FEMS Microbiology Letters 121 (1994) 181-188
© 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00
Published by Elsevier
181
FEMSLE 06115
Role of iron in the pathogenicity of Vibrio damsela
for fish and mammals
B. Fouz
a,b,*, A.E.
Toranzo
a
E.G. Biosca b, R. Mazoy c and C. A m a r o b
a Departamento de Microbiologfa y Parasitologfa, Facultad de Biologla, Universidad de Santiago de Compostela, Santiago de
Compostela, 15706, Spain
b Departamento de Microbiologfa, Facultad de Biologfa, Universidad de Valencia. Burjassot, 46100, Spain, and
c Departamento de Microbiolog{a y Parasitologfa, Facultad de Ciencias de Lugo, Universidad de Santiago de Compostela, Lugo, 27002,
Spain
Abstract: The ability to obtain iron of 14 isolates of l/ibrio damsela with different degrees of virulence for mice and turbot
(Scophthalrnus maximus) has been evaluated in artificial and natural iron-restricted environments. All strains were capable of
utilizing haemoglobin (Hb) and ferric ammonium citrate (FAC) as the sole iron sources in vitro. However, only virulent V. damsela
strains were able to resist the bacteriostatic and bactericidal effects of human and turbot sera, their growth being enhanced by the
addition of Hb and FAC. The inhibitory effect of these sera on the growth of the non-pathogenic strain (ATCC 35083), however,
was reversed by heat treatment (56°C for 60 rain). The role of iron-availability on the virulence was investigated in iron-overloaded
animals. The iron-treatment before the infection resulted in a significant reduction in the LDs0 of virulent strains. This fact
demonstrates a positive correlation between iron availability in host fluids and degree of virulence in the species Vibrio damsela.
Key words: Vibrio damsela; Iron acquisition; Serum resistance; Virulence
Introduction
The halophilic Gram-negative bacterium V/b-
rio damsela has been commonly associated with
wound infections in humans. This species can
also cause disease in warm and cold water fishes,
such as damselfish (Chromis punctipinnis), brown
shark ( Carcharhinus plurnbeus ), yellowtail ( Seriola
quinqueridata ), seabream ( Sparus aurata ) and turbot (Scophthalmus maximus) [1,2]. Some virulence factors of V. damsela, such as haemolysin
* Corresponding author. Tel.: (081) 563 100 (ext. 3255); Fax:
(081) 596 904.
SSDI 0 3 7 8 - 1 0 9 7 ( 9 4 ) 0 0 2 6 5 - S
and cytolysin production, have been already characterized [3,4]. However, nothing is known about
its ability to override the iron limitation imposed
by hosts, which represents one of the first lines of
defence against bacterial infection since virtually
all bacteria have an absolute requirement for this
nutrient. In vertebrates, most of this metal is not
available to invading microorganisms: or located
intracellularly (as ferritin or haem compounds) or
strongly bound to transferrins and lactoferrins in
body fluids. Therefore, the ability of bacterial
pathogens to overcome the iron-withholding capacity of the host has been shown to be a critical
factor in establishing many infections [5]. Bacteria
have developed different strategies to acquire this
nutrient from hosts, the siderophore-mediated
182
iron uptake systems [5] being the best studied.
However, other alternative mechanisms of iron
acquisition independent of the synthesis of iron
chelators, such as utilization of haem compounds,
are also very important for certain bacterial
pathogens [6]. It has been shown that some Vibrio
species as V. cholerae [7] and V. c,ulnificus [8,9]
can use a variety of haem compounds as a source
of iron and other species as V. anguillarum can
obtain the metal from both serum transferrins
and haem compounds present in body tissues
[10]. In a previous study we have established the
degree of virulence for mice and turbot of several
V. damsela isolates from clinical and environmental sources [2,4]. These isolates, mainly those from
epizootic outbreaks, were able to develop a septicalaemic infection. Because of the lack of information about the role of iron in the virulence of
V. damsela strains, we have evaluated the effect
of iron availability and serum resistance in the
pathogenicity of this species.
Materials and Methods
Bacterial strains and growth conditions
A total of 14 strains of V. damsela were used
in this study: ten were isolated by us from different epizootic outbreaks of vibriosis which occurred in spanish turbot and seabream farms [1,2]
and four, coming from culture collections, were
used as reference strains. Their virulence categories [4], as well as their host and geographic
origins, are listed in Table 1. All strain~ were
routinely cultured in tryptic soy agar (Difco)
(TSA) supplemented with 1% (w/v) NaC1 (TSA1) for 24 h at 25°C. They were maintained in
tubes of soft half-strength marine agar (/VIA)
(Difco) and stored at -80°C in Tryptic Soy broth
with 1% NaC1 added (TSB-1) containing 15%
(v/v) glycerol.
Iron-restricted growth conditions were achieved using M9 broth or M9 agar supplemented with the non-assimilable iron chelator
ethylenediamine di-(O hydroxyphenyl-acetic acid)
( E D D H A , Sigma, St. Louis, MO) (M9-E) [10].
Minimal inhibitory concentrations (MIC) of EDD H A for each strain were determined as previ-
ously described [11]. All assays were performed in
triplicate.
Stock solutions of bovine haemoglobin (Hb)
(Sigma) (4 raM) and ferric ammonium citrate
(FAC) (10 raM) were prepared in distilled water
[10]. Both solutions were sterilized by filtration
(0.45/zM pore size, Millipore membranes).
Utilization of different iron-containing molecules
The utilization of Hb and FAC as well as the
estimation of the minimal concentration of each
iron source that stimulates bacterial growth
(MSC) were undertaken in M9-E and M9-E agar
media, both containing E D D H A at the MIC [8].
The procedure on solid medium was as as previously described [8]. Briefly, a volume of 20 /zl of
sterile solution of Hb or FAC (at concentrations
of 0, 0.05, 0.1, 0.5, 1, 5 and 10/~M) was pippeted
onto 6-ram sterile filter paper disks (Oxoid Ltd.)
which, after drying, were placed onto M9-E plates
containing bacterial test inoculum (5 × 106 colony
forming units (cfu) per ml). Plates were then
incubated for 48 h at 25°C and examined for
positive growth around the disks. At the same
time, microorganisms (10 6 cfu m l - 1 ) w e r e inoculated into M9-E supplemented with the same
amounts of Hb or FAC and bacterial growth was
determined by plate counting on TSA-I (cfu ml - 1)
after 24-48 h of incubation. The effect of iron
addition on growth rate and cell yield was evaluated in M9-E supplemented with Hb or FAC (at
final concentration of 0.5 /zM and 10 /zM, respectively) by measurement of the optical density
at 600 nm (OD600) at hourly intervals. Viable
counting on TSA-1 was performed at 3, 6 and 10
h of incubation. The experiments were performed
in triplicate and average values as well as standard deviations (SD) of each dataset were calculated.
Isolation of erythrocytes and sera from turbot and
humans
Fish and humans were bled by venipuncture
and each type of blood from various donors was
pooled. For the isolation of erythrocytes, blood
was washed three times in sterile physiological
saline solution (SS, 0.9% NaC1, pH 7.5), resuspended in this solution at 5% (v/v) final concen-
183
tration and stored at 4°C for a maximal period of
24 h. For obtaining fresh serum, blood was allowed to clot at room temperature for 2 h and
then kept at 4°C overnight. Pooled serum was
stored at - 2 0 ° C until used.
with a chloronaphthol/hydrogen peroxide substrate mixture (HRP reagent, Biorad) at room
temperature for 5-20 rain. Escherichia coli Hb
101 were used as negative control for Hb receptor activity.
Assay for utilization of Hb from erythrocytes as the
sole iron source
The acquisition of iron from haemoglobin was
tested in a plate assay in which the sole source of
nutrients, including iron, for bacterial growth was
turbot or human erythrocytes [8]. To minimize
iron contamination, all saline solutions employed
were previously deferrated by extraction with 3%
8-hydroxyquinoline in chloroform as described by
Schwyn and Neilands [12]. Volumes of 5 /xl of
bacterial suspensions (up to 108 cfu ml -x) in
deferrated M9 salts were spotted directly onto
freshly made minimal blood agar (MBA) plates,
which contained 1% of agarose (Oxoid) plus 1%
of erythrocytes (resuspended in deferrated M9
salts). After incubating the plates for 48 h at
25°C, the spot areas were examined for growth
and halo of haemolysis.
Growth in turbot and human sera
Resistance of V. damsela strains to the inhibitory effect of turbot and human sera was
tested as follows: bacterial cells in sterile SS
(1 x 106 cfu m1-1) were inoculated into normal
and freshly heated serum (56°C for 60 min) (diluted 1:1 with sterile SS) and incubated at 25°C
for 24 h. Sampling was made at hourly intervals
and bacterial growth was measured as described
above. In order to test the effect of iron supplement on growth rate and cell yield of strains,
similar experiments were performed using fresh
serum with iron added as FAC (10 p~M) or Hb
(0.5 tzM). T h e saturation level of transferrins
present in both sera was estimated with a commercial kit from Sigma (kit for measuring the
unsaturated iron-binding capacity) [10].
Haemoglobin-binding assay
In order to detect whether V. damsela possesses haemoglobin-binding activity, a solid-phase
dot-binding assay was performed [13]. Bacterial
cells were grown in M9 with and without 10/zM
E D D HA until mid-exponential phase and washed
with Tris-buffered saline (TBS) (50 mM Tris-HC1,
pH 7.4, 0.9% NaCI). 20 /zl of each bacterial
suspension (2 x 107 cfu m1-1) was filtered on to
cellulose acetate paper (0.45-/xm HA paper, Millipore Corporation, Bedford, MA) using a 96-well
filter manifold (Minifold I, Schleicher and
Schuell). The paper was allowed to air dry before
blocking the non-specific protein binding sites for
1 h with TBS containing 2% gelatin. After removing the blocking solution, paper was washed with
distilled water prior to incubation with biotinylated haemoglobin (at a concentration of 50 nM).
The binding mixture was incubated at 37°C for 3
h, the solution removed and paper washed. After
a further incubation at 37°C for 1 h with the
peroxidase conjugate (Boehringer), followed by
washing several times, the blot was developed
Virulence assays with iron-ouerloaded mice and fish
To evaluate the effect of iron as adjuvant for
pathogenicity of V. damsela for turbot and mice,
these animals were injected intraperitoneally (i.p.)
with different iron sources 2 h before bacterial
infection. The amounts (/xg) of iron administered
to animals (per gram of body weight) were: (i) 9
and 0.9/xg as FAC to mouse and turbot, respectively [14]; and (ii) 0.8 /xg as Hb [9]. Infectivity
trails with selected virulent strains were performed with B A L B / c mice (4-5-week-old) and
turbot (8-10 g) in groups of six animals. 2 h after
the iron treatment, mice were inoculated with 0.2
ml and turbot with 0.1 ml of ten-fold dilutions of
bacterial suspensions in SS as described [2]. At
the same time, groups of animals were inoculated
with bacteria in the absence of iron supplement.
Injected bacteria (cfu ml-1) were enumerated by
plating 0.1-ml aliquots of appropriate dilutions of
these suspensions onto duplicate TSA-1. Mortalities were recorded daily for 14 days and the
degree of virulence (50% lethal dose LDs0) was
calculated according to the method of Reed and
Muench [15]. Control groups of animals, inocu-
184
lated with SS or with each iron source, were also
maintained during the challenge period.
~
08
Results and Discussion
M
9
- - 4- - M9-E
MgE,.b
--,
+ FAC
~:
• ,-M9-E
"~
0.6
The capability of a great number of pathogenic
bacteria to cause disease has been related to their
ability to remove iron from host fluids. The influence of iron on the growth of Vibrio damsela was
determined in an artificially iron-depleted medium (M9-E). In this medium, the inhibitory conditions are imposed by the iron chelator E D D H A
at the minimal inhibitory concentration (MIC).
These MIC values ranged between 20 and 40
~ M , depending on the strain. No growth was
obtained in M9-E but all isolates could multiply
in this medium when Hb or FAC was added (Fig.
1). The minimal concentrations that stimulated
bacterial growth (MSC) were 0.1 and 0.5 ~ M for
Hb and FAC, respectively. No significant differences in the MSC values were observed among
strains, but when assays were performed on solid
medium (M9-E agar) the MSCs were always
higher (data not shown). The growth kinetics of a
representative strain in M9-E medium and M9-E
,,d
<
L)
~
0.4
0.2
0
I
I
I
I
I
2
4
6
Time (h)
8
10
12
Fig. 1. Utilization of Hb and FAC as iron sources by a
representative Fibrio damsela (RG-191) strain in artificial
iron-limiting conditions. M9 medium containing 50 p~M of
E D D H A (M9-E) was supplemented with 0.5 and 10 ~ M of
Hb and FAC, respectively. The data correspond to the average values of three experiments with the respective standard
deviations.
supplemented with H b or F A C , as an example of
the general behavior of the species, is illustrated
in Fig. 1; growth rate and cell yield were practically the same as those in M9 medium. These
Table 1
Origin, degree of virulence and results of haemolysis in MBA of I/ibrio damsela strains
Strains
Origin
Haemolysis in MBA b
Virulence for a
Turbot
Mice
Turbot
human
Turbot, Spain
Turbot, Spain
Turbot, Spain
Turbot, Spain
Turbot, Spain
Turbot, Spain
Human, USA
Marine fish, USA
+
+
+
+
+
+
+
+
++
++
++
++
++
++
++
++
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Damselfish, USA
Seabream, Spain
+ +
+ +
+
Brown shark, USA
-
(+)
Highly virulent strains
RG-91
RG-151, RG-153
RG-191, RG-192, RG-193
RG-214
RM-71
RI-162
CDC-2227-81
CDC-1421-81
+
+
+
+
+
+
Moderately virulent strains
ATCC 33539
LD-07
Non-virulent st/'ain
ATCC 35083
a
+
(+)
Degree of virulence, expressed as range of mean lethal dose 50% (LDs0), for fish ( + + , 103-104; + , 104-105; -, > I08) and for
mice ( + + , 106-107; -, > 108). Data taken from Fouz et al. [1,2].
b Ratio of halo of haemolysis to inoculum diameter in minimal blood agar (MBA) medium containing turbot or human
erythrocytes: + , > 1.25; ( + ) , < 1.25; - , 0 (no haemolysis) [8].
185
results clearly demonstrate that all strains, irrespective of their degree of virulence, are able to
use Hb or F A C as iron sources in artificial conditions.
Haemoglobin is the oxygen-binding protein of
red blood cells of vertebrates and the most important haem-containing protein in the body. A
bacterial strategy for obtaining it from hosts, prior
to be used as source of iron, is the destruction of
erythrocytes, as described in both biotypes of V.
vulnificus [8]. To evaluate this possibility in V.
damsela, we performed the M B A plate assay the
results of which demonstrated that: (i) virulent
strains produce haemolysins in iron-restricted
conditions; and (ii) the haemolytic activity of the
non-virulent strains was very low or negative (Table 1). A positive correlation between haemolysin
production in iron-rich conditions and degree of
virulence had been previously detected in this
species [3,4]. The haemolysins of V. damsela could
function in vivo to produce an increase in free
Hb, thereby providing a source of iron for bacteria to multiply in blood. In fact, using a biotinylated haemoglobin probe in a solid-phase dotbinding assay, we have detected the presence of a
receptor for haemoglobin on the surface of all V.
darnsela strains grown both under iron-limited
and iron-sufficient conditions, but the mechanism
of removing iron from this protein remains to be
investigated.
T h e ability to grow in serum is important for
microorganisms which cause septicaemic infections. In the serum of vertebrates, transferrins
function as bacteriostatic agents by creating an
iron-restricted environment and complement attacks bacterial cells, causing them to lyse. Therefore, bacteria proliferate in blood only if they
overcome both kinds of defences. When we tested
the resistance of V. damsela to turbot and human
sera (two of its potential hosts), we found that all
virulent strains, irrespective of their origin, were
able to grow in both sera. As might be expected,
the h u m a n isolate (CDC-2227-81) grew better in
human serum whereas fish isolates grew optimally in turbot serum (Fig. 2a, c). F r o m the
decrease in absorbance and viable counts observed in the case of the non-virulent strain A T C C
35083 (Fig. 2b), it is deduced that this isolate was
~RG-191
•- " ~ - . R G -
in Turbot S e r u m
191 in H e a t e d Turbot S e r u m
0.8
0.6
0.4
L)
0.2
0
0
I
2
I
4
I
6
I
8
10
6
8
to
Time (h)
0.4
Normal TS
~Heated
TS
- - ~ - - Normal HS
0.3
0.2
0.1
0
-0.1
0
2
4
T i m e (13)
0.5
~RG-191
in normal HS
~RG-191
in heated HS
....
CDC-2227-81 in nomaal HS
- . • . - C D C - 2 2 2 7 - 8 1 in heated HS
" •0.4
Y&
--_~,"
L'
0.3
0.2
0.1
0
i
2
i
4
i
6
i
8
10
Time (h)
Fig. 2. Growth of representative Vibrio damsela strains in
normal and inactivated by heating turbot serum (TS) and
human serum (HS). (a, c) Virulent strains RG-191 and CDC2227-81. (b) Non-pathogenic strain ATCC 35083. Each point
in the curve expresses the average value of three experiments
with the respective standard deviation.
unable to resist the bactericidal action of sera.
Bacterial death started at time zero in h u m a n
serum and after 6 h in turbot serum (Fig. 2b). By
viable counting, decreases in the number of cells
from 10 6 to 10 3 cfu ml -a and from 10 6 to 10 5 cfu
ml - t were detected in h u m a n and turbot sera,
respectively, after 9 h of incubation. Therefore,
the pathogenicity of V. damsela is correlated to
186
the ability to overcome the antibacterial activity
of serum. A similar correlation has also been
found in other fish pathogens such as V. vulnificus [8] and Edwarsiella tarda [14]. Whilst the
bactericidal action of normal sera was completely
abolished by heating, this treatment did not destroy the bacteriostatic effect of transferrins, and
the optical density and viable counts increased
more than two-fold in the case of virulent strains
(Fig. 2a, c). Thus, virulent strains were able to use
iron bound to transferrin to grow in serum by a
mechanism that remains to be elucidated. The
avirulent strain multiplied successfully only in
heated turbot serum (Fig. 2b), suggesting that
complement could play an important role in the
growth inhibitory effect observed in turbot. Other
mechanisms, different from complement activity,
seem to be involved in the antibacterial action of
human serum, in which this strain was unable to
multiply even after the serum had been inactivated by heating. In our experiments, the saturation level of turbot transferrins was higher
(around 50%) than that of human transferrins
(around 17%). The non-virulent strain could have
a less efficient mechanism of iron-uptake than
that of virulent strains. However, when iron restriction in human serum was abolished, by adding
FAC or Hb, the growth response was still related
0.5
z
0.4
~ R G - 1 9 1 in normalHS
RG-191 in HS+ Hb
- - -~- - ATCC35083 in normalHS
/
0.3
0.2
0.1
0
" - t - - - t ; - - - t - . . . . . -i
-0.1
I
I
I
I
I
2
4
6
Time (h)
8
10
12
Fig. 3. Growth curves of representative Vibrio damsela
pathogenic (RG-191) and non-pathogenic (ATCC 35083)
strains in normal human serum (HS) and HS supplemented
with Hb (0.5 /~M). Each point in the curve represents the
average value of three experiments with the respective standard deviation.
to the degree of virulence of the strain: while the
avirulent strain was unable to grow, the cell yield
of virulent strains was increased in iron-enriched
conditions (Fig. 3). The growth kinetics in human
serum supplemented with iron, using Hb as an
iron source, is shown in Fig. 3. Therefore, only
those strains that are resistant to the bactericidal
Table 2
Effects of iron overload on susceptibility of fish and mice to infection with Vibrio damsela strains
Host
Strain
Untreated animals
Treated animals with b
(LDso) a
FAC
Poikilotherms
(turbot)
RG-191
RM-71
ATCC 33539
CDC-2227-81
ATCC 35083
5 x 103
1 X 104
6 x 103
3 x 10 s
> 108
Homoiotherms
(mice)
RG-191
RM-71
ATCC 33539
CDC-2227-81
ATCC 35083
1.3 x 106
2.8 X 10 6
> 5 x 107
2.8 x 106
> 10s
Haemoglobin
4x
2x
2x
5x
5x
101
102
10 a
103
107
4×
2x
5×
1×
8x
102
103
102
104
107
1x
2x
5x
3×
10s
106
106
106
104
5x
3x
2x
1×
> l0 s
102
103
104
102
a Mean lethal dose 50% of iron-untreated animals.
b Animals were injected with ferric ammonium citrate (FAC) or haemoglobin two hours prior to bacterial infection and LDs0
values were determined in these iron-overloaded animals.
187
action of serum responded satisfactorily to exogenous iron by utilizing it to improve their growth,
as found with other fish pathogens [8].
Finally, we investigated the influence of iron
overload on the disease-causing ability of V.
damsela in animal models. The results of the
LDs0 determinations for turbot and mice, as well
as the effect of treatment with Hb and F A C on
these LDs0 values, are summarized in Table 2.
When mice were pretreated with iron compounds, their susceptibility to infection with virulent V. damsela strains was enhanced considerably: LDs0 values were reduced by 3 or 4 log units
after treatment with Hb and by 1 - 2 log units with
FAC. Infectivity trails with turbot demonstrated
that pathogenic strains achieved the lowest LDs0
values when F A C was administered before bacterial infection. In terms of iron-response in vivo,
only the behavior of the non-pathogenic strain
(ATCC 35083) was different, since its 50% lethal
dose for both hosts was hardly reduced, irrespective of the iron treatment. Its inability to multiply
in fresh serum could explain this negative response to exogenous iron. Control groups of animals, inoculated with SS or the respective iron
source, survived and did not show symptoms of
toxicity after 14 days. All these data demonstrate
that injection of iron compounds increases the
lethality of virulent V. damsela strains, probably
by allowing a rapid growth of the bacteria in body
fluids. Similar findings have been reported for
other fish pathogens such as V. vulnificus [8,9]
and E. tarda [14], in which iron treatment (administered as haemin, haemoglobin or FAC) increased host susceptibility to infection by virulent
strains.
The results of this study indicate that resistance to the bactericidal effect of serum is correlated with pathogenicity in V.. damsela species
and that the availability of iron may play an
important additional role on the degree of virulence of this organism, enabling bacteria to survive and multiply successfully in host fluids. However, several factors seem to be necessary for the
development of the infectious disease. In fact, the
degree of virulence of the V. damsela strains has
been also demonstrated to be strongly correlated
with the level of exotoxins secreted [3,4].
Acknowledgements
B.F. and R.M. thank Conselleria de Educaci6n,
Xunta de Galicia for their predoctoral research
grants. B.F. also thanks for a research fellowship
to partially perform this study in the University of
Valencia, Spain. This work was supported by
Grants M A R 91-1206 and M A R 91-1133-CO2-O1
from the Comisi6n Interministerial de Ciencia y
Tecnologia (CICYT), Spain.
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