OPEN 3 ACCESS Freely available online
<P P L O S Io h .
Does Virulence Assessment of Vibrio anguillarum Using
Sea Bass (Dicentrarchus labrad Larvae Correspond with
Genotypic and Phenotypic Characterization?
Ingeborg Frans1'2'4'5, Kristof D ierckens5, Sam C rauwels1'2, A do Van A ssch e1'2, Jorgen Leisner3,
M arianne H. Larsen3, Chris W. M ichiels4, Kris A. W illem s1'2, Bart L ievens1'2, Peter B ossier59,
Hans Rediers1'2*9
1 Laboratory for Process Microbial Ecology and Bioinspirational M anagem ent (PME&BIM), Thomas More Mechelen, Campus De Nayer, D epartm ent of Microbial and
Molecular Systems (M2S), KU Leuven Association, Sint-Katelijne-Waver, Belgium, 2Scientia Terrae Research Institute, Sint-Katelijne-Waver, Belgium, 3 D epartm ent of
Veterinary Disease Biology, University o f Copenhagen, Frederiksberg, Denmark, 4 Centre for Food and Microbial Technology, M2S, KU Leuven, Heverlee, Belgium,
5 Laboratory of Aquaculture and Artemia Reference Center, D epartm ent of Animal Production, Ghent University, Gent, Belgium
Abstract
B ackg rou nd : Vibriosis is one o f the m ost ubiquitous fish diseases caused by bacteria belonging to the genus Vibrio such as
Vibrio (Listonella) anguillarum . Despite a lo t o f research efforts, the virulence factors and mechanism o f V. anguillarum are
still insufficiently known, in part because o f the lack o f standardized virulence assays.
M e th o d o lo g y /P rin c ip a l F ind in gs: We investigated and com pared the virulence o f 15 V. anguillarum strains obtained from
d ifferent hosts o r non-host niches using a standardized g n o to b io tic bioassay w ith European sea bass (Dicentrarchus labrax
L) larvae as m odel hosts. In addition, to assess potential relationships between virulence and genotypic and phenotypic
characteristics, the strains were characterized by random am plified polym orphic DNA (RAPD) and repetitive extragenic
palindrom ic PCR (rep-PCR) analyses, as w ell as by phenotypic analyses using Biolog's Phenotype MicroArray™ technology
and some virulence factor assays.
C onclusions/S ignificance: Virulence testing revealed ten virulent and five avirulent strains. W hile some relation could be
established between serotype, genotype and phenotype, no relation was found between virulence and ge no typ ic or
phenotypic characteristics, illustrating the com plexity o f V. anguillarum virulence. Moreover, the standardized g n o to b io tic
system used in this study has proven its strength as a model to assess and com pare the virulence o f different V. anguillarum
strains in vivo. In this way, the bioassay contributes to the study o f mechanisms underlying virulence in V. anguillarum .
C itation : Frans I, Dierckens K, Crauwels S, Van Assche A, Leisner J, e t al. (2013) Does Virulence A ssessment of Vibrio anguillarum Using Sea Bass (Dicentrarchus
labrax) Larvae Correspond with Genotypic and Phenotypic Characterization? PLoS ONE 8(8): e70477. doi:10.1371/journal.pone.0070477
Editor: D ongsheng Zhou, Beijing Institute o f Microbiology and Epidemiology, China
Received April 14, 2013; A ccepted June 10, 2013; Published August 6, 2013
C opyrigh t: © 2013 Frans e t al. This is an open-access article distributed under th e term s of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided th e original author and source are credited.
Funding: This study was supported by European FP7 project 'Promicrobe - Microbes as positive actors for m ore sustainable aquaculture' (Project Reference
227197) and by Ghent University project "Host microbial interactions in aquatic production" (BOF12/GOA/022). This work was financially supported by the
"Agency for Innovation by Science and Technology in Flanders (IWT)(www.iwt.be)". The funders had no role in study design, data collection and analysis, decision
to publish, or preparation o f th e manuscript.
C om peting Interests: The authors have declared th a t no com peting interests exist.
* E-mail: hans.rediers@biw.kuleuven.be
9 These authors contributed equally to this work.
V ibriosis, caused by Vibrio (Listonella) anguillarum, has b een
re p o rte d as one o f the m ost im p o rta n t infectious diseases affecting
m an y econom ically im p o rta n t fish, bivalves a n d crustaceans [3-5].
Sym ptom s are re d spots o n the ventral a n d lateral areas o f the fish
a n d swollen a n d d ark skin lesions th a t can ulcerate a n d bleed.
F u rth e rm o re , the eyes are often infected, initially resulting in
opacity, a n d later on in u lceration a n d exophthalm ia. Internally,
the intestines m ay be distended a n d filled w ith a clear, viscous
liquid. O u tb reak s o f this disease often result in high m ortality rates
o f infected fish. M oreover, in acute epizootics, infection spreads so
rapidly th a t the m ajority o f infected fish die w ithout show ing any
clinical signs [5-7]. B ecause o f this high m orbidity a n d m ortality
rate, the disease is responsible for severe econom ic losses in b o th
larviculture a n d a q u acu ltu re w orldw ide. D isease outbreaks can be
Introduction
O v e r the last five decades, the w orld aq u acu ltu re industry has
grow n considerably a n d according to the F ood a n d A griculture
O rg a n iza tio n (FAO), a q u acu ltu re represents the fastest-grow ing
anim al-food-producing sector [1], T his developm ent has been
acco m p an ied b y a transition to m ore intensive farm ing m ethods
supporting a n increased profitability. A side-effect o f these
intensified p ro d u c tio n systems is a n increased th rea t by diseases
caused b y a variety o f m icroorganism s, including bacteria, fungi,
viruses a n d protozoa. D espite all research activity th a t has been
carried out in o rd e r to develop ra p id diagnostic tests a n d effective
disease p rev en tio n strategies [2], large-scale disease outbreaks are
still causing considerable econom ical losses [1].
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Virulence Assessment o f Vibrio anguillarum
influenced b y w ater quality a n d tem p eratu re, the a m o u n t o f stress
im posed u p o n the fish, a n d the strain a n d virulence o f the b acteria.
O u t o f 23 identified O -serotypes ( 0 1 - 0 2 3 ) , only serotypes O l
a n d 0 2 , a n d to a lesser e x ten t serotype 0 3 , have b e en linked to
vibriosis in fish [8]. T h e o th er V. anguillarum serotypes represent
e nvironm ental isolates from sedim ent, p lankton o r seaw ater th a t
are m ainly n o n-pathogenic. O v e r the last couple o f years,
know ledge o f the virulence o f this b a cteriu m has b e en largely
increased by the use o f m olecular a n d biochem ical analysis
approaches [9,10]. In general, virulence factors in V. anguillarum
have b e en classified into those th a t are necessary for chem otaxis
a n d m otility, for adhesion a n d invasion, including proteases
[11,12], hem olysins [13-16], lipopolysaccharides (LPS) [17-19],
a n d those th a t are re q u ire d for b acterial proliferation a n d
persistence. R e g ard in g the latter, it is for exam ple know n th a t
sid erophore-m ediated iron-sequestering systems, enabling grow th
in iron-lim iting conditions, con trib u te significantly to the virulence
o f this path o g en . T w o different sid erophore-m ediated systems
have b e en described in V. anguillarum strains. In m ost pathogenic
strains o f serotype O l , the system is m ediated b y the 65 kb
virulence plasm id p J M l, h a rb o u rin g genes for the biosynthesis o f
the siderophore an guibactin a n d its cognate tran sp o rt system [20].
In contrast, serotype 0 2 strains a n d som e plasm idless serotype O l
strains p ro d u c e a chrom osom ally encoded siderophore vanchrob actin [21-24], N evertheless, despite these studies, the exact role
a n d con trib u tio n o f m an y o f these virulence factors in full V.
anguillarum virulence rem ain still largely unknow n, e.g. b y the lack
o f epidem iological studies a n d standardized virulence assays.
R ecently, a gnotobiotic sea bass (.Dicentrarchus labrax) larvae
m odel system has b e en developed a n d optim ized for studying hostp ath o g e n interactions a n d virulence assessm ent o f opportunistic
pathogens such as Aeromonas hydrophila a n d V. anguillarum [25]. T h e
E u ro p ea n sea bass was chosen as host because o f its high econom ic
im p o rtan ce for larviculture a n d aq u acu ltu re [26]. Briefly, in this
system, gnotobiotic larvae are challenged via im m ersion in
inoculated w ater or en capsulated Artemia sp., follow ed b y an
assessm ent o f the survival o f the sea bass larvae. In this p a p er, we
investigated a n d c o m p a red the virulence o f 15 V. anguillarum strains
o b tain e d from different hosts or n o n-host niches using this
standardized gnotobiotic bioassay. H ere, the assay has b e en used
for the first tim e to evaluate the virulence o f a com prehensive set o f
different V. anguillarum strains. In addition, to assess p otential
relationships betw een virulence a n d genotypic a n d phenotypic
characteristics, the strains w ere c h aracterized by two com plem en­
tary P C R -b ased genotyping m ethods, including repetitive extragenic p a lindrom ic P C R (rep-PC R ) a n d ra n d o m am plified
polym orphic D N A (RAPD) P C R , a n d phenotypic analysis using
Biolog’s P henotype M ic ro A rra y ™ technology [27].
a n d U K . In previous studies, m ost isolates have b e en assessed for
the presence o r absence o f the p J M l virulence plasm id (Table 1)
[8,28-33]. N evertheless, as bacterial strains m ay lose plasm ids over
tim e, presence o f the virulence plasm id has b e en confirm ed in this
study by isolation a n d sequencing. In addition, bacterial identities
w ere confirm ed as V. anguillarum by 16S rR N A gene sequencing,
follow ed by B L A ST analysis against G enB ank (see phylogenetic
analysis). S trains w ere stored in trypticase soy b ro th (TSB; O xoid,
E rem bodegem , Belgium) c o ntaining 1% N a C l a n d 15% (v/v)
glycerol a t - 8 0 ° C .
V irulenc e T e stin g
T h e 15 selected isolates w ere m ad e rifam picin (Rif) resistant by
inoculating the strains a t a final density o f 10 C F U m L _ in
m arin e b ro th w ith ad d itio n o f a n increasing c o n ce n tra tio n (1—
100 m g L ) o f Rif. A fter 24 hrs culturing o n a shaker a t 28°C ,
1 % o f the old culture is used as inoculum for the next culture untill
a resistance for 100 m g L 1 R if b y n a tu ra l m u tatio n is reached.
N ext, the 15 isolates w ere subjected to the virulence assay
previously developed by D ierckens e t al. [25], consisting o f a
standardized gnotobiotic m odel system w ith axenic E u ro p e a n sea
bass [D. labrax L.) larvae as hosts. T o this end, D. labrax eggs w ere
o b tain e d from n a tu ra l spaw ning a t the h a tc h ery o f Ecloserie
M a rin e de G ravelines (France). Follow ing egg disinfection [25],
germ -free eggs w ere allow ed to h a tc h for 60 h. Subsequently, 12
freshly h a tc h e d sea bass larvae w ere aseptically transferred one by
one into a tra n sp a re n t sterile screw cap vial w ith 10 m L filtered
(0.2 ftm), autoclaved sea w ater (FASW) co n tain in g 10 m g L
R if
a n d sterile fish hom ogenate equal to th ree d e a d sea bass larvae,
p roviding som e n utrients to the b a cteria in a gnotobiotic
environm ent. Vials w ere ro ta te d a t 4 rp m w ith a n axis tangential
to the axis o f the vials, providing a era tio n a n d avoiding
sedim entation aw aiting b acterial inoculation. Five days after
h a tc h in g (D A H 5), sea bass larvae w ere co u n ted for the first tim e.
In addition, b acterial strains w ere grow n in 10% m arin e b ro th
c o ntaining N aC l (resulting in the sam e salinity as the w ater in the
sea bass larvae experim ent, i.e. 36 g L ), a n d in cu b ated on a
h o rizontal shaker a t 120 rp m a t 1 6 ± 0 ,5 °C for two days. O n D A H
7, the gnotobiotic sea bass larvae w ere challenged w ith a
suspension o f approxim ately IO5 cfu m L 1 V. anguillarum (as
d e term in ed b y a sp ectrophotom eter a t 550 nm ). O n D A H 9, 11,
a n d 13 (i.e. 2 -6 days post-exposure), survival o f the sea bass larvae
was m o n ito re d b y m icroscopic analysis, i.e. by counting the
n u m b e r o f living sea bass larvae in relation to the situation a t D A H
7. F or each strain, 10 replicates w ere evaluated. N on-challenged
larvae, kept u n d e r sim ilar conditions, w ere used as a control. In
o rd e r to verify the gnotobiotic status o f the assay, axenity was
tested o n D A H 3 b y platin g fish larvae hom ogenates a n d w ater
sam ples as described by D ierckens et al. [25]. N o b a cteria could be
d etected after 72 h incubation. In addition, after the larvae
survival experim ent, hom ogenized fish larvae w ere p lated o n 10%
m arin e a g ar (MA; Difco L aboratories, D etroit, USA) to check for
m icrobial co n ta m in a tio n as well as to verify the identity o f the
inoculated strains by D N A analysis (RAPD fingerprinting; see
further). T h ro u g h o u t the entire experim ent, eggs a n d (challenged)
larvae w ere kept a t a salinity o f 36 g L
in a tem p eratu recontrolled ro o m a t 1 6 ± 0 .5 °C in co n stan t dim light (10 candela
steradian m - 2 ). Statistical analysis o f the larval survival d a ta was
p erfo rm ed by m eans o f R v2.12.1. Survival was re p o rte d as m ean
values ± sta n d ard e rro r o f the m ean (SEM). D a ta w ere tested for
norm ality a n d subjected to n o n -p a ra m etric tests. K ruskal-W allis
one-w ay analysis was used to co m p are the survival o f the sea bass
larvae. B onferroni test was used for m ultiple com parisons am o n g
M aterials and M ethods
Ethics S t a t e m e n t
All necessary perm its w ere o b tain e d for the described virulence
studies. T h e ex p erim en t was ap p ro v ed by the ethical com m ittee o f
G h e n t U niversity (no. E C 2 0 0 5 /9 5 ).
Bacterial S trains
Fifteen V. anguillarum strains, representing the m ajo r pathogenic
serotypes O l , 0 2 a n d 0 3 , w ere used in this study (Table 1).
F o u rteen strains have b e en isolated from different species o f fish,
including sea bass, rainbow tro u t (Oncorhynchus mykiss) a n d cod
[Gadus morhua L.), w hile one isolate was recovered from sedim ent.
F u rth e rm o re , the isolates w ere originating from six geographical
regions, including D en m ark , N orw ay, F inland, G reece, F rance
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Virulence Assessment o f Vibrio anguillarum
T able 1. Vibrio anguillarum
s t r a i n s u s e d in th i s s t u d y .
Strain
Host or e n v iro n m e n t
Origin
Sero typ e
V irulen ce
p las m id 3
Reference
VIB15
Sea bass
Greece
01
+
28
VIB93
Rainbow tro u t
Denmark
01
+
28
87-9-116
Rainbow tro u t
Finland
01
-
30
87-9-117
Rainbow tro u t
Finland
01
+
30
VaNT1
Rainbow tro u t
Denmark
01
+
8
S3 4/9
Mucus, Rainbow trout
Denmark
01
-
29
JLL237
Rainbow tro u t
Hjarno, Denmark
01
-
33
43
Sea bass
UK
01
-
31
VIB12
Sea bass
Greece
02
-
28
VIB103
Cod
Denmark
02
_b
28
VIB160
Sedim ent
Denmark
02
-
28
JLL143
Rainbow tro u t
Denmark
02
-
33
HI610
Cod
Norway
02
-
Parisvannet, Norway
VIB113
Rainbow tro u t
Denmark
03
-
28
CNEVANB11008
Sea bass
France
03
-
32
a+ = Present; —= Absent (also confirmed in this study).
bPresence of plasmid > 2 0 0 kb.
doi:10.1371 /journal.pone.0070477.t001
m eans in case o f non-hom ogeneity. Significance was accepted at
p < 0 .0 5 .
alignm ent p erfo rm ed using C lustalW a M ax im u m Likelihood tree
was constructed using the M E G A S software. T h e sequences
o b tain e d in this study w ere deposited in G enB ank u n d e r the
accession num bers K F 1 5 0 7 7 4 to K F 150818.
P h y l o g e n e t i c Analysis
G enom ic D N A was extracted follow ing overnight incubation on
m arin e ag ar at 28"C , using the p h e n o l/c h lo ro fo rm extraction
m eth o d as described b y Lievens et al. [34], Subsequently, the 16S
ribosom al R N A (rRNA) gene, as well as the genes encoding an Nacetylm uram oyl-L -alanine am idase involved in the separation o f
d a u g h te r cells after cell division (amiB) [35] a n d a n extracellular
m etalloprotease involved in virulence in V. anguillarum (empA) [11]
w ere partially am plified w ith the prim ers 63F a n d 1492R, am i8
a n d am i417, a n d em pA F a n d em pA R , respectively [36-39]. P C R
am plification was p erfo rm ed in a total volum e o f 20 pi containing
0.3 p M o f each p rim er, 0.3 m M o f each deoxynucleoside
trip h o sp h ate (Invitrogen, M erelbeke, Belgium), 2.0 U Taq D N A
polym erase (Bioké, L eiden, T h e N etherlands), 10X T h erm o P o l
R eactio n Buffer (Bioké), a n d 1 n g genom ic D N A (as m easu red by
a N a n o d ro p spectrophotom eter). Before am plification, D N A
sam ples w ere d e n a tu re d a t 94"C for 2 m in. Subsequently, 35
cycles o f the follow ing steps w ere run: 45 s at 94"C , 45 s a t 59"C
(16S rR N A gene, empA) o r 54"C (amiB), a n d 45 s a t 72"C, followed
by a final extension step at 72"C for 10 m in. S equencing o f
purified P C R products was p erfo rm ed using the forw ard p rim e r
used for D N A am plification for the empA a n d amiB am plicons a n d
using the forw ard a n d reverse p rim e r for the 16S rR N A gene
am plicons. F or the latter, forw ard a n d reverse sequences w ere
individually trim m ed for quality based on the o b tain e d electrop h ero g ram , using a P hred-score o f > 2 0 (i.e. 0.01 % e rro r rate) as a
cut-off value. P aired sequences w ere th en aligned using the
C lustalW algorithm w ithin the M E G A S software package [40],
follow ed b y m an u a l sequence editing based o n the p a ire d
electropherogram s, leading to a n accu rate consensus sequence.
Subsequently, a phylogenetic analysis was p erfo rm ed based on the
in silico c o n ca te n a te d nucleotide sequences com prising 16S rR N A ,
amiB a n d empA. T o this end, follow ing m ultiple sequence
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DNA F in g e r p rin t in g
D N A e x tracted from all isolates studied was subjected to two
fingerprinting techniques, including R A P D a n d re p -P C R . W ith
re g ard to the R A P D analysis, first 20 d ecam er oligonucleotides,
ran d o m ly chosen from the O p e ro n p rim e r kits (O peron T ec h n o l­
ogies Inc, A lam eda, CA, LTSA), w ere screened o n a subset o f five
b acterial strains from T ab le 1 to select the m ost discrim inative
R A P D prim ers. T w o prim ers, O P V -12 (5 '-A C C C C C C A C T -3 ')
a n d O P N -0 8 (5 '-A C C T C A G C T C -3 '), resulted in a clear a n d
discrim inating fingerprint, a n d these w ere selected for further
experim ents. Likewise, for the re p -P C R analysis, two prim ers a n d
one p rim e r set w ere first tested o n the sam e set o f five isolates,
including the B O X A 1 R p rim e r (S '-C T A C G G C A A G G C G A C G C T G A C G -3 '),
the
(G T G )5
p rim e r
(5'G T G G T G G T G G T G G T G -3 ') a n d the p rim e r p a ir R E P 1 R -I
(5 '-IIIIC G IC G IC G IC A T C IG G C -3 ') a n d R E P 2-I (5 '-IC G IC T T A T C IG G C C T A C -3 ') [41], As the B O X A 1 R a n d (GTG)5
prim ers gen erated only very w eak b ands o r an insufficient n u m b er
o f fragm ents, only R E P 1 R -I a n d R E P 2-I, yielding a clear a n d
discrim inating fingerprint, was m ain tain ed for analysis o f the
w hole collection. All am plifications w ere p erfo rm ed using a BioR a d T 1 0 0 th erm al cycler in a reaction volum e o f 20 p i containing
0.5 p M o f each p rim e r, 0.15 m M o f each deoxynucleoside
trip h o sp h ate (Invitrogen), 2.0 LT Taq D N A polym erase (Bioké),
10X T h erm o P o l R eactio n Buffer (Bioké), a n d 1 n g genom ic D N A
(as m easured by a N a n o d ro p spectrophotom eter). T h e reaction
m ixture was initially d e n atu red at 94"C for 2 m in, follow ed b y 35
cycles o f 1 m in at 94"C , 1 m in a t 35"C (RAPD) o r 40"C (repPC R ), a n d 2 (RAPD) o r 4 m in (rep-PC R ) at 72"C, w ith a final
extension step at 72"C for 10 m in. O b ta in e d P C R products w ere
separated by loading 7 pi o f the reaction volum e on a 1% (w/v)
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Virulence Assessment o f Vibrio anguillarum
Results
agarose gel follow ed b y 120 m in electrophoresis a t 4 V /c m in
1 x T ris /a c e ta te -E D T A (TAE) buffer. Gels w ere stained w ith
ethidium b ro m id e a n d visualized w ith U V light. A 1 kb D N A
lad d er (Sm artladder; E urogentec, Seraing, Belgium) was used as
m olecular w eight m arker. G el im ages w ere a cq u ired w ith the
B ioC hem i System (UVP, U p lan d , CA, USA). O b ta in e d im ages
w ere processed b y using G elC o m p a r software, version 6.6.4
(Applied M aths, Sint-M artens-L atem , Belgium). Follow ing n o r­
m alization a n d b ack g ro u n d subtraction, fingerprint sim ilarities
based on the co m b in ed dataset w ere calculated using the Pearson
correlation coefficient. C luster analysis was p erfo rm ed b y the
unw eighted p a ir g roup m eth o d w ith arithm etic averages (UPGM A) [42]. All reactions w ere p erfo rm ed a t least twice to check
reproducibility, a n d yielded sim ilar results. In all analyses, sterile
distilled w ater was used as a negative control.
V irulenc e T e stin g
In o rd e r to assess the virulence o f 15 V. anguillarum isolates,
gnotobiotic sea bass larvae w ere challenged w ith V. anguillarum,
w h e reu p o n the larval survival was m o n ito red by m icroscopical
analysis a t D A H 9, 11 a n d 13 (Fig. 1; T ab le 2). A fter D A H 13 the
sea bass larvae in the control tre a tm e n t died o f starvation, m aking
us to decide to term in ate the ex p erim en t a t D A H 13 (six days after
inoculation). Survival o n D A H 13 o f the sea bass larvae challenged
w ith the serotype O l strains V a N T l, V IB 93, S3 4 / 9 a n d 87-9-116
(ranging from 9 3 ± 3 % to 7 8 ± 8 % survival) was n o t significantly
different from the control group (8 9 ± 4 % ). T re a tm e n t w ith the 0 2
strain V IB 12 (6 7 ± 5 % ) was significantly different from the axenic
control b u t n o t significantly different from inoculations w ith S3 4 /
9 a n d 87-9-116. As such, based o n these observations these five
strains w ere classified as avirulent strains, a t least in the gnotobiotic
system used here. T h e o th er ten strains caused a significantly
h igher m ortality c o m p a red to the avirulent strains as well as the
axenic control a n d w ere classified as virulent strains. Strains
V IB 15 a n d 87-9-117, two O l serotype strains c o ntaining the
virulence plasm id, show ed high virulence tow ards sea bass larvae
(33 ± 10% a n d 1 3 ± 8 % survival, respectively). R em arkably,
alth o u g h strain 87-9-117 was originally isolated from rainbow
trout, it also caused high m ortality in sea bass larvae, indicating
th a t th ere is no stringent host-specificity for vibriosis. T w o o th er
O l serotype strains (43 a n d JL L 237), alth o u g h lacking the
virulence plasm id, w ere also virulent (2 2 ± 5 % a n d 4 ± 2 % survival,
respectively). S trains V IB 103, JL L 1 4 3 , a n d V IB 160, belonging to
serotype 0 2 also caused high m ortality (2 9 ± 3 % , 2 3 ± 4 % , a n d
6 ± 2 % survival, respectively). A pparently, alth o u g h strain V IB 160
was n o t isolated from fish b u t from sedim ent, it show ed high
virulence tow ards sea bass larvae. In addition, all tested 0 3 strains
(VIB113 a n d C N E V A N B 11008) a p p ea red to be highly virulent
(3 ± 3 % a n d 8 ± 4 % survival, respectively). A n in d ep e n d en t
replication o f this gnotobiotic sea bass challenging ex p erim ent
confirm ed the results show n in Fig. 1. F o r each experim ent, the
identity o f the inoculated strains was confirm ed b y isolating the
b a cteria again a t the e n d o f the ex p erim en t (D A H 13) follow ed by
R A P D fingerprinting, confirm ing th a t m ortality was caused by the
tested V. anguillarum strains a n d n o t b y n o n -a d d ed strains.
P h e n o ty p in g
F o r each strain, carb o n source oxidation was d e term in ed by
P henotype M ic ro A rra y ™ (PM) technology (Biolog, Flayw ard,
C A) using P M p late 1. U sing this technology, kinetic profiles are
gen erated by continuously m on ito rin g the m etabolic activity
d u rin g incubation [27]. T h e inoculum was p re p a re d by grow ing
strains for 24 hours a t 25°C o n trypticase soy ag ar supplem ented
w ith 1% N aC l. Cells w ere suspended in 10 ml inoculation fluid
(IF-0 supplem ented w ith 1 % (w/v) N aC l, Biolog) until a n optical
density (600 nm ) o f 0.38 (± 0 .0 2 ) was reached, using a SPEC T R A m a x P L U S 384 UV -vis sp ectrophotom eter (M olecular D evic­
es). T h e inoculum was diluted (1:5) in inoculation fluid containing
dye m ix D (Biolog). E ac h well was inoculated w ith 100 pi. Plates
w ere in cu b a ted in the O m niLog® a u to m a te d in cu b a to r-re a d er o f
Biolog for eight days a t 25°C a n d w ere re ad every 15 m inutes.
R esulting d a ta w ere analyzed using O m niLog® P M K inetic
Analysis software (version 1.6) according to the m an u fa ctu re r’s
instructions. C o m p ariso n o f the isolates was p erfo rm ed by
n u m erical analysis using the P earson p ro d u c t-m o m e n t correlation
coefficient a n d hierarchical clustering w ith U P G M A . T h e
clustering results w ere validated using cophenetic correlation
[43]. A selection o f reactions was p erfo rm ed a t least twice to check
reproducibility, a n d yielded sim ilar results.
A dditional phenotypic assays associated w ith virulence have
b e en perform ed. All enzym atic a n d hem olytic assays w ere done
a ccording to N a tra h et al. [44], Briefly, overnight cultures o f each
b acterial strain w ere diluted to a n O D 6 0 0 o f 0.5. F o r each assay,
5 p i o f d iluted culture was spotted in the m iddle o f the test plate.
M A plates supplem ented w ith 1 % T w een 80 (Sigm a-A ldrich) or
1 % egg yolk em ulsion (Sigm a-A ldrich) w ere used for the lipase
a n d phospholipase assays, respectively. T h e d evelopm ent o f
opalescent zones a ro u n d the colonies was observed a n d the
d iam e te r o f the zones was m easu red after 2 -4 days o f incubation
a t 28°C. T h e caseinase assay plates w ere p re p a re d by m ixing
double strength M A w ith a 4% skim m ilk p ow der suspension
(Oxoid), sterilized separately a t 121°C for 5 m in. C learin g zones
su rro u n d in g the bacterial colonies w ere m easured after 2 days o f
incubation. G elatinase assay plates w ere p re p a re d by m ixing 0.5%
gelatin (Sigm a-A ldrich) into M A . A fter 7 days o f incubation,
satu rated am m o n iu m sulfate (80%) in distilled w ater was p o u re d
over the plates a n d after 2 m in, the diam eters o f the clearing zones
a ro u n d the colonies w ere m easured. Flem olytic assay plates w ere
p re p a re d b y supplem enting M A w ith 5 % d efibrinated sheep blood
(Oxoid). C learin g zones w ere m easu red after 2 days o f incubation.
All assays w ere done a t least in triplicate.
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DNA Analysis
T o assess a poten tial relation b etw een virulence a n d genotypic
background, all strains w ere subjected to b o th a phylogenetic
analysis a n d D N A fingerprinting. First, a phylogenetic tree was
constructed based on the in silico c o n ca te n a te d sequences o f the
16S rR N A gene (1365 bp), amiB (365 bp) a n d empA (371 bp)
sequences (Fig. 2). H ow ever, due to the high degree o f
conservation o f these genes (98-100% similarity), n o t all strains
could be discrim inated from each other. In particular, five o u t o f
the eight O l strains (87-9-116, 87-9-117, V a N T l, V IB 93 a n d
V IB15) show ed identical 16S rR N A , amiB a n d empA sequences, as
did the two 0 3 strains (VIB113, C N E V A N B 11008) (Fig. 2).
N ext, tw o R A P D analyses (prim ers O P V -12 a n d O P N -08) a n d
one re p -P C R analysis (prim er p a ir R E P 1 R -I a n d R E P2-I) w ere
perform ed. R A P D a n d re p -P C R p attern s w ere o b tain e d yielding 5
to 14 distinct bands. T h e U P G M A d e n d ro g ra m derived from
Pearson correlation based on the co m bined datasets show ed high
congruence w ith the phylogenetic tree (Fig. 3; T ab le 2). H ow ever,
the discrim inative pow er displayed was considerably h igher w ith
the fingerprinting m ethods. O n ly strain V IB 12, belonging to
serotype 0 2 , clustered differently in b o th trees. Based o n a
sim ilarity level o f 50% , 5 distinct clusters could be identified in the
4
August 2013 | Volume 8 | Issue 8 | e70477
Virulence Assessment o f Vibrio anguillarum
120
110
100
90
80
sp
ds
to
70
60
>
50
3
40
1/1
30
20
10
0
0
1
2
3
4
6
5
8
7
10
9
11
12
14
Time (day after hatching)
-* -8 7 -9-116 — S3 4 /9
— Axenic
-1-43
^ r-87-9-117 — VIB160
JLL143
-» -V a N T l
-M /IB 1 2
— VIB103
-*-H I610
-«-VIB15
-*-CNEVA
-^JLL 237
-»-VIB113
-*-VIB93
Figure 1. Survival (in percentage) o f sea bass larvae during th e g no to bio tic e x p erim ent (unfed larvae) before and a fte r challenge (7
days a fte r hatching) w ith the 15 selected Vibrio anguillarum isolates. Error b a rs r e p re s e n t m e a n ± SEM (n = 10).
do i:1 0.1371 /jo u r n a l.p o n e .0 0 7 0 4 7 7 .g 0 0 1
U P G M A d endrogram . C luster I only con tain ed O l serotype
strains (5 out o f the eight studied), while cluster II was com prised o f
the tw o 0 3 strains (fingerprints sharing 100% similarity), tw o 0 2
serotype strains (VIB12 a n d V IB160) a n d one O l strain (S3 4 /9 )
T a b le 2 . G enotypic and Phenotypic properties o f the Vibrio anguillarum strains used in this study.
Strain
Virulence
assay
(%
Cluster
Cluster
Serotype survival)3 G enotypingb Phenotypingc
d
Enzyme assays
Lipase Phospholipase
Caseinase
Hemolytic
Gelatinase
VIB15
01
33 ± 10
1
A
-
+
+
+
+
VIB93
01
89±5
1
A
+
+
+
+
+
87-9-116
01
78±8
1
A
+
+
+
-
+
87-9-117
01
13 ± 8
1
A
+
+
+
+
+
VaNT1
01
93±3
1
A
+
+
+
+
+
S3 4/9
01
83±7
II
B
+
+
+
+
+
JLL237
01
4±2
IV
B
+
+
+
+
+
43
01
22±5
V
C
+
+
+
-
+
VIB12
02
67±5
II
E
+
+
-
-
+
VI Bí 03
02
29±3
V
B
+
-
+
+
+
VI Bí 60
02
6±2
II
B
+
+
+
+
+
JLL143
02
23±4
III
D
+
+
+
+
+
HI610
02
23±4
V
C
+
-
+
+
+
VIB113
03
3±3
II
D
+
+
+
+
+
CNEVA
NB11008
03
8±4
II
B
+
+
+
-
+
aResults from DAH 13, th e m e an % survival ± SEM Is p re s e n te d (n = 10) (see also Fig. 1).
C lu s te rin g results (co m b in ed d a ta se ts) a t a sim ilarity level o f 50% (see also Fig. 3).
C lu s te rin g results a t a sim ilarity level o f 90% (see also Fig. 4).
dEnzyme activity:-!- = Present; — = A bsent.
dol:10.1371/joum al.pone.0070477.t002
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Virulence Assessment o f Vibrio anguillarum
12 c arb o n sources (L-arabinose, 2'-deoxyadenosine, adenosine, Dglucose-1-phosphate, D -glucose-6-phosphate, D -fructose-6-phosp ate, L-lyxose, pyruvic acid, inosine, fum aric acid, L -asparagine,
a n d D-ribose). T hese findings w ere confirm ed in a n ind ep en d en t
experim ent. O u t o f 95 c arb o n sources tested in this study, 40 w ere
consum ed b y all o th e r 14 V anguillarum strains (L-arabinose, Dxylose, acetic acid, L -aspartic acid, L-lactic acid, L -alanine, 2 'deoxyadenosine, adenosine, D -glucose-6-phosphate, D ,L -m alic
acid, D -fructose-6-phospate, succinic acid, citric acid, L-lyxose,
uridine, pyruvic acid, D -sorbitol, inosine, D -gluconic acid, L-m alic
acid, L-serine, fum aric acid, ß-m ethyl-D -glucoside, L -asparagine,
glycerol, A la-Gly, L -threonine, G ly-Pro, L -glutam ic acid, Dalanine, D -fructose, D -m annose, a-D -glucose, sucrose, D -m annitol, L -glutam ine, L -proline, D -galactose, D -ribose, a n d N -acetylD -glucosam ine), while 20 w ere only m etabolized b y som e strains
a n d 35 b y none o f the strains tested (D -glucuronic acid, Dglucosam inic acid, 1,2-propanediol, a-m ethyl-D -galactoside, Lrham nose, D -th reo n in e, m ucic acid, D -aspartic acid, L-fucose, ahydroxyglutaric acid-g-lactone, D -galactonic acid-y-lactone, Dsaccharic acid, m -tartaric acid, adonitol, glyoxylic acid, glycolic
acid, a-D -lactose, N -acetyl-D -m annosam ine, acetoacetic acid,
lactulose, tricarballylic acid, tyram ine, glucoronam ide, D -m elibiose, m -hydroxyphenyl acetic acid, D -m alic acid, L -galactonic acidy-lactone, m ono-m ethylsuccinate, p-hydroxyphenyl acetic acid, Dpsicose, dulcitol, thym idine, 2-am inoethanol, D -galacturonic acid,
a n d phenylethylam ine) (Table SI).
N ext, the V anguillarum strains w ere clustered based on the
ability to utilize different c arb o n sources. As can be seen from the
U P G M A d e n d ro g ra m constructed from the PM 1 profiles (area
u n d e r the curve), clear differences can be observed betw een the
studied V anguillarum strains (Fig. 4; T ab le 2). Based on 90%
sim ilarity, five clusters can be identified, o f w hich cluster A
perfectly m atc h ed cluster I o b tain ed b y genotyping. T h e two 0 3
strains, C N E V A N B 11008 a n d V IB 113, w hich clustered to g eth er
b y genotyping (cluster II), w ere separated in this analysis (cluster B
a n d D , respectively). Strains H I6 1 0 ( 0 2 serotype) a n d 43 (O l
serotype) clustered to g eth er in cluster G, w hich is in agreem ent
w ith the genotyping results (clusters V). C luster B c ontained two
O l strains, JL L 2 3 7 a n d S3 4 /9 , tw o 0 2 strains, V IB 103 a n d
VIB15
VIB93
99
87-9-117
87-9-116
78
VaNT1
VIB113
CNEVA_NB11008
JII143
93
-VIB12
VIB103
43
HI610
JLL237
S3 1
----- VIB160
I----------- 1
0.001
Figure 2. M axim um Likelihood p hylogram based on a 2101 bp
concatam er o f 16S rRNA, amiB and empA gene sequences o f the
Vibrio anguillarum isolates investigated in this study. The
percentage o f trees from 1000 bootstrap resamples supporting the
topology is indicated when above 50.
doi:10.1371/journal.pone.0070477.g002
as well. C luster V is rep resen ted by tw o 0 2 strains (H I610 a n d
V IB103), supplem ented w ith one O l strain (43). Isolates J L L 2 37
(O l serotype) a n d JL L 1 4 3 ( 0 2 serotype) lan d e d in a separate
cluster, cluster III a n d IV , respectively (Fig. 3). A lthough th ere is
som e correspondence betw een genetic clustering a n d serotype, o u r
results do no t suggest a correlation w ith the results o b tain ed in o u r
virulence assay, as b o th the avirulent a n d virulent strains a p p e a r
scattered th ro u g h o u t the d e ndrogram .
P h e n o t y p i c C h a ra c t e riz a t io n
F o r all V anguillarum strains, high m etabolic activity was
observed, except for the 0 2 strain V IB 12, w hich only consum ed
I 1
III) 1 ! 1
mi l i 1
1
i I
11
lii I
in I
<1 I I u i i
'Hilli
M il 1
I I I il
lí 1 11
i 1 1
i
II
1 Ul
1
1
lili
II H i
1 1
1
1 1
1
11
1
1i 1
II
1
1n
i
Ml 1 1
1 II 1 1
1 1 1
m
ip
m
II
II 1
HI
1 II
II
II
II
ip
ip
i » ti
i m
i in
» p i
m »
Uii
»
f t
1 U1
1# 1
Ipl
Strain
Serotype
87-9-116
01
87-9-117
Ol
VaNTl
01
VD393
01
VEB15
01
CNEVA NB11008
03
VEB113
03
S3 4/9
01
VIB12
02
VEB160
02
JLL143
02
JLL237
01
43
01
HI-610
02
VIB103
02
*
'
-
IV
V
Figure 3. RAPD (tw o gels on the left) and rep-PCR (gel on th e right) fing erprints and corresponding den drog ram (com bined
datasets) d erived from UPG M A linkage o f Pearson correlation coefficients o f th e Vibrio anguillarum isolates investigated in this
study. The percentage o f trees from 100 bootstrap resamples supporting the topology is indicated when above 50.
doi:10.1371/journal.pone.0070477.g003
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Virulence Assessment o f Vibrio anguillarum
V IB 160, a n d one 0 3 strain, C N E V A N B 11008. C luster C
contain ed strains JL L 1 4 3 ( 0 2 serotype) a n d V IB 113 (0 3
serotype). As previously m entioned, the 0 2 strain V IB 12 show ed
very low m etabolic activity a n d therefore clustered separately.
Sim ilar to the genotypic analysis, no correlation could be observed
betw een virulence a n d the phenotypic analysis.
In addition, the p ro d u c tio n o f extracellular enzym es, w hich are
possibly involved in virulence a n d invasion o f host tissue [44], o f
the 15 V anguillarum strains was d e term in ed on a g a r plates
co ntaining various substrates (Table 2). S train V IB 12 lacked b o th
caseinase a n d hem olytic activity, w hich m ay explain its avirulent
b ehavior. R em arkably, although strain V IB 15 was identified as a
virulent strain tow ards sea bass larvae, it was the only strain w hich
did not contain lipase activity. Likewise, tw o virulent 0 2 strains,
V IB 103 a n d H I6 1 0 , w ere deficient in phospholipase activity.
Finally, no hem olytic activity was d etected in the avirulent strain
87-9-116, a n d the virulent strains 43 a n d C N E V A N B11008,
belonging to serotype O l , 0 2 a n d 0 3 , respectively (data not
shown).
general, o u r results w ere in line w ith o th e r virulence c h a ra c te r­
ization studies o f V anguillarum. F o r exam ple, strains V IB15,
V IB 103, 87-9-117, JL L 1 4 3 , JL L 2 3 7 a n d C N E V A N B 11008,
w hich w ere found to be virulent in o th e r studies ([28,32,33], M .
H a lb erg L arsen, unpublished data), w ere also virulent in o u r study.
A dditionally, strains V IB 93, 87-9-116, V a N T l a n d S3 4 / 9 w hich
w ere avirulent in earlier studies ([8,28,30,45], M . H a lb erg L arsen,
unpublished data), w ere avirulent here as well. O n the o th e r hand,
the virulence characteristics o f strains V IB 160 a n d V IB 1 13, w hich
w ere b o th virulent in o u r study, w ere in contrast to previous
studies [28]. T his discrepancy m ay be explained by a different host
specificity com bined w ith the use o f a different infection m odel
system, i.e. in tra p erito n ea l injection o f A tlantic salm on versus
challenge o f sea bass larvae b y im m ersion [46,47]. A dditionally,
despite strain V IB 12 was originally isolated from a diseased sea
bass, this strain was found to be avirulent in o u r screening system.
Potentially, this could be explained by the developm ental stage o f
the fish o r potential co-infections facilitating e n try o f V anguillarum
in the adult fish.
A lthough the virulence plasm id p JM l is generally reg ard ed as
a n im p o rta n t virulence factor in V anguillarum, no strict relation
betw een the presence o f the plasm id a n d virulence was d etected in
o u r study. F o r exam ple, serotype O l strains V a N T l a n d V IB93,
b o th contain in g the virulence plasm id [8,28], w ere found to be
avirulent in o u r study. It is well know n th a t b a cteria can be
deprived o f th e ir plasm ids d u rin g storage o r passage o f the strains
[48], T herefore, presence o r absence o f p J M l was verified for all
o u r strains based on plasm id isolation a n d sequencing, confirm ing
the strain characteristics. T h e avirulent c h a ra c te r o f these strains
on sea bass larvae, b o th originally isolated from rain b o w trout,
D iscussion
In this study, a set o f 15 V anguillarum strains from different
origins (sea bass, tro u t a n d cod, o r sedim ent) w ere subjected to a
previously designed standardized virulence assay using axenic
E u ro p ea n sea bass larvae as hosts [25], H ere, the utility o f the
m eth o d to assess a n d com pare the virulence o f different V
anguillarum strains was d em o n strated on a larg er scale for the first
tim e. O u t o f the 15 tested strains ten isolates w ere categorized as
virulent strains, w hereas five isolates w ere found to be avirulent. In
io
o
LO
I I L
LO
LO
_L_l
o
LO
__I__ L
LO
LO
_ L l
IO
o
oo
.............
io
o
IO
VaNTl
VIB93
-081
87-9-116
99
87-9-117
VIB15
VI B10 3
-0 7 8
VI B16 0
■<5 78
JLL237
S 3 4/9
-Q 76
<582
C NE VA N B 1 1 0 0 8
43
HI610
JLL143
VI B11 3
VIB12
Figure 4. Biolog PM1 phenotypes w ith in Vibrio anguillarum strains. The dendrogram was derived by UPGMA cluster analysis o f the Biolog
PM1 data (i.e. area under the curve) of the 15 selected V. anguillarum strains. The results were validated using cophenetic correlation.
doi:10.1371 /journal, pone.0070477.g004
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Virulence Assessment o f Vibrio anguillarum
In ad d itio n to the O m n ilo g P henotyping, the p ro d u c tio n o f
extracellular enzym es potentially involved in virulence was
d e term in ed for each isolate. Indeed, it was d e m o n stra ted th a t
the avirulent strain V IB 12 lacked b o th caseinase a n d hem olytic
activity. H ow ever, no clear relation b etw een enzym atic activity
a n d virulence could be observed in this study. F o r exam ple,
alth o u g h phospholipase activity has b e en previously described to
be a n im p o rta n t virulence factor for V. vulnificus, two virulent V.
anguillarum strains, V IB 103 a n d H I6 1 0 , lacked phospholipase
activity [58]. In addition, a lthough strain V IB 15 was identified as a
virulent strain tow ards sea bass larvae, it was the only strain
lacking lipase activity. Finally, no hem olytic activity was detected
in avirulent strain 87-9-116, a n d in virulent strains 43, C N E V A
N B 11008, belonging to serotype O l , 0 2 a n d 0 3 , respectively.
A ltogether, the genetic a n d p h enotypic properties o f the 15 V.
anguillarum isolates assessed in this study could n o t b e co rrelated to
the virulence tow ards sea bass larvae. T his illustrates the
com plexity o f the virulence m echanism s in V. anguillarum a n d
suggests th a t virulence in V. anguillarum is highly m ultifactorial a n d
c a n n o t be assigned to one o r a few crucial virulence factors. I t is
clear th a t fu rth er research is necessary to elucidate the underlying
m echanism s o f virulence differences in V. anguillarum. U sing
standardized virulence assays such as the one used in this study,
to gether w ith w hole genom e sequencing [59], sequencing o f w hole
transcriptom es [60] or epigenetics research [61], w e should b e able
to increase o u r u n d erstan d in g o f V. anguillarum virulence. H ow ever,
anticip atin g on the different advantages o f using a gnotobiotic
screening m ethod, it is acknow ledged th a t real-life conditions will
always be far m o re com plex a n d th a t findings m ad e u n d e r
gnotobiotic conditions will need to be validated.
m ay be explained by a stringent host specificity, w hich m ay differ
from strain to strain (see earlier). In addition, the lack o f virulence
o f V a N T l m ay b e explained b y its “ ro u g h ” colony m orphology
[8]. It has b e e n recognized th a t ro ugh strains, w hich lack the
lipopolysaccharide O -antigen, are m o re susceptible to com ple­
m en t-m ed iated killing, resulting in a n avirulent p h en o ty p e [17,49].
In contrast, two o th er O l serotype strains th a t contain the
virulence plasm id (VIB15 a n d 87-9-117) did result in a high
m ortality o f the sea bass larvae, an d , rem arkably, strains lacking
the virulence plasm id w ere found to cause high m ortality in our
study system (43 a n d JL L 2 3 7 ). H ow ever, as plasm idless O l strains
m ay have a chrom osom e-encoded iron sequestering system
instead o f the p J M l plasm id-encoded iron uptake system, its
function m ay have rem a in e d intact, c o n trib u tin g to its virulence
[50,51]. T hese d a ta strongly suggest th a t presence o f the virulence
plasm id is n o t crucial for full virulence tow ards sea bass larvae.
In o rd e r to assess poten tial relationships betw een virulence a n d
genotypic a n d phenotypic characteristics, the strains w ere
subjected to b o th genotypic a n d p h enotypic ch aracterization.
N ext to sequence analysis o f a n u m b e r o f housekeeping genes,
strains w ere genotyped using R A P D a n d re p -P C R , b o th used
successfully for epidem iological studies o f fish p athogens previously
[52-55]. T h e clustering based o n the genetic fingerprints show ed
high sim ilarity w ith the phylogenetic clustering, b u t h a d a m uch
h igher discrim inatory pow er. H ow ever, w hereas som e correspon­
dence betw een genotype a n d serotype could be observed, no
relation could b e found w ith virulence. In addition, sim ilar to
V an d en b e rg h e e t al. [56], a high p h enotypic heterogeneity w ithin
the V. anguillarum isolates was observed. N evertheless, again no
correlation could be m ade w ith virulence. O n the contrary, despite
som e exceptions, in general th ere was a good ag reem en t betw een
the phenotypic a n d genotypic clustering. O n e exception involves
the tw o 0 3 strains V IB 1 13 a n d C N E V A N B 1 1008 w hich grouped
in different clusters based o n the phenotypic ch aracterization,
because o f a few differences in carb o n utilization. S train V IB 113
was for exam ple u nable to m etabolize m altose a n d m altotriose in
co n trast to strain C N E V A N B 11008. F u rth e rm o re , the distinct
ecological niches from w hich these two strains w ere isolated, i.e.
sea bass in F ran ce (C N E V A NB11008) versus rainbow tro u t in
D e n m ark (V IB 113), m ight explain the differences in carb o n source
utilization p atterns. K ey m er et al. [57] also observed a phenotypic
diversity betw een coastal Vibrio cholerae strains isolated from
different environm ents. A n explan atio n for the low m etabolic
activity o f V IB 12 could n o t b e found. T his strain did n o t show any
differences in grow th characteristics in T SB +1 % N a C l c o m p a red
to the o th er V. anguillarum strains using the indirect R a p id
A u to m ated B acterial Im p ed a n ce T ec h n iq u e (RABIT) from D o n
W hidey Scientific (Shipley, U K ) (data n o t shown), dem o n stratin g
sim ilar grow th kinetics am o n g the different isolates.
Supporting Information
T a b le SI R esults o f the O m niLog® P M K inetic analysis o f 15
selected V ibrio anguillarum strains grow n on a PM 1 Phenotype
M ic ro A rray T M plate. F or each strain a n d each carb o n source, the
a rea u n d e r the curve is presented. V alues below 5000 a n d above
8000 are indicated in re d a n d green respectively.
(XLS)
A ck n ow led gm en ts
T he authors would like to thank Brigitte V an M offaert for her excellent
technical support.
Author C ontributions
Conceived and designed the experiments: IF K W BL PB C M FIR.
Perform ed the experim ents: IF. A nalyzed the data: IF SC AVA.
C ontributed reagents/m aterials/analysis tools: J L M L C M PB K D FIR.
W rote the paper: IF BL FFR.
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