Microbiol. Immunol. Vol. 22 (6), 357-360, 1978
Transmissible Citrate-Utilizing Isolated from Pigeons,
Ability in Escherichia Pigs and Cattle
coli
Gihei SATO, Masayoshi ASAGI, Chiaki OKA, Naotaka ISHIGURO, and Nobuyuki TERAKADO Departmentof VeterinaryPublicHealth,ObihiroUniversityof Agriculture and Veterinary Medicine,Obihiro,and National Instituteof AnimalHealth,Kodaira, Tokyo (Receivedfor publication, October 17, 1977)
Isolation of citrate-utilizing variants of Escherichia coli has been demonstrated (5), but no genetic study of citrate-utilizing ability (Cit+) has been reported. This paper deals with isolation of Cit+ E. coli strains from pigeons, pigs and cattle, and the instability and transferability of citrate-utilizing ability in the strains, indicating that the ability is controlled by a plasmid. Thirteen of 36 strains of E. coli isolated from cloacal swabs of 22 pigeons in a pigeonry in Sapporo, Hokkaido, grew on Simmons' citrate agar (Eiken) within 1 to 3 days. Twelve of 33 E. coli strains obtained from 33 composite fecal samples of 33 pens in a piggery in the same district utilized citrate. In addition, one of 24 E. coli strains isolated from feces of seven cattle on a farm in Hokkaido was citrate-positive. These three kinds of animals were kept in epidemiologically-unrelated places. In basic biochemical reactions except citrate utilization, the Cit+ strains could not be differentiated from the Cit- strains and a typical E. coli strain (ATCC11775). The Cit+ strains were resistant to three to six antimicrobial agents including chloramphenicol (CM), and carried thermosensitive R plasmids (3). A genetic study of the R plasmids of the representative strains indicated that the R plasmids showed no fertility inhibition and no restriction against phage lambda, and were classified into incompatibility group H (4). Three Cit+ E. coli strains [HT58 from pigeons, carrying R plasmid conferring resistance to tetracycline (TC), CM, streptomycin (SM) and sulfadimethoxine (SA); KE10 from pigs, carrying R plasmid conferring resistance to TC, CM, SM, SA and kanamycin (KM) ; and C53 from cows, carrying R plasmid conferring resistance to TC, CM, SM and SA] were used in this genetic study. Two methionine-requiring F- derivatives [ML1410 resistant to nalidixic acid (NA) and ML1410RFP resistant to rifampicin (RIF)] of E. coli K-12 strain were employed as recipients. In this paper, only the data of the HT58 strain are tabulated. The effect of passaging Cit+ strains in penassay broth (Difco) at 25 C, 37 C and 42 C was investigated. After each passage, the cultures were streaked onto desoxycholate-hydrogen sulfide-lactose agar plates (DHL, Eiken), and more than 100 colonies grown on the plates were impressed on Simmons' citrate agar plates contain357
358
G. SATO Table
ing
methionine
made
on
SA
at
The
or
in
very
high
E.
transconjugant and
ment.
Also
character
in
the
strains.
the
three
resistance
C53
in
used
was
and
of
the
the
strains
strains,
obtained
and
recipient
less
of
table,
of
loss
elimi-
temperatures. deter-
transfer of
experi-
citrate-utilizing corresponding
than
strain
character
in
ability,
with
both
and
Cit+R+,
the
3
frequency
frequently
Cit-R+
citratewas
a later
citrate-utilizing
subcultures
citrate-
that
citrate-utilizing in
this
of the
shows
above
received
transconjugants
in
the
also
(12.5 ƒÊg/ml),
transconjugant
of citrate-utilizing
colonies
were
the
a
of
in
were
SM
1
increasing
but
Replicas
coexistence
strain
shown
stability
their
each
HT58
with
of
in
which
the not
on
yielded
Cit-R-
at
passage
instability
and
the
and
ML1410 from
experiments
(2).
(25 ƒÊg/ml),
Table
origin
temperature,
the
method
ascertain
passage
coli
each
in E. coli
CM
to
pigeon
5th
E.
C53
indicate
order
ability
determinant.
of
after
determinants
Colonies
in
HT58
incubation
original
(25 ƒÊg/ml),
determinant
in
In
replica-plating
resistance
rates
KE10
findings
coli
coli
observed
increase
the
(25 ƒÊg/ml)
resistance
was
These
HT58. the
3
E.
subcultures
of
citrate-utilizing but
and
not
transconjugants
of
Cit+R-. KE10
and
strains. Next,
transferability
examined.
Donor
gentle shaking. of fresh penassay 37
KM
of citrate-utilizing
TC
and
ability
minant
by
containing
determinant
nated
to
plates
(800 ƒÊg/ml)
utilizing
Stability
(50 ƒÊg/ml)
agar
utilizing
1.
EL AL
C
not
from
and method.
or grow
the
minant gants
at
citrate
(50 ƒÊg/ml) did
Then broth,
(precultured
Simmons'
as were
RIF
estimated
to
coli 1,
were
checked As
37
C)
with
were
shown
When
for
of
strains. 2,
4,
applied citrate in
Table
6, for
and for
1 ml 24 hr
of at
The
it was frequency
and two
24
hr
after
single-colony and
citrate-utilizing
drug
25
C
of
that the
incubation. isolations
ability
were
either
NA
selective
media
were
derived.
they
many
on by
was
used
and
the the
with 10 ml C) or
citrate-utilizing As
resistance
C in 25
at
the
was
37
media
on
thought
strains or
mixed
(50 ƒÊg/ml) grown
Transfer
Cit+
at
selective
methionine
transconjugants
three
6 hr
recipient were 25 C (precultured
shaking. added
the
for
methionine,
utilization 2,
in
precultured
gentle
which
addition E.
ability
were
(50 ƒÊg/ml).
without
possible
citrate-utilizing
0.1 ml of donor and incubated
agar
recipient was
of
detertransconjusame
media,
replica-plating
transmitted
with
a
NOTES Table
considerable with
frequency
the
These
same
that
ability
transfer
in
E.
coli
frequencies
the
cells
of
E.
coli.
and
C53
in
further
the
In
in
first
that
such
is
in
all
citrate(2.6•~
cases,
strain
10-6
the
same
KE10,
both
However,
transfer
ability
ability
strain
and
(Cit+R-).
citrate-utilizing
indicatplasmid.
jointly
these In
the
C, R
transferred.
determinant of
25
their
HT58
C.
jointly
transconjugants
citrate-utilizing
indicates
the
37
observed.
the
transferred
transfers. were
at
transmitted
than
transfer
was
as
were
at
further
efficiently
strains
not
Also recipient
thermosensitive
frequently but
coli.
other
more
as
resistance
determinants of
the
but
is
and
C,
transferability
This
C,
drug
less 25
obtained
only
and
37
in E. coli
E. to
and
KE10
resistance
carried
instability
recipient
ability
at
three-fourths
second
above,
C
ML1410
were and
strain,
25
Also
respectively)
citrate-utilizing C53
at
ability
to
the
ability
transconjugant
citrate-utilizing
tested.
5.0•~10-4,
of citrate-utilizing
to
a
both of
citrate-utilizing
utilizing
the
HT58
from
transfer
transconjugants
and
from
occurred
the
Moreover,
Transfer
frequency
transfers
ing
2.
359
As
were
mentioned
observed
plasmid-mediated
in
one-ninth
in
the
in
host
3
Cit.
strains. As
described
together
with
if both
determinants
conducted utilizing
and on
minant
were carried
citrate-utilizing
found vii
developed the
indicating as
determinant
on (1)
in the
mentioned determinant
on
that above,
agar
strain
C53 transfer.
To
which No
could
CM.
many
All
is
located
on
transductdeter-
the
transconjugants these
be
However,
citrate-utilizing
From
citrate-
transductant
the
gave
was
acquired
media.
containing
not
confirm
experiment
without
determinant
during
transmitted
strains.
transduction
selective
plates
determinant this
a
HT58. as
always
KE10
(Cit+R+)
used
DHL
resistance
replicon,
strain
plates
was
and
ML1410 from
agar
HT58
same
using
determinants citrate
only
(Cit-R+),
citrate-utilizing determinant
P1
resistance
Furthermore, the
phage
Simmons'
transductants tested
the
resistance are
by
obtained
ants
above, the
R
plasmid.
carrying results,
it
only can
be
360
G. SATO ET AL
concluded that the citrate-utilizing ability is controlled by Cit-plasmids which are different from R plasmids. Whether the Cit-plasmid is conjugative or nonconjugative is not yet apparent. The transfer phenomenon of citrate-utilizing determinant observed in this study may be due to mobilization by R plasmids which coexist in the same bacteria. Further research is in progress. Transfer of biochemical characters is important from a taxonomical point of view. Coliforms are classified according to several biochemical characters including citrate utilization, and if this character can be spread by plasmids, classification may be hindered. This work was supported in part by a Grant (No. 211912) from the Scientific Research Fund of the Ministry of Education, Science and Culture of Japan. REFERENCES
1) 2) 3)
4) 5)
Ikeda, H., and Tomizawa, J. 1965. Transducing fragments in generalized transduction by phage Pl. III. Studies with small phage particles. J. Mol. Biol. 14: 120-129. Lederberg, J., and Lederberg, E.M. 1952. Replica plating and indirect selection of bacterial mutants. J. Bacteriol. 63: 399-406. Sato, G., Oka, C., and Terakado, N. 1977. Detection of thermosensitive R plasmids in chloramphenicol-resistant Escherichia coli isolated from cattle, pigs and pigeons. Japan. J. Bacteriol. 32: 135 (in Japanese). Terakado, N., and Sato, G. 1978. Demonstration of the so-called Mexican type R plasmids in Escherichiacoli isolated from domestic animals and pigeons. Microbiol. Immunol. 22: 227-229. Washington, J.A., II, and Timm, J.A. 1976. Unclassified, citrate-positive member of the family Enterobacteriaceaeresembling Escherichia coli.J. Clin. Microbiol. 4: 165-167.
Requests for reprints should be addressed to Dr. Gihei Sato, Department of Veterinary Public Health, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080, Japan.