Download Characterization and Identification of Enterococcus Species Isolated

INTERNATIONAL
JOURNAL OF SYSTEMATIC
BACTERIOLOGY,
July 1987, p. 257-259
0020-7713/87/030257-03$02.00/0
Copyright 0 1987, International Union of Microbiological Societies
Vol. 37, No. 3
Characterization and Identification of Enterococcus Species Isolated
from the Intestines of Animals
L. A. DEVRIESE,'* A. VAN DE KERCKHOVE,l R. KILPPER-BALZ,* AND K. H. SCHLEIFER2
Faculty of Veterinary Medicine, University of Ghent, B-9000 Ghent, Belgium,' and Lehrstuhl fiir Mikrobiologie,
Technische Universitat, Munich, Federal Republic of Germany2
Tests useful for the identification of Enterococcus strains were applied to a collection of isolates from animal
intestines and to reference strains, all of which were capable of growth on 40% bile and in 6.5% NaCl. Most
strains could be identified as known species, and their characteristics corresponded, with a few exceptions of
minor importance, with those described for Enterococcus hirae, Enterococcus durans, Enterococcus mundtii,
Enterococcus gallinarum, Enterococcus avium, and Enterococcus casseliflavus. However, some diagnostically
important carbohydrate reactions of Enterococcus faecalis and Enterococcus faecium strains differed from
those given in the species descriptions and in recent reports. Production of acid from D-raffinose and D-xylose
by E . faecium varied with the host species from which the strains were isolated. E . durans and E . gallinarum
were isolated only from poultry, whereas E . avium was found only in mammals.
Enterococci are an important group of intestinal bacteria
whose taxonomy has undergone important changes in the
last few years. The enterococci (sensu Sherman [9]), Streptococcus bovis, and Streptococcus equinus have been classified traditionally as serological group D streptococci. However, nucleic acid studies have shown that Streptococcus
faecalis and Streptococcus faecium are only distantly related to S. bovis and S. equinus (5,7). These studies resulted
in the proposal to transfer S. faecalis and S . faecium to a
new genus, Enterococcus, as E. faecalis and E. faecium (8).
Other group D streptococci which belong to the enterococcal
MATERIALS AND METHODS
Strains. A total of 264 strains (Table 1) were selected
solely on the basis of their ability to grow in the presence of
40% bile (bile-esculin agar, Difco Laboratories, Detroit,
Mich.) and in 6.5% NaCl in brain heart infusion agar (Oxoid
Ltd., London, England). The selection was made from a
larger collection of catalase-negative, gram-positive cocci
and short rods isolated from the intestines of farm animals on
mitis salivarius agar (GIBCO, Paisley, Scotland) or brain
heart infusion agar (Oxoid) with 10 pg of oxolinic acid
TABLE 1. Number of strains belonging to different Enterococcus species isolated from animal hosts
~
~~
No. of strains isolated from:
Enterococcus
species
Poultry
Cattle
Pigs
Dogs
E. faecalis
E. faecium
E. hirae
E. durans
E. gallinarum
E. avium
E. mundtii
E. casselgavus
Unknown
25
39
25
13
4
0
0
1
2
21
15
6
0
0
2
1
0
0
22
11
11
17
5
0
0
1
1
0
2
group have since been transferred to this genus (3), and new
species have been added (2, 4).
In the present study, the biochemical characteristics of
Enterococcus strains isolated from the intestines of animals
were studied, and an attempt was made to identify them as
known Enterococcus species. When necessary, the results
were confirmed by deoxyribonucleic acid (DNA) homology
studies.
* Corresponding author.
Horses
Sheep
Goats
Rabbits
4
0
0
0
0
0
2
(Sigma Chemical Co., St. Louis, Mo.) per ml. All strains
originated from different animals and from different farms or
owners except when more than one species was isolated
from a single animal. In such cases, one representative of
each species was included.
The following Enterococcus strains were obtained from
the Czechoslovac Collection of Microorganisms, Brno, the
Deutsche Sammlung von Mikroorganismen, Gottingen, Federal Republic of Germany, and the National Collection of
Dairy Organisms (now National Collection of Food Bacteria), Reading, United Kingdom: E. faecalis CCM 2541, E.
faecium DSM 20477T, CCM 2123, and CCM 2308, E. hirae
257
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258
INT.J. SYST.BACTERIOL.
DEVRIESE ET AL.
TABLE 2. Characteristics of Enterococcus strains from farm animals
No. of strains positive"
Characteristic
E. faecium
(76)
E . hirae
E. durans
(13)
E . gallinarum
(4)
E. avium
(3)
E . mundtii
E . casseliJlavus
0
0
94
85
0
0
8
96
0
0
76
62
68
0
69
76
0
0
59
32
54
0
50
59
0
0
13
0
0
0
0
13
4
0
4
3
4
0
4
4
0
0
3
0
0
0
0
0
0
3
3
0
3
0
3
3
1
1
1
0
1
0
1
1
0
96
96
96
96
0
0
96
2
92
25
70
18
72
63
0
34
74
71
0
76
76
0
30
71
0
70
0
7
73
2
20
0
0
0
0
0
59
55
0
15
50
0
57
0
0
59
0
0
0
12
13
0
0
10
0
0
0
0
0
0
0
0
0
4
4
0
4
4
0
4
4
0
3
0
0
4
0
4
0
0
3
3
3
3
3
0
0
0
0
3
3
3
0
2
0
3
3
3
3
3
2
2
2
1
3
0
3
3
2
3
0
0
1
1
0
1
1
1
1
1
0
1
0
1
1
1
1
0
0
E . faecalis
(96Y
Motility
Pigment (yellow)
Voges-Proskauer reaction
Hippurate hydrolysis
a-Galactosidase
P-Glucuronidase
p-Galactosidase
Arginine dihydrolase
Acid from:
L- Arabinose
Mannitol
Sorbitol
Lactose
Trehalose
Inulin
Raffinose
Amidon
Glycogen
Sucrose
Sorbose
Rhamnose
Melibiose
Melezitose
D - x ylose
Adonitol
Production of gelatinase
(59)
0
1
0
3
2
0
(3)
(1)
a All strains grew in 6.5% NaCl and 40% bile; all produced pyrrolidonylarylamidase and leucine arylamidase. None was alkaline phosphatase positive; all
hydrolyzed esculin and produced acid from ribose.
The numbers in parentheses are the numbers of strains tested.
CCM 2423, CCM 2424, DSM 2O16OT, and NCDO 2708, E.
durans CCM 5612T, E. gallinarum NCDO 2313T, NCDO
2311, and CCM 2518, E. casselijlavus CCM 247gT,E. avium
DSM 20063, and E. malodoratus NCDO 847. All of these
strains, except CCM 2541, CCM 2518, and DSM 20063, had
been studied previously by DNA-DNA hybridization analyses (3, 4, 5, 8).
Biochemical tests. Acid production from 0.5% sucrose,
sorbose, rhamnose, melibiose, D-xylose, adonitol, and
melezitose was tested in phenol red broth base (GIBCO),
and the results were read after 1,2, and 3 days at 37°C. Other
carbohydrates were tested in API Strep galleries (API,
Montaillieu-Vercier, France). Pigment production was examined on tryptic soy agar (Oxoid), and motility was tested
by stab inoculation into medium containing 1% tryptose,
0.5% NaC1, and 0.25% Noble agar (Difco). Dextran and
levan formation were tested on agar medium containing 5%
sucrose (SAC agar; API), and gelatinase production was
tested on a medium containing 1% tryptone, 0.1% yeast
extract, 0.3% meat extract, 0.3% gelatin, and 1.5% agar, to
which Frazier reagent was added after 2 days of incubation.
Other tests were performed by using API 20 Strep galleries
in accordance with the instructions of the manufacturer.
DNA-DNA hybridization studies were performed as previously described (7).
RESULTS AND DISCUSSION
Of the 264 isolates from animals, 255 could be identified as
known Enterococcus species. E. durans and E . gallinarum
were found only in poultry, whereas E. avium was isolated
from two cows and one pig but not from poultry (Table 1). E.
faecalis, E . hirae, and E. faecium, the most frequently
occurring species, were isolated from all, or nearly all, of the
animal hosts investigated.
The biochemical reactions of the animal bacterial strains
are shown in Table 2. Some of these reactions differed with
the hosts from which the strains were isolated. This was
particularly noticeable for acid production from D-raifinose
by strains identified as E . faecium. Of the 39 E. faecium
strains isolated from poultry, 30 produced acid from Draffinose (D-raffinose positive), whereas all 37 of the other E.
faecium strains isolated from different mammalian host
species and the 3 collection strains studied were D-raffinose
TABLE 3. DNA homology values for various enterococcia
% Homology with 35S-labeled
Source of
filter-bound DNA
Enterococcus sp. strain 804acb
Enterococcus sp. strain AlOa
E. faecium DSM 20477T
E. gallinarum CCM 2518
Enterococcus sp. strain A20
Enterococcus sp. strain A13
E. gallinarum NCDO 2313T
E. casselifavus CCM 2479
E. faecalis DSM 20478T
E. avium DSM 20063
Lactococcus lactis DSM 20481T
Streptococcus pyogenes NCTC 8198T
DNA from:
Enterococcus E . gallinarum
sp. strain 804acb CCM 2518
16
14
13
100
89
96
84
21
10
11
6
6
100
78
82
13
13
12
13
12
10
10
4
5
~~
Hybridization was performed under optimal conditions (25OC below the
melting point of DNA).
Identified as E. faecium by biochemical tests.
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CHARACTERIZATION AND IDENTIFICATION OF ENTEROCOCCI
VOL.37, 1987
TABLE 4. Differences between species descriptions and recent
reports and results obtained in this work with strains
from animals and international collections
Species
(reference[s])
E. faecalis
(3, 8)
Characteristic
Acid from:
L-Sorbose
D-Xylose
Melibiose
Amidon
Sorbitol
Hydrolysis of
hippurate
Production of:
a-Galactosidase
P-Galactosidase
Acid from:
D-Rafbose
Amidon
Rhamnose
D-Xylose
E. hirae (4)
Acid from melezitose
E. durans
(3, 8)
Acid from amidon
E. gallinarum Acid from inulin
(1, 3)
E. rnundtii (2) Acid from glycogen
Species
Other
description" reports"
this
study
Of
V
V
V
V
25/97b
63/97
19/97
96/97
97/97
+
+
62/79
NI
NI
+
-
68/79
69/79
-
24/79'
71/79
8/79
20179'
V
0163
10114
+d
217
113
+ ,Positive reaction; -, negative reaction; NI, not indicated; V, variable.
Number of strains positive/number of strains tested.
Dependent on origin of the strains (see the text).
From Collins et al. (3).
negative. The identification of one of the raffinose-positive
poultry strains, AlOa, was confirmed as E . fuecium by
DNA-DNA hybridization (Table 3). There was also a correlation between acid production from D-xylose by E. faecium
and the origin of the strains: most strains from cattle (12 of
15) and dogs (4 of 5) were xylose positive, whereas few
strains from poultry (2 of 39) and pigs (2 of l l ) , none of the
strains from other species, and none of the collection strains
produced acid from D-xylose. The relationship of D-xylosenegative (DSM 20477T and W a c ) and -positive (AlOa)
strains was confirmed by DNA-DNA hybridization (Table
3).
The results of a DNA homology study of two nonpigmented motile strains from poultry (A20 and A13) and a
collection strain, CCM 2518, which was originally classified
as motile E. fueculis (6) showed that all of these strains
259
should be identified as E. gallinarum. With few exceptions,
the characteristics of the collection strains studied corresponded to the descriptions given in the literature (3,4,5,8).
In our hands, the type strain of E . fuecium, DSM 20477, was
rhamnose positive and weakly amidon positive and E.
fuecium CCM 2123 was hippurate negative and a-galactosidase positive. The motility, arginine dehydrolase, and sorbitol reactions of the E. gallinarum strains did not correspond with those given in the original description (1)but did
agree with those given by Collins et al. (3), who transferred
this species to the genus Enterococcus. Only the negative
inulin reactions of the four animal strains investigated and of
strain CCM 2518 differed from the descriptions given by the
latter investigators.
Most of the differences between our results and those
published in the species descriptions and in other recent
reports can probably be explained by the fact that a much
larger number of strains from different origins were included
in our study. Only acid production from amidon appears
significantly more common among the enterococci we examined, irrespective of species and origin. Differences between
results obtained in this work and species descriptions are
given in Table 4. Only the few E. avium and E . casselijavus
strains examined here did not differ from the published
descriptions.
LITERATURE CITED
1. Bridge, P. D., and P. H. A. Sneath. 1982. Streptococcus gallinarurn sp. nov. and Streptococcus oralis sp. nov. Int. J. Syst.
Bactenol. 32:410-415.
2. Collins, M. D., J. A. E. Farrow, andD. Jones. 1986. Enterococcus
mundtii sp. nov. Int. J. Syst. Bacteriol. 36:&12.
3. Collins, M. D., D. Jones, J. A. E. Farrow, R. Kilpper-Balz, and
K. H. Schleifer. 1984. Enterococcus avium nom. rev., comb.
nov.; E. cusselifluvus nom. rev., comb. nov.; E. durans nom.
rev., comb. nov.; E. gallinarum comb. nov.; and E. malodoratus
sp. nov. Int. J. Syst. Bacteriol. 34:220-223.
4. Farrow, J. A. E., and M. D. Collins. 1985. Enterococcus hirae, a
new species that includes amino acid assay strain NCDO 1258
and strains causing growth depression in young chickens. Int. J.
Syst. Bacteriol. 3573-75.
5. Farrow, J. A., D. Jones, B. A. Phillips, and M. D. Collins. 1983.
Taxonomical studies on some group D streptococci. J. Gen.
Microbiol. 129:1423-1432.
6. Graudal, H. 1952. Motile streptococci. Acta Pathol. Microbiol.
Scand. 31:46-50.
7. Kilpper-Balz, R., B. L. Williams, R. Lutticken, and K. H.
Schleifer. 1984. Relatedness of "Streptococcus milleri" with
Streptococcus anginosus and Streptococcus constellatus. Syst.
Appl. Microbiol. 5596500.
8. Schleifer, K. H., and R. Kilpper-Balz. 1984. Transfer of Streptococcus faecalis and Streptococcus faecium to the genus
Enterococcus nom. rev. as Enterococcus faecalis comb. nov.
and Enterococcus fuecium comb. nov. Int. J. Syst. Bacteriol.
34: 31-34.
9. Sherman, J. M. 1937. The streptococci. Bacteriol. Rev. 1:3-97.
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