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JOURNAL
OF
CLINICAL
MICROBIOLOGY,
Apr.
1994,
p.
1050-1053
0095-1137/94/$04.00+0
Copyright
C
1994,
American
Society
for
Microbiology
NOTES
Molecular
Analysis
of
rRNA
and
Cholera
Toxin
Genes
Carried
by
the
New
Epidemic
Strain
of
Toxigenic
Vibrio
cholerae
0139
synonym
Bengal
SHAH
M.
FARUQUE,l*
A.
R.
M.
ABDUL
ALIM,'
SANJIT
K.
ROY,1
FERDOUSI
KHAN,'
G.
BALAKRISH
NAIR,2
R.
BRADLEY
SACK,1
AND
M.
JOHN
ALBERT1
Molecular
Biology
Laboratory,
International
Centre
for
Diarrhoeal
Disease
Research,
Bangladesh,
GPO
Box
128,
Dhaka
1000,
Bangladesh,1
and
National
Institute
of
Cholera
and
Enteric
Diseases,
Beliaghata,
Calcutta,
India2
Received
1
October
1993/Returned
for
modification
18
November
1993/Accepted
22
December
1993
Vibrio
cholerae
0139
synonym
Bengal
recently
caused
large
epidemics
of
cholera-like
disease
in
Bangladesh
and
India.
We
compared
the
restriction
fragment
length
polymorphisms
of
ctrA
and
rRNA
genes
(ribotypes)
in
27
isolates
of
V.
cholerae
0139
from
patients
in
Bangladesh
and
India
with
those
of
48
isolates
of
V.
chokrae
01
from
patients
and
21
V.
chokrae
isolates
from
surface
waters
in
Bangladesh,
which
included
2
0139
and
19
other
non-O1
isolates.
Ribotyping
of
the
isolates
with
BglI
revealed
that
all
29
isolates
of
0139
vibrios
belonged
to
a
single
ribotype,
suggesting
a
clonal
nature
of
the
infection.
However,
the
0139
vibrios
comprised
two
cxA
genotypes
and
carried
three
or
more
copies
of
the
cx4
gene,
and
the
chromosomal
locations
of
these
copies
were
unlike
those
of
the
El
Tor
or
classical vibrios.
Analysis
of
the
restriction
fragment
length
polymorphisms
of
the
rRNA
genes
suggested
that
V.
chokrae
0139
isolates
are
more
closely
related
to
El
Tor
strains
of
V.
cholerae
01
than
were
19
other
non-Ol
vibrios
and
33
classical
V.
chokrae
01
isolates
that
were
studied.
However,
further
studies
are
needed
to
determine
whether
V.
cholerae
0139
originated
from
mutations
and
genetic
changes
in
a
V.
chokrae
01
strain
or
was
due
to
the
acquisition
of virulence
genes
by
a
previously
unknown
V.
chokrae
non-Ol
strain.
In
December
1992,
an
epidemic
of
severe
acute
watery
diarrhea
clinically
resembling
cholera
occurred
in
southern
Bangladesh;
it
mainly
affected
adults
and
later
spread
to
other
parts
of
the
country,
including
the
capital
city,
Dhaka
(1,
3).
Similar
cholera-like
outbreaks
have
also
been
reported
from
several
places
in
neighboring
India
(14).
The
bacterium
re-
sponsible
for
the
outbreaks
in
Bangladesh
and
India
resembled
Vibrio
cholerae
01
in
cultural
and
biochemical
characteristics,
but
did
not
agglutinate
with
V.
cholerae
01
antisera
(1,
3,
14).
The
new
epidemic
strain
of
V.
cholerae
was
later
serogrouped
as
0139
and
was
given
the
suggested
name
Bengal
(3,
17).
Primers
specific
for
the
cholera
toxin
(CT)
operon
from
V.
cholerae
01
amplified
sequences
corresponding
to
CT
in
these
strains
in
PCR
(18),
and
all
the
strains
tested
were
also
positive
for
CT
production
by
standard
bioassays
for
CT
(1,
9,
21).
V
cholerae
non-C1
serogroups
were
not
known
to
be
associated
with
such
a
large
outbreak
of
diarrhea
before
the
present
epidemic.
Moreover,
they
were
known
to
produce
CT
at
a
very
low
frequency
(9),
unlike
the
serogroup
0139
isolates,
for
which
it
was
found
that
all
tested
isolates
produced
the
toxin.
We
attempted
to
characterize
the
epidemic
isolates
of
V.
cholerae
0139
from
the
recent
outbreaks
in
Bangladesh
and
India
by
analyzing
the
restriction
fragment
length
polymor-
phisms
(RFLPs)
of
their
rRNA
genes
and
ctxA
genes
to
determine
the
clonal
nature
of
the
isolates.
The
RFLPs
of
the
*
Corresponding
author.
Mailing
address:
Molecular
Biology
Labo-
ratory,
Laboratory
Sciences
Division,
International
Centre
for
Diar-
rhoeal
Disease
Research,
Bangladesh,
GPO
Box
128,
Dhaka
1000,
Bangladesh.
Telex:
675612
ICDD
BJ.
Fax:
880
2
883116.
genes
were
also
compared
with
those
of
other
V.
cholerae
non-O1
and
V
cholerae
01
isolates
to
investigate
the
relation-
ships
among
the
three
groups
of
isolates
and
to
find
out
clues
about
the
origin
of
V.
cholerae
0139.
A
total
of
29
V.
cholerae
0139
isolates
from
Bangladesh
and
India
were
studied
and
included
27
clinical
and
2
environmen-
tal
isolates.
All
non-O1,
non-0139
V.
cholerae
isolates
and
the
two
environmental
isolates
of
the
0139
serogroup
were
from
surface
waters
in
Dhaka,
and
V
cholerae
01
isolates
were
from
patients.
The
details
of
the
bacteria
are
given
in
Table
1.
All
bacteria
were
stored
in
Trypticase
soy
broth
(Difco
Laborato-
ries,
Detroit,
Mich.)
with
15%
glycerol
at
-
70°C
as
part
of
our
culture
collection.
The
identities
of
all
isolates
were
reconfirmed
by
standard
methods
as
described
previously
(3,
23).
The
RFLPs
of
the
ctxA
and
rRNA
genes
were
studied
by
hybridizing
Southern
blots
(20)
of
BglI-digested
total
DNA
by
using
radioactively
labeled
probes
for
the
ctxA
and
rRNA
genes,
respectively.
The
ctx;A
probe
was
a
550-bp
EcoRI
fragment
of
pCVD27
(11),
and
the
rRNA
gene
probe
was
a
7.5-kb
BamHI
fragment
of
pKK3535
described
previously
(2,
5).
The
recombinant
plas-
mids
were
prepared
and
digested
with
appropriate
restriction
enzymes
(Bethesda
Research
Laboratories,
Gaithersburg,
Md.),
and
the
inserts
were
purified
by
electroelution
from
agarose
gels
(12).
The
probe
DNAs
were
labeled
by
random
priming
(6)
by
using
[a-32P]dCTP
(3,000
Ci/mmol;
Amersham
International
plc,
Ayelsbury,
United
Kingdom)
and
a
random
primer
DNA
labeling
system
(Bethesda
Research
Laborato-
ries).
Southern
blots
were
hybridized,
washed
under
stringent
conditions,
and
autoradiographed
as
described
previously
(4,
1050
Vol.
32,
No.
4
NOTES
1051
TABLE
1.
Ribotypes
and
CT
genotypes
of
V
cholerae
01,
V.
cholerae
0139,
and
V.
cholerae
non-Ol,
non-0139
isolates
Strain
No.
of
Source
Countrv
Yr
of
RibotypexA
strains
isolation
genotype"
V
cholerae
01
Classical
4
Patient
Bangladesh
1961-1962
IA
I
Classical
6
Patient
Bangladesh
1963
IB
I
Classical
6
Patient
Bangladesh
1964-1966
IA
I
Classical
1
Patient
Bangladesh
1966
IA
3
Classical
3
Patient
Bangladesh
1967-1968
IA
I
Classical
11
Patient
Bangladesh
1982-1989
IA
I
Classical
2
Patient
Bangladesh
1991
IA
2
El
Tor
15
Patient
Bangladesh
1969-1989
IIA
4
V.
cholerae
non-01
0139
18
Patient
Bangladesh
1993
IIB
5
0139
2
Patient
Bangladesh
1993
IIB
6
0139
7
Patient
India
1993
IIB
5
0139
2
S.
water"
Bangladesh
1993
IIB
5
Non-0139
6
S.
water
Bangladesh
1986
III
Negative
Non-0139
1
S.
water
Bangladesh
1987
IV
I
Non-0
139
1
S.
water
Bangladesh
1987
IV
Negative
Non-0139
2
S.
water
Bangladesh
1989
V
Negative
Non-0139
9
S.
water
Bangladesh
1992-1993
VI
Negative
"Ribotypes
and
ctix:A
genotypes
are
based
on
Bgll
cleavage
patterns.
h
S.
water,
surface
water.
5).
The
CT
genes
in
different
isolates
were
further
compared
by
PCR
amplification
of
a
302-bp
portion
of
the
CT
subunit
A
gene
by
using
two
primers
corresponding
to
nucleotides
712
to
735
and
990
to
1013
of
the
ctx
operon
of
V
cholerae
01
(13,
18)
and
analyzing
the
PCR
products
by
digestion
with
the
restric-
tion
endonucleases
BamHI,
BglI,
EcoRI,
Hindlll
Hinfl,
or
TaqI.
The
different
ribotypes
and
ctxA
genotypes
were
desig-
nated
on
the
basis
of
the
differences
in
the
numbers
and
electrophoretic
mobilities
of
chromosomal
bands
generated
in
the
Southern
hybridization
experiments.
All
isolates
belonging
to
either
serotype
01
or
serotype
0139
were
positive
for
the
CT
genes
in
the
PCR
assay.
Southern
blot
hybridization
of
BglI-digested
chromosomal
DNAs
revealed
six
different
cleavage
patterns
of
the
CT
genes
(CT
genotypes
1
through
6;
Fig.
1)
among
the
isolates.
The
cleavage
patterns
consisted
of
one
to
four
bands
of
between
10.5
and
3.2
kb
in
size.
Thirty
of
the
33
(90.9%)
classical
V.
cholerae
isolates
studied
belonged
to
CT
genotype
1,
2
isolates
belonged
to
genotype
2,
and
1
isolate
belonged
to
genotype
3.
All
15
El
Tor
isolates
belonged
to
CT
genotype
4.
Of
the
29
isolates
of
the
0139
serotype,
27,
including
the
2
environmen-
tal
isolates,
belonged
to
CT
genotype
5,
and
the
remaining
2
isolates
belonged
to
genotype
6.
Only
1
of
the
19
environmen-
tal
non-01,
non-0139
isolates
contained
CT
genes,
and
this
isolate
belonged
to
genotype
1
(Table
1).
The
PCR
assay
produced
a
302-bp
product
for
all
CT-positive
isolates.
In
agreement
with
the
published
sequence
of
the
ctx
operon
(13),
the
PCR
products
from
all
CT-positive
strains
could
be
cleaved
with
TaqI
and
Hinfl
at
the
expected
positions
(see
Fig.
3)
and
did
not
react
with
the
other
four
restriction
enzymes
used.
Ribotyping
of
the
V
cholerae
isolates
produced
reproducible
restriction
patterns,
and
the
strains
could
be
differentiated
as
belonging
to
eight
different
ribotypes
on
the
basis
of
the
BglI
cleavage
patterns
of
their
rRNA
genes
(Fig.
2).
The
cleavage
patterns
consisted
of
7
to
10
bands
between
27
and
1.5
kb
for
different
ribotypes.
The
classical
vibrios
comprised
two
ri-
botypes,
ribotypes
IA
and
IB,
whereas
all
isolates
of
the
El
Tor
biotype
belonged
to
a
single
ribotype,
designated
ribotype
IIA.
All
0139
vibrios
isolated
from
patients
in
Bangladesh
and
1
2
3
4
5
6
kb
-
12.2
-
p
-10-2
-
5.1
-
460
.
so
-
3.0
-
2.0
FIG.
1.
Southern
hybridization
analysis
of
genomic
DNA
from
V.
cholerae
digested
with
BglI
and
probed
with
a
550-bp
fragment
of
the
A
subunit
of
CT
derived
from
pCVD27.
Bgll
restriction
patterns
corresponding
to
CT
genotypes
1
through
6
(lanes
1
through
6,
respectively)
are
shown.
Numbers
indicating
the
molecular
sizes
of
bands
correspond
to
a
1-kb
DNA
ladder
(Bethesda
Research
Labora-
tories).
VOL.
32,
1994
1052
NOTES
1
2 3
4
5
6
7
8
kb
.
..-12.2
VO-10.2
-8.1
-7.1
-5.1
4.0
3.0
2.0
-1.0
FIG.
2.
Southern
hybridization
analysis
of
genomic
DNA
from
V
cholerae
digested
with
BglI
and
probed
with
a
7.5-kb
BamHI
fragment
of
the
E.
coli
rRNA
clone
pKK3535.
BglI
restriction
patterns
of
the
rRNA
genes
produced
by
strains
belonging
to
ribotypes
IA
(lane
1),
IB
(lane
2),
IIA
(lane
3),
IIB
(lane
4),
and
III
through
VI
(lanes
5
through
8,
respectively)
are
shown.
Numbers
indicating
the
molecular
sizes
of
bands
correspond
to
a
1-kb
DNA
ladder
(Bethesda
Research
Labora-
tories).
India
and
from
environmental
waters
also
belonged
to
a
single
ribotype,
designated
ribotype
IIB.
This
suggested
that
a
single
clone
of
V.
cholerae
0139
was
responsible
for
the
outbreaks
in
Bangladesh
and
India.
Of
the
19
non-01,
non-0139
isolates,
6
belonged
to
ribotype
III,
2
belonged
to
each
of
ribotypes
IV
and
V,
and
the
remaining
9
isolates
belonged
to
ribotype
VI
(Table
1).
The
possible
mechanisms
for
the
emergence
of
this
new
toxigenic
strain
may
include
the
following:
(i)
V
cholerae
0139
emerged
as
a
result
of
mutations
in
an
El
Tor
vibrio
which
changed
its
serotype-specific
antigens
(7),
(ii)
a
nontoxigenic
environmental
strain
of
a
previously
undetected
non-01
sero-
type
acquired
the
array
of
virulence
genes
including
the
CT
genes
possibly
from
an
El
Tor
strain
to
emerge
as
a
new
toxigenic
serotype
of
V
cholerae,
and
(iii)
the
0139
vibrio
existed
in
the
environment
in
its
present
toxigenic
form
but
in
very
low
numbers
and
hence
was
never
detected
in
the
past,
but
some
undefined
environmental
changes
have
caused
the
vibrio
to
multiply
rapidly
and
dominate
over
existing
epidemic
strains.
The
0139
vibrios
belonged
to
ribotype
IIB,
which
was
distinctly
different
from
the
ribotypes
of
the
other
non-01
vibrios
(ribotypes
III
through
VI)
and
the
classical
vibrios
FIG.
3.
Restriction
analysis
of
a
302-bp
PCR-generated
fragment
of
the
ctxA
gene
from
V.
cholerae
01
and
0139
strains.
The
Hinfl
cleavage
patterns
of
the
302-bp
fragment
derived
from
three
classical
isolates
(lanes
2
through
4,
respectively),
three
El
Tor
isolates
(lanes
5
through
7,
respectively),
and
3
0139
isolates
(lanes
8
through
10,
respectively)
and
the
TaqI
cleavage
patterns
of the
302-bp
fragment
derived
from
three
classical
isolates
(lanes
11
through
13,
respectively),
three
El
Tor
isolates
(lanes
14
through
16,
respectively),
and
three
0139
isolates
(lanes
17
through
19,
respectively)
are
shown.
Numbers
indicating
the
molecular
sizes
of
bands
correspond
to
low-molecular-
mass
fragments
of
a
1-kb
DNA
ladder
(BRL)
are
shown
in
lane
1.
(ribotypes
IA
and
IB),
but
was
closely
related
to
that
of
El
Tor
vibrios
(ribotype
IIA).
Ribotype
IIB
differed
from
ribotype
IIA
in
the
BglI
restriction
pattern
of
rRNA
genes,
which
showed
an
additional
band
of
2.6
kb
in
ribotype
IIB
(Fig.
2).
Although
the
cleavage
patterns
of
the
rRNA
genes
in
0139
strains
were
similar
to
those
of
the
rRNA
genes
in
El
Tor
strains,
the
patterns
were
not
identical.
The
occurrence
of
the
additional
band
of
2.6
kb
for
all
0139
strains
was
very
consistent.
Moreover,
probing
of
the
BglI
restriction
fragments
of
the
chromosome
for
the
ctxA
gene
revealed
differences
among
the
classical,
El
Tor,
and
0139
vibrios.
Thus,
the
differences
in
the
RFLPs
of
the
rRNA
and
ctxA
genes
between
01
vibrios
and
0139
vibrios
suggest
that
a
mutation(s)
in
the
serotype-specific
genes
alone
of
a
V.
cholerae
01
strain
cannot
account
for
the
emergence
of
the
0139
vibrios,
as
suggested
in
a
recent
report
(7).
However,
the
possibility
that
the
0139
vibrios
arose
as
a
consequence
of
a
number
of
mutations
and
genetic
exchanges
in
an
El
Tor
strain
of
V
cholerae
01
cannot
be
ruled
out.
The
302-bp
PCR-generated
fragment
of
the
ctxA
gene
from
the
01
and
0139
isolates
showed
similar
restriction
patterns
(Fig.
3)
and
agreed
with
the
published
sequence
(13)
of
the
ctxA
gene
when
tested
with
a
large
number
of
restriction
enzymes.
This
indicates
that
the
302-bp
portion
of
the
ctxA
gene,
if
not
the
entire
CT
operon,
is
identical
in
these
strains.
The
differences
in
the
cleavage
patterns
of
the
ctxA
genes
detected
by
Southern
hybridization
of
total
genomic
DNAs
may
have
resulted
from
differences
in
the
copy
numbers
and
chromosomal
locations
of
the
genes
among
the
classical,
El
Tor,
and
0139
vibrios.
This
is
supported
by
previous
reports
indicating
that
CT
genes
may
be
deleted
or
inserted
at
different
locations
within the
bacterial
genome
(22),
possibly
because
of
the
presence
of
direct
repeat
sequences
flanking
the
CT
genes
(13).
Moreover,
it
has
been
suggested
in
a
recent
report
(8)
that
the
ctx
genes
are
unusually
unstable
in
the
chromosomes
of
0139
vibrios.
This
may
account
for
the
genetic
polymor-
J.
CLIN.
MICROBIOL.
NOTES
1053
phisms
in
the
ctxA
genes,
and
the
RFLPs
demonstrated
may
have
arisen
within
the short
time
span
of
the
epidemic
spread
of
infection.
This
suggests
that
the
infecting
isolates
were
indeed
-ional,
as
interpreted
from
the
ribotype
data
and
previously
reported
data
on
biochemical
and
cultural
charac-
teristics
(3),
although
RFLPs
in
ctxA
were
observed.
The
transformation
of
a
nontoxigenic
vibrio
to
toxigenicity
through
the
acquisition
of
toxin
genes
has
been
suggested
previously
for
the
origin
of
U.S.
Gulf
Coast
El
Tor
vibrios
(22).
A
similar
mechanism
may
have
transformed
an
ancestral
nontoxigenic
strain
of
0139
vibrio
which
acquired
virulence
genes
from
a
strain
of
toxigenic
V.
cholerae
01.
However,
it
is
necessary
to
demonstrate
the
presence
of
the
ancestral
non-
toxigenic
strain
of
0139
in
the
environment
to
confirm
this
assumption.
In
this
regard,
it
is
interesting
that
a
CT-negative
but
heat-stable,
toxin-positive
V.
cholerae
0139
strain
was
isolated
from
a
patient
with
diarrhea
in
Argentina
(15).
Another
possibility,
that
0139
vibrios
existed
in
their
present
virulent
form
in
very
low
numbers,
and
hence
were
never
detected
in
the
past,
cannot
be
ruled
out.
There
are
documentations
that
different
clones
of
toxigenic
V
cholerae
dominated
at
different
times
and
replaced
old
epidemic
strains
(10,
16,
19),
and
the
reasons
for
the
appearance
and
disap-
pearance
of
different
clones
have
never
been
explained.
More-
over,
the
appearance
of
new
clones
has
sometimes
gone
undetected
because
systematic
genetic
typing
methods
were
not
available.
We
have
reported
previously
(4)
the
transient
appearance
of
a
clone
of
classical
vibrios
in
1963
which
was
not
detected
either
before
or
after
the
end
of
1963
and
suggested
that
possible
undefined
environmental
changes
might
influ-
ence
the
emergence
and
domination
of
particular
clones.
The
survival
of
the
new
epidemic
strain
and
its
domination
over
01
strains
remain
to
be
monitored
in
the
future.
In
addition,
the
possibility
remains
that
more
new
strains
of
toxigenic
V.
cholerae
with
epidemic
potential
may
emerge
in
the
future,
and
the
factors
associated
with
the
emergence
and
domination
of
epidemic
strains
need
to
be
identified
and
monitored.
This
study
was
funded
by
the
U.S.
Agency
for
International
Devel-
opment
under
grant
DPE-5986-A-00-1009-00
with
the
International
Centre
for
Diarrhoeal Disease
Research,
Bangladesh
(ICDDR,B).
ICDDR,B
is
supported
by
the
aid
agencies
of
the
governments
of
Australia,
Bangladesh,
Belgium,
Canada,
Denmark,
France,
Japan,
The
Netherlands,
Norway,
Saudi
Arabia,
Sweden,
Switzerland,
the
United
Kingdom,
and
the
United
States;
international
organizations
including
the
United
Nations
Children's
Fund,
the
United
Nations
Development
Program,
the
United
Nations
Population
Fund,
and
the
World
Health
Organization;
and
private
foundations,
including
the
Ford
Foundation
and
the
Sasakawa
Foundation.
We
thank
Manzurul
Haque
for
secretarial
assistance.
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