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Applied and Environmental Microbiology, March 1999, p. 1005-1008, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Biodiversity of Lactococcus garvieae
Strains Isolated from Fish in Europe, Asia, and
Australia
Avi
Eldar,1
Mariella
Goria,2
Claudio
Ghittino,2
Amir
Zlotkin,3 and
Herve
Bercovier3,*
Department of Poultry and Fish Diseases, The
Kimron Veterinary Institute, Beit Dagan,1 and
Department of Clinical Microbiology, The Hebrew
University-Hadassah Medical School, Jerusalem
91120,3 Israel, and Experimental
Institute for Zooprophylaxis, 10154 Turin, Italy2
Received 6 August 1998/Accepted 8 December 1998
 |
ABSTRACT |
Lactococcus garvieae (junior synonym,
Enterococcus seriolicida) is a major pathogen of fish,
producing fatal septicemia among fish species living in very diverse
environments. The phenotypic traits of L. garvieae strains
collected from three different continents (Asia, Europe, and Australia)
indicated phenotypic heterogeneity. On the basis of the acidification
of D-tagatose and sucrose, three biotypes were defined. DNA
relatedness values and a specific PCR assay showed that all the
biotypes belonged to the same genospecies, L. garvieae. All
of the L. garvieae strains were serotyped as Lancefield
group N. Ribotyping proved that one clone was found both in Japan,
where it probably originated, and in Italy, where it was probably
imported. PCR of environmental samples did not reveal the source of the
contamination of the fish in Italy. Specific clones (ribotypes) were
found in outbreaks in Spain and in Italy. The L. garvieae
reference strain, isolated in the United Kingdom from a cow, belonged
to a unique ribotype. L. garvieae is a rising zoonotic
agent. The biotyping scheme, the ribotyping analysis, and the PCR assay
described in this work allowed the proper identification of L. garvieae and the description of the origin and of the
source of contamination of strains involved in outbreaks or in sporadic cases.
| |
INTRODUCTION |
During the last decade, sporadic and
epidemic outbreaks of fish diseases due to gram-positive cocci have
been reported from different parts of the world, including Japan
(18), Korea (14), Italy (11), Spain
(8, 24, 26), France (22), Australia (5), Israel (9, 10), and the United States
(2, 10). Taxonomic studies have indicated that at least six
different species of gram-positive cocci are associated with fish
diseases: Streptococcus parauberis (8),
Streptococcus iniae (10), Streptococcus
difficile (9), Lactococcus piscium
(29), Vagococcus salmoninarum (22), and Lactococcus garvieae (junior synonym, Enterococcus
seriolicida (7, 11, 18). Some pathogens are well
adapted to a specific host; others, like L. garvieae,
are ubiquitous. L. garvieae, originally isolated in the
United Kingdom from a mastitic udder (6), has been recovered
from fish species living in very diverse conditions: yellowtail
cultured in a marine environment in the Far East and trout raised in
temperate freshwater in Europe and in Australia. L. garvieae strains also have been isolated from humans, indicating the expanding importance of this bacterium (12).
Little is known about the ecological distribution, the source of
infection, and the modalities by which L. garvieae is
transmitted between fish. The epidemiological relationship between
strains isolated from fish has never been investigated. The published identification scheme for L. garvieae, based on
biochemical and antigenic characteristics (6, 7, 12, 17),
can barely differentiate L. garvieae from
Lactococcus lactis and from
"Enterococcus-like" strains isolated from diseased fish
(24, 26). A clindamycin test (13) and a PCR assay
(30) are the only tests able to differentiate L. garvieae from L. lactis definitively. The paucity of knowledge prompted us to investigate this organism. Different L. garvieae strains collected from diseased fish from
three different continents (Asia, Australia, and Europe) were analyzed
by phenotypic, genetic, and molecular methods. DNA relatedness values
and PCR analysis confirmed the wide geographical distribution of this species, and phenotypic traits demonstrated the existence of different biotypes. Restriction fragment length polymorphism (RFLP) ribotyping was found to be an efficient tool for studying the epidemiology of
L. garvieae in fish, suggesting possible routes of infection.
| |
MATERIALS AND METHODS |
Bacterial strains.
The strains used in this study are listed
in Table 1. The eight Italian
L. garvieae strains were randomly taken from a
collection of over 100 strains recovered from different outbreaks in
trout between May 1992 and August 1995. Japanese strains, isolated from diseased yellowtail, were kindly supplied by F. Salati. Spanish and
Australian strains, all isolated from trout, were kindly provided by A. Cacho and J. Carson, respectively. S. iniae ATCC
29178T, E. seriolicida ATCC 49156T,
and L. garvieae ATCC 43921T were purchased
from the American Type Culture Collection, Rockville, Md. L. piscium NCFB 2778T, L. lactis NCFB
604T, and V. salmoninarum NCFB 2777T
were purchased from the National Collection of Food Bacteria, Reading,
United Kingdom. S. difficile CIP 103769T was
from our own collection. Bacteria from stock cultures stored in 10%
glycerol at 
70°C were grown on Columbia agar base (Difco) plates
supplemented with 5% (vol/vol) defibrinated sheep blood at 24°C for
24 h, when characteristic alpha-hemolytic colonies became visible.
Biochemical and enzymatic tests.
Biochemical tests
(acidification of carbohydrates) and enzymatic tests were performed
with the API 20 Strep and API 50CH systems (bioMerieux S.A., Marcy
l'Etoile, France). Tests were done in accordance with the instructions
of the manufacturer, except for the temperature of incubation, which
was set at 24°C. Additional tests included the ability to grow on
skim milk (Difco) agar plates supplemented with 0.1 and 0.3%
(wt/vol) methylene blue (Sigma); growth at 10, 42, and 45°C;
growth in the presence of 10 and 40% (wt/vol) bile salts agar (oxgall;
Difco); growth on tryptic soy agar (Difco) containing 4 and 6.5% NaCl;
and growth on brain heart infusion agar at a range of pH values (7.0 to
9.6).
Hyperimmune sera and group antigen.
New Zealand White
rabbits, 1 kg each, were immunized with formalin-fixed E. seriolicida ATCC 49156T and L. garvieae ATCC 43921T, ITP2001, and S1449 whole cells
by methods described by Lancefield (19, 20) and MacCarthy
and Lancefield (21). Specific group antigens were extracted
by exposure of the bacteria to 0.2 N HCl for 10 min at 100°C
(19). L. lactis NCFB 717 was used as a
positive control for type N antigen. Capillary precipitin tests were
performed by the method of Lancefield (19), and
immunodiffusion tests were performed by the agar gel method of
Ouchterlony (25).
DNA-DNA hybridizations and PCR assay.
The methods used to
lyse gram-positive cocci and to extract the DNA content have been
described elsewhere (9). DNA-DNA hybridizations were done by
the hydroxyapatite (Bio-Rad) method (3) with modifications
regarding the volumes used as described previously (11).
Reactions were done in duplicate, and each run was performed twice. The
levels of DNA relatedness (relative binding ratio [RBR]) and the
differences in melting points between the homologous reactions and the
heterologous reactions for the labeled reference strains and other
strains [
Tm(e)] were calculated as described before
(3, 9). The rate of reassociation of the labeled DNA was
routinely 5 to 6%; this value was subtracted from the absolute ratios
of the hybridizations. An L. garvieae species-specific
PCR assay was performed on all the strains as described previously
(30). Ten water samples (100 ml) from Italian trout pounds
where L. garvieae infection was active were centrifuged at 8,000 × g for 20 min. The resulting pellet was
boiled and submitted to the PCR assay (30).
Ribotyping.
Genomic DNAs (4 µg) were digested with 50 U
each of restriction enzymes HindIII and EcoRI
(Promega) at 37°C for 16 h under the conditions specified by the
manufacturer. DNA fragments were separated by electrophoresis on a 1%
agarose gel (20 cm long) at 55 V for 17 h in 49 mM Tris acetate-2
mM EDTA and blotted onto a positively charged nylon membrane
(Boehringer GmbH, Mannheim, Germany). The DNA was fixed by UV
cross-linking. Prehybridization and hybridization for 3 and 20 h,
respectively, were carried out with 50 mM Tris-HCl (pH 7.5)-1 M
NaCl-1% sodium dodecyl sulfate-10% dextran sulfate-0.1% blocking
reagent. The DNA probe used was a 7.5-kb BamHI fragment of
pKK3535 (kindly supplied by G. Glazer, The Hebrew University of
Jerusalem), which is a pBR322-derived plasmid comprising the complete
Escherichia coli rRNA B operon (4). The DNA probe
was labeled by use of a random oligopriming kit (Boehringer) with a
mixture of nucleotides and reverse transcriptase in the presence of
0.35 mM digoxigenin-11-dUTP (Boehringer). After hybridization, the
filters were washed twice with 0.15 M sodium citrate solution
containing 0.1% sodium dodecyl sulfate for 15 min at room temperature.
Chemiluminescence was detected as recommended by the manufacturer
(Boehringer) by incubating the membranes in the presence of an
antidigoxigenin antibody linked to alkaline phosphatase and the
alkaline phosphatase substrate
AMPPD [3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)-phenyl-1,2-dioxe-tane]. Filters were autoradiographed by exposure to X-Omat AR 5 films (Kodak,
Rochester, N.Y.) from 10 to 120 min at room temperature. Each different
combination of patterns was considered a ribotype.
| |
RESULTS |
DNA-DNA hybridizations and PCR assay.
RBR and
Tm(e) values for the DNAs of the various L. garvieae strains and E. seriolicida ATCC
49156T DNA hybridized with labeled L. garvieae ATCC 43921T DNA ranged from 71 to 87% and
0.3 to 2.3°C, respectively (Table 1). The values for L. lactis DNA hybridized with labeled L. garvieae ATCC 43921T DNA were 39% and 6°C, confirming that
L. lactis and L. garvieae are
genetically different species (Table 1). All of the L. garvieae strains (field and reference strains) submitted to
the PCR assay produced a characteristic fragment of 1,100 bp
(data not shown). No water sample was found positive by the PCR assay.
Phenotypic studies and serotyping of L. garvieae
isolates.
All the strains had several common characteristics:
growth at 10 and 42°C, in the presence of 40% bile salts, at pH 9.6, and on 0.3% methylene blue-milk agar. Although scant, growth also was
observed on 6.5% NaCl agar and at 45°C. All isolates were alpha-hemolytic. Acetoin production (Voges-Proskauer reaction) and the
presence of the enzymes pyrallidonylarylamidase, leucine arylamidase,
and arginine dehydrolase were common to all isolates. The following
tests were negative for all the L. garvieae strains: reduction of hippurate; production of 
- and 
-galactosidase, -glucoronidase, and alkaline phosphatase; and utilization of erythritol, D-arabinose, D- and
L-xylose, m-xyloside, L-sorbose, rhamnose, dulcitol, inositol, -m-D-glucoside,
inulin, melezitose, D-raffinose, glycogen, xylitol,
D-turanose, L-lyxose, D- and
L-fucose, D- and L-arabitol,
gluconate, and 2- and 5-ketogluconate. All the L. garvieae strains produced acid from ribose, galactose, D-glucose, D-fructose, D-mannose,
mannitol, N--glucosamine, amygdalin, arbutin, esculin,
salicin, cellobiose, maltose, trehalose, and -gentobiose.
The Italian, Japanese, and reference ATCC strains which acidified the
above-mentioned carbohydrates were characterized as biotype 1 strains.
The Australian strain 88/1400 acidified, in addition,
D-tagatose (biotype 2 strain). The Spanish strains
acidified sucrose (biotype 3 strains) in addition to the sugars
acidified by the biotype 2 strain.
The capillary precipitin test and the Ouchterlony test
showed that all the strains were part of Lancefield group
N.
RFLP ribotyping.
EcoRI- and
HindIII-digested L. garvieae DNAs
resulted, respectively, in two and seven different patterns (ribotypes
a and b and c to i) (Fig. 1 and
2). After Southern blot
hybridization, EcoRI-digested DNAs resulted in three or four
detectable fragments ranging from 23 to 0.3 kb. The distribution of the
bands allowed the definition of two ribotypes. Italian and Japanese
strains, as well as the Australian strain and the two ATCC reference
strains, belonged to the same ribotype, a (Fig. 1, lanes 1 to 10 and 15 to 17). Spanish strains were designated ribotype b strains (Fig. 1,
lanes 11 to 13).
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FIG. 1.
RFLP ribotyping of EcoRI digests. Numbers on
the left are in kilobases. Lanes: 1 to 8, ITP1072, IPT1313, IPT1545,
IPT1794, IPT1814, IPT1964, IPT2001, and IPT1036; 9 and 10, S1449 and
S014; 11 to 13, Sp1, Sp2, and Sp3; 14, L. piscium NCFB
2778T; 15, 88/1400; 16, E. seriolicida ATCC
49156T; 17, L. garvieae ATCC
43921T.
|
|
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FIG. 2.
RFLP ribotyping of HindIII digests.
Numbers on the left are in kilobases. Lanes are as defined in the
legend to Fig. 1.
|
|
HindIII digests generated more polymorphisms, producing
six to nine reproducible restriction fragments ranging from 9.4 to
0.5 kb. Seven Italian strains and one Japanese strain had a common
pattern
(ribotype c strains) (Table
1 and Fig.
2, lanes 1 to
7 and 9). One
Italian strain (ITP2036) and one Japanese strain
(S014) differed only
slightly from ribotype c strains. Strain
ITP2036 (ribotype d) had an
additional 0.5-kb restriction fragment
(Fig.
2, lane 8), whereas strain
S014 (ribotype e) was characterized
by the presence of an additional
4.0-kb restriction fragment (Fig.
2, lane 10). The restriction
pattern of all Spanish strains was
identical, displaying six
restriction fragments (ribotype f) (Fig.
2, lanes 11 to 13). The
Australian strain and the two ATCC reference
strains
(
E. seriolicida ATCC 49156
T and
L. garvieae ATCC 43921
T) had different
patterns, which were designated, respectively,
ribotype g (Fig.
2, lane
15), ribotype h (Fig.
2, lane 16), and
ribotype i (Fig.
2, lane
17).
All the ribotypes obtained with the
L. garvieae strains
were different from those obtained with the reference strain of an
unrelated species,
L. piscium NCFB 2778
T
(Fig.
1 and
2, lanes
14).
| |
DISCUSSION |
Based on phenotypic characteristics, gram-positive cocci isolated
from fish and able to grow at 10 and 42°C, in the presence of 40%
bile salts, at pH 9.6, and on 0.3% methylene blue-milk agar (with
scant growth also on 6.5% NaCl agar and at 45°C) should be
classified as L. garvieae. None of the other five
species of gram-positive cocci pathogenic for fish grow under these
conditions. However, the existence of different L. garvieae biotypes emphasizes the difficulties of definitive
identification based on phenotypic traits alone. Therefore, final
identification cannot be determined without the support of genetic data.
Indeed, the physiological features of a defined bacterial species
should be expressed in terms of percentages rather than as clear-cut
characteristics. Murray (23) found that 56% of lactococci
tested were able to grow on 6.5% NaCl and that 25% of them were also
able to grow at 45°C and at pH 9.6. In the 9th edition of
Bergey's Manual of Determinative Bacteriology
(17), the inability to grow at 45°C and at pH 9.6 is
considered a cardinal feature enabling lactococci to be distinguished
from enterococci (which do grow under these conditions). The
implementation of rigid criteria might therefore lead to inaccuracy.
Serological data are also not conclusive, as a serogroup can encompass
various bacterial species. The criteria of DNA relatedness levels of
70% or more, accepted as the major criterion for delineating
genospecies (16, 28), proved that all the strains studied in
this work belonged to the L. garvieae genospecies,
regardless of the biotype. The species-specific PCR assay
confirmed that all the studied strains belonged to the same species,
L. garvieae (30).
Phenotypic traits indicated that a single L. garvieae
clone was involved in each of the outbreaks which occurred in Italy and
Japan (biotype 1) and that another clone was responsible for the
Spanish outbreak (biotype 3). rRNA gene restriction patterns (RFLP
ribotyping), first proposed by Grimont and Grimont
(15), allowed us to refine the discrimination among
strains of the same species. EcoRI digests of Italian and
Japanese isolates were found to be identical, substantiating the
hypothesis of a straightforward correlation between phenotype and
epidemiological source. However, HindIII digests
revealed that of the three Japanese strains, only one had the same
ribotype as the Italian strains (ribotype c), the second was only
closely related, and the third was a totally unrelated epidemiological
clone. The variety in ribotypes among Japanese strains is not
surprising given that the disease appeared in Japan years
(18) before being diagnosed in Europe (7) and
that no control measures were available. The Italian and Japanese strains with identical biotypes and identical HindIII
and EcoRI ribotypes raised the possibility that the disease
spread from Japan to Italy through the import of livestock or fish
food. Unfortunately, the PCR assay applied to water samples could not
provide an adequate answer regarding the source of contamination in
Italy (environment or food). The Spanish strains belonged to a single
biotype (biotype 3) and to a single ribotype, indicating that the
outbreak in Spain was due to a single epidemiological clone which
evolved separately. Since only one Australian isolate was included in
this study, no similar conclusion can be drawn regarding L. garvieae infection in Australia. Finally, the RFLP ribotyping
showed that L. garvieae ATCC 43921T,
originally isolated from a mastitic udder, is a clone different from
the Italian and Japanese strains, although it is phenotypically identical.
The data presented here emphasize the biodiversity of L. garvieae strains isolated from various animal sources and
continents and show that ribotyping and biotyping can be efficient
tools for tracing possible methods of dissemination of this emerging pathogen.
| |
ACKNOWLEDGMENT |
This work was supported by a joint American-Israeli grant (BARD
IS-2307-93).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Clinical Microbiology, The Hebrew University-Hadassah Medical School, POB 12272, Jerusalem 91120, Israel. Phone: 972-2-6758256. Fax: 972-2-6784010. E-mail: HB{at}cc.huji.ac.il.
| |
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Applied and Environmental Microbiology, March 1999, p. 1005-1008, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
