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Arch Microbiol (1995) 163 : 366-372
ORIGINAL
9 Springer-Verlag 1995
PAPER
F. Ascencio 9 A. Ljungh - T. Wadstr6m
Cell-surface properties
of the food- and water-borne pathogen Aeromonas hydrophila
when stored in buffered saline solutions
Received: 26 December 1994 / Accepted: 11 January 1995
A b s t r a c t Aeromonas hydrophila, a ubiquitous inhabitant
of aquatic environments, commonly expresses several
cell-surface properties that may contribute to virulence.
Since many aquatic microorganisms in hostile environments can withstand starvation conditions for long periods, we examined the effect of storage under nutrientpoor conditions on the expression of cell-surface properties of this pathogen. Phenotypes studied were: (1) cellsurface hydrophobicity and charge, and (2) the ability to
bind connective-tissue proteins and lactoferrin. Our results suggest that the response of A. hydrophila to nutrient-poor conditions is regimen specific. Generally, A. hydrophiIa cells became more hydrophobic and significantly
increased their ability to bind the iron-binding glycoprotein lactoferrin when the bacterium was stored under nutrient-poor conditions; however, under these conditions,
the cells seemed to lose their ability to bind connectivetissue proteins.
K e y w o r d s Starvation - Cell-surface hydrophobicity 9
Cell-surface charge 9 Connective-tissue proteins -
Aeromonas hydrophila
Introduction
Aeromonas hydrophila and other Aeromonas species are
ubiquitous inhabitants o f aquatic environments and are
commonly isolated from freshwater, sewage, coastal waters, lakes, rivers, drinking water, and in association with
aquatic animals (Krovacek et al. 1992; Krovacek et al.
F. Ascencio (N:~)
Department of Marine Pathology, Center for Biological Research,
PO Box 128, La Paz, Baja California Sur 23000, Mexico
Tel. +52-112-5-3633; Fax +52-112-5-4710
e-mail ascencio@ cibnor.conacyt.mx
A. Ljungh 9T. Wadstr6m
Department of Medical Microbiology,
University of Lund, S-22362 Lund, Sweden
1994; Sugita et al. 1994). In addition, A. hydrophila has
long been known to cause different types of infections in
poikilothermic animals, such as fish, reptiles, and amphibians (Hazen et al. 1978; Sugita et al. 1985; Austin and
Austin 1987; Pasquale et al. 1994). Although freshwater
fish are intensively cultured worldwide, little is known
about the occurrence of A. hydrophila in the intestinal
tract of fish. The study ofA. hydrophila in fish intestine is
important for the efficient control of fish diseases. This
pathogen has also been associated with several categories
of human infections, such as gastroenteritis, peritonitis,
endocarditis, meningitis, septicemia, urinary tract infections, and wound infections (Janda and Duffey 1988;
Janda 1992).
A number of putative virulence factors have been ascribed to A. hydrophila to explain the pathogenic processes of these organisms. Such factors include the production of exotoxins, hemolysis, the ability to bind proteins of the connective tissue and to invade epithelial
cells, and the potential to assimilate host iron complexed
in iron-binding glycoproteins (Ljungh and Wadstr6m
1986; Ascencio et al. 1991, 1992; Neves et al. 1994). Although A. hydrophila has a cosmopolitan occurrence in
aquatic environments, it is frequently subjected to adverse
environmental conditions, such as starvation, high temperature, osmotic stress, or oxidative stress, all of which
can affect their ability to grow and survive. To overcome
these problems, A. hydrophila metabolizes a variety of organic compounds such as amino acids, carbohydrates,
fatty acids, and carboxylic acids, even when the nutrients
are present in drinking water at a concentration of only a
few micrograms per liter (Van der Kooij and Hijnen
1988). Furthermore, recent studies indicate that A. hydrophila is able to survive in natural waters under constant thermic and osmotic stress for more than 1 month
(Hasan et al. 1991) and is an important epizootic agent in
aquatic ecosystems (Fliermans et al. 1977).
Recent research on the metabolic and physiological
changes that accompany entrance into the stationary
phase reveal an array of metabolic and physiological
changes that occur as the result of carbon and energy
367
source starvation in bacteria (Siegel and K o l t e r 1992). In
addition, several bacterial species synthesize stress-response proteins w h e n e x p o s e d to e n v i r o n m e n t a l stimuli
(Matin 1991), t h e r e b y increasing the survival o f the cells
f r o m p r o l o n g e d nutrient deprivation. S o m e o f the bacterial r e s p o n s e s to e n v i r o n m e n t a l stimuli i n c l u d e the p r o d u c tion of: (1) c a r b o n - s t a r v a t i o n proteins (Cst), w h i c h are
m a i n l y i n v o l v e d in the u p t a k e and utilization o f alternative c a r b o n sources to p r e p a r e cells for survival ( A l e x a n der and D a m e r a u 1993); (2) postexponential proteins (Pex)
that l e a d to a general resistance state that r e s e m b l e s the
survival characteristics o f stationary phase cells ( S i e g e l e
and K o l t e r 1992); (3) other proteins that are i n d u c e d b y
o x i d a t i v e stress, o s m o t i c stress, heat s h o c k (i.e., D n a K ,
G r o E L and G r p E chaperonins); and (4) stationary phase
i n d u c i b l e proteins (Sip) ( A l e x a n d e r and St. Johns 1994).
T h e e x p r e s s i o n o f starvation proteins is a sequential
process, i.e., s o m e are s y n t h e s i z e d transiently early during
the starvation response, while others occur for e x t e n d e d
p e r i o d s later in the r e s p o n s e ( G r o a t et al. 1986). Starvation is one o f the e n v i r o n m e n t a l stresses that induce nonculturable bacterial s u b p o p u l a t i o n s that r e m a i n p h y s i o l o g ically active ( O l i v e r et al. 1991), a p h e n o m e n o n that has
serious r e p e r c u s s i o n s for p u b l i c and a n i m a l health since
s o m e o f these d o r m a n t cells also m a i n t a i n their infective
and p a t h o g e n i c potential (Magarifios et al. 1994). T h e s e
virulence factors during the starvation process have not
been d e f i n e d adequately.
Taking these findings into consideration, the aims o f
the p r e s e n t study were: (1) to evaluate w h e t h e r p u t a t i v e
v i r u l e n c e factors, such as surface h y d r o p h o b i c i t y and
charge, and the ability to b i n d c o n n e c t i v e - t i s s u e proteins
and lactoferrin, are e x p r e s s e d during a nutrient starvation
r e g i m e n and (2) to d e t e r m i n e h o w starvation affects the
culturability o f the selected strain o f A. hydrophila.
Materials and methods
Bacterial strains and storage conditions
Aeromonas hydrophila strain A205, isolated from a diseased fish
(skin ulcer infection), expresses cell-surface components that bind
to extracellular matrix proteins when grown in nutrient-poor media
(Ascencio et al. 1990). This strain was grown for 24h at 32~ in
minimal medium broth (Meverech and Werczberg 1985) composed of (g/l): NaH2PO 4 (2.0), NH4NO 3 (1.6), KC1 (0.5), Na2SO 4
(0.5), NaC1 (8.7), and glycerol (1%) (pH7.4). The bacteria were
washed once with phosphate-buffered saline; 0.02M K-phosphate
buffer, pH 7.2, 0.15 M NaC1), and the cells were resuspended (OD540
of 1.0) in the different buffered saline solutions that represented a
spectrum of metabolic stresses: (1) chemically defined broth, a
medium that included carbon, nitrogen, and essential nutrients for
Aeromonas growth (Pazzaglia and Sack 1987) at pH7.4, composed
of the following in (g/l): NaC1 (8.7), anhydrous MgSO 4 (36.9),
KC1 (0.18), MnC12 (0.12), NH4C1 (0.14), Na-succinate (0.5), CaC12
(0.5); and in (mg/l): MnC12 - 4H20 (0.3), ZnSO 4 9 7Ha0 (0.44),
FeSO4 97H20 (2.3), CuSO4 95H20 (0.05); (2) minimal medium broth,
a medium lacking a carbon source (Meverech and Werczberg
1985), at pH7.4, composed of the following in (g/l): NaH2PO4
(2.0), NH4NO 3 (1.6), KC1 (0.5), anhydrous Na2SO4 (0.5), NaC1
(8.7); (3) phosphate-buffered saline, a medium lacking sources
of carbon and nitrogen, at pH 7.4, composed of the following in
(g/l): K2HPO 4 (3.48), KH2PO 4 (2.72), NaC1 (8.7); and (4) distilled
water. The cell suspensions were stored at 22~ for various periods.
125I-Labeled protein-binding assay
Purified collagen (types I and IV), human plasma fibronectin,
laminin, and lactoferrin from human milk were labeled with 0.2
mCi of 125Iusing Iodo-beads (Markwell 1982). Protein-binding assays were performed as described previously (Ascencio et al.
1991). Briefly, 50 pl of 125I-labeled protein (ca. 25,000 cpm) solution in phosphate-buffered saline containing 0.1% bovine serum
albumin was incubated with 100gl of an A. hydrophila cell suspension in a polystyrene centrifuge tube for 1 h at 20~ Incubation
mixtures without bacteria were used as background cpm controls.
After adding 2 ml of ice-cold phosphate-buffered saline containing
0.1% Tween 20, the mixtures were centrifuged (4,500g, 10min,
4C), and the radioactivity of the pellets was measured in a gamma
counter. The amount of 125I-labeled protein bound to the bacteria
was expressed as a percentage of the added radiolabeled protein.
Cell-surface charge and hydrophobicity
Cell-surface charge and hydrophobicity of bacterial cells stored in
the various nutrient-poor media were determined by an aqueous
two-polymer phase-partitioning assay based on techniques described previously (Johansson 1974). An aqueous polymer twophase system containing 7.13% (w/w) polyethylene glycol and
8.75% (w/w) dextran in 0.015M NaC1 (pH6.8) was prepared as a
partition reference control (system I). To determine the cell-surface charge of bacterial cells, negatively charged dextran sulfate at
a concentration of 0.4% (w/w; replacing an equivalent amount of
dextran) was included in the phase system (system II). Hydrophobic affinity partitioning was performed after replacing part of the
polyethylene glycol with monosubstituted polyethylene glycolpalmitate (0.4% w/w) (system III). Differences in the cell-surface
charge and hydrophobicity of A. hydrophila cells maintained in the
various starvation media are expressed as A log G, which is defined
by the equation:
G value of system II or system IlI
G value of system I
% cells in the top phase
where G =
% cells in the lower phase
A log G = log
Concentrations of cells in the top and lower phases were calculated
spectrophotometrically by measuring the OD540 n m of each of the
phase-cell suspensions. Partitioning coefficients depended on the net
charge of the bacterial cell-surface interacting with the sulfate groups
of the phase-forming polymer dextran sulfate, and on the affinity of
the bacterium for the hydrophobic groups of the phase-forming polymer polyethylene glycol-palmitate. Thus, a A log G value of 0 indicates a hydrophobic or negatively charged character (Johansson 1974).
Data analysis
Samples were tested in duplicate, and each set of experiments was
performed at least three times. Background residual radioactivity
from incubation mixtures without bacteria was less than 5%. Each
figure represents the average values of each set of experiments;
standard deviations were less than 10%.
Results
The f o l l o w i n g describes the cell-surface h y d r o p h o b i c i t y
and charge o f A. hydrophila cells stored in various nutrie n t - p o o r media.
368
Cells stored in water
values recorded at time zero (Fig. 1B). There were two
peaks of high cell-surface hydrophobicity, one at day6
and the other at day 15 of starvation. There were also two
low points of cell-surface hydrophobicity at day 12 and
day24 of starvation (Fig. 1B, insert). From day24 to day
60, increasing A log G values of cell-surface hydrophobicity
were observed (Fig. 1B, insert).
Regarding cell-surface charge, A logG values of 0-1
were observed during the first 24h of the starvation regimen (Fig. 1B). From 24h to day24, and from day36 to
day 60, the cell-surface charge followed a pattern similar
to the cell-surface hydrophobicity (Fig. 1B, insert).
The cell-surface hydrophobicity values (A log G) of cells
stored in water reached a peak at 3 h and remained almost
constant within the first 24h of the starvation regimen
(Fig. 1A). The A logG values followed a positive tendency
of increase, reaching a maximum value after day 20 of
starvation (Fig. 1A, insert), followed by a decrease, reaching the original A log G values that occurred at time zero
(Fig. 1A, insert); however, starting from day36, the cellsurface hydrophobicity of the starved cells increased,
reaching high A logG at day60 (Fig. 1A, insert).
Within the first 24h of the starvation regimen, cell-surface charge followed a discontinuous pattern in which
high and low A log G values were registered (Fig. 1A). From
24h to day 60 of starvation, the cell-surface charge followed a pattern similar to the cell-surface hydrophobicity
(Fig. 1A, insert).
Cells maintained in chemically defined broth
The cell-surface hydrophobicity of cells maintained in
chemically defined broth dropped at 3 h of the starvation regimen from a A logG value of 7-0 (Fig. 1C) and re-
Cells maintained in phosphate-buffered saline
Fig. IA-D Partition behavior of Aeromonas hydrophila A205
maintained in various starvation solutions. A Water, B phosphatebuffered saline, C chemically defined broth, D minimal medium
broth. Filled circles cell-surface hydrophobicity, open circles cellsurface charge. Values are the means obtained from three similar
experiments (SD < 10%)
Except for the A log G values registered at 6 h of starvation,
the cell-surface hydrophobicity during the first 24 h of the
starvation regimen of the bacterial ceils maintained in
phosphate-buffered saline remained almost constant at the
10
10
B
A
6
4
10
~8
L~
O
o
--J
,~
10
C
10
D
8:
6i
10
-
-
8
12 24 36 48 60
O
0
3
6
9
12
15
18
21
24
6
T I M E (h)
0
3
0
0
0
(~
9
1"2
12 24 36 48 60
0
0
0
O
1'5
1'8
21
24
369
mained at a value of 1.0 over the next 60days (Fig. 1C, insert).
The starved cells were not negatively charged during
the first 24h of the starvation regimen (Fig. 1C); from 24
h to day 60, the cell-surface charge followed a pattern similar to the cell-surface hydrophobicity, with A log G values
comparable to those registered for cell-surface hydrophobicity (Fig. 1C, insert).
For cell-surface charge, k log G of 0 was registered after
the first 5 days of the starvation experiment (Fig. 1D, insert), and only from day5 to day24 and from day36 to
day 60 did the cell-surface charge follow a pattern similar
to the cell-surface hydrophobicity (Fig. 1D, insert).
Cells maintained in minimal medium broth
Different connective-tissue protein-binding patterns were
observed among A. hydrophila cells stored in the various
starvation media: (1) cells stored in water expressed a
general connective-tissue protein-binding pattern: the
bacterium bound 125I-labeled collagen (type I and IV), fibronectin, and laminin at a percentage ranging from 10 to
20% during the first 24h, and from 10 to 30% from day3
to day 60 of the starvation regime (Fig. 2A); (2) during the
first 24 h of the starvation regime, cells stored in chemically defined broth, minimal medium broth, and phosphate-buffered saline seemed to lose the ability to bind
t25I-labeled collagens (type I and IV) and fibronectin, but
not laminin, since the percentages of protein-binding were
less than 10% (Figs.2B-D). However, after day3 of the
starvation period, the bacterium lost its ability to bind
connective-tissue proteins (Figs. 2B-D), except for the
Binding of 125I-labeled connective-tissue proteins
by A. hydrophila maintained under starvation conditions
The cell-surface hydrophobicity of cells maintained in
minimal medium broth was constant for the first 3 h, then
rapidly increased, reaching a high A log G after 12 h of the
starvation regimen (Fig. 1D). The cell-surface hydrophobicity reached high A logG values at 3, 15, 30, and 60days
of starvation (Fig. 1D, insert)
Fig.2A-D Binding of ~aSl-labeled connective-tissue proteins by
Aeromonas hydrophila A205 maintained in various starvation so-
lutions. A Water, B phosphate-buffered saline, C chemically defined broth, D minimal medium broth. Filled circles collagen type
I, open circles collagen type IV, filled triangles fibronectin, open
squares laminin. Values are the means obtained from three similar
experiments (SD < 10%)
40
Z
.E 3 0 t
A
30
. . . .
3~
B
12 2 4 3 6 4 8 6 0
12 2 4 3 6 4 8 6 0
O
Time (days)
LLI
I-0
1
"E;
cc 2 0
o.
w
10
ILU
~>
I0
w
3ol
40
C
Z
Z
0
0
I
O
Z
E3
z_
m
3o!
O
2ok .
7t
30
20
10
0
3
6
w
1'2
1"5
1'8
~;1
24
0
T I M E (h)
3
6
9
12
15
1"8
21
24
370
r
50
4O
..,I .E 20 ,
v
~
z~ 30
Z
20
m
0
.
.
3
.
6
.
9
Fig.3 Binding of [~SI-lactoferrinbyAeromonas hydrophila A205
maintained in various starvation solutions. Open circles water,
filled circles phosphate-buffered saline, filled triangles chemically
defined broth, open squares minimal medium broth. Values are the
means obtained from three similar experiments (SD < 10%)
cells stored in minimal medium broth, which retained
their ability to bind 125I-labeled collagen type ! (Fig. 2D).
125I-lactoferrin-binding by A. hydrophila
maintained under starvation conditions
The lactoferrin-binding ability of bacterial cells maintained in the various starvation media was more or less
constant during the first 24 h of the starvation regime, except at 12h when a decrease in the percentage of lactoferrin-binding was observed (Fig. 3). Nevertheless, the percentage of lactoferrin bound by cells maintained in water
and in minimal medium broth was lower than that by cells
stored in phosphate-buffered saline and in chemically defined broth (Fig. 3). However, the lactoferrin-binding patterns of bacterial cells stored in the various starvation media were quite different after the day 3 of the starvation
regime (Fig. 3); the percentage of lactoferrin bound by
cells stored in the poorest starvation media (phosphatebuffered saline and water) was higher than that of bacterial cells stored in chemically defined broth and in minimal
medium broth (Fig. 3).
.
12
15
1"8
21
4
Discussion
Starvation is a common physiological state for microorganisms living in ecosystems where nutrients are insufficient for growth. Thus, copiotrophic bacteria exposed to
oligotrophic waters commonly express certain surface
properties to ensure adhesion to solid surfaces where nutrients accumulate (Dawson et al. 1981; Kjelleberg et al.
1983; Kefford et al. 1986). These properties may also enhance adhesion to mucosal surfaces and other host tissues,
allowing the microbes to scavenge nutrients during the
colonization of animal hosts. However, the expression of
putative virulence factors by bacterial cells exposed to
hostile environments may be a strategy that microbes use
to become successful commensal and pathogenic organisms. Thus, virulent determinants may be expressed not
because they are needed in the environment in which they
are induced, but because they allow efficient transition
into a new environment (Mekalanos 1992).
Recently, it has been demonstrated that A. hydrophila
cells, both clinical and environmental isolates, can survive
under nutrient stress conditions for long periods (Hasan et
al. 1991). These bacteria express various starvation-survival patterns similar to those of other pathogens (Amy
and Morita 1983).
Bacterial. cells under starvation conditions express high
cell-surface hydrophobicity, which may facilitate the uptake of surface-localized substrates (Kjelleberg et al.
371
Table 1 Composition of the starvation media
Chemically defined
broth (CDB)
Minimal
medium broth
(MMB)
Chemical
(g/l)
Chemical
(g/l)
Chemical
(g/l)
NaC1
MgSO4
KC1
MnC12
NH4C1
Na-Succinate
CaC12
MnC12
ZnSO 4
FeSo4
CuSO 4
8.70
36.90
0.18
0.12
0.14
0.50
0.50
300 ~tg/l
440 gg/1
2.3 ~tg/1
50 ~tg/1
NaHzPO4
NH4NO3
KC1
Na2SO 4
NaC1
2.0
1.6
0.5
0.5
8.7
K2HPO 4
KH:PO4
NaCI
3.48
2.72
8.70
pH 7.4
pH 7.4
Phosphate
buffer
(KPB)
pH 7.4
1983). S t a r v e d A. hydrophila cells in H 2 0 , p h o s p h a t e buffered saline, and m i n i m a l m e d i u m broth e x p r e s s e d
high h y d r o p h o b i c i t y (Fig. 1). H o w e v e r , this was not the
case for cells starved in c h e m i c a l l y d e f i n e d m e d i u m (Fig.
1C), w h i c h s h o w e d a d e c l i n e in cell-surface h y d r 0 p h o b i c ity as has b e e n also n o t e d in Streptococcus salivarius
g r o w n in continuous culture in a c h e m i c a l l y defined
m e d i u m l i m i t e d for g l u c o s e (Harry and H a n d l e y 1989).
W e f o u n d that the ability o f A. hydrophiIa to b i n d connective-tissue proteins p e r s i s t e d during the first 2 4 h o f
nutrient-stress conditions. H o w e v e r , w h e n the b a c t e r i a
were stored in n u t r i e n t - p o o r m e d i a for r e l a t i v e l y long p e riods (up to 60 days), their ability to b i n d 125I-labeled connective-tissue proteins was d r a s t i c a l l y reduced.
A l t h o u g h there is no e v i d e n t relationship b e t w e e n cellsurface h y d r o p h o b i c i t y , cell-surface charge, and c o n n e c tive-tissue p r o t e i n - b i n d i n g , our findings indicate that A.
hydrophila cells under nutrient-stress conditions b e c o m e
m o r e h y d r o p h o b i c , but lose the ability to b i n d connectivetissue proteins.
A n u m b e r o f reports h a v e d e m o n s t r a t e d that various
bacterial p a t h o g e n s g r o w n under iron-restricted conditions synthesize specific outer m e m b r a n e proteins that
function as receptors for i r o n - c a r r y i n g c o m p l e x e s and for
the transport o f c o m p l e x e s across the cell e n v e l o p e (Crosa
and H o d g e s 1981; Chart et al. 1988; D e n e e r and Porter
1989; M o r t o n and W i l l i a m s 1989). T h e results p r e s e n t e d
here show that A. hydrophila r e s p o n d s to an i r o n - l i m i t e d
e n v i r o n m e n t b y e n h a n c i n g its ability to b i n d lactoferrin.
U n d e r s t a n d i n g the regulation o f v i r u l e n c e properties
m a y help us to define w h a t constitutes a potential virulence factor and, indeed, can facilitate the identification o f
n e w v i r u l e n c e factors on the basis o f o n l y their r e g u l a t o r y
properties ( M e k a l a n o s 1992). S o m e o f the cell-surface
properties studied here m a y be the result o f a p r o c e s s required for survival. F u r t h e r studies are r e q u i r e d to determ i n e w h e t h e r these starvation-elicited c h a n g e s contribute
to the survival and v i r u l e n c e o f the f o o d - and w a t e r - b o r n e
p a t h o g e n A. hydrophila.
Acknowledgements This study was supported by grants from the
Swedish Medical Research Council (16X-04723), the Swedish
Board for Technical Development, and the Swedish Institute. We
thank A. Kreger, J. D. Oliver, and S. Kjelleberg for valuable comments and discussions, and to Roy Bowers for improving the English grammar.
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