Genotypic and Phenotypic Characteristics of Staphylococcus aureus Isolates from Lettuces and Raw Milk

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(1)KOREAN J. FOOD SCI. TECHNOL. Vol. 37, No. 1, pp. 134~141 (2005). ©The Korean Society of Food Science and Technology. çºf öFö* ªÒ ï

(2) êç\~ F*; 5 R*; ßû ;BêÁ&¢Á;W\Á~çêÁ^1ÁBÆ^2ÁÇfS3 ;{z4ÁB" 5ÁB76ÁBW* %&v ®", 1Ú&v ~ã", 2ÿ&v ~"& zv , ~&v ~"& Ö¦"v , 4ãç&v ®", 5«ö, 6Ï§&v ®". 3. Genotypic and Phenotypic Characteristics of Staphylococcus aureus Isolates from Lettuces and Raw Milk Hye-Jin Jung, Joon-Il Cho, Sung-Hee Park, Sang-Do Ha, Kyu-Ho Lee1, Cheol-Ho Kim2, Eun-Seop Song3, Duck Hwa Chung4, Min-Gon Kim5, Kwang-Yup Kim6, and Keun-Sung Kim* Department of Food Science and Technology, Chung-Ang University Department of Environmental Science, Hankuk University of Foreign Studies 2 Department of Biochemistry and Molecular Biology, College of Oriental Medicine, Dongguk University 3 Department of Obstetrics and Gynecology, College of Medicine, Inha University 4 Division of Applied Life Science, Gyeongsang National University 5 Laboratory of Integrative Biotechnology, Korea research Institute of Bioscience and Biotechnology 6 Department of Food Science and Technology, Chungbuk National University 1. To characterize genotypic and phenotypic traits of Staphylococcus aureus isolates (n = 86) from lettuces and raw milk, major virulence-associated genes and antibiotic susceptibility were detected using PCR-based methods and disk diffusion method, respectively. All isolates possessed coagulase gene and showed five polymorphism types [500 bp (2.4%), 580 bp (17.4%), 660 bp (61.6%), 740 bp (17.4%), and 820 bp (1.2%)] due to variable numbers of tandem repeats present within the gene. Two or three different loci of hemolysin gene family were dominant in isolates, 47 of which (55%) possessed combination of hla / hld / hlg-2 genes as the most prevalent types. Among enterotoxin-encoding genes, sea was detected from 32 isolates (37%), sed from 1 isolate (1%), and sea and sed genes were co-detected from 4 isolates (5%), whereas seb, sec, and tsst-1 genes were not detected. All isolates were susceptible to ciprofloxacin, trimethoprim/sulfamethoxazole, oxacillin,and vancomycin, 85 isolates (99%) to penicillin G, 54 isolates (63%) to chloramphenicol, 51 isolates (59%) to erythromycin, and 7 isolates (8%) to clindamycin. Among resistant isolates, seven displayed multiantibiotic-resistance against two different antibiotics. Key words: Staphylococcus aureus, coagulase, hemolysin, enterotoxin, antimicrobial susceptibility.. *. . aureusö ~ 7ëf 9.62% Ë"j2ÒJ, Salmonella spp. ö Ú ^ ® ô B~º 7ëj ¢V~º ö b rJ^ ® (1-5). S. aureusº ?"~ ·Ïj O~ ÷öW êç ÿ;~ 7º æ& >º ÚW~ extracellular enzyme coagulasef exotoxin hemolysin(Ï.ë²), 7ë~ ö >º enterotoxin(Ë&ë²), Ò toxic shock syndromej ¢ bÊº toxic shock syndrome toxin 1(TSST-1, Wë²) · ëW¶¢ Ö~º ©b rJ^ ® (3,6). ëW¶ 7 staphylococcal enterotoxinf S. aureus& ® ÚöB Ã~º ";ÿn ÖB ©b Clostridium botulinum 7ë" îR&æ enterotoxinj SbB B> º &' ë²; 7ë FBbî(7,8), 1 µg ~~ ë ²ê ÚÚöB 7ëj BÒ > ® . S. aureus& Ã. Staphylococcus aureusº V, Æ· ö 6Ò ª~, ß ® Wî, ê>zb ôf ®ö J"F &ËW Ö ¸ , ®b~ J"ãê Ö ·~ 7ë~ öb ® *ç 7º~² *æ ® . $ "ö ^ê ' öB S. aureusö ~ 7ë B nê& ¸jæ ® Ö Ò ¾¢~ ãÖ, 2003jê Ú 7ë B Ûêö V S. *Corresponding author: Keun-Sung Kim, Department of Food Science and Technology, College of Industrial Sciences, Chung-Ang University, 72-1, Ansung-si, Gyunggi 456-756, Korea Tel: 82-31-670-3032 Fax: 82-31-675-4853 E-mail: [email protected] 134.

(3) çºf öFöB ªÒ ï êç~ b' ßû. r ÖB enterotoxin f ößWö V¢ A, B, C1, C2, C3, D, E, G, H, I, J~ 11«b ª>, S. aureusf . ë²;7 &æ $º v &æ; ç~ ë²¢ Ö~º ©b rJ^ ® . " A;¾ D;ö ~ 7ë Òf& ôf ©b > ®, $ enterotoxin Bº 7 & Ë ; ÚWj &ê ©b rJ^ ® (9,10). $, S. aureusº ~òª¢öB B ÚW Ã& ~ z× ² ^B> ® . >j *¦V ï êçö ~ 6"Ã ~ò¢ * >ôf bî BB>Ú zb¾, çÏ Bö & Â n®öö V¢ ~ &¦ ª «~~ Bö & 6>Wj ç ~ ÚW _ f ¸f &Wj æî² >î . ß® 1960j&¦V ªçöB methicillin-resistant S. aureus(MRSA)~ ªÒ& >V ·~ B *Òræ *Ú S. aureus 7öB MRSA~ ªÒnêº 6 6 ¸jæ ®º ; (11,12). Ò " ªçöBò ¦ Â>î~ MRSA& ®öB ªÒ>î º (13) Ú j ® /¶ 5 ²j¶ö² MRSAö & 6" B * þ ¸jæ ®j ©b .ç> MRSA ö ÚÊ Böê &>æ pº vancomycin-resistant S. aureus(VRSA) ~ Â*b 'Wf 6N & ®º ; . V¢B öBº, Ñ ÖF 5 çº~ ®j ²j ê S. aureusö ~~ 7ë B~&~ Òf& B(14) ãÖ& ®bæ ÆÒ' 7ë FB^bB çºf öF ¦V S. aureus¢ ªÒ~& . ~ ªÒ S. aureus "¢ &çb ~ ÷öW" &ê& pf ©b rJê coagulase f hemolysin, enterotoxin, TSST-1(toxic shock syndrome toxin) F*¶¢ PCR O»j Ï~ {~& . Ò îæïb "~ B ÚW ;ê¢ ÚÚ¶ vancomycinj j 8«~ Bö & 6>W ¦Ò¢ ~ S. aureus ª Ò"~ b' ßWj Ò~¶ ~& .. Òò 5 O» Staphylococcus aureus ªÒ 5 { 2003j 7ú" 9ú, ãVê nW~ ~¾ îÞf BÞ &£ ÿ ³Öb ËöB « çºf nW ²Ò ÏË~ <² ¦V OFB öF¢ '' 25 g _f 25 mLO Z'b ~ 225 mL~ 0.1% phosphate bufferö I stomacher(Casta Brava, Spain)¢ Ï~ 2ª* îz Î ê ç[ 7 1 mLj brain heart infusion(BHI, Difco Laboratories, MI, USA) brothö 7«~ 37oCöB 24* V·~& . V· 1 mLj 10% sodium chloride& FB tryptic soy broth (TSB, Difco Laboratories, MI, USA)ö 7«~ 37oCöB 24 * Ã V·~& . ÃB j Baird Parker plate(BPP, Difco Laboratories, MI, USA)ö ³F V·~ 37oCöB 24 * V· ê ¦; ÷£" clear zone ¾æÂ ÷£ö & { þj ~& . S. aureus~ { þf *f ? ªÒ V·B ï6 Væ ç~ ÷£ö &~ r" ? ~& . *;j {~ º O»b gram staining, catalase test, gelatinase production, egg-yolk reaction, mannitol" glucose fermenting test Ò Maduuxf Koehne(15)~ O»ö V API-Ident System(API Staph, Bio-merieux, France)j ~& . $ F*;j { ~º O»b S. aureus& &æº femA F*¶(16), nucA F* ¶(17) Ò S. aureusò F' Sa442 F*¶(18)¢ ¦Â. 135. ~º PCRj >¯~&, methicillin-resistant S. aureus(MRSA) æ ¦¢ 6;~V * mecA F*¶(19)¢ ¦Â~º PCR ê ÷¯~ ~& . PCRj Ï coagulase, hemolysin, enterotoxin F*¶ ¦Â R: þö staphylococcal enterotoxin typeê 4« ~ S. aureus &" ÒÏ>îb, SEAº KN556, SEBº H288a, SECº ATCC 19095, Ò SED 1634/ 93 '' ÒÏ>î . Total DNA ºÂ: S. aureus ªÒ "¢ Luria-Bertani(LB) broth 2 mLö 7« ê 37oCöB 180 rpmb êû V·~&. . Ã V·j ELISA reader(Molecular Devices, CA, USA) 600 nmöB 7ê¢ G;~ O.D. 8j 0.5& >² ; ê '' 1 mLO ~ 15,000 rpmb 5ª* öª Ò~& . V·b¦V ç[j ªÒ áf pelletj 4% Chelex 100(Bio-Rad Laboratories, CA, USA) 400 µL¢ I *çV . Ò *çj 95oC water bathöB 15 ª* y ê 15,000 rpmöB 5ª* öªÒ~ ç[ j îÚ þ&b TV ¢ PCR ªCj * template DNA ÒÏ~& . PCR ª+ # {: PCR >w bf 5 µL~ 10Ü reaction Buffer(« ³ê-400 mM KCl, 15 mM MgCl2, 100 mM Tris-HCl, pH 9.0), 5 µL~ dNTP, 1 U~ Taq DNA polymerase (Bioneer, Daejeon, Korea), 25 pmol ' primer 1 µL 5 DNA template 20 µL~ bö Ã~>¢ « 50 µL& >ê Î &~ PTC-100(MJ Research, Inc., MA, USA)öB ÃV . þö ÒÏ primersº Table 1ö ¾æÚîb, PCR Ö bf 1.5% agarose(1ÜTAE) gelöB 120V 1* *V'ÿ~ ethidium bromide(5 µg/mL) "ï ê transilluminator {~& . Öb~ Vº 100 bp Plus DNA Ladder(Bioneer, Daejeon, Korea)¢ ÒÏ~ G;~& . Coagulase F*¶ ¦Â: Hookey (20)ö ~ nB coagulase F*¶ ¦ÂÏ primer¢ ÒÏ~&, r ÒÏ PCR ~ *" Nê f r" ? . 94oCöB 4ª* & ê, denaturationº 94oCöB 1ª, annealingf 55oCöB 1ª, Ò extensionf 72oC 1ªb 30 cycle >~&, îæïö 72oCöB 10ª* z >wV . Hemolysin F*¶ ¦Â: Jarraud (21) n α-hemolysin (hla), β-hemolysin (hlb), γ-hemolysin (hlg-2), δ-hemolysin (hld) F*¶¢ ¦Â > ®º PCRj ~ hemolysin typej Ö; ~& . Hemolysin F*¶ ¦Âj * PCR f 95oCöB pre-denaturationj 5ª ê denaturationj 94oCöB 1ª, annealingj 55oCöB 1ª 5 extensionj 72oCöB 1ªj 30 cycle >~&b 72oCöB « extensionj 10ª~ ;æ ~& . Staphylococcal enterotoxin F*¶ ¦Â: Staphylococcal enterotoxinj A-D;b ª > ®º Rosec (22) n primers¢ &æ PCRj ~&, PCR f r" ? . 94oCöB pre-denaturation 3ª Ê 94oC denaturation 30.

(4) ®"²æ B 37 ² B 1 ^ (2005). 136. Table 1. Sequences of the primers used in this study and sizes of their amplicons Genes. Primer names. femA. femA-1 femA-2. nucA. Amplicon sizes (bp). References. 5'-CTTACTTACTGGCTGTACCTG 5'-ATGTCGCTTGTTATGTGC. 686. 16. nucA-1 nucA-2. 5'-GCGATTGATGGTGATACGGTT 5'-AGCCAAGCCTTCACGAACTAAAGC. 280. 17. Sa442. Sa442-1 Sa442-2. 5'-AATCTTTGTCGGTACACGATATTCTTCACG 5'-CGTAATGAGATTTCAGTAGATAATACAACA. 108. 18. mecA. mec1 mec2. 5'-AAAATCGATGGTAAAGGTTGGC 5'-AGTTCTGCAGTACCGGATTTGC. 533. 19. coa. coa-1 coa-2. 5'-ATAGAGATGCTGGTACAGG 5'-GCTTCCGATTGTTCGATGC. size polymorphisms. 20. hla hlb hld hlg-2. hla-1 hla-2 hlb-1 hlb-2-2 hld-1 hld-2 mphlg-1 mphlg-2. Primer sequences. 5'-CTGATTACTATCCAAGAAATTCGATTG 5'-CTTTCCAGCCTACTTTTTTATCAGT 5'-GTGCACTTACTGACAATAGTGC 5'-GTTGATGAGTAGCTACCTTCAGT 5'-AAGAATTTTTATCTTAATTAAGGAAGGAGTG 5'-TTAGTGAATTTGTTCACTGTGTCGA 5'-GACATAGAGTCCATAATGCATTYGT 5'-ATAGTCATTAGGATTAGGTTTCACAAAG. 209 309 21 111 390. seu sea seb sec sed. SE-f sea-r seb-r sec-r sed-r. 5'-TGTATGTATGGAGGTGTAAC 5'-ATTAACCGAAGGTTCTGT 5'-ATAGTGACGAGTTAGGTA 5'-AATTGTGTTTCTTTTATTTTCATAA 5'-TTCGGGAAAATCACCCTTAA. 270 165 102 306. tsst-1. tst-1 tst-2. 5'-ATCGTAAGCCCTTTGTTG 5'-GTGGATCCGTCATTCATTG. 578. ., 40oC annealing 30. 5 72oC extension 1ªj 30 cycle > ~&b îæïö 72oCöB extensionj 2ª~ ;æ~& . Toxin shock syndrome toxin 1 (tsst-1) F*¶ ¦Â: Moore (23) n primer¢ ÒÏ~ PCRj ~&, PCR f 94oCöB pre-denaturation 3ª Ê 94oC denaturation 30., 40oC annealing 30. 5 72oC extension 1ªj 30 cycle >~&b 72oCöB « extensionj 2ª~ ;æ~& . B 6>9 ¦Ò Bauer (24)~ disc diffusion methodö ~~ B 8«ö &~ 6>W þj ~& . ªÒ "¢ Muller-Hinton broth(MHB, Difco Laboratories, MI, USA)ö 7«~ 37oCö B 24* V·~& . V·j ELISA reader(Molecular Devices, CA, USA) 600 nmöB 7ê¢ G;~ O.D. 8 j 0.5 C~ ; ê 15ª Úö B /j Ï~ 4 mm vþ &j Muller-Hinton Agar(MHA, Difco Laboratories, MI, USA) plateö ¢~² êö~& . êö ê 5ª* ï6Væ¢ Î r B fqb ''~ B 6>W ¦ÒÏ :Ê(Fluka, MO, USA)¢ 20 mm ç Ò¢ Fæ~B ï6Væ *ö ;V . Disk& ;B ï6Væ¢ 37oC V·VöB 24* V· ê G &æ~~ çãj G;~& . G &æ~ çãö V Ö"º National Committee for Clinical Laboratory Standardization(NCCLS)ö &~ 6>W ¦¢ 6ë~& (25).. 22. 23. Ö 5 8 S. aureus ªÒ 5 { ¢>'b S. aureusº gram staining" catalase testö ·W gelatinj z~ WË > ® egg-yolkö ~ G &>æ pb 6V' öB glucosef mannitolj Ï~ ¾ WË~º ©b rJ^ ® . öFf çºöB F Væ¢ Ï~ '' ªÒ 124Bf 100B~ ªÒ"ö &~ *Û' O»ö ~ S. aureus { þj Ö" º Table 2öBf ? Gram stainingf 142"(63.4%)öB · W>wj &, catalase testº 90"(40.2%), gelatinase Ö f 131"(58.5%), Egg-Yolk reactionj ®jr ¾ WË~ º "º 208"(92.9%), Ò glucosef mannitolj BÎ ~ WË&Ë "º 162"(72.3%)& . ¾ çöB ö 6«~~ S. aureus { O»f þ"; ÿ÷£' , « >&~ ªÒöBº ª¶b' O»¾ PCRj Ï O»ö j~ ;{W Ôbæ öFf çºöB ªÒ ªÒ"¢ &çb S. aureus& &æº femA(16), nucA(17), mecA F*¶(19) Ò S. aureusò F' Sa442 F*¶ (18)¢ Ï PCRj ~& . Ö" S. aureus~ target F*¶7 Sa442~ ãÖ 86"(38.4%)öB {>î, femA 86"(38.4%)öB {>îb, nucAº 144"(64.3%) öB {>î . þöBº Sa442, femA, nucA F*¶& Îv {> S. aureus Fê~&º C 86"(38.4%)& S. aureusª {>î . ¾ MRSA& &æº mecA F*.

(5) çºf öFöB ªÒ ï êç~ b' ßû. 137. Table 2. Numbers of bacterial isolates from lettuces and raw milk showing various phenotypic and genotypic characteristics No. of isolates (%). Typing methods. Lettuces. Raw milk. Total. 100(100.0) 63(63.0) 39(39.0) 59(59.0) 88(88.0) 73(73.0). 124(100.0) 79(63.7) 51(41.1) 72(58.1) 120(96.8) 89(71.8). 224(100.0) 142(63.4) 90(40.2) 131(58.5) 208(92.9) 162(72.3). API-kit. 37(37.0). 49(39.5). 86(38.4). nucA gene Sa442 gene femA gene mecA gene. 85(85.0) 37(37.0) 37(37.0) 0(0.0). 59(47.6) 49(39.5) 49(39.5) 0(0.0). 144(64.3) 86(38.4) 86(38.4) 0(0.0). Selective media Gram staining Catalase test Gelatinase production Egg-Yolk reaction glucose and mannitol fermentation. Table 3. Results of PCR detections on coa gene of S. aureus isolates from lettuces and raw milk Amplicon sizes. No. of S. aureus isolates (%) Lettuces (n=37). Raw milk (n=49). Total (n=86). 500 bp 580 bp 660 bp 740 bp 820 bp. 1 (2.7) 0 36 (97.3) 0 0. 1 (2.0) 15 (30.6) 17 (34.7) 15 (30.6) 1 (2.0). 2 (2.4) 15 (17.4) 53 (61.6) 15 (17.4) 1 (1.2). Total. 37 (100.0). 49 (100.0). 86 (100.0). ¶¢ ¦Â~º PCRöBº ÚÊ "öBê &ç F*¶& ¦ Â>æ pj þöB çºf öF¦V ªÒ S. aureus "º Îv MSSA ©b 6«>î . $ *Û' O»" PCR O»öB ¢~B Ö"¢ 86B~ ªÒ"ö &B API staph kit¢ Ï~ S. aureusªj { Ö", ''~ Î "& S. aureus (P = 97.8%)ª {>î . Ö"'b öBº öF¦V 49B "(39.5%)f çº¦V 37 B "(37.0%)¢ S. aureus ÿ;~&b, ~ ï ªÒ nêº 38.4%î . º Kang (2) bF¦V áf 24.6% ¸f ªÒnê . Coagulase F*¶ ¦Â Coagulaseº ÚW~ extracellular enzymebB ÷öW êç ÿ;~ 7º æ& > ?"~ ·Ïj O ~ ëW¶ ·Ï . - staphylococci¢ ÿ;~º &Ë FÏ O» coagulase testöº Ò¢»" þ&» ® . ¾ Ò¢»f *·W $º rWÖ"¢ .¾ >ê ®b, þ&»f £ ·W >wj ¾æâ >ê ®, >w*ê J¾ Òº 6 ® . æ ;{~ *Úö 6ë > ®º coagulase F*¶¢ ¦ï~º PCR» n>îb, þöBº Hookey (19) n coagulase F*¶ ¦ÂÏ PCR primer¢ &æ coagulase test¢ >¯~& . coa(coagulase) F*¶º 81 bp short tandem repeat sequence ¢ &æ ®b "î >>º Å>& ·~ Ö"' b amplicon size& "î. ·~² ¾æÂ (26). *çf $ "ö Scherrer (27) "²f ·~ öF¦V ªÒ S. aureus~ coa F*¶ö & amplicon size& 500 bp. (2.7%), 580 bp(15.4%), 660 bp(32.4%), 740 bp(23.2%) 5 820 bp (23.5%) ·~² ¦Â>î B { > ® . þöB çºf öF¦V ªÒ 86B~ S. aureus ªÒ "¢ &æ coa F*¶ ¦Â test¢ Ö"ö ~~ Table 3öBf ? Î ªÒ"öB coa F*¶ö ·W>w j ¾æÚî . Ò $ PCR Öb~ amplicon sizeº 500 bp(2.4%), 580 bp(17.4%), 660 bp(61.6%), 740 bp(17.4%) 5 820 bp(1.2%) U «~& ¦Â>î . Hemolysin F*¶ ¦Â S. aureus& &æ ®º ë² bî7ö exotoxin hemolysin (Ï.ë²) ® . S. aureus~ hemolysin typef 5%~ sheep blood& FB blood agar plate(BAP)öB '.¢ Ï~º Wîö V¢ 3b ª~>º α-hemolysinf ®j* Ï .j "Ú Ï.&& § β-hemolysinf j* Ï.j "Ú clear hemolysis zonej ;W~ γ-hemolysinf Ï.W ìÚB Ï.&& æ pº . ¾ 3b ~ ª~V&f 6; ®{ ÷£ ôjB ª~ ôf ^ B6j ¢V . 6j j~¶ *;ö ~~ æ p PCRj Ï~ hemolysin F*¶¢ ¦Â~º O» " ô ÒÏ>Úæ ® . þöB 86B~ S. aureus ªÒ"ö & hemolysin F*¶ ¦Â ¦¢ Ò Ö"º Table 4f ? Î "öB hemolysin F*¶& ¦Â>î . $ öF¦V ªÒ 4B~ "(hld F*¶ F")¢ B Î ªÒ"º hemolysinö &~ 2« ç «~ ~ multiple genej ²F~&, 7 47B "(54.7%)& hla / hld / hlg-2 F*¶¢ Îv ²F "B &Ë ôf ª¢ ¾æÚî . º Kang (15) öFöB ªÒ S. aureus ª.

(6) ®"²æ B 37 ² B 1 ^ (2005). 138. Table 4. Results of PCR detections on hla, hlb, hld, and hlg-2 genes of S. aureus isolates from lettuces and raw milk Hemolysin genes. No. of positive S. aureus isolates (%) Lettuces (n=37). Raw milk (n=49). Total (n=86). hld hla and d hld and g-2 hla, d and g-2 hlb, d and g-2. 0 17 (45.9) 1 (2.7) 19 (51.4) 0. 4(8.2) 14 (28.6) 0 28 (57.1) 3 (6.1). 4 (4.7) 31 (36.0) 1 (1.2) 47 (54.7) 3 (3.5). Total. 37 (100.0). 49 (100.0). 86 (100.0). Table 5. Results of PCR detections on sea, seb, sec, sed, and tsst-1 genes of S. aureus isolates from lettuces and raw milk Enterotoxin genes. No. of positive S. aureus isolates (%) Lettuces (n=37). Raw milk (n=49). Total (n=86). sea seb sec sed sea and sed tsst-1. 11 (29.7) 0 0 0 0 0. 21 (42.9) 0 0 1 (2.0) 4 (8.2) 0. 32 (37.2) 0 0 1 (1.1) 4 (4.6) 0. Total. 11 (29.7). 26 (53.1). 37 (43.0). Ò" 7 multiple hemolysin gene ²F"& 55.6%& º f Salasia (28) êJj~ central JavaæO~ öFö B ªÒ S. aureus ªÒ" 7 multiple hemolysin gene ²F "& 27.8% ªÒ" 7 &Ë ¸f ªÒjNj ¾æÚî. Ö"f FÒ~& . Enterotoxin, TSST-1 F*¶ ¦Â Staphylococcal enterotoxinf Ò²ö² gastroenteric syndrome j ¢bÊ¾ toxic shockj ¢bÒ > ®º extracellular protein . S. aureus& Ã r ÖB enterotoxinf öß Wö V¢ SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, SEG, SEH, SEI 5 SEJ~ 11&æ ö; >Ú ®b, SEF($ º TSST-1)º 1.Ó; >Ú ® . $ "ö Orwin (29)f superantigenicity, pyrogenicity, enterotoxin~ ~ÒÎ"¢ Ã&Ê Æ¢öB ~ÒÎ"¢ &æº SEK¢ ªÒ~& ~& . Staphylococcal enterotoxinf in vivo $º in vitroöB b ' Wö ~ ¦ÂF > ®bæ þ ÚöB *~² latex agglutinationö ~ ¦Â O» ¢>'b 6Ò Ï> Ú z . ¾ post-genome & º¾öº *öB B staphylococcal enterotoxin F*¶~ "VB «>Ú . j Ï PCRO» B® >Ú &ÚO»b Ï > ® . ¢ *~ çºf öF¦V ªÒ S. aureus¢ PCR O»j Ï~ staphylococcal enterotoxin F*¶¢ ¦ï Ö ", Table 5öBf ? 86B~ S. aureus ªÒ" 7 37" (43.0%)& enterotoxin F*¶¢ ²F~& . F*;ê sea F*¶& 32"(37.2%)B Ö6;b ¾æÒb, sed F*¶& 1"(1.1%), seaf sed F*¶& Îv ¦ÂB ªÒ "º 4"(4.6%)& . º Lim (30) öF¦V ªÒ S. aureus " 7 sea F*¶¢ ²F "& 19.3% &Ë ¸² ¾æÒ º Ö"f ¢~~& . $ ÚÊ ªÒ. "¦V ß; enterotoxin F*¶& ¦Â> " Ú öB F*¶¦V >º enterotoxin B*>º ©b rJ^ ®bæ(31), PCRj Û~ áf þ~ Ö"º ¢ >'b ®öB ªÒB "7 enterotoxin type A& ô ¦*B V~ þÖ" " ¢~ > ®. . Casman (32)f Ö ïÿ®" öFöB ªÒ ï êçb ë² Ö þj Ö" enterotoxin type A f type~ b; 50% çj Næ~ ·<öBº enterotoxin type Af D& *Ú~ 35%¢ Næ~&b, ß® » ÖB®" GB®¢ ãÖ 96%& enterotoxin type Af type~ b;¢ ~& . $, Sokari (33)f ¾æÒ j GB®öB enterotoxin type A, B, D 5 C~ jN ' ' 57%, 15%, 6% 5 5%¢ ~& ÖÒ¾¢~ ãÖê fKöB ªÒ S. aureus "ö & enterotoxinj þ Ö"ö ~~ enterotoxin type A& 42.5% &Ë ¸² ¾æ ¾ þöB sea F*¶& ¸f nê ¦ÂB Ö"f ¢ ~~& (5). Þ, þöBº sebf sec 5 tsst-1 F*¶º ¦Â>æ p~º, º GB®öB sec 5 sed F*¶& ô ¦Â B º r~ fº ç~& . Hazariwala (34)~ &. ~ F¾ S. aureus ªÒ"ö &~ enterotoxin F*¶ ¦Â þj Ö"ö ~~ sea, seb, sec 5 sed F*¶& '' 12.2%, 2.4%, 22%f 24.4% '' ¦Â>Ú sec 5 sed F* ¶~ ¦Â nê& ¸~ . z× Salasia (28)f ë¢~ Hesse æO öFöB ªÒ S. aureus " f secf sed F*¶¢ '' 57.9%f 15.8%O F~ ® ~& . $ Sharma (35)ö ~~ ·<b¦V ªÒB S. aureus ªÒ "öBº sea F*¶& 3.0%, sec F*¶º 61.2% Ò sea f sec F*¶& 9.0%O '' ¦Â>î, &.~öB ªÒB S. aureus ªÒ"öBº sed F*¶& 50.0% ¦Â>î ~& . Þ Klotz (36)f ªçöB ªÒ S. aureus ªÒ "öBê sea, seb, sec 5 sed F*¶~ ¦Â jN 12.9%,.

(7) çºf öFöB ªÒ ï êç~ b' ßû. 139. Table 6. Results of antibiotic susceptibility tests on S. aureus isolates from lettuces and raw milk Antibiotics. Sources. Chloramphenicol. No. of isolates (%) susceptible. intermediate. resistant. Lettuce (n=37) Raw milk (n=49) Subtotal. 23 (62.0) 31 (63.0) 54 (62.8). 13 (35.0) 15 (31.0) 28 (32.6). 1 (3.0) 3 (6.0) 4 (4.7). Ciprofloxacin. Lettuce (n=37) Raw milk (n=49) Subtotal. 37 (100.0) 49 (100.0) 86 (100.0). 0 0 0. 0 0 0. Clindamycin. Lettuce (n=37) Raw milk (n=49) Subtotal. 3 (8.0) 4 (8.0) 7 (8.1). 2 (5.0) 6 (12.0) 8 (9.3). 32 (87.0) 39 (80.0) 71 (82.6). Erythromycin. Lettuce (n=37) Raw milk (n=49) Subtotal. 23 (62.0) 28 (57.0) 51 (59.3). 12 (32.0) 11 (22.0) 23 (26.7). 2 (6.0) 10 (21.0) 12 (14.0). Oxacillin. Lettuce (n=37) Raw milk (n=49) Subtotal. 37 (100.0) 49 (100.0) 86 (100.0). 0 0 0. 0 0 0. Penicillin G. Lettuce (n=37) Raw milk (n=49) Subtotal. 37 (100.0) 48 (98.0) 85 (98.8). 0 0 0. 0 1 (2.0) 1 (1.2). Trimethoprim+ sulfamethoxazole. Lettuce (n=37) Raw milk (n=49) Subtotal. 37 (100.0) 49 (100.0) 86 (100.0). 0 0 0. 0 0 0. Vancomycin. Lettuce (n=37) Raw milk (n=49) Subtotal. 37 (100.0) 49 (100.0) 86 (100.0). 0 0 0. 0 0 0. 9.7%, 21.5% Ò 14.0% ¾æ¾ secf sed F*¶~ ¦Â nê& ç&'b ¸ ~& . Ö'b ¢^öB çºf öF¦V ªÒ S. aureus ªÒ"~ enterotoxin F*¶ ¦Â þöB sea F*¶& & Ë ¸f nê ¦ÂB Ö"º Lim (30)~ Ö"f ¢~>î b¾, &.~f F F¾ Ò ªçöB ªÒ S. aureus ªÒ"öB secf sed F*¶& " ¦Â>î º Hazariwala (34), Salasia (28), Sharma (35) Ò Klotz (36)~ fº ç~& . ¾ ®öB ªÒB " ~ ãÖ &¦ª enterotoxin type A¢ " W º (5,32,33) ê ® . æ ÚÊ «~~ enterotoxin typej W~º S. aureus& ÚÊ ®ê " J">ºæö & Ò& ¦ z B® Ú^¢ ©b 'B . B 6>9 þ S. aureusº β-lactamase¢ Ö~æ β-lactamaseö ~~ ®Wz>º Bö & ÚW Ö ¸b º βlactamase Öö &~º ÚW plasmid(R-plasmid)~ ³ö ~ " BB . β-lactamase Ö S. aureus~ Î" ' ~ò¢ *~ Î²ö ª>æ pº methicillin ~. · B& BB>Ú penicillin ÚW"~ ~òö ÒÏ> îb¾ *Òº methicillinj · Bö & Ú W"~ Â*b S. aureus 6"Ã ~òö ôf ÚJæj Ð ®º ; . $ Jones (13)f " ªçöBò ¦Â >î~ MRSA& :jvB "æVf coleslawöB ªÒ>î. ©b Ú j ® /¶ 5 ²j¶ö² MRSAö & 6"B *þ ¸jæ ®j ©b .ç> Ú, * ªÒ"~ ÚW ;ê¢ 2k~¶ B 6>W. þj ~& . öFf çºöB ªÒ S. aureus 86B "~ chloramphenicol, ciprofloxacin, penicillin G, oxacillin, erythromycin, trimethoprim/sulfamethoxazole, vancomycin 5 clindamycin 8«~ Bö & 6>W þj Ö"ö ~~ Table 6" ? Î "& ciprofloxacin, oxacillin, vancomycin 5 trimethoprim/sulfamethoxazoleö & 6>Wj ¾æÚî penicillin G(98.8%)ö & ¸f 6>Wj &b¾ chloramphenicol(63%), erythromycin(59%)ö & 6>Wf Ô~ . ß® chloramphenicol" erythromycinö & 7* ;ê~ ÚWj ¾æÞ "º '' 32.6%f 26.7%î . Chloramphenicol, clindamycin, erythromycin 5 penicillin Gö &~º '' 4.7%, 82.6%, 14.0% 5 1.2%~ "& ÚWj &æ ®î, þ S. aureus 86" 7öB 81"(94.2%)º 'Úê bîö &~ ÚWj ¾æÚî . 2&æ bîö &~ ÚWj ¾æÞ "º 7"(8.1%) 7 4"(4.7%)º chloramphenicol" clindamycinö ÚWîb, 3"(3.5%)º clindamycin" erythromycinö &~ ÚWj ¾æÚîb¾ 3&æ ç ~ bîö ÚWj º "º ¾æ¾æ p~ . º Speller (37) '" ¢®öB ªÒ êç~ methicillin ÚWf 1989-1991jræº 1.5%îb¾ 1995jöº 13.2% /Ï® Ã&~&b, ÿö erythromycin, clindamy-.

(8) ®"²æ B 37 ² B 1 ^ (2005). 140. cin, ciprofloxacin, gentamicin, trimethoprim, rifampicinö & ÚW Ã&~& ©" Baiocchi (38) oxacillin ÚW 1980j& 39%öB 1990j& 69% Ã&~&b, ciprofloxacin, clindamycin, rifampicinö & ÚW Ã&~&. ÚÏ" FÒ Ö" . $, ªÒB S. aureus7öB MRSAæ jòæ ¦¢ 6 ;~V * mecA F*¶¢ Ï PCRj Ö" Î "& methicillin-susceptible S. aureus(MSSA) C&r, B þ Ö" * "& vancomycin" oxacillinö 6>Wj ¾æÚÚ jçræ ®öB VRSA¾ MRSA~ ¦Â nê&. Ö Ôf ©b ÒòB . ¾, Ha (9)~ ¢^öB . v /²~ >, Ò «Ò¶~ ¶, ~î, ïË ö B ªÒ S. aureus "º Îv B ampicillin" penicillin ö ÚWj &, ß® /²~ >¦V ªÒ S. aureus "~ ¢¦º oxacillinö ÚWj &æº MRSA " ©b ¾æÒ º r > ® B~ Ìf Â ~ ªçöBöò jî¢ ®~ã¾ ¢> "~ãöBê 6N B ÚWj <º "~ Â* Ã& º^ö ®Ú ª¢ö & Úê' æ³' & ºÓ> ® .. º. £. S. aureusº ¶êö 6Ò çÒ ®b ÖÒ ¾¢~ ãÖ Salmonella spp.ö ~ 7ë rb ô B~º 7ë ~ öb rJ^ ® . V¢B þöBº çºf ö F¦V ªÒ S. aureus(n = 86)~ *; 5 F*; ßûj 2k Ï'b coagulase, hemolysin, enterotoxin 5 toxic shock syndrome toxin 1 ö & F*¶¢ &çb PCR O »j Ï~ typing~&b, $ 8&æ Bö &~ B 6>W þj >¯~ ªÒ" ~ B ÚW ;ê¢ Ö;~& . çºf öF¦V ªÒ S. aureus " º Îv coagulase F*¶¢ F~ ®î, $ PCR Öb ~ amplicon sizeº 500 bp(2.4%), 580 bp(17.4%), 660 bp(61.6%), 740 bp(17.4%) 5 820 bp(1.2%) U «~& ¦Â>î . Hemolysin F*¶~ ãÖº öF¦V ªÒ 4B~ hld F* ¶ F"¢ B~ Î ªÒ"öB multiple hemolysin genej F~&b, 7 47B "(54.7%)& hla / hld / hlg2 F*¶¢ Îv ²F "B &Ë ôf ª¢ ¾æÚî. . Þ, enterotoxin F*¶¢ ²F S. aureus ªÒ"º 37 "(43.0%)B 7 32"(37.2%)öB sea F*¶& ¦Â >î, 1"(1.1%)öB sed F*¶, Ò 4"(4.6%)öB seaf sed F*¶& Îv ¦Â>îb¾, seb, sec _f tsst-1 F *¶º ¦Â>æ p~ . Ò B 6>W þ Ö"öB º Î "& ciprofloxacin, oxacillin, vancomycin Ò trimethoprim/sulfamethoxazoleö &~ 6>Wj ¾æÚî penicillin G(98.8%)º ¸f 6>Wj &b¾ chloramphenicol (63%)" erythromycin(59%)f 6>W Ô~, chloramphenicol, clindamycin, erythromycin 5 penicillin Gö &~º '' 4.7%, 82.6%, 14.0% 5 1.2%~ "& ÚWj &æ ®î . $ B BÚW ·çj ÚÚ Ö"ö ~~ ÒÏB 8 «~~ bî 7 2&æ bîö &~ ÚWj ¾æ Þ "º 7B(8.1%), 3« ç~ bîö &~ B ÚWj º "º ¾æ¾æ p~ .. 6Ò~ º æ¦ ~òVFêÒë~ æöö ~~ >¯>îb, ö 6Òãî (03-PJ1-PG1-CH11-0003).. ^. ò. 1. Chung GS, Tak RB. Effects of pH, temperature and food additives on staphylococcal growth and enterotoxin production. Korean J. Vet. Publ. Hlth. 17: 13-38 (1993) 2. Kang HJ, Choe HG, Son WG. Incidence and characterization of coagulase-positive Staphylococcus aureus in bulk milk from dairy farms. Korean J. Vet. Publ. Hlth. 14: 9-13 (1990) 3. Kim SG, Lee YS, Lee TJ, Lee TY, Kim HS. Identification and antimicrobial susceptibility aspects of pathogenic Staphylococcus aureus. J. Korean Soc. Microbiol. 28: 251-259 (1993) 4. 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Screening of MRSA (Methicillin Resistant Staphylococcus aureus) and seb Gene in producing strains isolated from food service environmental of elementary schools. J. Food Hyg. Safety 18: 79-86 (2003) 10. Park SG, Hwang YO, Jung JH, Lee KM. Biological characteristics of Staphylococcus aureus isolated from food-borne patients in seoul. J. Food Hyg. Safety 16: 159-167 (2001) 11. Hussain Z, Stoakes L, Garrow S, Longo S, Fitzgerald V, Lannigan R. Rapid detection of mecA-positive and mecA-negative, coagulase-Negative staphylococci by an anti-penicillin binding protein 2a slide latex agglutination test. J. Clin. Microbiol. 38: 2051-2054 (2000) 12. Reischl U, Linde HJ, Metz M, Leppmeier B, Lehn N. Rapid identification of methicillin-resistant Staphylococcus aureus and simultaneous species confirmation using real-time fluorescence PCR. J. Clin. Microbiol. 38: 2429-2433 (2000) 13. Jones TF, Kellum ME, Porter SS, Bell M, Schaffner W. An outbreak of community-acquired foodborne illness caused by methicillin-resistant Staphylococcus aureus. Emerg. Infect. Dis. 8: 82-4 (2002) 14. Bergdoll MS. Foodborne infections and intoxication. 2nd ed. Bryan FL. Academic Press, NY, USA (1979) 15. Maddux RL, Koehne G. Identification of Staphylococcus hyicus with the API Staph. J. Clin. Microbiol. 15: 984-986 (1982) 16. Vannuffel P, Gigi J, Ezzedine H, Vandercam B, Delmee M, Wauters G, Gala GL. Specific detection of methicillin-resistant Staphylococcus species by multiplex PCR. J. Clin. Microbiol. 33: 2864-2867 (1995) 17. Brakstad OG, Aasbakk K, Maeland JA. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nucA gene. J. Clin. Microbiol. 30: 1654-1660 (1992) 18. Martineau F, Picard FJ, Roy PH, Ouellette M, Bergeron MG. Species-specific and ubiquitous-DNA-based assays for rapid iden-.

(9) çºf öFöB ªÒ ï êç~ b' ßû tification of Staphylococcus aureus. J. Clin. Microbiol. 36: 618623 (1998) 19. Barski P, Piechowicz L, Galinski J, Kur J. Rapid assay for detection of methicillin-resistant Staphylococcus aureus using multiplex PCR. Mol. Cell. Probes 10: 471-475 (1996) 20. Hookey JV, Richardson JF, Cookson BD. Molecular typing of Staphylococcus aureus based on PCR restriction fragment length polymorphism and DNA sequence analysis of the coagulase gene. J. Clin. Microbiol. 36: 1083-1089 (1998) 21. Jarraud S, Mougel C, Thioulouse J, Lina G, Meugnier H, Forey F, Nesme X, Etienne J, Vandenesch F. Relationships between Staphylococcus aureus genetic backgroud, virulence factors, agr groups (alleles), and human disease. Infect. Immun. 70: 631-641 (2002) 22. Rosec JP, Giguad O. Staphylococcal enterotoxin genes of classical and new types detected by PCR in France. Int. J. Food Microbiol. 77: 61-70 (2002) 23. Moore PCL, Lindsay JA. Genetic variation among hospital isolates of methicillin-sensitive Staphylococcus aureus: evidence for horizontal transfer of virulence genes. J. Clin. Microbiol. 39: 2760-2767 (2001) 24. Bauer AW, Kerdy WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Path. 45: 493-496 (1966) 25. NCCLS. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. 6th ed. M7-A6. NCCLS, Wayne, PA, USA (2003) 26. Goh S, Byrne SK, Zhang JL, Chow AW. Molecular typing of S. aureus on the basis of coagulase gene polymorphisms. J. Clin. Microbiol. 30: 1642-1645 (1992) 27. Scherrer D, Corti S, Muehlherr JE, Zweifel C, Stephan R. Phenotypic and genotypic characteristics of Staphylococcus aureus in bulk-tank milk damples of goats and sheep. Vet. Microbiol. 101: 101-107 (2004) 28. Salasia SI, Khusnan Z, Lammler C, Zschock M. Comparative studies on pheno- and genotypic properties of Staphylococcus aureus isolated from bovine subclinical mastitis in central Java in Indonesia and Hesse in Germany. J. Vet. Sci. 5: 103-109 (2004). 141. 29. Orwin PM, Leung DY, Donahue HL, Novick RP, Schlievert PM. Biochemical and biological properties of staphylococcal enterotoxin K. Infect. Immun. 69: 360-366 (2001) 30. Lim SK, Joo YS, Moon JS, Lee AR, Nam HM, Wee SH, Koh HB. Molecular typing of enterotoxigenic Staphylococcus aureus isolated from bovine mastitis in Korea. J. Vet. Med. Sci. 66: 581584 (2004) 31. Olsen JE, Aabo S, Hill W, Notermans S, Wernars K, Granum PE, Popovic T, Rasmussen HN, Olsvik O. Probes and polymerase chain reaction for detection of food-borne bacterial pathogens. Int. J. Food Microbiol. 28: 1-78 (1995) 32. Casman EP, Bennett RW, Dorsey AE, Issa JA. Identification of a fourth staphylococcal enterotoxin, enterotoxin D. J. Bacteriol. 94: 1875-1882 (1967) 33. Sokari TG, Anozie SO. Occurence of enterotoxin producing strains of Staphylococcus aureus in meat and related samples from traditional markets in Nigeria. J. Food Prot. 53: 1069-1070 (1990) 34. Hazariwala A, Sanders Q, Hudson CR, Hofacre C, Thayer SG. Distribution of staphylococcal enterotoxin genes among Staphylococcus aureus isolates from poultry and human invasive staphylococcal disease. Avian Dis. 46: 132-136 (2002) 35. Sharma NK, Rees CE, Dodd CE. Development of a single-reaction multiplex PCR toxin typing assay for Staphylococcus aureus strains. Appl. Environ. Microbiol. 66: 1347-53 (2000) 36. Klotz M, Opper S, Heeg K, Zimmermann S. Detection of Staphylococcus aureus enterotoxins A to D by real-time fluorescence PCR assay. J. Clin. Microbiol. 41: 4683-4687 (2003) 37. Speller DC, Johnson AP, James D, Marpes RR, Chalett A, George RC. Resistance to methicillin and other antibiotics in isolates of Staphylococcus aureus from blood and cerebrospinal fluid, England and Wales, 1989-95. Lancet 359: 323-325 (1997) 38. Baiocchi P, Galie M, Santini C, Czarfagna P, Cassone M, Tarasi D, Venditti M. In vitro susceptibility of Staphylococcus aureus isolated from blood to currently used antistaphylococcal drugs. J. Chemother. 10: 25-28 (1998) (2004j 12ú 2¢ %>; 2005j 2ú 1¢ j).

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