Genetic properties of conjugative R plasmids in Escherichia coli and Salmonella isolated from feral and domestic pigeons, crows and kites

Genetic properties of conjugative R plasmids in Escherichia coli and Salmonella isolated from feral and domestic pigeons, crows and kites

ZbI. Bakt. Hyg., 1. Abt. Orig. A 255,271-284 (1983) Genetic Properties of Conjugative R Plasmids in Escherichia coli and Salmonella Isolated from Fer...

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ZbI. Bakt. Hyg., 1. Abt. Orig. A 255,271-284 (1983)

Genetic Properties of Conjugative R Plasmids in Escherichia coli and Salmonella Isolated from Feral and Domestic Pigeons, Crows and Kites Genetische Eigenschaften konjugativer R-Plasmide aus Escherichia coli und Salmonellen, die von Wild- und Haustauben, Krahen und Schwarzmilanen isoliert wurden MASASHI NIIDA r, SOUICHI MAKINO \ NAOTAKA ISHIGURO r, GIHEI SATOr, and TAKAMASA NISHI0 2 Department of Veterinary Public Health, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080, Japan 2Hiroshima Prefectural Institute of Public Health, Hiroshima 734, Japan

1

With 1 Figure' Received February 25, 1983

Summary A total of 62 conjugative R plasm ids detected in 48 Escherichia coli and 19 conjugative R plasmids in 16 Salmonella strains from domestic (carrier and emerald) and feral pigeons, crows (Japanese jungle crows and carrion crows) and black kite, were examined for genetic properties. Out of the 62 E. coli R plasmids examined for fertility inhibition (Fi), 29 (46.8%) were Fi+, and the remainder were Fi-. Among the 62 R plasmids, 16 (25.8%) were classified into incompatibility (Inc) group FIl, 15 (24.2%) into Itx, 7 (11.3%) into HI and 2 (3.2%) into N. The remaining 23 R plasmids could not be classified in this study. Among the 19 Salmonella R plasmids, 4 showed thermosensitive (ts) transfer. Out of the 18 plasmids tested, 13 (72.2%) including 4 ts-R plasmids were Fi-, and the remaining 5 (27.8%) were Fi+. Five R plasmids containing 4 Fi- were IncItX, while 4 ts-R plasmids belonged to IncHI (1 R plasmid) and H2 (3 R plasmids). The remainings were not tested. In E. coli strains, IncFIl and ItX were widely (4 of 6 lots of birds, respectively) distributed, followed by N (2 lots) and HI (1 lot). IncN R plasmid was detected from domestic pigeons and crows, but not from feral pigeons. In Salmonella strains from 5 lots of birds, IncFII and N plasmids which were detected from E. coli were not found, but IncH2 was detected from domestic pigeons (1 lot) and kite (1 lot). IncItX was detected from feral pigeons (1 lot) and kite (1 lot). The potential transfer of R plasmids between Salmonella and E. coli, and of dissemination of the plasmids among the free-flying birds, animals and humans is discussed.

Zusammenfassung In der vorliegenden Arbeit wurden die genetischen Eigenschaften von 62 konjugativen R-Plasmiden untersucht, die in 48 Escherichia coli Stammen nachgewiesen wurden, sowie

272

M. Niida, S. Makino, N.Ishiguro, G. Sato, and T. Nishio

19 konjugative R-Plasmide aus 16 Salmonella-Stammen. Die Bakterien wurden von Haustauben (Brieftauben und Smaragdtauben), Wildtauben, Krahen (japanische Dschungelkrahen und Aaskrahen) und einem Schwarzmilan isoliert. Unter den 62 E. coli R-Pasmiden, die auf ihre Fertilitatshemmung (Fi) untersucht wurden, erwiesen sich 29 (48,8%) als Fi+, die restlichen als Fi-. Von 62 R-Plasmiden konnten 16 (25.8%) der Inkompatibilitats(Inc)Gruppe FII zugeordnet werden, 15 (24.2%) der Inc-Gruppe la, 7 (11,3%) in Gruppe HI und 2 (3.2%) in Gruppe N. Die restlichen 23 Plasmide waren nicht bestimmbar. Von den 19 Salmonella R-Plasmiden zeigten 4 einen thermosensitiven (ts) Transfer. Unter den 18 geprliften Plasmiden erwiesen sich 13 (72,2%; darunter 4 ts-Plasmide) als Fi-, die restlichen 5 (27,8%) als Fi+. Flinf R-Plasmide, darunter 4 Fi-, lieRen sich in die Inc-Gruppe Ia einordnen, wogegen 4 ts-R-Plasmide zu IncH 1 (1 R-Plasmid) bzw. IncH2 (3 R-Plasmide) gehorten. Die restlichen wurden nicht getestet. Bei E. coli-Stammen waren IncFII und IncIa weit verbreitet (bei 4 von 6 Vogelgruppen), gefolgt von IncN (in 2 Vogelgruppen) und IncHI (in einer Gruppe). IncN R-Plasmide traten bei Haustauben und Kriihen auf, nicht aber bei wilden Tauben. Bei den Salmonella-Stammen, die bei 5 der untersuchten Vogelgruppen isoliert wurden, kamen die bei E. coli-Stammen dieser Tiere nachgewiesenen IncFII und IncN-Plasmide nicht vor, wohl aber IncH2 bei Haustauben (1 Vogelgruppe) und bei einem Schwarzmilan. IncIa wurde bei Salmonellen von wilden Tauben und bei dem Schwarzmilan nachgewiesen.

Introduction Many investigation of conjugative R plasmids of enterobacteria of human and domestic animal origins have been reported, but reports on R plasmids from wild birds not directly exposed to antimicrobial agents are few. In Japan, Sato et al. (25) detected very frequently conjugative R plasmids in drug resistant Escherichia coli strains isolated from feral and domestic pigeons (62.3%) and crows (73.3%), while drug resistant Salmonella typhimurium was isolated infrequently from feral pigeons (18, 24, 25) and conjugative R plasmids was not detected (24, 25). Conjugative R plasmids were detected less frequently from drug resistant E. coli isolated from feral pigeons (25.0%) (13) and crows (19.1%) (22), and at rates of 9.2-13.9% in resistant E. coli from wild birds (Japanese tree sparrows, green pheasants and bamboo partridges) (12). Recently, it was reported (14, 20) that the conjugative R plasmids were found in more than half of resistant E. coli strains isolated from the pet birds imported into Japan.

Table 1. Bacterial strains employed Strain designation Escherichia coli K-12 ML1410 SG1 SG3

Row

Relevant characters'

F-, met, nal rifampin-resistant mutant of W1895 (Hfr, met) rifampin-resistant mutant of E. coli 921, met, thi, thr, leu, lac, rk - mksensitive to colicin

Reference 8 8

7

23

a met, methionine; thr, threonine; thi, thiamine; leu, leucine; nal, nalidixic acid; rk-mk-, restriction and modification deficient.

Genetic Properties of R Plasmids from Birds

273

Genetic properties of R plasmids were useful as an epidemiological marker in calf salmonellosis (16,24) or assessing the potential transfer of R plasmids between strains of Salmonella and E. coli (6,9,26). From the epidemiological point of view, it is interesting to compare the genetic properties of conjugative R plasmids from E. coli and Salmonella from different kinds of wild or pet birds. Isolation of R plasmids from S. typhimurium derived from domestic pigeons (24, 27) and wild birds (1,24) has been reported. However, there has been no report on the genetic properties of conjugative R plasmids in E. coli and Salmonella isolated from wild or pet birds, except those of Nakamura et al. (21,22). This paper deals with the genetic properties of conjugative R plasmids in both E. coli and Salmonella strains isolated from domestic and feral pigeons, crows and a kite. Materials and Methods Bacterial strains, plasmids and phages used for genetic experiments The E. coli K-12 derivatives used in this study for genetic experiments of R plasmids are shown in Table 1. The reference plasmids used for incompatibility test are shown in Table 2. These reference plasmids belong to 22 incompatibility groups (11). Phages f2, PI,

Plvir, Avir, T4 and T7 were used in this study. R plasmid-carrying (R+) E. coli strains

A total of 48 E. coli strains isolated from pigeons and crows in Japan were examined (Table 3). These strains were isolated in our laboratory (25). As a rule, one representative strain per bird was used in this study.

R+ Salmonella strains Details of sixteen R + Salmonella strains derived from feral and domestic pigeons, a crow and a kite are shown in Table 4. These strains excepting one S. litchfield strain are S. typhimurium subserovar copenhagen (14 strains) and S. typhimurium (1 strain). Concerning Lot I strains (18), R plasmid mediation was indicated by us.

Media Nutrient broth used were penassay broth (PAB, Difco) and L broth (15). Heart infusion (HI, Eiken) and Mueller Hinton (MH, Eiken) agars were also used as basic media for selection of R+ strain according to the method of Ishiguro et a!. (8). To the HI or MH agar as selection media, 4 ml of 0.2% bromothymol blue and 1.5 g lactose per 100 ml were added. The selection media used for citrate utilization (Cit) was Simmons citrate agar plates, supplemented with methionine (50 fig/ml) and nalidixic acid (50 fig/ml).

Drug sensitivity test Drug sensitivity test was done by using the agar dilution method (9) and 10 antibiotics at the following concentrations (fig/ml): ampicillin (Ap), 25; chloramphenicol (Cm), 25; kanamycin (Km), 25; streptomycin (Sm), 12.5; sulfadimethoxine (Su), 800; tetracycline (Tc), 25; furatrizine (Ft), 6.3; nalidixic acid (Na), 25; rifampin (Rif), 25; and colistin (Cl), 12.5 U/m!.

Detection of conjugative R plasmids Detection of conjugative R plasmid was done by the method of Ishiguro et a!. (9). E. coli ML1410 was used as recipient. The broth mating was carried out at 28°C and 37 DC

274

M.Niida, S.Makino, N.lshiguro, G.Sato, and T.Nishio

Table 2. Reference plasmids employed Plasmid designation

Incompatibility group

Phenotype&

Reference

RA1 RI4 R4Da R386 F'lac RIOD ColB-K98 RI24 Folac R27 R478 Tp116 R144 Tp114 R621a R391 R387 R446b RN3 RP4 Rtsl R753 Rs-a R6K

A B C FI FI FlI FIJI FlV FV

SuTc ApSmSuTc ApKmSm Tc lac CmSmSuTcHg Cba Tc lac Tc CmKmTcTer CmSmSuTcHgTer Km(Tc) Km Tc Km CmSu SmTc SmSuTc ApKmTc Km ApCmSmSu CmKmSmSu Ap

11 11 11 11 11

ApCm KmTcCit+ CmKmSmSu Sm ApCmKm KmSmTc Cm ApCmKmSm

16 8 32 16 9 9 7 Unpublished b

pOH1477 pOH3123 RSTlO-l pOH3149 pOH3047-2 pOH3032 pOH806-2 pOH3153

Hi H2 H2 la 12 Iy

J

K M N P T V W X HI HI Hl H2 FII FII Ia

N

11 11 11 11 11 11 11 11 11 11 11 11 11 11

11 11 11

11 11

a Ap, ampicillin; Cm, chloramphenicol; Km, kanamycin; Sm, streptomycin; Su, sulfadimethoxine; Tc, tetracycline; Hg, mercury resistance; Ter, tellurite resistance; Cit, citrate utilization. b Detected from piggery sewage (Hanzawa, Y., Master thesis of Obihiro University of Agriculture and Veterinary Medicine, 1981).

(6). If the transconjugants were more frequently detected by the mating at 28°C than at 37 °c, they were considered to carry thermosensitive (ts) conjugative R plasmids. The selection was carried out by using all the drugs to which each donor strain was resistant, in order to detect as many transconjugants with different resistant patterns as possible. To examine their resistance patterns, five transconjugants on each selection plate were purified onto the same selection agar. The purified colonies were examined for their resistance pattern and the colonies showing different resistance patterns were used for genetic experiments.

Genetic Properties of R Plasmids from Birds

27S

Transconjugants of E. coli ML1410 obtained in the transfer experiments were tested for resistance to mercury (Hg) and tellurite (Ter), according to the methods of Nakahara et aI. (19), and Summers and Jacoby (29), respectively. Mercury compound used was HgCl 2 and its concentration was 20 ,ug/ml. Tellurite compound used was potassium tellurite diluted at the final concentrations of 10-3 , 10- " 10- 5 , and 10- 6 M. Citrate-utilizing ability of E. coli ML1410 carrying R plasmids was examined (9) .

Experiments of genetic properties of conjugative R plasmids The fertility inhibition (Fi) character of R plasmids was tested by the n:ethod of lshiguro et al. (9), using phage f2. Phage inhibition experiment was done according to the method of Taylor and Grant (3), using phages Avir, T4 and T7 in host strain SG3. The test for incompatibility (Inc) grouping of R plasmids derived from E. coli and Salmonella strains was done by the method of Ishiguro et al. (9). The reference R plasmids used are shown in Table 2. E. coli ML1410 and SG3 were used as the donor and recipient strain, respectively. Subgrouping of incompatibility group H was done using F'lac (28). Sextuple resistance R plasmids of which resistance marker overlapped with those of all reference plasmids used were applied for the acridine orange treatment (4), or transduction experiment by PI phage (17) to obtain the appropriate segregant plasmids for incompatibility test. If R plasmids to be tested could not be examined for incompatibility because of overlapping of their resistance marker with that of all or a part of reference plasmids, they were recorded as NO (could not be determined) .

Preparation of plasmid DNA, restriction enzyme reactions and electrophoresis Plasmid ONAs were prepared from wild strains and transconjugants, and centrifuged to equilibrium in a CsCI-ethidium bromide density gradient as described previously (10). Restriction enzyme of EcoR! was purchased from Takara Shuzo (Kyoto) and used according to the manufacturer's recommendations to examine the EcoRI-digested patterns of R plasmids. Agarose gels (1%) in electrophoresis buffer (0.089 M tris-0.002S M disodium EOTA-0.089 M boric acid, pH 8.3) were run in the horizontal apparatus (10).

Results

Antibiotic resistance E. coli: As shown in Table 3, most of the 48 strains examined were resistant to all or a part of drugs except N a, Rif, Cl and Ft. Majority of E. coli strains showed multiple resistance. The most frequent resistance pattern was quadruple (19 strains -39.6%), followed by sextuple (10-20.8%), single (8-16%), quintuple (5-8.3%), double (3-6.3%) and triple (3-6.3 %) . CmSmSuTc resistance pattern (17-35.4%) was most frequent. Cm resistance was found in all lots but Lot E. Ap resistance was not detected in Lots A, Band E. The resistance pattern was relatively uniform in each of Lots A, C and E. Salmonella: As shown in Table 4, 16 Salmonella strains isolated from pigeons, a crow and a kite were resistant to one or more drug(s) such as Ap, em, Km, Sm, Su und Tc. Quadruple resistance pattern (KmSmSuTc) was widely distributed in Salmonella strains obtained in Lots G, J and K, whereas Sm resistant strains were found in Lot H alone. Also, Sm and Ap resistance were limited to Lot I strains.

Detection of conjugative R plasmid E. coli: As indicated in Table 3,62 R plasmids were obtained from the 48 strains tested. Transferred resistance patterns were very variable, but they were relatively

2

10

3

9

7

Lot B, domestic pigeons (carrier, Columba livia domestica) Obihiro, 1975

Lot C, feral pigeons (Columba livia domestica) Iwamizawa, 1976

Lot D, feral pigeons (Columba livia domestica) Otaru, 1976

Lot E, feral pigeons (Columba livia domestica) Sapporo, 1977

Lot F, carrion crows (Corvus carone orientalis) and Japanese jungle crows (Corvus macrorhynchos ;aponensis) Horonobe, 1977

b

" No. of pigeons and crows examined. Including strain D-7-4. e Including transconjugant OH3198. d Including transconjugant OH3197.

17

)"

1 1 1

2. 2

1 2

CmSmSuTc ApCmSmSu SmSuTc SmTc Tc ApCmSmSuTc CmKmSmSuTc ApSmTc SmTc

(3)

(4)

1 1 7

lOb

ApCmSmSuTc

(10)

(3)

1 1 1

16

CmKmSmTc Cm ApCmKmSmSuTc

SmSuTc

CmSmSuTc

Resistance pattern

(1)

(17)

(

No. of E. coli strains tested

Lot A, domestic pigeons (carrier, Columba Ii via domestica) Sapporo, 1975

Source of birds

Table 3. Conjugative R plasmids in E. coli strains isolated from pigeons and crows

CmSmSuTcCit CmSmSuTcHg SmSuTc KmSmTc Cm ApCmKmSmSuTcHg ApCmKmSmSu SmSu ApCmSmSuTcHg ApCmSmSu SmSu CmSmSuTcHg CmSmSu ApCmSmSu SmSu Sm Tc ApCmSmSuHg Tc SmSu Tc ApSmTc SmTc Tc

Resistance pattern of R plasmids

1 1 1

1

1 2 1

1 1 7

1

1

1

9c 1 10 d 1 1 1

7 9 1 1 1



Z e;;. ::r

~

::l 0-





'" ?

C)

~o

.., =

~ -. en ::r qq.

~

0



:-;-



~

'"

~ 5: J'

~

0\

N

"

Genetic Properties of R Plasmids from Birds

277

uniform in Lot A and Lot C. CmSmSuTc resistance pattern was most frequent and it was isolated from 17 strains. In multi-drug resistant E. coli strains obtained from Lots C, D and F, 2 or 3 different transferred resistance patterns were found in a single parent strain (Table 3). It should be noted that 7 R plasmids in Lot A showed thermosensitive (ts) transfer and mediated citrate utilization (Table 5). Twentytwo (35.5%) R plasmids from crows and pigeons (3 of Slots) carried Hg resistance. Hg resistance was accompanied frequently with Cm, Sm, Su and T c resistance. However, none of the conjugative R plasmids mediated Ter resistance. Salmonella: Nineteen conjugative R plasmids were detected from the 16 strains as shown in Table 4. Four strains from pigeons (Lots G and I) and a kite (Lot K) carried ts R plasmids. The segregation of transferred pattern was uncommon compared with those derived from E. coli. Six R plasmids (31.6%) from Lot I feral pigeons and Lot K kite carried Hg resistance, but no R plasmid coded for citrate utilization. Cm resistance was obtained in only one strain but Ap resistance was not. SmSuTc R plasmids showing ts transfer and both Hg and Ter resistances were derived from 2 strains of kite origin.

Genetic properties of conjugative R plasmids E. coli: A total of 62 conjugative R plasmids were tested for Fi character and incompatibility, and the results were listed in Table 5. Twenty-nine (46.8%) R plasmids were Fi+ and 33 (54.8 %) were Fi-. Of these 29 Fi+ R plasmids, 16 (55.2%) were classified into Inc group FII, one plasmid from Lot B was classified into la, and the remaining 12 plasmids were could not be classified in this study. On the other hand, the 33 Fi- R plasmids were divided into 4 groups, i. e. IncIa (14 plasmids), IncN (2), IncHl (7) and ND group (10). All Fi+ and Fi- plasmids noted as NO were compatible with reference R plasmids of Inc FII, IncIa and IncH!' ALthough nine sextuple resistance (ApCmKmSmSuTc) R pJasmids from Lot C were applied for acridine orange treatment and transduction with PI phage, no appropriate segregant for Inc test was obtained. In Lots C and 0, IncIa plasmids conferring SmSu resistance were thought to coexist in the host cell together with multi-resistance R plasmids with different genetic properties, being showed in this paper (Table 5). R plasmids belonging to IncFII and Ia were widely distributed in E. coli strains of bird source regardless of isolation place. IncN plasmid was detected from domestic pigeons and crows, but not feral pigeons. Salmonella: Of 18 plasmids tested for Fi character, 13 (72.2%) including 4 ts-R and non ts-R plasmids were Fi- , and the remaining 5 (27.8%) were Fi+ (Table 6). Four R plasmids were classified into IncH2 (3 plasmids) and IncH1 (1 plasmid). Three R plasmids belonging to IncH2 in Lots G and K inhibited the development of phages A vir and T7 (data not shown). Four Fi- plasmids and one Fi+ plasmid were identified as Inda .. Since 9 Sm resistance R plasmids from Lots H and I showed low transfer frequency, they could not be tested for Inc. Moreover one KmSmSuTc resistance R plasmid isolated from crow in Lot J was not sufficiently transferred to recipient E. coli K-12 strains for the Fi and incompatibility test. In Salmonella strains, Fi- R plasmids were detected at higher frequency than in E. coli. IncFII and N plasmids which were detected from E. coli were not found in Salmonella, but IncH2 was detected in both domestic pigeon (1 lot) and kite (1 lot) Salmonella strains. IncIa was distributed in feral pigeons (1 lot) and kite (1 lot).

1 1

S. typhimurium subserovar copenhagen

S. typhimurium subserovar copenhagen

Lot J, Japanese jungle crow (Corvus macrorhynchos), Otaru, 1976 (24)

Lot K, black kite (Milvus migrans lineatus) Tokachi, 1976 (24)

b

a No. of pigeons, crows and a kite examined. Strain OH1612. e Transconjugant OHI612-1.
(1)

3

S. typhimurium subserovar copenhagen S. typhimurium S. litchfield

Lot I, feral pigeons (Columba Livia domestica) Hiroshima, 1978-1979 (18)

2

1

(3)

7

S. typhimurium subserovar copenhagen

Lot H, domestic pigeons (emerald dove, Chalcophaps indica) Gifu, 1980 (27)

(1)

(1)

(1)

(6)

(1)

1

S. typhimurium subserovar copenhagen

Lot G, domestic pigeon (carrier, Columba Livia domestica) Tokyo, 1970 (24)

No. of strains tested ( )&

Serovar

Source of birds

Ib 1

KmSmSuTc

1

2 1 1

1

7

1

SmSuTc

KmSmSuTc

CmKmSmSuTc SmSuTc ApSm SmSuTc

Sm

KmSmSuTc

Resistance pattern

Table 4. Conjugative R plasmids in Salmonella strains isolated from pigeons, crows and a kite

SmSuTcHgTer Tc SmSuTcHgTer Tc

KmSmSuTc

CmKmSmSuTcHg SmSuTcHg Sm SmSuTcHg Sm

Sm

KmSmSuTc

Ie 1" 1 1

1

1 2 1 1 1

7

1

Resistance pattern of R plasm ids

p-



;i Z. ;n

P-

::l

w

.,

8

Vl

.,

0

9

...c:

ati·

'"p-

~ ......

S· 9

?"

.,~

:n

~ 0.: J'

~

00

N 'I

CmSmSuTcCit CmSmSuTcHg SmSuTc KmSmTc Cm

ApCmKmSmSuTcHg ApCmKmSmSuHg SmSu ApCmSmSuTcHg CmSmSuTcHg ApCmSmSu CmSmSu SmSu SmSu Sm Tc ApCmSmSuTcHg ApSmTc SmSu SmTc Tc

Lot A, domestic pigeons Sapporo, 1975

Lot C, feral pigeons Iwamizawa, 1976

4

-

+

+

1 1 1 1

+ + + +

+ +

+

+

Fi

+

+

ts

1 1 7

9 1 10 1 1 2 1 1

7 9 1 1 1

No. of R plasm ids

(2)" (2)

FII ND ND FIl N ND ND ND Ioe

ND ND Ioe FII FII FJI FJI loe

HI FII N Ioe Ioe

Inc

A, B, C, 12, J, K, T, X A, FIV, Iy , M, N A, B, FlV, M, N, V, W, Iy

A, B, FIV, M, N, V, W A, B, FIV, Iy, P

All 22 reference plasmids A, B, C, 12, J, K, T, X

Inc reference plasm ids could not be tested b

ts = R plasmid showing thermosensitive transfer. Fi = Fertility inhibition. Inc = Incompatibility group. ND = Tested but not determined. " No. of R plasmids showing each character. b Incompatibility grouping could not be made because of overlap of resistance marker.

Lot E, feral pigeons Sapporo, 1977 Lot F, crows Horonobe, 1977

Lot D, feral pigeons Otaru, 1976

Lot B, domestic pigeon Obihiro, 1975

Resistance pattern

Source

Table 5. Genetic properties of conjugative R plasm ids derived from E. coli from pigeons and crows

\D

'-.J

tv

'"

0-

tl:I ;:; .

~

;;;
~ '"0

~

0


~ (b '

(b

'"

0

'"0 ....

n

::s (b

n'

Cl (b

280

M. Niida, S. Makino, N.Ishiguro, G. Sato, and T. Nishio

Table 6. Genetic properties of conjugative R plasmids derived from S. typhimurium and S. litchfied of bird origin Source

Resistance pattern

No.ofR plasmids

ts

LotG, domestic pigeon Tokyo, 1970 Lot H, emerald doves Gifu,1980 Lot I, feral pigeons Hiroshima, 1978-1979 Lot J, crow Otaru,1976 Lot K, kite Tokachi, 1976

KmSmSuTc

1

+

Sm

7

CmKmSmSuTcHg SmSuTcHg

1 3c

Sm KmSmSuTc

2C 1

SmSuTcHgTer Tc

2 2

Fi

Inc H2

+ + +

(4)a (3) (2) (1)

NP

+

NP NT HI IiX IiX NT NT H2 IiX

a No. of R plasmids showing each character. Could not be tested because of low transferability. C Including one R plasmid from S. litchfield.

b

A

B 2

3

4

5

2

112 plosmid I", plosmid Cl1ro --

Fig. 1. Agarose gel electrophoresis of plasmid DNA contents in the cells and plasmid DNAs cleaved by EcaR!. A) Bacterial plasmid contents of E. coli D-7-4 (lane 1), a transconjugant OH3197 showing IncIiX (lane 2) and a transconjugant OH3198 showing Inc ND (lane 3). Digested with EcoRI: (lane 4) pOH3197; (lane 5) pOH3198. Chro: chromosomal DNA band. B) Bacterial plasmid contents of S. typhimurium OHI612 (lane 1), a transconjugant OH1612-1 showing IncH2 (lane 2) and a transconjugant OH1612-1 showing IncIiX (lane 3).

Genetic Properties of R Plasmids from Birds

281

Assay of bacterial plasmids The transferred resistance patterns were found to be segregated in transconjugants from wild E. coli strains (Lots C, D and F) and Salmonella strains (Lots I and K) during mating experiments (Tables 3 and 4). To confirm whether the segregated resistance patterns of transconjugants are mediated by different R plasmids contained in a single cell, analytical gel electrophoresis of plasmid DNA contents was carried out. The representative gel electrophoresis for E. coli strain D-7-4 and 2 transconjugants of OH3197 and OH3198 (Table 3), and Salmonella strain OH1612 and 2 kinds of transconjugants (OH1612-1 and OH1612-2) from OH1612 (Table 4) were shown in Fig. 1, A and B, respectively. As shown in Fig. lA, the large broad band was found in E. coli strain D-7-4 (lane 1), and also 2 transconjugants (OH3197 and OH3198) showing different resistance patterns contained one large plasmid band (lanes 2 and 3). To examine if each plasmid (designated pOH3197 and pOH3198) in OH3197 and OH3198 are originated from an ancestor plasmid, both plasmid DNAs were digested by EeoRI enzyme and electrophoresed (Fig. lA lanes 4 and 5). Since the different EcoRIdigested patterns of R plasmids were observed, the plasmids of pOH3197 and pOH3198 were different each other as well as their Inc properties, indicating that D-7-4 contained different 2 plasmids with almost similar molecular weight. In contrast to E. coli, Salmonella strain OH1612 (Lot K) contained 3 different plasmids, while one transconjugant OH1612-1 contained a large faint plasmid (not clearly visualized in Fig. lB, lane 2) and the other OH1612-2 possessed only IIX plasmid band (Fig. lB, lane 3). Discussion

Sato et al. (25) stated that further studies on conjugative R plasmids detected in E. coli from domestic and feral pigeons and crows are needed to clarify source of the R plasmids and cause of difference of resistance status between Salmonella and E. coli isolated from pigeons. This genetical study was made for these purposes. The resistance pattern of E. coli and Salmonella strains and plasmid-mediated resistance pattern varied from single to sextuple, but sextuple resistance pattern was not found in Salmonella strains. As shown in Tables 3 and 4, the drug resistance pattern of Salmonella strains were more multiply than those of E. coli. In a part of E. coli and Salmonella strains, 2 or 3 R plasmids with different Fi and Inc characters were detected from the same strain, for example, Fi+ unclassified R plasmid (i.e. pOH3198) coding for sextuple-drug resistance and Fi- IncIa plasmid (i.e. pOH3197) showing SmSu resistance coexisted in E. coli (D-7-4) in Lot C feral pigeons (Fig. 1). Comparing the genetic properties of conjugative R plasmids derived from E. coli to those from Salmonella strains, first, isolation frequencies of Fi+ and Fi- R plasmids derived from E. coli were almost the same, however, in Salmonella Fi- R plasmids were more prevalent (72.2%). The same tendency in the character in Salmonella R plasmids has been recognized in domestic animals (31), and in human (36). Secondly, the most common incompatibility group R plasmids derived from E. coli was IncFII (25.8%) and followed by IncIlX (24.2%), while among those from Salmonella, IncIlX (27.8%) was the most common group and followed by H2 (16.7%). However, 50% of R plasmids in Salmonella could not be classified.

282

M.Niida, S.Makino, N.lshiguro, G.Sato, and T.Nishio

In this study, H1 and IIX R plasmids were detected from both E. coli and Salmonella. Especially, Fi- Tc resistance plasmids belonging to IncllX were distributed both in E. coli (Lot F crows) and Salmonella (Lot K kite) (Tables 5 and 6). This finding may suggest the possibility of R plasmid transfer between E. coli and Salmonella strains, although animal hosts were not the same. Concerning R plasmid from E. coli thought to be as pathogens or meat contaminants in domestic fowls IncllX, N, FI and X have been reported in Australia (2), and IncllX, P and FII in France (3). Inc groups H1 and IIX were detected from Salmonella of chicken origin in Japan (28). Inc groups H1, IIX, FII, Land W were detected in E. coli from imported mynahs (21). In Japan Inc groups of H1, H2, IIX, Nand FII were detected in E. coli of bovine origin (9, 26, 32) and IncH1, H2 and IIX in Salmonella (9, 16, 26, 31, 33) of the same origin, and the same Inc groups except N were also detected in E. coli (6,34) and Salmonella (6,7,31) of swine origin. There are not sufficient data on Inc groups of R plasmids in E. coli of human origin in Japan, but Yoshida et al. (35) reported that IncH2 was detected from Salmonella strains. Moreover, IncH1, IIX and FII R plasmids were detected among S. typhimurium strains isolated from humans with acute gastroenteritis in Hiroshima City (unpublished) where Lot I feral pigeons giving Salmonella strains carrying H1 and IIX plasmids (Table 6) inhabited during the same period (18). Davies and Stewart (5) reported that IncllX, FII and P were detected in coliform from humans and pets (dogs and cats) in Australia. As indicated above, Inc groups detected in E. coli (H1, IIX, FII and N) and Salmonella (H1, H2 and IIX) from pigeons and other birds in this study have been already distributed in animals and in humans in Japan. This suggests that wild birds may play an important role as pool of R plasmids among animals and humans. It is not strictly concluded that domestic and feral birds such as pigeons, crows and kites have a role of dissemination of R plasmids in animals and humans, because number of R plasmids isolated from Salmonella strains is small in this study. However, it is of interest to know how these free-flying birds, especially feral ones which are not exposed directly to antimicrobial agents are contaminated with R plasmids from the environments. Acknowledgments. We wish to thank Dr. M.Shinagawa for his helpful advice, and Dr. K. Hirai, Department of Veterinary Microbiology, Faculty of Agriculture, Gifu University, for Salmonella strains.

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with frozen chicken carcasses and characterization of conjugative R plasmid derived from such strains. Antimicrob. Agents Chemother. 16 (1979) 701-709 3. Chaslus-Dancla, E., J. F. Guillot et J. P. Lafont: Evolution de l'antibio-resistance bacterienne dans des elevages avicoles. Ann. Rech. Vet. 10 (1977) 77-86 4. Datta, N., V. M. Hughes, M. Hugent, and H. Richhard: Plasmids and transposons and their stability and mutability in bacteria isolated during an outbreak of hospital infection. Plasmid 2 (1979) 182-196 5. Davies, M. and P. R. Stewart: Transferable drug resistance in man and animals: Genetic

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Examination of 32 H 2 S positive strains isolated during the years 1950-1970. J. gen. Microbiol. 77 (1973) 487-499 24. Sato, G., N.Ishiguro, M. Asagi, C.Oka, T. Kawanishi, and T. Inoue: Biochemical characteristic and in-vitro drug sensitivity of Salmonella typhimurium Copenhagen variety isolated from domestic and feral pigeons, crows, a kite chicken and animals in Japan. Jpn. J. Vet. Sci. 39 (1977) 609-617 25. Sato, G., C. Oka, M. Asagi, and N.Ishiguro: Detection of conjugative R plasmids conferring chloramphenicol resistance in Escherichia coli isolated from domestic and feral pigeons and crows. Zbl. Bakt. Hyg., LAbt. Orig. A 241 (1978) 407-417 26. Sato, G. and N. Terakado: R factor type found in Salmonella typhimurium and Escherichia coli isolated from calves in a confined environment. Amer. J. vet. Res. 38 (1977) 743-747 27. Sawa, H. and K. Hirai: An outbreak of Salmonella typhimurium subserovar Copenhagen infection in pigeons (Chalcophas indica) imported from Hong Kong. Jpn. J. Vet. Sci. 43 (1981) 277-279 28. Smith, H.R., D.F. Grindley, G. O.Humphreys, and E.S.Anderson: Interaction of group H resistance factor with the F factor. J. Bact. 115 (1973) 623-628 29. Summers, A.G. and G.A.Jacoby: Plasmid-determined resistance to tellurium compounds. J. Bact. 129 (1977) 276-281 30. Taylor, D. E. and R. B. Grant: Incompatibility and bacteriophage inhibition properties of N-1, a plasmid belonging to the H2 incompatibility group. Molec. Gen. Genet. 153 (1977) 5-10 31. Terakado, N., T.Ohya, H. Ueda, Y.Isayama, and K. Ohmae: A survey of drug resistance and R plasmids in Salmonella isolated from domestic animals in Japan. Jpn. J. Vet. Sci. 42 (1980) 543-550 32. Terakado, N. and G.Sato: Demonstration of so-called mexican type R plasmids in Escherichia coli isolated from domestic animals and pigeons. Microbiol. Immunol. 22 (1978) 227-229 33. Terakado, N. and G.Sato: Detection of R plasmid of incompatibility group H in Salmonella typhimurium of bovine origin. Nat. Inst. Anim. Hlth Quart. 18 (1978) 180-181 34. Terakado, N., H.Ueda, Y.lsayama, and S.Arai: Trimethoprim R plasmids in Escherichia coli isolated from pigs. Microbial. Immunal. 24 (1980) 1233-1236 35. Yoshida, Y., Y. Terawaki, and R. Nakaya: R plasmids with thermo sensitive transferability in Salmonella strains isolated from human. Microbiol. Immunol. 22 (1978) 735743 36. Yoshikawa, M., S. Nagashima, and S. Matushima: Genetic destinction of R factor derived from Shigella and Salmonella. Jpn. J. Microbiol. 15 (1971) 425-436 Dr. G. Sato, Department of Veterinary Public Health, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080, Japan