Identification and characterization of shiga toxin-producing Escherichia coli isolated from the feces of slaughtered pigs

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(1)³¤¥Èó. * .* a. (2004) 44 4 Korean J Vet Res(2004) 44(4) : 551~559. ê»îËªæbR¦84IJHB5PYJO1SPEVDJOH &TDIFSJDIJBDPMJ~ªÒB9ç. Ç~ÁBæÁjãÁ;c ÁB«ÆÁ*Z;

(2). ÏÎL >~ ÏÎL ;î*~Bæ\>8 (²Òß: 2004j 10ú 5¢). 1. Identification and characterization of shiga toxin-producing Escherichia coli isolated from the feces of slaughtered pigs Young-hwan Song, Ji-young Kim, Mi-kyung Chae, Chang-sik Park1, Myung-chul Kim and Moo-hyung Jun* College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Korea Research Center for Transgenic Cloned Pigs, Chungnam National University, Daejeon 305-764, Korea (Accepted= October 5, 2004). 1. Abstract : Shiga toxin-producing Escherichia coli (STEC) causes various clinical signs in human and animals, and has been indicated as a global enteropathogen with zoonotic importance. In this study, the feces of healthy pigs were collected from the slaughtered pigs of Daejon abattoir during the period from December 2001 to October 2002. Of 326 specimens, 13 STEC were confirmed by culture, PCR and colony hybridization. The isolates were further studied for toxin types, pathogenic factors, plasmid profiles, and antimicrobial resistance to characterize the genetic and toxigenic properties. In PCR, all of 13 isolates were evident to have shiga toxin gene (stx). Of 13 isolates stx1 gene was detected in 4 and stx2 gene in 9. The genes of eaeA, hlyA and rfbE were not present in any isolates. In colony hybridization using shiga toxin common primer (STXc), 2 to 9 per 100 colonies subcultured from 13 isolates showed the positive reaction. In the examination for plasmid profiles of the isolates, one to eleven plasmids with varying sizes of 1.0 Kb to 100 Kb were detected, and the 13 STEC could be classified into four groups by the plasmid patterns. The antimicrobial resistance patterns of the isolates were comparably corresponded with the plasmid profile patterns. Key words : shiga toxin-producing E. coli, fecal samples, pigs, genetic and toxigenic factors. * . 6"Ã j ¢V . f?f ÷V*f E. coli "~ ÷öW" &7 &ê& ® [17, 18]. JÒ¢ ¢bÊº E. coli~ ÷V*" &NB ¶ º shiga toxin(Stx), heat-stable toxin, enterohemolysin, intestinal adherence factor, pO157 plasmid, iron transport system, lipopolysaccharides rJ^ ® [10, 18]. &Ú, Stxº stx, intestinal adherence factor(intimin)f eaeA, enterohemolysinf hlyA, Ò lipopolysaccharides. &Ë(E. coli)f ÿb~ Ë&Úö " B~º Û W6VWb < ÚÂ ÿb~ *Ë&ÚöB B ~ ?"f &ê¢ Fæ~¾ î÷j FB. [17]. E. coliº ?"~ OÚV* &~>¾ * Ë& êÛ~ OÚËã ¶çj «îj ãÖ Ë6ïö B &ï Ã~ 6"j FB~ JÒ, N.Ã, º. \º ]Ò Ö>\>8(R11-2002-100-0000-0)~ æöj ¢¦ )j >¯>îr. *Corresponding author: Moo-hyung Jun College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Korea [Tel: +82-42-821-6753, Fax: +82-42-822-4216, E-mail: [email protected]]. 551.

(3) 552. Ç~ÁBæÁjãÁ;cÁB«ÆÁ*Z;. º rfbE F*¶ö '' z^>Ú ®b, Stxº shiga toxin-producing E. coli(STEC)f enterohemorrhagic E. coli(EHEC)~ ÷öWj æV~º "º ¶ C&^ ®. [10, 18]. STEC, ¢« vero cytotoxin-producing E. coli(VTEC)º Stx ë²¢ Ö~ attaching/effacing(A/ E)÷æj FB~ 60 MDa plasmid¢ &æº ;;' EHECf Stx ë²¢ Ö~Bê A/E ÷æj FB~ æ p 60 MDa plasmid& ìº j;; EHEC ª B [10, 17, 18]. STECöº Stx1 _f Stx2¢ Ö~º ©" Stx1" Stx2 Îv¢ Ö~º ©b ªF > ®b Îv Ò ²" ÿböB JÒÃ" &7 &ê& ®b, EHEC~ ÷V*öº Stx öò jî¢ intimin, enterohemolysin, lipopolysaccharide &ê& ® [17, 22, 23]. 7 eaeAö ~ z^>º intiminf 97-kDa A/E Wî E. coli& ?"~ Ë6ïö ¦O > ®ê ~º j . $ &¦ª~ EHECö Ò~º 90 Kb V ~ plasmidö ®º hlyA F*¶ö z^>Ú ®º enterohemolysin ®b, ©f Â.W &Ë"" Â .W ºëÃ Ãê~ ÷V*ö 7º j . [18, 23]. STECöº 43«~ .Ó; ®b, O26, O11 5 O157 .Ó;f Ò²öBº Â.W JÒÃ, Â.W &Ë ", /WËî~, Â.W ºëÃ Ãê, .*W .² 66²W ¶>÷j ¢bÊ "æöBº JÒÃ 5 ¦ «÷, ÇjæöBº F ê JÒÃj FB [9, 14, 18]. ß® " O157:H7 .Ó;f Ò² Â.W JÒÃ j FB~º ÷Ú "Ïj A ® [5, 10]. V¢B STECf Ú'b "º >Û*"÷~ ÷öÚ B ;>Ú, &»" »Ö®ö & STEC~ ªÒ 5 ÿ;" Ò & ¾¢öB >¯>îb , " F*¶ ¦ï V»j Ï ë² &N F* ¶~ Wçö & & B® ê¯> ® [7-9, 12, 21]. ÚöBº ;ç ÿb ªæb ¦V E. coli O157:H7 5 Stx Ö ªÒ þj >¯~ O157:H7 .Ó; j ¦Â : ®b [6], ê»ËöB ¾ÒB Gö & E. coli O157 J"¦¢ ¦Ò~ V, îG 5 êGöB ªÒ>î B : ® [1]. $ E. coli O157:H7 ³¦Âj * multiplex-PCR V»j {ã~ Vö & ¦Âþj Ö" stx1" stx2 F*¶¢ &ê E. coli¢ ¦Â : ® [3]. STEC f Ò²" ç7 $º *7'b 7/ &Ë Öëÿ b 5 jÿbö 7º*~² >Ú ®b, ªæb VJ>Ú ~ã" »Ö®ö J">Ú 7' ^B¢ ¢V > ®bæ ;ç ÿbö & STEC~. ª ¢ « jº& ®r æ'> ® [6, 7, 9, 21, 23]. öBº &*æ²Ò ê»ËöB ê»>º " æ~ Ëªæö & V·», PCR 5 colony hybridization j Ï~ STECj ªÒ~ reverse passive latex agglutination test¢ Ï~ Stx ë²~ F*¶;" ë²;j þ~& . $ enterohemolysin" intimin j z^~º hlyA 5 eaeA F*¶¢ ¦ï~ ªÒ "~ plasmid profileö & ¢N~ þj >¯~& .. Òò 5 O». &¦> j 2001j 12ú¦V 2002j 10úræ &*æ ê»Ë öB Z·* F; ;ç"æ(5~6Bú_, 90~110 kg) 326v~ ÖË¦*¢ Z'b .B ê ÚÏbj /b 3~5 g ;ê j~ Ò">& Î&B >ÇÏVö IÚ ïËç¢ Fæ~B þ Ú>~ ~& . " # 8æ E. coli ATCC 35150(STEC, O157:H7)j &Ï " ~& . Brain heart infusion broth(BHI, Difco), Luria-Bertani broth(LB broth, Difco), SIM Væ(Difco), MacConkey agar(Difco)¢ ~& . $ MacConkey agarö D-sorbitol(Difco) 1% >² Î& Sorbitol MacConkey agar¢ FVæ ÒÏ~& . 8· # QPMZNFSBTF DIBJO SFBDUJPO 1$3. jB ªæj '' 1 g O 10 ml~ BHIö 7«~ 37 CöB 20* ÿn êû V·~& . ê V· 0.1 mlj 5 ml~ LB brothö 7« ê 37 CöB 6* ÿn êû V·~& . ^V· 1 mlj 15,000 rpmöB 5ª* ö ê B phosphate-buffer saline(PBS, pH 7.1)ö 3® ^¿ ê 100 µl~ Ã~ > ;~² v>Ê ¦FV . ¦Fj 95 CöB 5ª* ¾Ò ê 15,000 rpmö B ö r ç[ 1 µlö STXc-Ff STXc-R '' 1 µl O, 10Üreaction buffer(100 mM Tris-HCl, 400 mM KCl, 15 mM MgCl , pH9.0) 5 µl, dNTPs mixture(10 mM, each dNTP 2.5 mM) 4 µl, taq polymerase(:Jîj) 1 µl(2 Unit) Ò Ã~> 37 µl¢ &~ C 50 µl & >ê b ê PCRj >¯~& . 1% ethidium bromide agarose gelj Ï~ *V'ÿ ~& Image Analyser(Pharmacia) 6ë~& [16, 24]. PCR Ö" stx F*¶ ·W>wj ¾æÞ ªæ~ V· o. o. o. 2.

(4) ê»î ËªæbR¦8 Shiga Toxin-Producing Escherichia coli ~ ªÒB Wç. 553. Table 1. Characteristics of the primers and reaction conditions for polymerase chain reaction Primersa. a. Sequences. Location. Sizes. PCR condition (oC/m'sec"). STXc-F STXc-R. 5'-GAGCGAAATAATTTATATGTG-3' 5'-TGATGATGGCAATTCAGTAT-3'. 280-300 778-797. 518. 94/5', 94/1' 58/1'30" 72/1'30". STX1-F STX1-R. 5'-GAAGAGTCCGTGGGATTACG-3' 5'-AGCGATGCAGCTATTAATAA-3'. 1022-1041 1132-1151. 130. 94/5', 94/1' 60/1'30' 72/1'30". STX2-F STX2-R. 5'-GCGTTTTGACCATCTTCGT-3' 5'-ACAGGAGCAGTTTCAGACAG-3'. 415-433 773-792. 378. 94/5', 94/1' 55/1'30" 72/1'30". EaeA-F EaeA-R. 5'-GTGGCGAATACTGGCGAGACT-3' 5'-CCCCATTCTTTTTCACCGTCG-3'. 853-873 1723-1743. 891. 94/5', 94/30" 65/30" 72/1'30". HlyA-F HlyA-R. 5'-GGTGCAGCAGAAAAAGTTGTAG-3' 5'-TCTCGCCTGATAGTGTTTGGTA-3'. 238-259 1767-1788. 1551. 94/5', 94/1' 59/1'30" 72/1'30". RfbE-F RfbE-R. 5'-GTCTGGACTCAACGTGGATT-3' 5'-AACTTGCTCATTCGATAGGC-3'. 181-200 1147-1166. 986. 94/5', 94/1' 55/1'30" 72/1'30". STXc for STEC, STX1 and STX2 for stx1 and stx2, EaeA and HlyA for virulence genes, RfbE for E. coli O157.. f MacConkey agarö êö~ 37 CöB 18* V · ê ªsïj ¾æÚº 100B~ ÷£j LB agarö patching method Ò 7«~ colony hybridizationö ÒÏ~& [24]. STEC" E. coli O157~ ¦Âj * STXc [16] 5 RfbE [26] primers¢, toxin typingj * STX1 [25] 5 STX2 [19] primers¢, Ò ÷öW ¶ intimin" enterohemolysin &N F*¶ ¦Âj * EaeA [13] 5 HlyA [15] primers¢ '' ·B~&. o. (Table 1).. $PMPOZ IZCSJEJ[BUJPO DIG DNA Labelling and Detection kit(Boehringer Mannheim, Germany) Masahisa et al. [20] . STXc primers 518 bp 10 denaturation DNA 100oC 2 µl hexaprimer 2 µl dNTPs(dATP, dCTP, dGTP, dTTP) digoxigenin dUTP 1 µl 18 Klenow enzyme 37oC 2 µl EDTA . 4M lithium chloride 100% ethanol 50 µl TE (100 mM TrisCl, pH 8.0, 1 mM EDTA) digoxigenin gene probe DNA . Nylon membrane. f ~ O»j w Ï~ >¯~&. ÃB ~ Î ª.j öB ª* y ê ~ f ~ 5 æB ¢ Î&~ ~ j &~ öB * ÿn > wÎ r ~ Ïj &~ >wj ;æ V ÚB Ï" b *Vb ©j ~ Ï ö ¦F~ æB ¢ B~&. (Boehringer Mannheim, Germany)j LB agarö ;WB ÷£ö ¿ 5ª* ;~Î ê âÚÚÚ filter paper (Whatmann, Germany) *öB æW(0.5N NaOH, 1.5M NaCl)b 15ª*, 7z(1.5M NaCl, 1M Tris)b 15 ª* '' ¾Ò~, 2X SSC(0.3M NaCl, 30 mM Sodium citrate, pH 7.0) 15ª* ¾Ò~& . ÏB ÷ £j 30ª* 80 C j & DNA¢ ;~& . ê kit ÒÏ O»ö V¢ hybridization >w" Bï>w þj >¯~&, STEC ·Wb Ã«B ÷£j F ~ r þö ~& . & DPMJ 0 ¦ï STXc primers¢ Ï PCRöB ·W>wj " j E. coli O157ö ß' RfbE primers¢ Ï PCRj >¯~& . $ Farmer et al. [11]~ O»ö V ¢ Sorbitol MacConkey agar ï6ö 7«~ 18~24 * V· ê sorbitol¢ ª~æ pº Wï ÷£j F B~ MacConkey agar ï6ö êö~ Fj ª~º ÷£j ¦ï~& . 3FWFSTF QBTTJWF MBUFY BHHMVUJOBUJPO 31-" þ VTEC-RPLA test kit(Senka Deiken, Japan)¢ Ï~ >¯~& [16]. ªÒB STECj BHI agarö 7«~ o.

(5) Ç~ÁBæÁjãÁ;cÁB«ÆÁ*Z;. 554. öB 16* V· ê W. 2V ;ê~ j j ~ Polymyxin B Ï(5000U/ml) 1 mlö ¦FÎ ê 10ª *Ïb êû~ 37 CöB 30ª* V·~& . ê 900 göB 15ª* ö~ ç[j þö Ò Ï~& . 96 well V-type microplate(Becton Dickinson, USA)¢ Ï~ 25 µl~ C &ê wellö V · ç[j &~ 2V ê C~& (1:2~1:128). Vö Stx1 Æ¢Úf Stx 2 Æ¢Ú& '' z+B ¢ sÊ«¶¢ 25 µl O &~& . rW& ;ç Æ¢ IgG& z+B ¢sÊ«¶¢ ~&, ·W& º verotoxin 1" 2¢ ~& . Microplateº ÛJç ¶ö IÚ NöB 18* >wÎ ê *;ê¢ G nb {~&b >w;êö V¢ 1+öB 3+ ~& . .Ó; þ E. coli antisera kit(Denka Seiken, Tokyo, Japan)¢ Ï~ >¯~& [23]. O.Ó; þf ªÒj BHI agarö 7«~ 37 CöB 18* V· ê W.~ 3~4V V~ j j~ 3 ml~ Ò">ö ¦FV . ¦Fj 121 CöB 15ª* ¾Ò ê 900 göB 20ª* öªÒ~ ç[j ªÒ 0.5 ml~ Ò">¢ &~ ÒÏ~& . Ò¢ &¢ Ê *ö ÷öW E. coli &N 43B O.Ój '' ÎÚ NÖ ê ö 10 µl Oj &~ b r w÷ F Z¢ &V~& . H;ê þf ªÒB STECj SIMV æöB 3~5² ê& V·~ ÚÿWj Ã&Î ê. ~& . þ&ö ÷öW E. coli &N 22B H.Ó 100 µlf ö 0.5 ml Oj '' b ê 50 C N >öB 1* >wÎ r w÷ç¢ &V~&. . Ö"~ 6;f Oê þf 1ªÚö ; w÷> wj ¾æÚº ©f ·Wb 6;~& . H;ê þ f Fç~ ß w÷j º ©j ·Wb ~& . 37oC. o. o. o. o. 1MBTNJE QSPGJMF LB broth 37oC 18 15,000 rpm 3 100 µl TAE buffer(40 mM Tris-acetate, 2 mM EDTA, pH 7.9) . Lysis buffer(3% SDS, 50 mM Tris-HCl, pH 12.6) 200 µl Phenol55oC, 45 chloroform solution(1:1 vol/vol) 15,000 rpm 10 , 0.7% ethidiumbromide agarose gel Image Analyser(Pharmacia) [24].. * ÿn êû V· j ö öB ª* ö ªÒ ê ç[j B~ ~ *j Ò¦FV. ¢ & ê DÚ ª* ¾Ò ê " N>öB j & v> ê öB ª* ö~&b ö r ç[ j ~ j Ï ~ *V'ÿ ~& 6 ë~&. B ÚW ¦Ò þ"¢ Mueller Hinton medium(Difco)ö 7« ê Sensi-Disc(Becton Dickinson, U.S.A.)j Ï~. amikacine(AN, 30 µg), amoxicillin-clavulanic acid(AMC, 20 µg+10 µg), ampicillin(AM, 10 µg), cefazolin (CZ, 30 µg), ceftiofur(CT, 30 µg), cephalothin(CF, 30 µg), ciprofloxacin(CIP, 5 µg), clindamycin(CC, 2 µg), colistin (CL, 10 µg), enrofloxacin(ENR, 5 µg), erythromycin(E, 15 µg), gentamicin(GM, 10 µg), kanamycin(K, 30 µg), lincomycin (L, 2 µg), linco-spectin(LS, 15 µg+200 µg), neomycin(N, 30 µg), streptomycin(S, 10 µg), sulfamethoxazoletrimethoprim(SXT, 23.75 µg+1.25 µg), tetracycline(TE, 30 µg), tylosin(TL, 150 µg) 20 [16].. ¦Ò~&. ~ &æ Bö & ÚWj. Ö . ~ ªÒ &*æ²Ò ê»ËöB j "æ ªæ&¦b 326 j V·~ Stx common primer STXc¢ Ï~ PCRj Ö" 13B(3.9%)~ ªæöB 518 bp~ ß Z& ÃNj &V > ®î (Fig. 1, Table 2). STXc primers¢ Ï PCRöB ·W>wj V· j MacConkey agarö 7« ê ªsïj ¾æÚº ÷£j LB agarö 7«~ colony hybridizationj Ö" 100B~ ÷£ 7 2~9B~ ÷£ STECb Ã «>î . ' ªæ&¦b¦V STECb Ã«B ÷£ 7 ~¾¢ F~ B>V·~&b ªÒ"¢ S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12 5 S13b ««~& (Table 2). & DPMJ 0~ ¦ï STECb ÿ;B 13"¢ Sorbitol MacConkey agarö V·~&~ : Îv ªsï ÷£j ¾æÚÚ O157 r Wî . $ RfbE primers¢ Ï~ PCRj >¯~ &~ : Îv rWîb, O 5 H;ö & .Ó; þöBê O157;f ¦ï>æ p~ (Table 2). $ intimin" enterohemolysin ¦Âj * EaeA 5 HlyA primers¢ '' Ï~ PCRj >¯ : 13"~ STEC ªÒ" Îv rWb ¾æÒ (Table 2). ë²; Ö; 13"~ STEC ªÒ"ö & STX1(STX1-F, STX1-R) 5 STX2(STX2-F, STX2-R) primers¢ Ï~ PCRj. ~&b, Reverse passive latex agglutination test ë²;j þ~& . Ö" Fig. 1 5 Table 2öB ¾ 45&$.

(6) ê»î ËªæbR¦8 Shiga Toxin-Producing Escherichia coli ~ ªÒB Wç. 555. Fig. 1. Agarose gel electrophoresis of the genes of stx(A), stx1(B) and stx2(C) amplified by PCR using STXc, STX1 and STX2 primers, respectively. Lane R; Reference strain E. coli ATCC35150(O157 : H7), Lane 1~2; STEC isolates, M; 1 Kb Plus Ladder marker. Table 2. Characterization of 13 strains of STEC isolated from the feces of healthy pigs. 1) 2). Strains. Sampling date. Serotype2). S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13. Dec. 2001 Dec. 2001 Dec. 2001 Dec. 2001 Dec. 2001 Dec. 2001 Jan. 2002 Feb. 2002 Mar. 2002 Mar. 2002 May. 2002 Aug. 2002 Sep. 2002. OUT:HUT OUT:H11 OUT:HUT OUT:HUT O166:NM OUT:NM O128:NM OUT:NM O166:NM OUT:NM OUT:NM O166:NM O128:NM. stx + + + + + + + + + + + + +. Strains S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 2). hlyA -. rfbE -. Plasmid patterns A A A A C D C C B B B B D. +: positive, -: negative OUT: O-untypable, HUT: H-untypable, NM: nonmorbility. Table 3. Stx-types in 13 strains of STEC isolated from the feces of healthy pigs. 1). stx1 + + + + -. PCR1) stx2 eaeA + + + + + + + + + -. VTEC-RPLA Stx1. Stx2. Sensitivity 1) /Titers2). + + + + -. + + + + + + + -. 3+ / (1:128) 3+ / (1:128) 3+ / (1:128) 3+ / (1:128) 1+ / (1:32) 1+ / (1:16) 1+ / (1:8) 1+ / (1:16) 1+ / (1:16) 1+ / (1:16) 1+ / (1:16) -. VTEC-RPLA test sensitivity End-point of positive reaction in VTEC-RPLA test. æÞ :f ? 13"~ STEC ªÒ" 7 S1, S2, S3 5 "º stx1 F*¶ö ~º 130 bp~ ßZ& Ã >îb S5, S6, S7, S8, S9, S10, S11, S12 5 S13 "öBº stx2 F*¶ö ~º 378 bp~ ßZ& Ã>îb, stx1" stx2¢ ÿö &æº STECf & V>æ p~ . Reverse passive latex agglutination testöBº S1, S2, S3 5 S4"º Stx1ö & ·W>wj &b w÷ &º 1:128î . Ò S5, S6, S7, S9, S10, S11 5 S13"º Stx2 6«>îb w÷&º 1:8~1:32 î. . PCRöB Stx2ö ·W>wj ¾æÞ S8 5 S13"º rWb ¾æÒ (Table 3). S4. 1MBTNJE QSPGJMFT 13 STEC plasmid profile (Fig. 2), 10~11 ( 100, 40, 20, 5.0, 4.0, 2.5, 2.3, 2.0, 1.8, 1.5. " ~ B£. j jv~. 5.

(7) 556. Ç~ÁBæÁjãÁ;cÁB«ÆÁ*Z;. Fig. 2. Plasmid profiles of 13 STEC isolates. Lane R; Reference strain E. coli ATCC35150(O157:H7), Lane 1~13; STEC isolates(1:S1, 2:S2, 3:S3, 4:S4, 5:S5, 6:S6, 7:S7, 8:S8, 9:S9, 10:S10, 11:S11, 12:S12, 13:S13), M; Lambda DNA/HindIII Markers. Table 4. Antimicrobial susceptibility of 13 strains of STEC isolated from the feces of healthy pigs Antimicrobial drugs Amikacin(AN) Amoxicillin-clavulanic acid(AMC) Ampicillin(AM) Cefazolin(CZ) Ceftiofur(CT) Cephalothin(CF) Ciprofloxacin(CIP) Clindamycin(CC) Colistin(CL) Enrofloxacin(ENR) Erythromycin(E) Gentamicin(GM) Kanamycin(K) Lincomycin(L) Linco-spectin(LS) Neomycin(N) Streptomycin(S) Sulfamethoxazole-trimethoprim(SXT) Tetracycline(TE) Tylosine(TL). V ~ Z¢ ¾æÚº 5 "º ; B£ 5 V ~ Z& & VB 5 "º ; Ò B £ 5 V ~ Z¢ º 5 "º ; b ª~& $ B £ 5 V ~ Z¢ ¾æÚº "f B £. 1.0 Kb ) S1, S2, S3 S4 A , 4 ( 100, 20, 4.0 2.0 Kb ) S9, S10, S11 S12 B , 2 ( 100 20 Kb ) S5, S7 S8 C . 7 ( 100, 80, 20, 4.5, 4.3, 3.0 2.2 Kb ) S6 4 ( 100,. No. of strains(%) Resistant. Intermediate. Susceptible. 0(0) 0(0) 13(100) 0(0) 0(0) 2(15.4) 0(0) 13(100) 0(0) 2(15.4) 13(100) 0(0) 4(30.8) 13(100) 0(0) 0(0) 13(100) 2(15.4) 13(100) 13(100). 0(0) 11(84.6) 0(0) 9(69.2) 10(76.9) 11(84.6) 1(7.7) 0(0) 10(76.9) 1(7.7) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 6(46.1) 0(0) 0(0). 13(100) 2(15.4) 0(0) 4(30.8) 3(23.1) 0(0) 12(92.3) 0(0) 3(23.1) 10(76.9) 0(0) 13(100) 9(69.2) 0(0) 13(100) 13(100) 0(0) 5(38.5) 0(0) 0(0). 5 3.3 Kb V)~ Z¢ º S13"º D; b ª~~& (Table 2). B ÚW" QMBTNJE QBUUFSOT Amikacinj 20B~ Bö & ÚW ¦Ò Ö" ªÒB 13"~ STEC Îv AM, CC, E, L, S, TE,. 20, 3.5.

(8) ê»î ËªæbR¦8 Shiga Toxin-Producing Escherichia coli ~ ªÒB Wç Table 5. Antimicrobial resistance patterns of 13 strains of STEC isolated from the feces of healthy pigs Strains. Plasmid patterns. Resistant antimicrobials. S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13. A A A A C D C C B B B B D. AM, CC, E, K, L, S, TE, TL AM, CC, E, K, L, S, TE, TL AM, CC, E, K, L, S, TE, TL AM, CC, E, K, L, S, TE, TL AM, CC, E, L, S, SXT, TE, TL AM, CC, ENR, E, L, S, TE, TL AM, CC, E, L, S, TE, TL AM, CF, CC, ENR, E, L, S, SXT, TE, TL AM, CC, E, L, S, TE, TL AM, CC, E, L, S, TE, TL AM, CC, E, L, S, TE, TL AM, CC, E, L, S, TE, TL AM, CF, CC, E, L, S, TE, TL. ~ 7&æ Bö & ÚWj ¾æîb CFö & 2"(15.4%), ENRö & 2"(15.4%), Kö & 4" (30.8%) Ò SXTö & 2"(15.4%)& '' ÚWj ¾æÚî . AN, GM, LS 5 Nö &Bº 13" Îv 6>W ®º ©b ¾æÒb ¾^æ Bö & Bº 7ê ç~ 6>Wj ¾æî (Table 4). ªÒB 13"~ STEC Îv þö ÒÏ 20B~ Bö & 7B çöB ÚWj ¾æÚî . "ê ÚW·çf 10«~ £Bö & ÚW 1", 8«~ £ Bö & ÚW 7", 7«~ £Bö & ÚW 5" ¾æÒ (Table 5). B ÚW" plasmid profile patternj jv : A; 4"º AM, CC, E, K, L, S, TE 5 TL, Ò B; 4"º AM, CC, E, L, S, TE 5 TL ö & &Wj j* ¢~~& . C; 3"º 80~87.5% Ò D; 2"º 87.5%~ ÚW BB~ ¢~Nj & . S8"º ß~² 10 B BBö & ÚWj ¾æî (Table 5).. TL. V. ~ ÷öW" &NB ¶ 7 shiga toxin f Ò²" ç7 $º *7'b 7/ &Ë Öëÿb 5 jÿbö 7º*~² >Ú ®b, ªæb VJ>Ú ~ã" »Ö® ö J">Ú 7' ^B¢ ¢V > ® [7, 9, 17, 23]. $ STEC~ ¦Ânêº ÒG~ã 5 æ ö V¢ ·b »Öb~ n;W {¢ * ê» îö & STEC~ ª ¢ « jº& ® [1, 6, 9, 21]. V¢B öBº &*²Ò ê»ËöB E. coli producing E. coli(STEC). 557. ê»>º ;ç "æ ªæj j~ STEC ªÒ þ j ~ 326B~ ªæ ÒòöB 13"¢ ÿ;~ ÷ö ' 5 F*' Wçj ~& . Bulte [9]f ë¢öB ÒG7 ; ÿb¦ V ªÒ 2,100"~ E. coliö & Stx1" Stx2~ ß F*¶ öïV»j Ï DNA-DNA colony hybridization V»j Ï~ STECj ¦Â : "æ öB 6.7%~ jN STEC Ò~ ®rj ~ &, Beutin [7]f ë¢öB ÒG7 ; ², ·, "², "æ 7«~ &» 720îÒöB çË 5 Ö Ëb¦V ªæj j~ colony hybridizatonb STECj ¦ï Ö" 208îÒöB STEC ªÒ>îb , ÿbê ·öB 66.6%, "²öB 56.1%, ²öB 21.1%, "æöB 7.5%~ jN Ò º Ö"¢ : ® . ~ W'f þöB áÚê STEC ªÒN 3.9%(13/326). ² ¸f ©b ¾æ Ò . ¾ Osek [21]º ¦öB F¶î 7 JÒ ¢ ¢bÊº BÚf ; BÚöB PCRV»j Ï STECj ¦Ò : 46îÒ~ ; ¶î 7 2îÒ(4.8%) öB Stx2 variant Stx2e& ªÒ>Ú W'" FÒ W'j : ® . ÚöBº N [6] ãÎæO~ ; ²f "æ ~ ªæöB STEC~ ¢« E. coli O157¢ ªÒ : ²öB 3" "æöB 2"~ O157 .Ó;~ E. coli& ª Ò>î ~&b, 5 s [1]f ãV 5 ;ö æ~ ê»ËöB ¾ÒB GöB E. coli O157:H7~ .Ó; ÖG, îG 5 êGöB 6.4%, 2.5% 5 1.1% & ªÒ>î ~& . ÿb ªæ¾ »Ö®ö & STEC~ ªÒNf æ , ÿb « 5 ÒG~ã ' º²ö V¢ N& ®b, ÒÏB ¦ïV»~ 6>W" ßWö V¢ ' Ëj Aj > ®j ©b 'B . Ú STEC~ ª ÒN ¾¢ö j jv' Ôb, ß® "æöB ªÒN Ô² ¾æÂ ©f ~ã' º ö ¦Òÿb ~ >f ÒÏ ¦Ò»~ 6>W º ·Ï Ö", ö & æ³' & >¯>Ú¢ © . STEC ªÒ"ö & ë²; þ Ö" Stx1 $º Stx2 ¢ '' Ö~º ªÒ"º ®îb¾ Stx1" Stx2¢ ÿ ö Ö~º ªÒ"º ìî . Ö"º Beutin [7] ; "æöB ªÒ 9"~ STECö & ë²;þ Ö", Stx1¢ Ö~º ªÒ"& 2", Stx2 ¢ Ö~º "& 7", Ò Stx1" Stx2 Îv¢ Ö ~º "º ìî Ö"f FÒ~& . Ò ªÒ" S8" S13f stx2 F¶¢ &æ ®îb¾ VTEC-RPLA þ Ö" rWb ¾æÂ *çf Ò².

(9) 558. Ç~ÁBæÁjãÁ;cÁB«ÆÁ*Z;. b¦V ªÒ~ STECöBê «ÃB : ®îb, ë ²F*¶f öWf N& ®rj "î [7]. V ¢B VTEC-RPLAf , Ú ë²; ÊÒ þ»æò ;{ Stx~ ¦Â 5 typingj *F PCR" VTEC-RPLA þj ÷¯B >¯~º © : ²ç ~ 'B . Stxf z®Ú enterohemorrhagic E. coli& î÷j FB Êº ·Ï~º 94 kDaV~ ËÚ¦O¶ intiminj z^~º F*¶f, 90 Kb V plasmidö *~~º enterohemolysin z^ F*¶¢ ¦Â~V * '' EaeAf HlyA primers¢ ·B~ PCRj ~ &b¾ 13 STEC ªÒ" ÎvöB rW>w ¾æ Ò . $ ªÒ"~ plasmid profile Ö"f jv~ " r 90 Kbf jÝ V~ plasmidº &V>îb¾ enterohemolysinj z^z ~º hlyA genef ìº ©b 'B . W'f Gannon [13] "æö B ªÒ 4"~ STECö & multiplex PCRV»b eaeA ¦Âþj : Îv rWî ©" ?f Ö"¢ & . ¾ Beutin [7] colony hybridizationj Ï~ ; "æöB ªÒ STEC 7 hlyA genej &æ ®º ªÒ"& 2.6% ò¢ Ö"f, Rowland [23] ^"öB ÒG7 <²öB ªÒ STECöB enterohemolysin" intimin~ FNj Ò Ö" £ 20%~ STEC ÷öW ¶ j &æ ®î º f jv r ² N& ®îb Wçf STEC~ ' ßWj «~º 7º ºb æ'> ® [7, 23]. 13"~ STECö & plasmid profilej plasmid~ > f Vö V¢ 4B b ª > ®îb, ; [4]" ; [2] "æ ªæb¦V ªÒ &Ë~ plasmid profile Wçj þ~ ªÒ" 1.0~125 Kb V~ · >~ plasmid& &V>î º Ö" f FÒ~& . $ 13"~ STEC~ plasmid NZ" Ú Wj ¾æÚº BB~ «~¢ plasmid ;" " * ö ç^ jv : Ö &W ¸ º Ò j r > ®î . ò S8"~ ãÖ ªÒ" ö j F ~ ®º plasmid~ >º 'æò 10«~ Bö Ú Wj ¾æÚº ©b j F~ ®º plasmid Ú ö ôf Bö & ÚW F*¶& Ò &ËW ®º ©b 'B . þö &¦b~ >ïf 'f Þ¾ Ú ; ê»î~ Ë&Úö B~ ®º STEC~ ë²; " F*' Wçj « > ®î . STECf Öëÿb 5 jÿbö 7º*~² >Ú ®b, 7 'b 7º >Û÷öÚ ;> ®V r^ ö »«, ê. 5 æê 7º* ÿbj &ç. b ª¶' ¢ >¯ jº& ® ' B .. Ö . 2001j 12ú¦V 2002j 10úræ &*²Ò ê»Ë öB ê»B "æ 326v~ ªæj j~ ªÒ V ·», PCR 5 colony hybridization O»j Ï~ 13 "(3.9%)~ shiga toxin-producing E. coli(STEC)¢ ªÒ ~& . STEC ªÒ"ö & PCRj : STX common primer STXcöBº 13" Îv 518 bp~ ß Z& ¾æÒ, STX1 primersöBº 4"öB stx1 F*¶ö >º 130 bp, Ò STX2 primersöBº 9"öB stx2 F*¶ö >º 378 bp~ ßZ& '' &V>î . stx1" stx2¢ ÿö &æº STECf &V>æ p~b, PCRöB Stx2ö ·W>wj ¾æÞ S8 5 S13"º rWb ¾æÒb, 13"~ STEC Î v eaeA, hlyA, rfbE F*¶& ¦Â>æ p~ . $ STXc primers¢ Ï PCRöB ßZ¢ ¾ æÚº ªæ &¦bö & colony hybridizationj >¯ : 100B 2~9B~ ÷£ STEC ß >wj & . 13"~ STEC ªÒ"ö & plasmid profile ªC Ö" 4&æ NZ &V>îb, 20«~ Bö & ÚWNZj plasmid profile NZ" jv : ¸f &Wj & .. ^^ò. Þ, I«. êÚ ~ ªæJ"" verotoxin W Escherichia coli O157:H7 ªÒö & . ®*n*W²æ 78-82. ; , ¾Ò, ;Ï^. ,1997, .Rç12,. JÒ ¶îbR¦8 2. ªÒ Escherichia coli~ ßWö &; B 6>W, Ëë² 5 RÎ~ F*;~ ª 5 plasmid ;profile. +4,&>~²æ ;÷, JËö., 2000, `Jç40,, B301-310. «", ;Ï^. 3. 8 7 Escherichia coli O157:H7 ³¦Âj * multiplex-PCR8» BB. &>~²æ. 1998, 38, 173-181. ; >, ;+4, Röj. æ F¾ &Ë~ > 4. ' ßW" plasmid profile ö &~. &>~² æ;. 1990, ê , N30,^287-295. BÏ~. JÒ~¶R¦8 verotoxin 5. , Ö&Ë~ ªÒ 5 > ' ëW. >~7 89-96. N²æ ^, . B1996, Ï~20, 6. . ÿ>ªæöB Escherichia coli O157:H7~ ªÒ 5 Ö~º verotoxin-2 ~ >z' ßW. &>~²æ. 1996, 36, 371-. 1.. 378. 7. Beutin, L. Geier, D. Steinruck, H., Zimmermann, S..

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