APPLIED
AND
ENVIRONMENTAL
MICROBIOLOGY,
May
1987,
p.
1199-1202
0099-2240/87/051199-04$02.00/0
Copy igh
©
1987,
Ame ican
Socie y
o
Mic obiology
Vol.
53,
No.
5
Suscep ibili y
o
Halobac e ia
o
Hea y
Me als
J.
J.
NIETO,*
A.
VENTOSA,
AND
F.
RUIZ-BERRAQUERO
Depa men
o
Mic obiology,
Facul y
o
Pha macy,
Uni e si y
o
Se ille,
Se ille,
Spain
Recei ed
8
Decembe
1986/Accep ed
18
Feb ua y
1987
Six y-eigh
halobac e ia,
including
bo h
cul u e
collec ion
s ains
and
esh
isola es
om
widely
di e ing
geog aphical
a eas,
we e
es ed
o
suscep ibili y
o
a sena e,
cadmium,
ch omium,
cobal ,
coppe ,
lead,
me cu y,
nickel,
sil e ,
and
zinc
ions
by
an
aga
dilu ion
echnique.
The
cul u e
collec ion
s ains
showed
di e en
suscep ibili ies,
clus e ing
in o
i e
g oups.
Halobac e ium
medi e anei
and
Halobac e ium
olcanii
we e
he
mos
me al
ole an ,
whe eas
Haloa cula
cali o niae
and
Haloa cula
sinaiiensis
had
he
highes
suscep ibili ies
o
he
cul u e
collec ion
s ains.
Di e en
pa e ns
o
me al
suscep ibili y
we e
ound
o
all
he
halobac e ia
es ed,
and
he e
was
a
uni o m
suscep ibili y
o
me cu y
and
sil e .
All
s ains
es ed
we e
mul iply
me al
ole an .
Halobac e ia
a e
ae obic
mic oo ganisms
which
equi e
high
NaCI
concen a ions
in
a
medium
o
g ow
and
su i e
(9)
and
which
a e
included,
oge he
wi h
me hanogens
and
he moacidophiles,
in
he
a chaebac e ia
(22).
This
mic obial
g oup
has
been
ex ensi ely
s udied
wi h
ega d
o
hei
physiology, biochemis y,
ecology
and,
ecen ly,
gene ics
(2,
9,
10).
Al hough
he
suscep ibili y
o
an ibio ics
and,
in
some
ins ances,
he
mechanisms
o
ac ion
o
some
an imi-
c obial
agen s
ha e
been
s udied
in
halobac e ia
(3,
4,
12),
o
da e
he e
a e
no
epo s
conce ning
hei
na u al
suscep i-
bili y
o
hea y
me als
and
o ganome allic
compounds,
le
alone
hei
abili y
o
de elop
hea y
me al
esis ances.
The
la e
subjec
migh
p o ide
use ul
in o ma ion
abou
ways
o
de eloping
an ibio ic
esis ance,
since
in
nonhalophilic
bac-
e ia
genes
de e mining
esis ance
o
an ibio ics
and
ce ain
me als
a e
o en
ca ied
on
he
same
plasmids
(20).
I
was
ound
ecen ly
ha
megaplasmids
o
unknown
unc ion
a e
ha bo ed
by
he
majo i y
o
halobac e ia
(6).
A
knowledge
o
me al
ole ance
may
e eal
some
unc ions
o
some
o
hese
plasmids
and,
in
addi ion,
he
possible
hea y
me al
esis-
ances
could
be used
in
halobac e ia
as
gene ic
ma ke s.
On
he
o he
hand,
me al
ole ance
could
be
ele an
o
he
ecology
and
physiology
o
halobac e ia,
since
hey
usually
g ow
in
habi a s
such
as
sola
sal e ns
o
hype saline
lakes
con aining
some
wa e s
which
ha e
ecen ly
been
ound
o
be
pollu ed
wi h
hea y
me als
(unpublished
da a).
The
aim
o
he
p esen
s udy
was
o
examine
he
suscep-
ibili y
o
a
la ge
numbe
o
halobac e ia,
including
bo h
cul u e
collec ion
s ains
and
eshly
isola ed
s ains,
o
se e al
hea y
me als.
A
o al
o
68
halobac e ial
s ains
we e
selec ed
o
his
s udy.
The
13
cul u e
collec ion
s ains
a e
lis ed
in
Table
1,
and
he
o he
55
s ains
ha e
been
cha ac-
e ized
in
a
p e ious
axonomic
s udy
and
iden i ied
as
membe s
o
he
genus
Halobac e ium
(19).
These
la e
s ains
ep esen
a
e y
he e ogeneous
g oup
o
halobac e ia
ha
can
be
commonly
isola ed
om
hype saline
habi a s
in
Spain.
The
cul u es
we e
g own
in
a
medium
( e e ed
o
as
SWYE)
con aining
a
inal
o al
sal s
concen a ion
o
ca.
25%
(w / ol)
and
0.5%
(w / ol)
yeas
ex ac
(Di co
Labo a o ies,
*
Co esponding
au ho .
De oi ,
Mich.).
The
composi ion
o
he
sal s
solu ion
was
as
ollows
(pe cen
[weigh / olume]):
NaCl,
19.4;
MgC92,
1.6;
MgSO4,
2.4;
CaCl2,
0.1;
KCl,
0.5;
NaHCO3,
0.02;
and
NaB ,
0.05
(6).
The
pH
was
adjus ed
o
7.2.
Incuba ion
was
done
a
37°C
in
an
o bi al
shake
(Lab-Line
Ins umen s,
Inc.,
Mel ose
Pa k,
Ill.)
a
200
s okes
pe
min.
The
10
hea y
me als
es ed
we e
p o ided
om
s anda d
comme cial
sou ces
as
Na2HAsO4,
AgNO3,
CdCI2,
CoC12,
CuSO4,
K2C O4,
HgCl2,
NiSO4,
Pb(NO3)2,
and
ZnSO4.
S ock
solu ions
we e
made
in
dis illed
wa e ,
s e ilized
by
il a ion
h ough
0.22-,um-po e
memb ane
il e s
(Millipo e
Co p.,
Bed o d,
Mass.),
and
kep
a
4°C
o
no
longe
han
1
day.
Suscep ibili y
was
de e mined
by
an
aga
dilu ion
me hod
(21)
wi h
a
S ee s
eplica o
(17).
Pla es
con aining
20
ml
o
SWYE
medium
solidi ied
wi h
2%
(w / ol)
Bac o-Aga
(Di co)
and
se e al
di e en
concen a ions
o
he
me al
inhibi o s
we e
p epa ed
on
he
days
o
he
expe imen s.
The
concen a ions
o
all
he
me als
we e
as
ollows
(millimola ):
0.005,
0.01,
0.05,
0.1,
0.5,
1,
2.5,
5,
10,
20,
40,
and
80.
This
ange
co e ed
hose
concen a ions
usually
used
in
s udies
o
me al
esis ance
in
eubac e ia
(7,
20).
Be o e
use,
he
pla es
we e
d ied
a
37°C
o
30
min;
hey
we e
hen
inocula ed
wi h
104
o
105
mic oo ganisms
om
exponen-
ially
g owing
cul u es
pe
spo .
Twen y-one
spo s
(each
om
a
di e en
bac e ial
s ain)
could
be
con enien ly
es ed
pe
pla e.
The
pla es
we e
hen
incuba ed
a
37°C
o
10
days.
Aga
pla es
wi hou
he
an imic obial
agen s
and
inocula ed
wi h
he
co esponding
es
mic oo ganisms
we e
used
as
con ols.
Simila
expe imen s,
in
which
he
sal
concen a ion
in
he
es
medium
was
15,
20,
o
30%
(w / ol),
we e
also
ca ied
ou .
A e
incuba ion,
he
MIC
was
de e mined
as
he
lowes
concen a ion
o
me al
which
p e en ed
g ow h.
The
MICs
o
all
he
s ains
we e
he
same
when
he
s ains
we e
es ed
in
a
leas
h ee
di e en
expe imen s.
Fo
he
pu pose
o
de ining
me al
ole ance,
hose
s ains
which
we e
no
inhibi ed
by
10
mM
As;
1
mM
Ag,
Cd,
Co,
C ,
Cu,
Ni,
Pb,
and
Zn;
and
0.1
mM
Hg
we e
ega ded
as
ole an .
These
concen a ions
ha e
been
used
o
his
pu pose
in
he
majo i y
o
s udies
ca ied
ou
wi h
eubac e ia
(11,
14,
20).
I
is
impo an
o
poin
ou
ha
he
a ailabili y
o
he
espec i e
me als
can
be
in luenced
by
chlo ides
o
o he
kinds
o
sal s
con ained
in
he
cul u e
1199
APPL.
ENVIRON.
MICROBIOL.
TABLE
1.
MICs
o
10
me al
ions
es ed
agains
13
halobac e ial
cul u e
collec ion
s ains
MIC
(mM)
o ':
As
Ag
Ni
Co
Pb
Cd
C
Hg
Zn
Cu
H.
medi e anei
ATCC
33500
20
0.5
2.5
1
20
2.5
5 0.01
0.5
1
H.
olcanii
DS2
20
0.5
2.5
1
20
0.5
5 0.01
0.5
1
H.
hispanicum
ATCC
33960
10
0.5
2.5
1
10
0.5
5
0.01
0.5
1
H.
allismo is
ATCC
29715
10
0.5
2.5
1
10
0.5
5
0.01
0.5
1
H.
gibbonsii
ATCC
33959
10
0.5
2.5
1
10
0.5
5
0.01
0.5
1
H.
saccha o o um
ATCC
2952
10
0.05
1
0.5
20
0.5
2.5
0.01
0.5
2.5
H.
halobium
CCM
2090
10
0.05
1
0.5
20
0.5 2.5
0.01
0.5
2.5
H.
apanicum
NCMB
767
10
0.05
1
0.5
5
0.5
2.5
0.01
0.5
2.5
H.
salina ium
CCM
2084
20
0.05
1
0.5
5
0.1
2.5
0.01
0.05
1
Haloa cula
sp.
s ain
WS-1
10
0.05
0.1
1
5 0.05
2.5
0.05
0.05
2.5
Haloa cula
sp.
s ain
GN-1
10
0.05
0.1
1
5
0.05
2.5
0.05
0.05
2.5
H.
cali o niae
ATCC
33799
10
0.05
1
0.5
10
0.1
1
0.05
0.5
2.5
H.
sinaiiensis
ATCC
33800
10
0.05
1
0.5
5
0.05
2.5
0.05
0.05
1
a
Bold ace
ype
indica es
concen a ions
in ol ing
ole ance
o
he
co esponding
me al
ion.
medium
used
in
he
p esen
s udy.
In
addi ion,
he
o ma ion
o
me al
complexes
in
his
cul u e
medium
may
de e mine
he
ue
soluble
me al
concen a ions,
and,
indeed,
he
oxici y
o
some
me als
could
be
a ibu ed
o
a
me al
complex
a he
han
a
me al
ca ion.
The
MICs
o
he
hea y
me als
es ed
agains
he
halobac e ial
collec ion
s ains
used
in
he
p esen
s udy
a e
shown
in
Table
1.
On
he
basis
o
he
simila
MICs
o
all
he
me als
es ed,
hese
s ains
can
be
g ouped
in o
i e
majo
g oups.
(i)
Halobac e ium
medi e anei
and
Halobac e ium
olcanii
appea ed
o
be
he
leas
suscep ible.
(ii)
Halobac-
e ium
hispanicum,
Halobac e ium
allismo is,
and
Halobac e ium
gibbonsii
had
he
same
esponse
o
each
me al
and
we e
equally
a ec ed
by
h ee
me al
ions
(C ,
Ni,
and
Pb).
(iii)
Halobac e ium
halobium,
Halobac e ium
sac-
cha o o um,
Halobac e ium
apanicum,
and
Halobac e-
ium
salina ium
had
a
simila
esponse
o
Ag,
C ,
Co,
Hg,
and
Ni
ions.
Howe e ,
he
MICs
o
Cd,
Cu,
and
Zn
ions
o
H.
salina ium
we e
lowe ,
bu
his
s ain
was
he
only
one
in
his
g oup
o
which
he
MIC
o
As
ion
was
highe .
O he -
wise,
all
hese
s ains
we e
ela i ely
ole an
o
C
and
Pb
ions
(MICs,
2.5
and
5
mM,
espec i ely),
and
h ee
o
hem
we e
also
ole an
o
Cu
ion
(MIC,
2.5
mM).
(i )
Haloa cula
sp.
s ain
WS-1
and
Haloa cula
sp.
s ain
GN-1
had
he
same
esponse
o
each
o
he
me als
es ed
and
we e
ole an
o
C ,
Cu,
and
Pb
ions.
( )
Haloa cula
cali o niae
and
Haloa cula
sinaiiensis
we e
inhibi ed
by
he
lowes
concen-
a ions
o
mos
o
he
me als
es ed.
TABLE
2.
Suscep ibili y
o
68
halobac e ial
s ains
o
10
me al
ions
Cumula i e
%
o
s ains
suscep ible
o
he
ollowing
me al
Me al
ion
ion
concn
(mM):
0.005
0.01
0.05
0.1
0.5
1
2.5
5
10
20
As
0
0
0
0
0
5
12
41
85
100
Ag
0
28
35
71
90
100
100
100
100
100
Cd
0
0
0
28
66
91
100
100
100
100
Co
0
0
0
7
38
75
100
100
100
100
C
0
0
0
0
0
32
41
85
100
100
Cu
0
0
0
0
5
20
98
100
100
100
Hg
0
70
100
100
100
100
100
100
100
100
Ni
0
0
0
7
10
55
86
100
100
100
Pb
0
0
0
0
0
0
6
20
66
100
Zn
0
0
10
43
84
94
100
100
100
100
All
he
cul u e
collec ion
s ains
we e
ela i ely
ole an
o
lead
and
ch omium
(excep
o
H.
cali o niae
o
he
la e
me al).
Only
he
s ains
belonging
o
g oup
iii
(excep
o
H.
salina ium)
and
he
Haloa cula
s ains
(excep
o
H.
sinaiiensis)
we e
ole an
o
coppe .
Only
h ee
s ains
(H.
medi e anei,
H.
olcanii,
and
H.
salina ium)
we e
ole an
o
a sena e.
H.
medi e anei
showed
he
highes
ole ance
o
cadmium
o
all
he
s ains
es ed.
The
cumula i e
pe cen ages
o
s ains
suscep ible
o
a -
ious
concen a ions
o
me al
ions
a e
shown
in
Table
2.
A
wide
ange
o
concen a ions
was
es ed
because
o
he
pauci y
o in o ma ion
on
he
suscep ibili y
o
halobac e ia
o
hea y
me als.
I
was
hoped
ha
an
inhibi o y
concen a-
TABLE
3.
Pa e ns
o
ole ance
o
10
hea y
me al
ions
in
68
halobac e ial
s ains
No.
o
di e en
No.
(%)
ole ances
o
s ains
8
As,
Cd,
Co,
C ,
Cu,
Ni,
Pb,
Zn
1
(1.5)
7
Cd, Co,
C ,
Cu,
Ni,
Pb,
Zn
1
(1.5)
6
C ,
Co,
Cu,
Ni,
Pb,
Zn
1
(1.5)
5
Co,
C ,
Cu,
Ni,
Pb
7
(10.3)
As,
C ,
Cu,
Ni,
Pb
2
(2.9)
As,
Cd,
C ,
Ni,
Pb
1
(1.5)
As,
C ,
Co, Cu,
Pb
1
(1.5)
Cd,
C ,
Cu,
Ni,
Pb
1
(1.5)
Co,
C ,
Cu,
Ni,
Pb
1
(1.5)
4
C ,
Cu,
Ni,
Pb
6
(8.8)
Co,
Cu,
Ni,
Pb
4
(5.9)
As,
C ,
Ni,
Pb
1
(1.5)
Cd,
C ,
Cu,
Pb
1
(1.5)
C ,
Cu,
Pb,
Zn
1
(1.5)
Cd, Co,
Cu,
Pb
1
(1.5)
3
C ,
Cu,
Pb
12
(17.6)
C ,
Ni,
Pb
4
(5.9)
As,
C ,
Pb
3
(4.5)
As,
Cu,
Pb
1
(1.5)
Cu,
Ni,
Pb
1
(1.5)
2
Cu,
Pb
12
(17.6)
C ,
Pb
5
(7.5)
1200
NOTES
NOTES
1201
ion
would
be
ob ained
which
could
be
used
as
a
guide
o
u he
in es iga ions,
despi e
he
ac
ha
he
oxici ies
o
some
me als
could
be
a ibu ed
o
me al
complex
o ma ion.
On
he
o he
hand,
in
expe imen s
in
which
he
sal
concen-
a ion
in
he
es
medium
was
15,
20,
o
30%
(w / ol),
ins ead
o
he
s anda d
25%,
no
signi ican
di e ences
in
he
MICs
we e
obse ed
in
he
ange
o
20
o
30%
sal s.
Howe e ,
a
he
lowes
sal
concen a ion,
an
enhanced
oxici y
o
he
hea y
me als
es ed
was
ound.
This
esul
may
ha e
been
due
o
a
highe
a ailabili y
o
cells
o
ake
up
he
me al
ions
in
he
medium
wi h
he
lowes
sal
concen a-
ion
(closely
ela ed
o
physiological
cellula
changes
which
ake
place
a
his
low
salini y)
o
o
a
lowe
deg ee
o
in e ac ion
be ween
he
ee
me al
ions
and
he
sal s
p esen
in
he
medium.
The
equencies
o
ole ance
o
each
me al
ion
in
all
he
s ains
es ed
we e
as
ollows:
As,
15%;
Ag,
0%;
Cd,
9%;
Co,
25%;
C ,
72%;
Cu,
80%;
Hg,
0%;
Ni,
45%;
Pb,
100%;
and
Zn,
6%.
All
halobac e ia
es ed
we e
ole an
o
lead,
and
a
g ea
pe cen age
o
hem
we e
ole an
o
e en
a
10
mM
concen a ion
o
his
me al.
Fu he mo e,
a
g ea
ac-
ion
o
he
s ains
we e
also
ole an
o
coppe
and
ch o-
mium.
Howe e ,
i
should
be
poin ed
ou
ha
his
high
ole ance
o
lead,
coppe ,
and
ch omium
could
ha e
been
due
o
he
binding
o
hese
me al
ions
o
some
o
he
componen s
o
he
es
medium,
as
has
been
epo ed
by
Ramamoo hy
and
Kushne
(13).
These
in es iga o s
showed
ha
di e en
componen s
o
he
medium
(yeas
ex ac ,
pep one,
yp one,
e c.)
exhibi ed
high
binding
o
lead,
coppe ,
and
cadmium.
Besides,
lead
can
be
accumu-
la ed
in
he
cell
wall
and
memb ane
(18).
Mo eo e ,
i
may
be
ha
he e
was
much
less
lead
a ailable
in
he
cul u e
medium
han
ini ially
added,
since
he e
was
a
g ea
amoun
o
sul a e
p esen
in
he
medium.
As
a
esul ,
he
soluble
lead
would
be
a
a
lowe
concen a ion.
Ne e heless,
since
e y
li le
esea ch
has
been
conduc ed
on
he
gene ic
basis
o
lead
esis ance
in
bac e ia,
he
ole ance
exhibi ed
by
halobac e ia
appea s
o
be
an
in e es ing
ield
o
addi ional
esea ch.
O he wise,
all
he
s ains
demons a ed
a
high
suscep ibil-
i y
o
me cu y
and
sil e
and
e y
li le
ole ance
o
zinc,
cadmium,
and
a sena e.
Me cu y
was
he
agen
ha
showed
he
highes
ac i i y
agains
he
halobac e ia
es ed;
all
we e
inhibi ed
by
a
0.05
mM
concen a ion
o
his
me al
ion,
and
mos
o
hem
we e
inhibi ed
by
0.01
mM.
The
suscep ibili y
o
halobac e ia
o
me cu y
has
been
p e iously
used
as
a
pheno ypic
ea u e
in
some
axonomic
s udies
on
halobac-
e ia
(5,
15).
Howe e ,
he
concen a ion
o
HgCl2
used
in
hose
epo s
was
highe
han
0.1
mM,
a
concen a ion
which
inhibi ed
all
he
s ains
es ed.
I
was
epo ed
(1)
ha ,
in
he
p esence
o
high
concen a ions
o
NaCl,
he
oxici y
o
zinc
was
inc eased,
and
his
was
he
esul
no
o
a
syne gis ic
in e ac ion
be ween
Zn2+
and
ele a ed
osmo ic
p essu es
bu
o
he
o ma ion
o
complex
anionic
ZnCl-
species,
which
exe ed
g ea e
oxici ies
han
did
ca ionic
Zn2+.
This
ac
could
be
esponsible
o
he
high
suscep i-
bili y
o
halobac e ia
o
zinc.
All
he
s ains
es ed
we e
ole an
o
mul iple
me al
ions:
17
we e
ole an
o
wo
me al
ions
(25%),
21
we e
ole an
o
h ee
(30.9%),
14
we e
ole an
o
ou
(20.7%),
13
we e
ole an
o
i e
(19.2%),
and
1
(1.5%),
1
(1.5%),
and
1
(1.5%)
we e
ole an
o
six,
se en,
and
eigh
me al
ions,
espec-
i ely.
S ains
wi h
iple
ole ance
we e
ound
mos
e-
quen ly,
ollowed
by
hose
wi h
double
ole ance.
Howe e ,
s ains
ole an
o
i e
o
ou
di e en
me al
ions
we e
signi ican ly
de ec ed
(abou
40%
o
he
o al
s ains).
The
pa e ns
o
ole ance
o
he
hea y
me als
es ed
in
he
68
halobac e ial
s ains
es ed
a e
shown
in
Table
3.
I
is
well
documen ed
ha
many
me al
esis ances
can
be
go e ned
by
plasmid-encoded
mechanisms
in
bo h
g am-
nega i e
and
g am-posi i e
eubac e ia
(8,
16,
20).
Since
we
ha e
ecen ly
ound
ha
75%
o
all
halobac e ia
used
in his
s udy
ca y
a
leas
one
plasmid
and
ha
in
he
majo i y
o
hese
s ains
h ee
o
ou
megaplasmids
can
be
de ec ed
(6),
addi ional
expe imen s
o
co ela e
he
p esence
o
plasmids
and
he
ole ance
o
some
hea y
me al
ions
in
each
indi id-
ual
s ain
a e
now
in
p og ess
in
ou
labo a o y.
This
in es iga ion
was
suppo ed
by
g an s
om
he
Comisi6n
Aseso a
pa a
el
Desa ollo
de
la
In es igaci6n
Cien i ica
y
Tecnica
and
om
he
Jun a
de
Andalucia.
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NOTES