1243

Drug Resistant Plasmid of Actinobacillus pleuropneumoniae Isolated from

Swine Pleuropneumonia in Thailand

Akio KIUCHI, Motonobu HARA and Kiyoshi TABUCHIVeterinary Microbiology, School of Veterinary Medicine, Azabu University, Kanagawa 229, Japan

(Received: April 9, 1992)(Accepted: May 12, 1992)

Key words: Actinobacillus pleuropneumoniae, drug resistant plasmid , swinepleuropneumonia

Abstract

Actinobacillus pleuropneumoniae, isolated from the pneumonic lung tissue of pigs, was resistant tostreptomycin, sulfadiazine and tetracycline. This isolate, TA5, possessed 2 plasmids (pTA51=3.7 Kb,pTA52=6.2 Kb); one of the plasmids (pTA52) was coded for resistance to SM and SA. Another isolate of A.pleuropneumoniae, TA8, also possessed 2 plasmids (pTA81=3.7 Kb, pTA82=4.4 Kb), with a plasmid(pTA82) coded for resistance to SM and SA.

All 4 plasmids were not transmissible and small.

Introduction

Actinobacillus pleuropneumoniae is a hemolytic gram-negative microorganism requiring nicotinamideadenine dinucleotide (NAD). It produces lobar pneumonia with fibrinous, hemorrhagic and necroticpleuritis and is especially common in young and rapidly growing swine1,2,3,4). Swine pleuropneumonia is amajor respiratory disease problem for swine production in many countries . Actinobacillus pleuropneu-moniae was susceptible to drugs during the 1970s, however it has become rapidly resistant to manydrugs5,6). Some of them carried R-plasmids that are responsible for antibiotic resistance7,8,9). It has beenpointed out that some of antibiotic resistance in animal strains may play a role in the spread of resistanceto human pathogens10). We would like to report here about the relationships between drug resistance and

plasmids that were isolated from drug resistant strains in Thailand in 1988.

Materials and Methods

Bacterial strains: A. pleuropneumoniae strain TA5 (serovar 1) and TA8 (serovar 5) was isolated from

pneumonic lungs of pigs at a Bangkok slaughter house in Thailand. A. pleuropneumoniae strain TA1 is aplasmidless strain of serovar 1, and strain TA1-1 is a rifampicin resistant mutant of TA1. E. coli strain K12ML1410-1 is a rifampicin resistant mutant of E. coli strain K12 ML1410.

Medium: A. pleuropneumoniae strains were grown in BHI (brain heart infusion)-broth or on agar

(DIFCO, USA) supplemented with 10 pg of fiNADH/m1 (ORIENTAL KOBO, JAPAN).The drugs used and the abbreviations: sulfaisoxazole (SA); streptomycin (SM); tetracycline (TC).Antibiotic susceptibility test: Minimum inhibitory concentrations (MICs) were determined by the agar

dilution method standardized by the Japan Society of Chemotherapy").Preparations of plasmid DNA: The alkaline lysis method was used for preparation of small amounts of

plasmid DNAl2). For large scale preparations of plasmid DNA, cesium chloride (CsC1)-ethidium bromide

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平成4年9月20日

1244 Akio KIKUCHI et al

(EtBr) equilibrium density gradient centrifugation was used. The final density was adjusted to 1.571 g/cm3

(Refractive index, 1.3875), and the solution was centrifuged with a vertical rotor at 60,000 rpm for 6 hours

at 20•Ž.

Mating: Mating of the various strains of A. pleuropneumoniae with A. pleuropneumoniae strain TA1-1

and E. coli K12 ML1410 was performed using a membrane filter technique.

Transformation: Transformation of bacterial cells with plasmid DNA was carried out by the method of

Hanahan13). After incubation, aliquots of transforming mixture were plated out onto the Mueller Hinton

agar containing SM (100 pg/ml) or SA (400 pg/ml).

Restriction endonuclease digestion: Purified plasmid DNA preparations were digested with restriction

endonucleases, Alul, BamHI, EcoRII, Fokl, HindIII, MboII, Pstl and PvuII. Restriction endonucleases were

purchased from Takara Shuzo Co., LTD. Purified plasmid DNA and restriction endonuclease digested

plasmid DNA were electrophoresed through a 0.8% horizontal agarose gel (FMC, USA) for 1 to 2 h at 100

volts in tris-acetate buffer. Gels were stained with EtBr and photographed under UV illumination with a

Polaroid camera.

Results

Both A. pleuropneumoniae strains TA5 and TA8 were resistant to SM, SA and TC (Table 1) .

The results of agarose gel electrophoresis of DNA purified from A . pleuropneumoniae TA5 and TA8 are

shown in Fig. 1. A. pleuropneumoniae TA5 kept two plasmids, 3 .7 Kb and 6.2 Kb. While strain TA8

possessed two plasmids, 3.7 Kb and 4.4 Kb.

Mating of A. pleuropneumoniae stain TA5 or TA8 with A . pleuropneumoniae TA1-1 or E. coli K12

ML1410-1 was unsuccessful as far as transfer of resistant genes was concerned .

Transformation of A. pleuropneumoniae TA1 with purified plasmid DNA obtained from TA8 gave a

transformant resistant to SM and SA. This transformant had two plasmids with DNA the same as that of

TA8 (data not shown). Transformats of E. coli K12 ML1410 with plasmid DNA obtained from TA5 and

TA8 gave transformants resistant to SM and SA but not TC (Table 2) . The results of agarose gel

electrophoresis of DNA prepared from transformed E . coli K12 ML1410 showed that the plasmid of 6.2 Kb

(hereafter designated pTA52) is coded for resistance to SM and SA, while the one of 4.4 Kb (pTA82 for the

plasmid from TA8) is also coded for SM and SA (Fig. 1). No transformants had the plasmid of 3.7 kb.

The results of restriction endonuclease analysis showed that pTA2 and pTA82 were digested by Fokl

into 2 segments, each of 0.4 Kb fragment size. Moreover, pTA52 was digested into 8 pieces and pTA82 into

6 pieces by restriction endonuclease MboII. Four of the pieces had the same electrophoretic mobilities (Fig.

2). However, BamHI, EcoRII, Pvull and Sall did not digest two plasmids at all .

Table 1 Susceptibility of A. pleuropneumoniae

isolates

a) MIC = Minimal inhibitory concentration

b) An antibiotic susceptible strain of A. pleuro-

pneumoniae

Table 2 Susceptibility of transformed A. pleuro-

pneumoniae and E. coli

a) A. pleuropneumoniae TA1 bearing pTA81 and

pTA82b) E. coli K12 ML1410 bearing pTA52

感染症学雑誌 第66巻 第9号

Drug Resistant Plasmid of Actinobacillus pleuropneumoniae 1245

Fig. 1 Agarose gel electrophoresis of plasmid DNA extracted from

strains of A. pleuropneumoniae and transformed E. coiiKl2 ML1410

TA5: A. pleuropneumoniae TA5

pTA52: E. coli K12 ML1410 bearing pTA52TA8: A. pleuropneumoniae TA8

pTA82: E. coli K12 ML1410 bearing pTA82

Fig. 2 Fragment patterns of pTA52 and pTA82 after digested with restriction endonucleases.

a/HindIII is a molecular size marker

Discussion

The genes responsible for resistance to SM and SA in A. pleuropneumoniae strain TA5 and strain TA8

are located on plasmids pTA52 (6.2 Kb in size) and pTA8 (4.4 Kb in size), respectively. None of these

plasmids possess the genes necessary for their conjugative transfer to A. pleuropneumoniae or to E. coli.Hirsh et al.91 isolated two plasmids, and Ishii et a1.14) isolated one plasmid from A. pleuropneumoniae

strains, encoding SM and SA resistance. These plasmids were different from pTA5 and pTA8 in size and in

restriction endonuclease analysis. We suppose that the TC resistance in two strains, TA5 and TA8, was

not dependent on plasmids, because transformed strains of A. pleuropneumoniae and E. coli did not express

TC resistance. Also, A. pleuropneumoniae strain TA9 that was TC resistant had no plasmid. We were

unable to assign this property to a 3.7 Kb plasmid that was born on A. pleuropneumoniae strains TA5 and

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1246 Akio KIKUCHI et al

TA8. Some genetical hypotheses have been put forth that a drug sensitive strain acquired resistant genesfrom external sources with the support of a resident cryptic plasmid and by mobilization with a conjugal

plasmid, like RP4, of broad host range14).It is not surprising that such resistant A. pleuropneumoniae have emerged because antibiotics have

been used not only for treatment, but also frequently for economical reasons. This intense selective

pressure by antibiotics caused a widespread increase in R plasmid containing bacteria15).Medeiros et al.16) suggested that there is a potential for spread of drug resistance from an animal

reservoir to human pathogens.It is of interest to us to known what the correlation is between our plasmid and the isolated plasmids

all over the world.We have to expand our studies on the molecular genetics of drug resistant plasmids.

References

1) Kume, K., Nagano, I. & Nakai, T.: Bacteriological, serological, and pathological examinations of Haemophilus

pleuropneumoniae infection in 200 slaughtered pigs. Jpn. J. Vet. Sci. 48: 965-970, 1986.

2) Shope, R. E.: Porcine contagious pleuropneumonia, I. Experimental transmission, ethiology and pathology. J. Exp.

Med. 119: 357-368, 1964.

3) Pohl, S., Bertschinger, H. U., Frederiksen, W. & Mannheim, W.: Transfer of Haemophilus pleuropneumoniae and the

Pasteurella haemolytica-like organism causing porcine necrotic pleuropneumonia to the genus Actinobacillus

(Actinobacillus pleuropneumoniae comb. nov.) on the basis of phenotypic and deoxyribonucleic acid relatedness. Int.

J. Syst. Bacteriol. 33: 510-514, 1983.

4) Linnabary, R. D. & Smith, A. G.: Isolation of Haemophilus pleuropneumoniae from infected swine. Vet. Med. Small

Anim. Clin. 78: 1768-1771, 1983.

5) Didier, P. J., Perino, L. & Urobance, J.: Porcine Haemophilus pleuropneumoniae: Microbiologic and pathologic

findings. J. Am. Vet. Med. Assoc. 184: 716-719, 1984.

6) Gilbride, K. A. & Rosendal, S.: Antimicrobial susceptibility of 51 strains of Haemophilus pleuropneumoniae. Can. J.

Comp. Med. 48: 47-50, 1984.

7) Kawahara, K., Asano, M., Nakai, T., Kume, K. & Danbara, H.: Antibiotic susceptibility of serotype 2 and 5 strains of

Actinobacillus (haemophilus) pleuropneumoniae isolated from swine from 1974 to 1986 . Jpn. J. Vet. Sci. 51: 359-363,

1989.

8) Gilbride, K. A., Rosendal, S. & Brunton, J. L.: Plasmid mediated antimicrobial resistance in Ontario isolates of

Actinobacillus (Haemophilus) pleuropneumoniae . Can. J. Vet. Res. 53: 38-42, 1989.

9) Hirsh, D. C., Martin., L. D. & Libal, M. C.: Plasmid mediated antimicrobial resistance in Haemophilus pleuropneu-

monie. Am. J. Vet. Res. 43: 269-272, 1982.

10) Levy, S. B.: Playing antibiotic pool: time to tally the score . N. Engl. J. Med. 311: 663-665, 1984.

11) Okoshi, M.: The revised standard method for determination minimal linhibitory concentrations of antibiotics

against bacteria. Chemotherapy (Tokyo) 22: 1126-1129, 1974 (in Japanese) .

12) Birnboim, H. C. & Doly, J.: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic

Acids Res. 7: 1513-1523, 1979.

13) Hanahan, D.: Techniques for transformation of E. coli. pp. 109-136, In: DNA cloning, vol I (Glover, D. M. ed.) IRL

Press, Oxford, 1985.

14) Ishii, H., Nakasone, Y., Shigehara, S., Honma, K., Arai, Y., Iyobe, S. & Hashimoto, H.: Drug-susceptibility and

isolation of a plasmid in Haemophilus (Actinobacillus) pleuropnemoniae. Jpn . J. Vet. Sci. 52: 1-9, 1990.

15) Silver, R. P., Leming, B., Garon, C. F. & Hjerpe, C. A.: R-plasmid in Pasteurella multocida . Plasmid 2: 493-497,1979.

16) Medeiros, A. A., Levesque, R. & Jacoby, G. A.: An animal source for the ROB-1 ƒÀ-lactamase of Haemophilus influenzae

type b. Antimicrob. Agents Chemother. 29: 212-215, 1986.

感染症学雑誌 第66巻 第9号

Drug Resistant Plasmid of Actinobacillus pleuropneumoniae 1247

タイ国 に おけ るブ タ胸 膜肺 炎 由来Actinobacillus

pleuropneumoniaeの 薬 剤 耐性 プ ラス ミ ド

麻布大学微生物学第一講座

木内 明男 原 元宣 田淵 清

(平成4年4月9日 受付)

(平成4年5月12日 受理)

要 旨

タ イ 国 で ブ タ 肺 炎 病 巣 よ り 分 離 さ れ た

Actinobacillus pleuropneumoniaeに,ス トレプ ト

マ イ シ ン,サ ル フ ァ剤 お よび テ トラサ イ ク リン に

対 す る耐 性 が認 め られ た.Actinobacillus pleuro-

pneumoniae TA5株 は 二 個 の プ ラ ス ミ ドDNA

(pTA51= 3.7Kb, pTA52= 6.2Kb)を 保 有 して お

り,形 質 転 換 の 結 果 か ら プ ラ ス ミ ド(pTA52)は,

ス トレ プ トマ イ シ ン とサ ル フ ァ剤 耐 性 を コー ドし

て い た.一 方,Actinobaoillus pleuropneumoniae

TA8株 も 二 個 の プ ラ ス ミ ドDNA(pTA81= 3.7

Kb,pTA82= 4.4Kb)が あ り,プ ラ ス ミ ド

(pTA82)が ス ト レプ トマ イ シ ソ とサ ル フ ァ剤 耐

性 に 関与 して い た.四 個 の プ ラス ミ ドは非 伝 達 性

で,い ず れ も小 型 で あ った.制 限 酵 素 に よ る消 化

パ タ ー ン か らpTA52とpTA82は 一 部 共 通 し た

DNA断 片 が あ る こ とが 明 らか に な った.家 畜 が

薬 剤 耐 性 菌 の ヒ トの リザ ー ノミー と な っ て お り,注

目す る必 要 が あ る と思 わ れ る.

平成4年9月20日