Download H ydrop hobicity-hydrop hilicity of staphylococci

J . Med. Microbiol. - Vol. 24 (1987), 65-73
01987 The Pathological Society of Great Britain and Ireland
Hydrop hobicity-hydrop hilicity of staphylococci
F. REIFSTECK, S. WEE* and B. J. WlLKlNSONt
Microbiology Group, Department of Biological Sciences, Illinois State University, Normal, Illinois 6 1 76I , USA
Summary. The hydrophobicity-hydrophilicity of various strains of Staphylococcus has
been studied by a technique involving partitioning of the cells between aqueous and
hydrocarbon phases. S. aureus was typically hydrophobic, and to a greater degree in
stationary- than in exponential-phase cultures. Mutants that lacked teichoic acid,
protein A or coagulase production were hydrophobic, indicating that none of these
factors was responsible for hydrophobicity. The presence of a capsule rendered
strains hydrophilic. Thus, determination of hydrophobicity may be a useful additional
test for capsulation. However, a non-capsulate S. aureus strain was hydrophilic.
Trypsin treatment converted strains from hydrophobic to hydrophilic. Isolated
bacterial cell wall preparation, either crude or purified, and peptidoglycan were
hydrophilic. These results indicate that the determinant of hydrophobicity is a protein
or protein-associated molecule localised at the cell surface of the organism, i.e., a
component of either the cell wall, cell membrane, or both. Twenty-five strains of
twelve coagulase-negative species were examined and most ( 1 8 ) were hydrophobic,
again indicating that protein A is not a major determinant of hydrophobicity in these
staphylococci. Four of seven hydrophilic strains were capsulate; three strains of S.
sciuri were hydrophilic but non-capsulate.
Introduction
The molecular nature of the bacterial cell surface
is critical in the interaction of micro-organism and
host. The hydrophobic or hydrophilic nature of the
bacterial cell surface is an important determinant
in the adherence of bacteria to living and nonliving surfaces (Rosenberg et al., 1980; Rosenberg,
1984). Rosenberg et al. (1980) described a simple
method for the determination of the hydrophobicity
of the bacterial cell surface that has been used
extensively with various species (Rosenberg, 1984).
However, despite considerable research, the nature
of the hydrophobicity determinant is not known
with certainty for any bacterial species. In the
original publication of Rosenberg et al. (1980),
Staphylococcus aureus was reported to be hydrophobic, whereas S. albus (S. epidermidis?) was hydrophilic.
During the course of our investigation, there
have been several reports on staphylococcal hydrophobicity. Hogt et al. (1983a,b, 1985, 1986) have
concentrated on S. epidermidis and S . saprophyticus.
They studied the influence of capsulation and slime
Received 1 Jul. 1986; accepted 1 Sep. 1986.
* Present address : Department of Biochemistry, University of
California, Berkeley, CA 94720, USA.
f Correspondence should be sent to B. J. Wilkinson.
65
production on the hydrophobicity of coagulasenegative staphylococci (Hogt et al., 1983b). Noncapsulate S. epidermidis strains showed different
degrees of hydrophobicity, and slime production
appeared to increase hydrophilicity. Surprisingly,
only one of eight capsulate strains was hydrophilic
(Hogt et al., 1983b), and only one of seven in a later
study (Hogt et al., 1985), which differs from the
findings of Jonsson and Wadstrom (1983) with one
capsulate strain of S. aureus, our findings (see
below), and the influence of capsulation on Acinetobacter calcoaceticus (Rosenberg et al., 1983). Hogt
et al. (1983a) reported that S . epidermidis was more
hydrophobic than S. saprophyticus ; pepsin treatment increased the hydrophilicity of s.epidermidis,
but hydrophilicity was not increased by exposure
to subinhibitory concentrations of penicillin. Using
different methods for assessing hydrophobicity,
Jonsson and Wadstrom (1983) suggested that
protein A was a major determinant of the hydrophobicity of S. aureus, and Malmqvist (1983)
reported that the hydrophobicity of S. aureus was
greatest in early stationary phase. Rozgonyi et al.
(1985) reported improvements in the salt aggregation test for hydrophobicity and its application to
various staphylococcalspecies.
Here we report on the hydrophobicity-hydrophilicity of various coagulase-positive (S. aureus)
and coagulase-negative staphylococcal strains, in-
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F. REIFSTECK, S. WEE AND B. J. WILKINSON
enzyme treatment of cells, and on studies with
isolated cell-wall preparations.
Materials and methods
Bacteria, growth conditions and harvesting
Strains were obtained either from the culture collection
of this laboratory (Gramoli and Wilkinson, 1978;
Wilkinson, 1983), or were supplied by W. E. Kloos,
North Carolina State University, Raleigh, NC, USA.
The bacteria were grown in PYK medium-Phytone
Peptone (Baltimore Biological Laboratory, Cockeysville,
MD, USA) 5 g/L-Yeast Extract (Difco Laboratories,
Detroit, MI, USA) 5 g/L-K2HP043 g/L-glucose 2 g/L,
pH 7.2. Strains were grown in 25 ml of medium in a 50ml Erlenmeyer flask at 37°C with shaking at 200 rpm,
generally for 18 h. The inoculum comprised a loopful of
culture from a Tryptic Soy Agar (Difco) slant, upon
which the strains were maintained. Cultures were
harvested by centrifuging (13 000 g for 5 min at 4°C or
31 000 g for 10 min at 4" for capsulate strains). Cells were
washed twice in cold distilled water.
Coagulaseproduction
Three drops of overnight culture were added to 0.5 ml
of coagulase plasma (Difco); tubes were incubated at
37°C for 4 h and examined for clotting.
Hydrophobicity assay
The procedure of Rosenberget al. (1980) was used with
slight modifications. Bacteria were suspended in 0.097 M
K2HPO44053M KH2P04 buffer, pH 7-1, containing
0.03 M urea and 0.18 mM MgS04.7H20to an A500nmof
0.5. Samples (4.8 ml) of bacterial suspension were placed
in 15 x 150 mm test tubes. Various amounts (0-0-8 ml) of
n-hexadecane,n-octane orpxylene were added to a series
of tubes which were then vortex mixed for 30 s. The tubes
were allowed to stand for 20 min, then the lower aqueous
phase was removed with a Pasteur pipette and the A500nm
of the suspension was measured in a spectrophotometer
(Bausch and Lomb Scientific Optical Products Div.,
Rochester, NY, USA). When the hydrophobicity of cellwall preparations was being assayed they were resuspended in the assay buffer to an A500nmof 0.5.
Trypsin treatment of cells
Washed cells from 25 ml of culture were resuspended
in 10 ml of 0.1 M Na2HP04buffer, pH 8, and, to 5 ml of
this suspension,trypsin (type 1 Worthington Biochemical
Corp., Freehold, NJ, USA) 1 mg/ml was added; this and
the control suspension were incubated at 37°C for 1 h.
The suspensions were then harvested and washed twice
in cold distilled water before determination of
hydrophobicity.
Pepsin treatment of cells
Clumpingfactor
A loopful of bacteria taken from a fresh slant-culture
was examined for visible clumping within a few seconds
of mixing the suspension and coagulase plasma.
Cells were treated with pepsin in a similar manner to
trypsin treatment except that incubations were in 0.1 M
sodium citrate buffer, adjusted topH 4.5 with acetic acid,
containing pepsin (Worthington Biochemical Corp.)
200 pg/ml or 1 mg/ml for 30 min at 37°C.
Serum soft agar
This test was performed as described by Finkelstein
and Sulkin (1958) in 3 ml of Brain Heart Infusion Broth
(Difco)-serum soft agar made with agar 0.15% w/v and
normal rabbit serum 1% v/v in 12 x 75 mm test tubes. A
loopful of culture from a slant or 1-2 drops of overnight
culture in PYK medium was used to make a suspension
in 5 ml of sterile saline 0.9%w/v. An inoculating needle
dipped into the bacterial suspension was used to inoculate
the serum soft agar tubes that were then incubated for
24 h at 37°C.
Indian ink preparations
To examine directly for capsulation, Indian ink
preparations were made as described by Cruickshank et
Cell-wall preparations
Crude cell walls (CCW), sodium dodecyl sulphatetreated CCW (SDS-CCW), purified cell walls (PCW) and
peptidoglycan (PG) were prepared as described previously (Peterson et al., 1978; Wilkinson et al., 1978).
Briefly, organisms were broken by shaking with glass
beads and CCW were harvested by differential centrifugation and were washed six times with cold water. SDSCCW were obtained by stirring CCW with sodium
dodecyl sulphate 2% w/v overnight at room temperature,
followed by washing with water and buffer to remove
sodium dodecyl sulphate. PCW were prepared from SDSCCW by treatments with RNAase, DNAase, trypsin
and phenol 40% w/v (Peterson et al., 1978). PG was
prepared by heating PCW at 60°C with trichloroacetic
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HYDROPHOBICITY-HYDROPHILICITY OF STAPHYLOCOCCI
acid 10% w/v for 90 min (Peterson et al., 1978).
Preparations were resuspended with the aid of brief
sonication when necessary. Lyophilised preparations of
S . aureus strain H, and freshly prepared, non-lyophilised
preparations, stored frozen overnight, from S . aweus
strain Cowan I, were examined.
Strains with values of 89% or less were subdivided
into three groups : 80-89%, weakly hydrophobic ;
20-79%, hydrophobic ; < 20% strongly hydrophobic.
Teichoic acid-altered strains. Strains H, HSmR,
52A5 and T were all found to be similarly
hydrophobic (table I). Strain H is the parent strain
of strain HSmR, which is the parent of teichoic
Results
acid-deficient strain 52A5, and of strain T which
produces a wall-associated teichuronic acid as well
Studies with S . aureus strains
as teichoic acid, which it masks (Park et al., 1974).
We had available various S. aureus strains Strain T, which may be regarded as micro-capsulate
including “typical” ones, mutants with teichoic (Park et al., 1974; Wilkinson, 1983), was also
acid alterations (Park et al., 1974), protein A- hydrophobic, in contrast to fully capsulate strains
deficient strains (Kronvall et al., 1970; Peterson et (see below). However, a mixed diffuse and compact
al., 1977), and capsulate and non-capsulate strains colony morphology of strain T (table I) may indicate
(Wilkinson, 1983). We first determined coagulase a heterogeneous population within this strain.
production, and tested for capsulation by lack of
Protein A-deficient strains. As noted above, strain
clumping factor (Smith et al., 1971), diffuse colony Cowan I was strongly hydrophobic. Strain Wood
morphology in serum soft agar (Finkelstein and 46 has been used extensively as a protein A-negative
Sulkin, 1958), and directly by examining Indian S. aureus strain (Forsgren and Sjoquist, 1966;
Kronvall et al., 1970) and was found to be
ink preparations (Wilkinson, 1983)(table I).
Strain Cowan I was regarded as a “typical” S. hydrophilic (fig. 2). The strain was clumping-factor
aureus strain and was strongly hydrophobic in tests negative but had a compact morphology in serum
with all three hydrocarbons after growth overnight soft agar and no capsule could be seen in Indian
(fig. la); exponential phase cells were still hydro- ink preparations. Although this suggests that
phobic, but less so (fig. 1b). The hydrophobicity of protein A is an important determinant of hydrophothe other strains was then determined and the bicity, another protein A-negative strain (EMS
percentage of initial ASOOnm
with 0-4ml of n- 252) (Peterson et al., 1977) was hydrophobic (table
hexadecane was used as the point of comparison I). Strain EMS 252 was coagulase- and clumping
between the strains (table I). If the value was 90% factor-negative, had diffuse colony morphology in
or more, the strain was designated as hydrophilic. serum soft agar and was identified as S. epidermidis
Table I. Characteristicsand hydrophobicityof S . aureus strains
S . aureus
strain
Coagulase Clumping
production
factor
Morphology
Percentage of
in serum
Capsule initial ASOOnm
with
soft agar (Indian ink)
hexadecane
Designation
-
Cowan I
H
HSmR
-
HB
HB
HB
HB
HB
HP
HB
HP
HB
+
100
HP
33
93
100
59
33
HB
HP
HP
HB
HB
-
-
52A5
T
Wood 46
EMS 252
M
M variant
Smith
Diffuse
Smith
Compact
NS58D
SA222
R-75
FB-1
27
42
41
56
56
96
83
102
54
+
+
+
-
+
+
C
D
S
C
C
-
+
+
-
C = compact; D =diffuse; S = spherical; HB = hydrophobic;HP = hydrophilic.
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F. REIFSTECK, S. WEE AND B. J WILKINSON
120-
120
a
100 -id
80
-
60
-
40
-
E
0
b
100
0
0
In
0
0
A
0
2ot
0
A
0.4
C
B
A
0.8
Volume of hydrocarbon ( ml )
a,
;
2c
n
0
0.4
Volume of hydrocarbon ( ml )
Fig. 1. Hydrophobicity of S. aureus strain Cowan I: (a) stationary phase cells, (b) exponential phase cells-0,
octane; 0,
p-xylene.
E
0
0
2
-a
..-
CI
.E
'64 8
0
Q
80
60
w-
0
&
a
40
CI
c
a,
2
a,
20
n
0
0 -4
n-hexadecane; A,n-
by the API-Staph-Ident system (Analytab Products,
Plainview, NY, USA) (Kloos and Wolfshol, 1982).
However, protein A-negative mutants, including
this one, often show pleiotropic effects (Peterson et
al., 1977; Jonsson et al., 1985). Growth of strain
HSmR in medium supplemented with NaCl 5%
w/v had a negligible effect on the hydrophobicity
of the organism. High concentrations of NaCl are
believed to inhibit protein A production (West and
Apicella, 1984).
Coagulase-negative S. aureus strains. Two coagulase-negative strains (Gramoli and Wilkinson,
1978) were hydrophobic (table I), indicating that
coagulase production is not responsible for hydrophobicity.
Capsulate strains. Capsulate strain M was hydrophilic whereas its non-capsulate derivative, M
variant, was hydrophobic (fig. 3). Similarly, capsulate strain Smith Diffuse was hydrophilic and noncapsulate strain Smith Compact was hydrophobic
(table I). Two other capsulate strains, NS58D and
SA222, were also hydrophilic.
120
100
08
08
VolumeOf hydrocarbon ( m'
Fig. 2. Hydrophobicity of S. aureus strain Wood 46; symbols as
in fig. I.
Efect of proteolytic enzyme treatment of cells on
hydrophobicity
The experiments were performed with hydrophobic strains Cowan I and H, capsulate hYdroPhilic
strain M and non-capsulate hydrophilic strain
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HYDROPHOBICITY-HYDROPHILICITY OF STAPHYLOCOCCI
a
120
12c
E 100
0
0
v)
4
a
80
.E
60
..-
a
80
(d
..-c
.-
Ew
0
A
0
$-r
c,
0
0
a,
a,
40
o
a
c
c
;
n
0
40
c
,
c,
A
60
Y-
w-
a,
b
a,
2
a,
20
20
n
0-4
0
0.4
0
0.8
Volume of hydrocarbon ( ml
0-8
Volume of hydrocarbon ( ml )
Fig. 3. Hydrophobicityof (a) S . aureus M (capsulate), (b) M variant (non-capsulate); symbols as in fig. 1 .
Wood 46. Trypsin treatment converted strain
Cowan I from hydrophobic to hydrophilic (fig. 4a).
Pepsin treatment increased the hydrophilicity of
strain Cowan I (fig. 4b), but not to the same extent
as trypsin, even when higher concentrations of
pepsin were used for longer incubation times (data
not shown). Similar results were obtained with
trypsin and pepsin treatment of strain H (data not
shown). Neither trypsin nor pepsin altered the
hydrophilicity of strains M or Wood 46.
120-
120-
Ec
0
100-AA
A
0
0
v)
a
-a
.‘E
.-
c,
80-
b
80
0
0
A
-O
A
60 -
60-
w-
0
a
c,
0
a,
40-
c
g
a,
C
a,
0
L
0
c
,
20-
20
A
n
a,
n
0
0
0-4
0.8
Volume of hydrocarbon ( ml )
0
0
0.4
0.8
Volume of hydrocarbon ( ml )
Fig. 4. Hydrophobicity of S . aureus Cowan I after treatment with (a) trypsin, (b) pepsin; n-hexadecane was used as the hydrocarbon
phase-0, control, untreated; trypsin or pepsin treated.
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F. REIFSTECK, S. WEE A N D B. J. WILKINSON
Hydrophobicity of cell-wall preparations
In an attempt to localise the hydrophobicity
determinant, various cell-wall preparations were
studied. These were: CCW, which were expected
to retain cell-wall proteins and cell membrane
fragments; SDS-CCW, from which membrane
fragments were expected to be stripped but covalently attached proteins should have been retained ;
PCW, which consisted of PG and teichoic acid;
and PG. PCW and PG from strain Cowan I were
hydrophilic, as expected (fig. 5). At the highest
concentration of n-hexadecane, CCW was weakly
hydrophobic, i.e., much less so than intact cells.
SDS-CCW was more hydrophobic than CCW,
possibly because of retention of small amounts of
SDS in the preparations. The Cowan I cell-wall
preparations were freshly prepared. Similar results
were found with strain H preparations which were
lyophilised (data not shown).
Hydrophobicity of coagulase-negativestaphylococcal
strains
Eighteen out of 25 strains were hydrophobic
(table 11). Four of the seven hydrophilic strains
were capsulate, whereas capsules could not be
120
100
B8 Q
A
80
€I
P
A
0
A
60
40
20
0
0.4
0 -8
Volume of hydrocarbon ( ml )
Fig. 5. Hydrophobicity of S. a u r m strain Cowan I cell-wall
preparations; n-hexadecane was used as the hydrocarbon
phase-0, CCW; A, SDS-CCW; 0,PCW; 0 ,PG.
detected in the other three, which were different
strains of S.sciuri. There was a fifth capsulate strain
that was not strongly hydrophobic (73%). It was
difficult to make further generalisations, other than
that both strains of S. haemolyticus were strongly
hydrophobic.
Discussion
From this study it appears that S. aureus is
typically hydrophobic and is more hydrophobic in
stationary phase than in exponential phase cultures.
This finding is in agreement with previous findings
(Rosenberg et al., 1980; Jonsson and Wadstrom,
1983; Malmqvist, 1983). Teichoic-acid deficiency
and lack of coagulase production had little influence
on hydrophobicity. Protein A does not appear to
play a major role in hydrophobicity. Of the two
protein A-deficient S. aureus strains studied, one,
Wood 46, was hydrophilic and one, EMS 252, was
hydrophobic. Jonsson and Wadstrom (1983), using
hydrophobic interaction chromatography, found
that the Wood 46 strain was hydrophilic and noted
a correlation between hydrophobicity and high
production of protein A. However, nine protein Anegative mutants had higher hydrophobicity than
the parent strain.. We found that a majority of the
strains of coagulase-negativespecies, which do not
contain protein A (Forsgren, 1970),were hydrophobic. Thus, although protein A may contribute to
hydrophobicity, it cannot be solely responsible for
this property.
Two pairs of capsulate and non-capsulate variants were hydrophilic and hydrophobic respectively, and two other capsulate strains were
hydrophilic. Jonsson and Wadstrom (1983) found
that the capsulate Smith Diffuse strain was hydrophilic. Our findings are similar to those of Rosenberg et al. (1983) on the correlation between
capsulation and hydrophilicity in A. calcoaceticus.
Thus a capsule apparently overcomes the tendency
of S. aureus to be hydrophobic. This is not surprising
in view of the chemical nature of capsules, which
are highly hydrophilic and acidic polysaccharides
(Wilkinson, 1958; Wilkinson, 1983).
Nevertheless, these findings are in contrast to
those of Hogt and his colleagues with capsulate S.
epidermidis and S . saprophyticus. Only one out of
eight capsulate strains was hydrophilic in an earlier
study (Hogt et al., 1983a) and one out of seven in a
later study (Hogt et al., 1985). Four out of five of
the capsulate coagulase-negative strains that we
studied were hydrophilic. The other strain was not
strongly hydrophobic. It is difficult to reconcile the
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HYDROPHOBICITY-HYDROPHILICITY OF STAPHYLOCOCCI
71
Table 11. Hydrophobicity and capsulation of coagulase-negative staphylococci
Species
Strain
S. epidermidis
S . saprophyticus
s. xylosus
S . simulans
S . lentus
S. hominis
S. capitis
S . cohni
S. haemolyticus
S. sciuri
S . auricularis
S. wameri
Percentage of
Capsule
initial ASOOnm
with
(Indian ink)
hexadecane
Designation
ATCC 14990
ATCC 15305
GH 196
TPC 2
TPC 1
ML-26
LH-A 12
76
FB-2
K6
ATCC 27844
DGS 73
ATCC 27840
LH-A3
DSM 20260
BB 3
KH A1 1
DSM 20263
ATCC 29059
ATCC 29062
GV 234
ATCC 33757
ATCC 33750
KL 105
GM28
-
+
+-
-
+-
-
+
-
-
-
-
+
30
93
73
25
69
58
58
100
45
8
6
63
98
64
77
89
6
7
93
98
94
79
73
54
93
HB
HP
HB
HB
HB
HB
HB
HP
HB
HB
HB
HB
HP
HB
HB
HB
HB
HB
HP
HP
HP
HB
HB
HB
HP
HB = hydrophobic;HP = hydrophilic.
results of the previous work with ours. Perhaps in
some capsulate strains, hydrophobic molecules,
e.g., proteins, are also present at the surface of the
capsule. Slime production tended to increase the
hydrophilicity of coagulase-negativestaphylococci
(Hogt et al., 1983a, 1986).
Some further approaches were taken to attempt
to identify the hydrophobicity determinant(s).
Treatment of cells with the proteolytic enzyme
trypsin converted the strains from hydrophobic to
hydrophilic, and pepsin increased their hydrophilicity. These results suggest that the hydrophobicity
determinant is a protein or protein-associated
molecule. Hogt et al. (1983a) found that pepsin
increased the hydrophilicity of S . epidermidis, but
trypsin was without effect. Proteolytic treatment of
M protein-containing group A streptococci increased their hydrophilicity (Ofek et al., 1983).
These authors favoured the idea that hydrophobicity in this organism was mainly due to glycolipids,
such as lipoteichoic acid, complexed with and
orientated by surface proteins. Miorner et al. (1983)
have also provided evidence that lipoteichoic acid
is mainly responsible for the hydrophobicity of
group A streptococci. Morris et al. (1985) noted a
correlation between loss of cell-wall proteins and
hydrophilicity of Streptococcus sanguis, which is
normally hydrophobic. However, they were unable
to eliminate a role for lipoteichoic acid in hydrophobicity.
None of the cell-wall preparations were as
hydrophobic as the cells from which they were
prepared. At first sight this might suggest that the
determinant is membrane-associated and exposed
through the cell wall at the surface of the bacteria.
Alternatively, the hydrophobicity determinant may
be removed from CCW during preparation, perhaps during washing with water. However, in cellwall preparations the inner surface of the wall is
exposed. If this were highly hydrophilic it might
decrease the observable hydrophobicity of cell-wall
preparations. Nesbitt et al. (1982) reported that S .
sanguis cell walls tended to be hydrophobic and
concluded that hydrophobic amino acids associated
with the cell wall contributed to the observed
hydrophobicity of intact cells. However, these
authors found that walls from Bacillus subtilis were
hydrophilic.
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F. REIFSTECK, S. WEE AND B. J. WILKINSON
Although staphylococci are typically hydrophobic they can be rendered hydrophilic either by loss
of surface proteins or protein-associated molecules,
or by the presence of a capsule. S. aureus strain
Wood 46 and three strains of S. sciuri were
hydrophilic but non-capsulate as observed in India
ink preparations. It would be interesting to know
whether these strains are hydrophilic because of
the absence of surface proteins or the presence of
hydrophilic microcapsules. The Wood 46 strain has
recently been shown to produce what appears to be
a surface exopolysaccharide(P. Vaudaux, personal
communication). A recent report suggests that
microcapsules might be very common in S. aureus
(Sompolinsky et al., 1985).
At this time we cannot describe the staphylococcal hydrophobicity determinant more precisely
than as a cell-surface protein or protein-associated
molecule that is a component of the cell wall or the
membrane or both.
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