-

-

-

Growth at10 °C

+

+

+

-

-

Growth at 45°C

+

+

+

+

+

Growth at 50°C

+

+

-

-

-

Growth at pH 9.6

+

+

+/-

-

-

Growth at 6.5% NaCl

+/-

+/-

+/-

-

-

Growth at 40% bile

+

+

+

+

+

Resists 60°C for 30 min

+

+

+/-

-

-

NH3 from arginine

+

+

+

-

-

Gelatin liquefied

-/+

-

-

-

-

Tolerates 0.04% Pot. tellurite

+

-

-

-

-

Acid from Glycerol

+

-

-

-

-

Acid from Mannitol

+

+

-

-/+

-

Acid from Sorbitol

+

-

-

-/+

-

Acid from L-arabinose

-

+

-

+/-

-

Acid from Lactose

+

+

+

+

-

Acid from Sucrose

+

+/-

-

+

+

Acid from Raffinose

-

-

-

+

-

Acid from Melibiose

-

+

-

+

-

Acid from Melezitose

+

-

-

-

-

Starch hydrolyzed

-

-

-

+

-

Tetrazolium reduced at pH 6.0

+

-

-

+/-

-



3. ISOLATION AND ECOLOGY
Most procedures employ presumptive media followed by confirmatory tests. Primary selective agents can be azide, tellurite, bile, neonycin, Tween 80, taurocholate, selenite, NaCl, alcohol, phenylethyl, and thallium. For isolation, the Association of Food and Drug Officials of the United States recommends KF agar medium. This is selective differential agar that contains sodium azide, that inhibits catalase positive organisms, and tetrazolium chloride which produces a red color in the colonies.

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).





4. PUBLIC HEALTH SIGNIFICANCE
The enterococci are used as a bacterial indicator for determining the extent of fecal contamination in foods and in recreational surface waters. Water quality guidelines based on enterococcal density have been proposed for recreational waters. The guideline is 33 enterococci/100 mL for recreational fresh waters. For marine waters, the guideline is 35 enterococci/100 mL. The guidelines are based on the geometric mean of at least five samples per 30-d period during the swimming season. There are two types of selection methods. The membrane filter technique is used for samples of fresh and saline waters; however, it is unsuitable for highly turbid waters. The multiple-tube technique is also applicable to fresh and marine waters, but is primarily used for raw and chlorinated wastewater.

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).



5. ADDITIONAL SOURCES OF INFORMATION

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



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