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.

Transmissible citrate-utilizing ability in Escherichia coli isolated from pigeons, pigs and cattle.

Microbiol. Immunol. Vol. 22 (6), 357-360, 1978 Transmissible Citrate-Utilizing Isolated from Pigeons, Ability in Escherichia Pigs and Cattle coli...
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