Isolation of

Enterococcus in
foods

1.
2.
3.
4.
5.
6.

Identifying
characteristics
Gram positive.
Cocci shape.
Non-motile.
Occur in pairs or short chains.
Cells are one micrometer in diameter.
Predominately inhabit human
intestines.

7. facultative anaerobes.
8. Complex and variable nutritional requirements.
9. Resistant to many Gram-positive antibiotics.
10. members of the lactic acid bacteria (LAB) group.
Perform simple fermentation.

11. Mechanism of pathogenicity unknown.

12. Used as indicators of fecal pollution in the
purification of water and dried and frozen
foods.
13. Members of genus streptococcus.
14. Belong to Lancefield's serologic group D
Streptococcus.
15. Catalase negative.
16. Can grow in 6.5% NaCl.
17. Can grow at a pH range of 9.6 to 4.6.
18. Can grow at temperatures ranging from 10
to 45C.
19. Optimum growth at 37C.
20. Sensitive to chlorination.

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
(45C) 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.

Isolation and ecology

Most procedures employ presumptive media
followed by confirmatory tests. Primary selective
agents can be azide, tellurite, bile, neomycin,
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
organisms, and tetrazolium chloride which
positive
Ethyl violet azide (EVA) broth can be used as a
produces
a red
color
in the from
colonies.
confirmation.
Fecal
enterococci
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.

Enterococci are able to grow in the presence of bile

and hydrolyze the esculin; the liberated
diphydroxycourmarin complexes (Esculetin) with ferric
citrate present in the media to form a dark
brown/black soluble compound.

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.. Some strains
produce high levels of the amines tyramine
and histamine.

They are capable of producing extracellular

proteinases and peptidases to hydrolyze large
peptides and transport them into the cell to convert
them to amino acids.
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.

 The genusEnterococcusis the most controversial

group of lactic acid bacteria. Studies on the
microbiota of many traditional cheeses in the
Mediterranean countries have indicated that
enterococci play an important role in the ripening
of these cheeses, probably through proteolysis,
lipolysis, and citrate breakdown, hence
contributing to their typical taste and flavour.
Enterococci are also present in other fermented
foods, such as sausages and olives. However, their
role in these products has not been fully
elucidated.

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
There are two types of selection methods. The
swimming season.
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
colonies of
thetechnique,
enterococcus
indicates
In the membrane
filter
the group.
sample is
filtered, the filter containing the colonies are
transferred to an agar medium which is incubated.
The filter is transferred to membrane-EnterococcusEsculin Iron Agar (mE-EIA) containing esculin and
ferric acid as selective agents. Pink to red
enterococci colonies develop a black or reddishbrown precipitate.

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 in
6.5% NaCl indicates presence of enterococcus
group.
For clinical or food samples, additional
tests that may be conducted include bile
solubility.

 Enterococcus organisms have been detected by

plate counts using either Packers crystal violet
azide blood agar or maltose azide agar ( KF
streptococcus agar).
Alternatively, for smaller concentration of
organisms, an MPN technique can be used
employing maltose azid broth ( KF streptococcus
broth ) or glucose azide broth.
The media used also tend to permit the growth of a
number of enterococci and other group D
streptococci that are not necessarily of fecal origin.
The presence of azide in the media helps to inhibit
staphylococci, as a result of its inhibitory action
against the cytochromes.

Glucose azide broth

1. This procedure employs the multiple tube technique, so that small
numbers of enterococci can be detected.
2. Pipette aseptically 1 ml of each of the prepared dilutions of the sample
into each of 3 or 5 tubes of glucose azide broth.
3. If very low concentrations of organisms are expected, 10 ml or even
100 ml amounts of the lowest dilution may be added to equal
volumes of double strength medium, three or five bottles being
prepared at each dilution.
4. Incubate the inoculated glucose azide broths at 35C for 72h and
for the tubes
production
acid.
5. examine
Record those
that of
are
positive and subculture
a loopful from each positive tube into fresh single
strength glucose azide broth ( 5ml per tube ) and
incubate for 48h, examining the tubes after 18 and 48h.
6. The production of acid within 18h indicates
enterococci, and can be confirmed rapidly by
microscopies examination for the presence of short
chained streptococci.
7. The most MPN of the enterococci can be

Kanamycin esculin azide agar

Surface inoculate plates of well dried medium
by spreading 0.1ml amounts of dilutions over
the medium.
Incubate at 37C for 24h.
This medium is made selective by the use of
azide and kanamycin.
Colonies of enterococci are small, white or
grey, and surrounded by large black haloes
Some strains of mesophilic lactobacilli may
grow on this medium, and some of these are
capable of splitting esculin and therefore
producing black haloes.

 SF broth

contains sodium azide, which inhibits

most bacteria other than enterococci.
The enterococci will grow in SF broth and ferment
the dextrose, turning the pH indicator from violet
to a yellow brown color.

Bile Esculin agar slant:

The enterococci will grow in the presence of the bile
salts in the medium.
They hydrolyze the esculin, producing esculetin
which reacts with the iron salts in the medium
turning the agar black.

Selected differential physiological characteristics for species

of the enterococci.

Hemolysis
Growth at10 C
Growth at 45C
Growth at 50C
Growth at pH 9.6
Growth at 6.5% NaCl
Growth at 40% bile
Resists 60C 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

E.equinu
s
+
+
-

E.
bovis
+
+
-

E.
durans
+/+
+
+/+/+
+/-

E.
faecium
+
+
+
+
+/+
+

E.faecalis

+
-

+
-

+
-/+
+

+
-

-/+
-/+
+/+
+
+
+

+
-

+
+
+
+/+

+
+
+
+
+
-

-/+
+
+
+
+
+/+
+

END OF LECTURE