Polyphasic study of Chryseobacterium strains isolated from diseased aquatic animals.

Abstract

Members of most Chryseobacterium species occur in aquatic environments or food products, while strains of some other species are pathogenic to humans and animals. A collection of 52 Chryseobacterium sp. strains isolated from diseased fish, one frog isolate and 22 reference strains were included in a polyphasic taxonomy study. Fourteen clusters of strains were delineated following the comparison of whole-cell protein profiles. Most of these clusters were confirmed when the phenotypic and RAPD profiles and the 16S rRNA gene sequences were compared. Fatty acid composition helped differentiate the Chryseobacterium strains from members of related genera. None of the fish isolates could be allocated to the two species previously reported from fish but two isolates belonged to C. joostei, while the frog isolate was identified as Elizabethkingia meningoseptica, a human pathogen previously included in the genus Chryseobacterium. Three clusters grouping from 3 to 13 isolates will probably constitute the core of new Chryseobacterium species but all other isolates occupied separate or uncertain positions in the genus. This study further demonstrated the overall high similarity displayed by most Chryseobacterium strains whatever the technique used and the resulting difficulty in delineating new species in the genus. Members of this bacterial group should be considered potential emergent pathogens in various fish and frog species, farming conditions and geographical areas.

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Systematic and Applied Microbiology 28 (2005) 640–660
Polyphasic study of Chryseobacterium strains isolated from
diseased aquatic animals
J.-F. Bernardeta,?, M. Vancanneytb, O. Matte-Taillieza, L. Grisezc,1, P. Tailliezd,
C. Bizete, M. Nowakowskie, B. Kerouaulta, J. Swingsb
aInstitut National de la Recherche Agronomique, Unite ´ de Virologie et Immunologie Mole ´culaires, Jouy-en-Josas, France
bGhent University, BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie, Ghent, Belgium
cZoological Institute, Laboratory for Ecology and Aquaculture, Leuven, Belgium
dInstitut National de la Recherche Agronomique, Universite ´ Montpellier II, Unite ´ d’ Ecologie Microbienne des Insectes et
Interactions Ho ˆte-pathoge `ne, Montpellier, France
eInstitut Pasteur, De ´partement Structure et Dynamique des Ge ´nomes, Collection de l’Institut Pasteur, Paris, France
Received 11 March 2004
Summary
Members of most Chryseobacterium species occur in aquatic environments or food products, while strains of
some other species are pathogenic to humans and animals. A collection of 52 Chryseobacterium sp. strains
isolated from diseased fish, one frog isolate and 22 reference strains were included in a polyphasic taxonomy
study. Fourteen clusters of strains were delineated following the comparison of whole-cell protein profiles. Most of
these clusters were confirmed when the phenotypic and RAPD profiles and the 16S rRNA gene sequences were
compared. Fatty acid composition helped differentiate the Chryseobacterium strains from members of related genera.
None of the fish isolates could be allocated to the two species previously reported from fish but two isolates belonged
to C. joostei, while the frog isolate was identified as Elizabethkingia meningoseptica, a human pathogen previously
included in the genus Chryseobacterium. Three clusters grouping from 3 to 13 isolates will probably constitute the
core of new Chryseobacterium species but all other isolates occupied separate or uncertain positions in the genus. This
study further demonstrated the overall high similarity displayed by most Chryseobacterium strains whatever the
technique used and the resulting difficulty in delineating new species in the genus. Members of this bacterial group
should be considered potential emergent pathogens in various fish and frog species, farming conditions and
geographical areas.
r 2005 Elsevier GmbH. All rights reserved.
Keywords: Chryseobacterium; Elizabethkingia; Fish disease; Frog disease; Polyphasic taxonomy; Phenotypic characteristics;
Randomly amplified polymorphic DNA; Whole-cell protein profiles; SDS-PAGE; Fatty acid analysis; 16S rRNA gene sequence
Introduction
The genus Chryseobacterium was proposed to accom-
odate six bacterial species previously included in the
genus Flavobacterium [41]. Following an extensive
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www.elsevier.de/syapm
0723-2020/$-see front matter r 2005 Elsevier GmbH. All rights reserved.
doi:10.1016/j.syapm.2005.03.016
Abbreviation: RAPD, randomly amplified polymorphic DNA
?Corresponding author. Tel.: +33134652587;
fax:+33134652591.
E-mail address: jean-francois.bernardet@jouy.inra.fr
(J.-F. Bernardet).
1Current address: Intervet Norbio Singapore Pte Ltd., Singapore.

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polyphasic study, the whole group of organisms related
to the genera Chryseobacterium and Flavobacterium was
included in the family Flavobacteriaceae [4,5]. The genus
Chryseobacterium currently comprises nine valid species
and a non-valid one representing a variety of ecological
niches [3,20–22,41,44,46]. Five species, namely C.
indoltheticum [10], C. defluvii [21], ‘‘C. proteolyticum’’
[44], C. formosense [46] and C. daecheongense [22] were
isolated from various soil and aquatic environments.
Chryseobacterium spp. strains are also part of the
psychrotrophic and proteolytic bacterial population
that causes a variety of defects in food products such
as processed meats, chilled fish and shellfish, and dairy
products [14,18]. The polyphasic study of a group of
dairy isolates recently resulted in the description of C.
joostei [19,20]. Other Chryseobacterium species are
pathogenic to humans and animals. Chryseobacterium
gleum and C. indologenes may cause nosocomial
infections in humans, usually in neonates or immuno-
compromised patients; they can also be retrieved from
hospital environments and equipments and are usually
resistant to many disinfectants and antibiotics [3]. The
16S rRNA gene sequences of the putative new clinical
species ‘‘C. massiliae’’ and ‘‘Candidatus Chryseobacter-
ium timonae’’ have recently been deposited in GenBank
[12]. Finally, C. balustinum [6,16] and C. scophthalmum
[28,29] were isolated from diseased fish. Chryseobacter-
ium spp. strains were also detected among the bacterial
flora in the gut of insects by direct amplification and
sequencing of 16S rRNA genes [13], but they have not
been fully characterized. Until recently, two more
bacterial species were allocated to the genus Chryseo-
bacterium, i.e. C. meningosepticum and C. miricola.
However, owing to their separate position compared to
other Chryseobacterium species in 16S rRNA gene
sequence comparison studies, their transfer to the new
genus Elizabethkingia was proposed [23]. Hence, the
new combinations Elizabethkingia meningoseptica and
E. miricola will be used thereafter. The latter species
was isolated from condensation water in the space
sation Mir [25] whereas the former is considered the
most serious human pathogen in the group [3]. Since
E. meningoseptica was also isolated from diseased
birds [39], frogs, turtles and cats [11,15 and referen-
ces therein,26], it may be the agent of zoonotic
infections.
Over the last few years, fish pathology laboratories in
Europe (Belgium, Finland, France) and Asia (Taiwan,
Singapore) have isolated an increasing number of
bacterial strains that shared the basic phenotypic
characteristics of the genus Chryseobacterium and that
were tentatively identified as Chryseobacterium sp., C.
indologenes or E. meningoseptica using commercial
identification galleries. In this paper, we present the
results of the polyphasic study [42] of 52 fish isolates and
one frog isolate.
Materials and methods
Bacterial strains
The bacterial strains used in this study are listed in
Table 1. Except one frog isolate, all isolates were
retrieved from diseased fish between 1994 and 2001.
The host fish represented a variety of fish species,
farming conditions, and geographical origins. About
one-third of the strains were isolated from superficial
lesions in otherwise apparently healthy fish while the
other strains were isolated from internal organs of
fish or frog suffering from hemorrhagic septicemia.
Bacterial strains were isolated from samples inocu-
lated on trypticase soy agar (bioMe ´ rieux sa, Marcy--
l0E´toile, France) incubated at 22 or 261C for 3 days.
After the basic common characteristics (i.e., Gram-
negative, non-motile rods, producing bright yellow-
orange flexirubin type pigments, a strong
catalase and oxidase, and yielding a Chryseobacter-
ium spp. profile in API 20E and 20NE galleries) of
these strains were identified, a retrospective screen-
ing of the culture collection of the Unite ´ de Virologie
et Immunologie Mole ´ culaires yielded three poorly
characterized fish isolates (i.e., JIP 105/82, JIP 108/
83, and SAVU 12/90), tentatively labelled ‘‘Flavo
bacterium-like’’ or ‘‘Flavobacterium sp.’’ at the time
of preservation, that shared the same basic char-
acteristics and were consequently included in the
study. The type strain of six valid Chryseobacterium
species and of E. meningoseptica were also studied, as
well as a second strain of each species. In the
particular case of C. scophthalmum, a very high
DNA homology had been demonstrated previously
between the seven strains available [29]. Hence, all
seven strains were included in this study in order to
test the repeatability of the techniques used there
after as well as their ability to reveal intra-specific
variability. Since Bergeyella zoohelcum, Riemerella
anatipestifer and R. columbina [40,41] form a distinct
separate branch together with the Chryseobacterium
and Elizabethkingia species in published 16S rRNA
genesequencesimilarity
3,4,20,22,25], the type strains of these three species
were also included in the study. The other members
of the Chryseobacterium–Elizabethkingia–Riemerella–
Bergeyella branch were not studied, either because
they were published after this study was completed
(C. defluvii [21], C. formosense [46], C. daecheongense
[22],
E.miricola
[25],
Sejongia antarctica and S. jeonii [45]) or because
they were not available (‘‘Haloanella gallinarum’’
[Pham, unpublished] and ‘‘Chryseobacterium proteo-
lyticum’’ [44]). However, their 16S rRNA gene sequen-
ces were included in the phylogenetic study (see
below).
smell,
dendrograms [e.g.,
Kaistellakoreensis
[24],
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Table 1.Bacterial strains included in this study
Name as receivedStraina
Cluster numberbSequence
numberc
Sourced
Elizabethkingia meningoseptica
LMG 4027T
CIP 60.57T
11AY468445 Cerebrospinal fluid, premature infant,
Massachusetts, USA, 1949
Elizabethkingia meningoseptica
LMG 12280 11AY468480Blood, newborn infant, Florida, USA,
1950
Chryseobacterium scophthalmum
LMG 13028T
CIP 104199T
12Gills, juvenile turbot (Scophthalmus
maximus) with hemorrhagic septicemia,
Scotland, UK, 1987
Chryseobacterium scophthalmum
LMG 1302912 Gills, diseased turbot, Scotland, UK,
1987
Chryseobacterium scophthalmum
LMG 1303012Diseased turbot, Scotland, UK, 1987
Chryseobacterium scophthalmum
LMG 13031 12Diseased turbot, Scotland, UK, 1987
Chryseobacterium scophthalmum
LMG 1303212Seawater, Scotland, UK, 1987
Chryseobacterium scophthalmum
LMG 13033 12Seawater, Scotland, UK, 1987
Chryseobacterium scophthalmum
LMG 1303412Gills, diseased turbot, Scotland, UK,
1987
Chryseobacterium balustinum
LMG 8329T
CIP 103103T
AY468447Heart blood, dace (Leuciscus leuciscus),
Dordogne river, Aquitaine, France, 1959
Chryseobacterium balustinum
LMG 128565AY468481Water, hospital, London, UK, 1978
Chryseobacterium indoltheticum
LMG 4025T
CIP 103168T
10
AY468448Marine mud
Chryseobacterium indoltheticum
LMG 1334210 AY468444London, UK
Chryseobacterium gleum
LMG 8334T
CIP 103039T
14 AY468449 High vaginal swab, London, UK, 1979
Chryseobacterium gleum
LMG 1245014 Wound swab, London, UK
Chryseobacterium indologenes
LMG 8337T
CIP 101026T
5 AY468450Human trachea at autopsy, 1958
Chryseobacterium indologenes
LMG 124525 No data available
Chryseobacterium joostei
LMG 18212T
CIP 105533T
3Raw cow milk, Ixopo district, Kwazulu-
Natal, South Africa, 1981
Chryseobacterium joostei
LMG 182083AY468479Raw cow milk, Heidelberg district,
Transvaal, South Africa, 1981
Bergeyella zoohelcum
LMG 8351T
CIP 103041T
Human sputum, Nebraska, USA
Riemerella anatipestifer
LMG 11054T
CIP 82.28T
Duck blood, USA
Riemerella columbina
LMG 11607T
CIP 106288T
Pigeon palatine cleft, Germany, 1989
JIP 105/82 AY468457Skin lesion, Koi carp, (Cyprinus carpio),
Ile-de-France, France, 1982 (Japan)
JIP 108/83AY468458 Extensive skin ulcer, snakehead (Channa
sp.), France, 1983 (Thailand)
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Table 1. (continued)
Name as received Straina
Cluster numberbSequence
numberc
Sourced
SAVU 12/90AY468459 Skin lesion, sea bass (Dicentrarchus
labrax), Mediterranean coast, France,
1990
UOF CO294AY427792Gills, brown trout (Salmo trutta),
Finland, 1994
UOF CR694
LMG 22906
CIP 108621
3 Tail lesion, Atlantic salmon (Salmo
salar), Finland, 1994
UOF CR1094
LMG 22907
CIP 108622
3AY468451 Deep ulcerative dorsal lesion, Atlantic
salmon, Finland, 1994
UOF CM295AY468452Kidney, brown trout, Finland, 1995
UOF CM895AY468453Kidney, Atlantic salmon, Finland, 1995
UOF CR29954AY468454Deep ulcerative dorsal lesion, Atlantic
salmon, Finland, 1995
UOF CR43954 AY468455 Peduncle lesion, Atlantic salmon,
Finland, 1995
UOF CR4095AY468483Deep ulcerative dorsal lesion, Atlantic
salmon, Finland, 1995
UOF CO496
LMG 22914
CIP 108623
6 Jaw erosion, brown trout, Finland, 1996
UOF CM1396
LMG 22905
CIP 108620
6AY468456Kidney, Atlantic salmon, Finland, 1996
UOF CM1996
LMG 22915
CIP 108624
6 Kidney, Atlantic salmon, Finland, 1996
UOF CM1796Kidney, brown trout, Finland, 1996
ZIL K4U1Skin ulcer, Koi carp, Belgium, 1996
ZIL K5U1 Skin ulcer, Koi carp, Belgium, 1996
ZIL K5M11 Muscle lesion, Koi carp, Belgium, 1996
ZIL K5M21 Muscle lesion, Koi carp, Belgium, 1996
ZIL K5BV1 Skin ulcer, Koi carp, Belgium, 1996
ZIL K3.2
LMG 19173
CIP 108619
1 AJ874979Tail lesion, Koi carp, Belgium, 1996
(Japan)
ZIL K4.31Tail lesion, Koi carp, Belgium, 1996
(Japan)
ZIL K6.11Skin lesion, Koi carp, Belgium, 1996
(Japan)
ZIL K12.11 Mouth lesion, Koi carp, Belgium, 1996
(Japan)
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Table 1. (continued)
Name as received Straina
Cluster numberbSequence
numberc
Sourced
ZIL K13.11Mouth lesion, Koi carp, Belgium, 1996
(Japan)
JIP 16/96AY468460 Internal organs, dwarf gourami (Colisa
lalia), France, 1996 (Singapore)
JIP 17/96
LMG 22908
CIP 108646
2 AY468461Kidney, leopard corydoras (Corydoras
julii), France, 1996 (USA)
LPAA 31209 AY468462 Skin lesion, rainbow trout (Oncorhynchus
mykiss), Brittany, France, 1996
LPAA 32309Skin lesion, rainbow trout, Brittany,
France, 1996
FRGDSA 1502/97 AY468463Kidney, sturgeon (Acipenser sturio),
Landes, France, 1997
FRGDSA 4034/97AY468464 Skin ulcer, rainbow trout, Landes,
France, 1997
FRGDSA 4580/97 AY468465Siberian sturgeon (Acipenser baeri) fry,
Landes, France, 1997
LDA39 G1966 4a13 AY468466Skin lesion, Asian catfish (Pangasius
bocourti), Kompong Chhnang,
Cambodia, 1997
LDA39 G1966 12a13AY468467 Fin lesion, Asian catfish (Pangasianodon
hypophthalmus), Kompong Chhnang,
Cambodia, 1997
LDA39 G1966 12c 13AY468468 Spleen, Asian catfish (Pangasianodon
hypoph-thalmus), Kompong Chhnang,
Cambodia, 1997
LDA39 G1966 13a 13AY468469Skin lesion, walking catfish (Clarias sp.),
Kompong Chhnang, Cambodia, 1997
FRGDSA 11/99
LMG 22909
CIP 108647
1 Skin ulcer, Koi carp, Landes, France,
1999
LDVH 33/998 AY468470 Goldfish (Carassus auratus), He ´ rault,
France, 1999 (P. R. China)
LDVH 1AY468472 Internal organs, goldfish, He ´ rault,
France, 2000 (P. R. China)
LDVH 2
LMG 22910
CIP 108648
1Internal organs, Koi carp, He ´ rault,
France, 2000 (Japan)
LDVH 38AY468484 Skin ulcer, goldfish, He ´ rault, France,
2000 (Italy)
LDVH 48AY468473 Internal organs, goldfish, He ´ rault,
France, 2000
LDVH 5
LMG 22911
CIP 108649
1Internal organs, goldfish, He ´ rault,
France, 2000 (Italy)
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