INTERNATIONAL
JOURNAL
OF SYSTEMATIC
BACTERIOLOGY,
July 1991, p. 406-409
O020-7713/91/030406-04$02
.OO/O
Copyright 0 1991, International Union of Microbiological Societies
Vol. 41, No. 3
Enterococcus seriolicida sp. nov. a Fish Pathogen
R. KUSUDA,'" K. KAWA1,l F. SALAT1,l C. R. BANNER,2 AND J. L. FRYER2
Fish Disease Laboratory, Faculty of Agriculture, Kochi University, Nankoku, Kochi 783, Japan, and
Department of Microbiology, Oregon State University, Corvallis, Oregon 97331-38M2
The properties and taxonomic position of bacterial strains isolated from diseased specimens of cultured
yellowtail and eels were examined. The isolates were gram-positive, short-chain-forming, catalase-negative,
facultatively anaerobic cocci. Growth at 10 and 45°C in 6.5% NaCl (pH 9.6) with 40% bile and in 0.1%
methylene blue-milk were both positive. The isolates could be distinguished from other species of the genus
Enterococcus by several biochemical characteristics and by Lancefield's group antigen. Guanine-plus-cytosine
content of DNA was 44 mol% as determined by the thermal melting temperature. The value for DNA-DNA
hybridization was sufficiently low to warrant distinguishing this species from reported Enterococcus species.
The name Enterococcus seriolicida is proposed. The type strain is YT-3 (=ATCC 49156).
An infectious disease of fish which has been called streptococcicosis is one of the most important diseases of yellowtail (Seriola quinqueradiata) that are cultured in Japan,
where the disease occurs throughout the year and with a high
prevalence during summer. The typical symptoms exhibited
by diseased yellowtail are exophthalmos, petechiae on the
inside of the opercula, and congestion of the pectoral and
caudal fins. Internally, there are congestion and hemorrhage
in the intestine, liver, spleen, and kidney. A bacterium was
isolated from marine and freshwater fishes and from seawater or mud around fish farms but not from mammals or
humans. The causative agent of the diseases in cultured
yellowtail (12) and eel (Anguilla japonica) (14) has been
described as a Streptococcus sp. because it does not fit any
of the described species as determined by biochemical and
cultural characteristics (12,13,14) and Lancefield's serotype
(11). For economic reasons, much effort has been taken to
clarify the pathogenicity (7, 19, 21), characterize toxins (8,
lo), apply antibiotics (15), and study the epizootiology (9)
related to this bacterium.
In this study, characteristics of the bacteria isolated from
diseased yellowtail and eels are described in order to determine their taxonomic status.
tested. Bacteria inoculated into BHI broth were observed to
grow in 0.0 to 6.5% NaCl at 0 to 50°C and pH 4.5 to 9.6.
Biochemical characteristics. Routine tests for biochemical
properties were done as described in the Manual of Methods
for General Bacteriology (20). Hemolysis on BHI agar
supplemented with horse, sheep, and rabbit blood was
tested.
DNA isolation and determination of moles percent G+C.
Strains Enterococcus seriolicida YT-3, E . faecalis ATCC
19433T, and E. faecium ATCC 19434T were cultured in BHI
broth and harvested by centrifugation. The cells were lysed
by the method of Garvie (9,and the DNA was purified by
the method described by Johnson (6). Moles percent G+C
was determined by the thermal melting method according to
an equation by Galau et al. (described in reference 6) with a
Beckman model DU-8 computing spectrophotometer.
Absorbance at 260 nm was recorded at 1.0"C increments
between 50 and 90°C. A reference DNA sample was prepared from Escherichia coli WP-2.
DNA-DNA hybridization. Strains E. seriolicida YT-3, E.
avium ATCC 14025T, E. casselifEavus ATCC 2578gT, E.
durans ATCC 19432T,E . faecalis ATCC 19433T,E . faecium
ATCC 19434T, E. gallinarum ATCC 35038T, E. hirae IF0
3181T, E. mundtii NCDO 2375T, E. pseudoavium NCDO
2138T, E. rafinosus NCTC 12192T, E. solitarius NCTC
12193T, and Escherichia coli ATCC 11775T were grown in
BHI broth. DNA was prepared from the harvested cells as
MATERIALS AND METHODS
Source of isolates. Sources of the strains used in this study
are summarized in Table 1. The tested strains were isolated
from the tissues of diseased yellowtail pen cultured in
shallow sea areas and of diseased eels cultured in freshwater
ponds in Japan. The bacteria were grown in brain heart
infusion (BHI) broth or on BHI agar at 25°C.
Morphological characteristics. The size and cell form of
cells stained by shadowing with gold-palladium were observed by light microscope and electron microscope. Motility was tested by the microscopic examination of wet mounts
of cells cultured in BHI broth at 25 and 37°C.
Cultural characteristics. Growth on nutrient agar, MacConkey agar, salmonella-shigella agar, 40% bile agar and PEA
azide agar (Eiken, Tokyo, Japan; composed of 1.5% tryptone, 0.5% beef extract, 0.15% phenylethanol, 0.5% sodium
chloride, 0.01% sodium azide, and 1.5% agar, pH 7.4),
methylene blue-milk, and Streptococcus faecalis broth was
* Corresponding
TABLE 1. List of E. seriolicida strains used in this study
E. seriolicidu
strain
Source
YT-3T ...............ATCC 49156; kidney of yellowtail; Kochi,
Japan, in 1974
SK-7 .................Liver of yellowtail; Kochi, Japan, in 1974
SK-11 ...............Liver of yellowtail; Kochi, Japan, in 1974
SK-12 ...............Heart of yellowtail; Kochi, Japan, in 1974
SK-13 ...............Heart of yellowtail; Kochi, Japan, in 1974
SK-14 ...............Liver of yellowtail; Kochi, Japan, in 1974
SE-1 .................Kidney of yellowtail; Ehime, Japan, in 1974
SE-5 .................Kidney of yellowtail; Ehime, Japan, in 1974
SY-1 .................Heart of yellowtail; Yamaguchi, Japan, in
1974
SY-2 .................ATCC 49157; heart of yellowtail; Yamaguchi,
Japan, in 1974
AK-1 ................Kidney of eel; Kochi, Japan, in 1973
MA.. .................Kidney of eel; Ehime, Japan, in 1973
author.
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VOL. 41, 1991
ENTEROCOCCUS SERIOLICIDA SP. NOV.
407
TABLE 2. Differential characteristics of E. seriolicidu and other Enterococcus speciesa
E . serioliCharacteristic
YT-3
Motility
Growth at:
45°C
+
Others
(n = 11)
+
50°C
Growth in:
+
+
6.5% NaCl
Telluri te (0.04%)
+
+
Tetrazolium (0.01%)
+
Methylene blue (0.1%)- +
milk
Pigment production
Hemolysis
H,S production
Ammonia from arginine
Arginine hydrolysis
Hippurate hydrolysis
Voges-Proskauer
Acid produced from:
D-Xylose
L-Rhamnose
Sucrose
Lactose
Melibiose
RafEnose
Melezitose
Glycerol
Adonitol
+
+
Sorbitol
+
+
Mannitol
Not D Not D
Lancefield group
44
G+C mol%'
c.mi- s e r i . i
Umc'd
vus
+
-
+-
+
+
-
c.au- c . j a e - c .
calisc*e ciurri
+
-
+
+
+
+
.
-(+)
+,+(- .)
+
+
+
+
-
+
Ih
ni
+
+
-
-
+
+(-I
+
+
-
-
-
+
+
+
+
+
+
V
-
+
-
-
-t
+
V
-
+
+
+
+
+
+
+
+
+(-I
+
+
+
+(-I
+
+
+
+
V
+
V
-
+(-)
-
-(+I
+
+
+(-I
-
+
+
+
+
V
-
+
+
+(-I
D
D
D
D(Q) D
39-40 4 1 4 5 38-40 37-40 3 7 4 0
a
+
+
+
+
+
+
+
+
+
+
+
-
V
D
39-40
D
37-38
-
+
+
+
+
+
+
+
+
+
+
+
-
V
-
+
+
+
+
D
4041
+
+
+
+
+
+(-I
+(-I
V
V
+
D
38-39
40
w-1
39-40
+
+
+
+
D
38
Symbols: +, positive; +(-), most strains positive; v, variable; -(+), most strains negative; -, negative; -@), some strains p; -(a),some strains a;D(-),
most strains D.
Data from this study. All of the 11 strains other than YT-3 showed the same characteristics.
Data from reference 22.
Data from reference 3.
Data from reference 17.
Data from reference 4.
Data from reference 2.
Data from reference 1.
As determined by the thermal melting temperature.
follows. Bacterial cells were homogenized with glass beads
in saline-0.1 M EDTA solution and extracted with a same
volume of phenol-chloroform-isoamyl alcohol (25:24:1) mixture. The supernatant was digested by RNase A and was
precipitated by adding 1/10 of a volume of 3 M sodium
acetate and a double volume of ethanol. The precipitated
DNA was washed with 70% ethanol. Determination of
DNA-DNA homology was performed with the Enhanced
Chemiluminescence Gene Detection System (Amersham) as
follows: DNAs were dissolved in water, boiled for 5 min,
chilled immediately, and spotted in equal amounts on nitrocellulose membranes. The membranes were pretreated in
hybridization buffers, dried, heated at 80°C for 3 h, and
reacted with peroxidase-labeled DNA probe of E . seriolicidu
YT-3 overnight at 32°C. After being rinsed with washing
buffers, the membranes were treated with a reaction solution
which included hydrogen peroxide, luminol, and enhacer to
produce chemiluminescence and were then attached to photosensitive films to expose them. The percentage of reassociation was determined by measuring the density of the dark
spots formed on the developed film with a densitometer (PN
39431; Gelman). The results were expressed as mean values
of triplicate data.
RESULTS AND DISCUSSION
Isolates showed gram-positive ovoid cells (1.4 by 0.7 pm)
forming short chains. The cells were elongated in the direction of the chain (Fig. 1). Endospores were not formed.
Isolates were facultatively anaerobic. Growth occurred at 10
to 45°C with 0 to 6.5% NaCl and pHs of 4.5 to 9.6, with
optimum growth at 37"C, 0% NaC1,-and a pH of about 7.5.
Methyl red test, Voges-Proskauer reaction, reduction of
2,3,5-triphenyltetrazolium chloride (tetrazolium), and hydrolysis of esculin were positive. Catalase, cytochrome
oxidase, casein digestion, and reduction of potassium tellurite were negative. Production of indole, hydrosulfide, and
gelatinase and reduction of nitrate and methylene blue were
negative. These results indicate that the isolates belong to
the genus Enterococcus and that they can be differentiated
from Streptococcus spp. and Luctococcus spp. by the following: positive growth at both 10 and 45"C, in 6.5% NaC1,
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408
KUSUDA ET AL.
INT. J. SYST. BACTERIOL.
TABLE 3. DNA-DNA hybridization of DNA probe of
E. seriolicida YT-3 with DNAs prepared
from Enterococcus strains
Strain of hybridized DNA
Densitometric
measurementa
(mm2)
Reassociationb
E. seriolicida TY-3
E . avium ATCC 14025T
E. casserijiavus ATCC 25788T
E. durans ATCC 19432T
E . faecalis ATCC 19433;
E. faecium ATCC 19434
E. gallinarum ATCC 35038=
E. hirae I F 0 3181T
E. mundtii NCDO 2375=
E. pseudoavium NCDO 2138T
E. rafinosus NCTC 12192T
E. solitarius NCTC 12193T
Escherichia coli ATCC 1177!iT
365.7 2 4.85
45.2 k 3.69
18.5 k 3.06
60.4 2 4.86
61.0 2 11.8
51.3 2.97
47.9 f 4.75
89.1 f 6.96
83.2 2 11.6
23.1 k 0.42
65.4 2 12.9
48.7 f 24.7
6.9 2 0.91
100
12
5
17
17
14
13
24
23
6
18
13
2
%
*
~~
~
Mean value from three trials 2 standard deviation. Each value is
expressed in square millimeters as determined from densitometric patterns of
dark spots which were formed on photosensitive films after hybridization and
development with the Enhanced Chemiluminescence Gene Detection System
a
FIG. 1. Electron micrograph of E. seriolicida YT-3 cultured on
BHI agar at 37°C for 24 h. Bar, 1 Fm.
and at pH 9.6, which are important characteristics of Enterococcus spp. (18). Therefore, it would be appropriate hereafter to call the disease caused by this etiological agent
‘ ‘enterococcal infection” instead of “streptococcal infection” or “streptococcicosis,” names which have been used
previously.
The isolates can be distinguished from other species of the
genus Enterococcus by biochemical characteristics, as
shown in Table 2. Serological tests using some of the
isolates, including strain YT-3, have been performed previously by Kusuda et al. (11).The antigen extracted from the
isolates by the hot acid method of Lancefield (16) was not
homologous with the antigens of groups A, B, C, D, E, F, G,
H, K, L, M, N, and 0. Since the isolates were shown not to
have Lancefield’s D group antigen, it can be distinguished
from E. casselijavus, E. durans, E. faecalis, E . faecium, E .
gallinarum, E . hirae, E . malodoratus, E. mundtii, and E .
solitarius.
The moles percent G+C of DNAs from E. seriolicida
YT-3, E. faecalis ATCC 19433T,E . faecium ATCC 19434T,
and Escherichia coli WP-2 were 44, 38, 39, and 51%,
respectively. The results of DNA hybridization, shown in
Table 3, indicate sufficiently low DNA homology values of
E. seriolicida YT-3 to warrant distinguishing this species
from Other ‘pecies Of Enterococcus’
the DNADNA homology datum between E . seriolicida and E. mafodoratus is absent in Table 3, considerable differences in
characteristics and moles percent G+C, shown in Table 2,
demonstrate that they can be discriminated as different
species.
On the basis of morphological, cultural, biochemical,
serological, and nucleic acid characteristics, it is concluded
that the isolates represent a new species within the genus
Enterococcus, for which we propose the name Enterococcus
seriolicida sp. nov.
Description of Enterococcus seriolicida sp. nov. Enterococcus seriolicida (se ri o li ci’ da. L. n. seriola, generic name of
the yellowtail; L. suffix cida from L. v. caedo, to kill; N. L.
adj . seriolicida, yellowtail killer), is a facultatively anaero-
c A ~ ~ ~of reassociation
~ ~ ~ g were
e sdetermined with respect to the homologous reaction of E. seriolicida YT-3, which was made to equal 100%.
bic, nonmotile, non-spore-forming, gram-positive coccus
that occurs in short chains of cells. The cells are ovoid and
about 1.4 by 0.7 Fm in size. E. seriolicida grows on nutrient
agar, 40% bile agar and PEA azide agar and in 0.1%
methylene blue-milk but does not grow on MacConkey agar
or salmonella-shigella agar or in S. faecalis broth. Growth
occurs at 45”C, 6.5% NaC1, and pH 9.6, E. seriolicida is
fermentative, producing acid from D-glucose, D-mannose,
galactose, D-fructose, trehalose, maltose, cellobiose, dextrin, sorbitol, mannitol, salicin, and esculin but not from
D-xylose, D-arabinose, L-rhamnose, sucrose, lactose, melibiose, raffinose, melezitose, starch, glycogen, glycerol,
inositol, or adonitol. Methyl red test, Voges-Proskauer reaction, and tetrazolium reduction are positive. Catalase
production, cytochrome oxidase production, casein digestion, and potassium tellurite reduction are negative. Production of gelatinase, indole, and hydrosulfide and reduction of
nitrate and methylene blue are negative. a-Hem01ysis occurs
on blood agar. E. seriolicida does not belong to Lancefield’s
groups A to H, K to N, and 0.
The type strain is YT-3 (=ATCC 49156). The moles
percent G+C of the DNA from the type strain is 44 mol%, as
determined by the thermal denaturation method.
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ENTEROCOCCUS SERIOLZCIDA SP. NOV.
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