分子疫学的手法を用いた食中毒原性黄色ブドウ球菌の遺伝学的解析

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(1)Title. 分子疫学的手法を用いた食中毒原性黄色ブドウ球菌の遺伝学的解析( 本文(Fulltext) ). Author(s). 佐藤, 祐介. Report No.(Doctoral Degree). 博士(獣医学) 甲第429号. Issue Date. 2014-09-24. Type. 博士論文. Version. ETD. URL. http://hdl.handle.net/20.500.12099/50399. ※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。.

(2) ÂĕǌėǔŎƟ@ǆ" ɾƖÚńʃȣqdFǁȥ-ɝ ėǔȸſ. 2014 ĳ. ᒱ㜧኱Ꮫ኱Ꮫ㝔㐃ྜ⋇་Ꮫ◊✲⛉. (ĩŎďė). ¦ȧǣ. .

(3) ǙƐ ǉɁȷ. . 5. ȅȹ. . 9. Ǯ 1 Ǭ ʃȣqdFǁȥ-Ŭȵɝ ėǔȸſŎƟ-ǡǫ . 13. 1-1ʇ. ĶɄ. . 13. 1-2ʇ. ŻŪ:0ŭƟ. . 14. . 14. 1-2-1. «ǆȥƄ)ăɿż 1-2-2.. Genomic elements ȸſǆ LA-PCR rEx-ȻȺ) . 14. 1-2-3.. Genomic DNA -ǵȰ) LA-PCR . 15. 1-2-4.. LA-PCR Ĉİǅƹ-ȸſ. . 16. 1-2-4-1.. ÇɯɡǺÃūɪĎāȸſ. . 16. 1-2-4-2.. ST jEqÊZU~ . . 16. 1-2-4-3.. ĆĄɟÄ-ƜĚ. . 17. 1-2-5.. Accession number. . 18. 1-2-6.. ěʀŏȿǊä. . 19. 1-3ʇ. Ȁƀ. . 19. 1-3-1.. ƊƯȥƄ@ǆ" SaPIs -Ĉİ-ǡȿ. . 19. 1-3-2.. ȞĵÂɴƄ,= SaPI rpBEȸſ . 19. 1-3-3.. H acMU ɝ ĕɬɘ genomic elements -ȸſ . 20. 1-4ʇ. Ȍġ. 22. Ĉİǅƹ-Ʈ. . .

(4) Ǯ 2 Ǭ Űź,=ɾƖÚńO -çĚ. . 45. 2-1ʇ. ĶɄ. . 45. 2-2ʇ. ŻŪ:0ŭƟ. . 46. 2-2-1.. ȽȥƄ

(5) ăɿż+;0, DNA ŖÁ. 2-2-2.. . 46. ʃȣqdFǁȥ- genotyping. . 46. 2-2-3.. SEA -ǅǄɧƮĚ. . 48. 2-2-3-1.. R ne c SEA ǵȰ. . 48. 2-2-3-2.. ELISA ǆwOeœ§-ǵȰ)ƊɆ . 48. 2-2-3-3.. SEA ƺǋǔ sandwich ELISA. . 50. 2-2-4.. Genomic elements -ȸſ. . 51. 2-2-5.. ěʀŏȿǊä. . 52. 2-3.. Ȁƀ. . 52. 2-3-1.. åǩÂɴƄ- Coa āÆ. . 52. 2-3-2.. åǩÂɴƄ- SE/SEl ¯ŷƻƞ. . 52. 2-3-3.. åǩÂɴƄ- MLST ȸſ)Ɨɏ . 53. 2-3-4.. åǩ CC - SEA ǅǄɧ. . 54. 2-3-5.. CC81 -ɝ ėǔȸſ. . 54. 2-4.. Ȍġ. . 55. .

(6) ȄŘ. . 72. ïũȳǸ. . 75. ȤũȳǸ (Summary). . 78. Ʌɐ. . 82. Ĺǆũƽ. . 83. .

(7) ǉɁȷ. att: attachment sequence ǜɟÄ BHI: Brain heart infusion ȗŁȝŖÁƦ CC: Clonal complex Coa: Coagulase RCPZ DNA: Deoxyribonucleic acid bJMUvƅɢ DDBJ: DNA Data Bank of Japan Űź DNA b]k O EDTA: Ethylenediaminetetraacetic acid H_ VCy ÷ɠɢ. egc: enterotoxin gene cluster ELISA: Enzyme linked immunosorbent assay EMBL: European Molecular Biology Laboratory `lÂĕǄƹėǞǪō EtBr: Ethidium Bromide H_VFzqxEd GS4B: Glutathione Sepharose 4B P]_J YpBW 4B GST: Glutathione S-transferase P]_J -S-c WpGZ LA-PCR: Long-accurate Polymerase chain reaction ɪɩƒǡń PCR ÞŃ LB: Luria bertany LB (ăĀ) MHC: Major histocompatibility complex ȳǿȈȱæ§ MLST: Multi locus sequence typing MRSA: Methicilline-resistant Staphylococcus aureus {_U ȎńʃȣqdFǁȥ MSSA: Methicilline-susceptible Staphylococcus aureus {_U ŉáńʃȣqdF ǁȥ NRS: Normal rabbit serum ƒįFSNȩƪ. .

(8) NTP: Nucleotide tri-phosphate gOJ_d 3 ɢ O.D.: Oprtical dencity ¸ėğķ ORF: Open reading flame PBS: Phosphate buffered saline ɢȇȭɾĆƙ PCR: Polymerase chain reaction. S. aureus: Staphylococcsua aureus ʃȣqdFǁȥ Sa3C: Sa3 Center region Sa3L: Sa3 Left region Sa3R: Sa3 Right region SaPI: Staphylococcus aureus pathogenicity island ʃȣqdFǁȥǎÚɝ ĕĪ SCCmec: Staphylococcal chromosome cassette mec SDS: Sodium dodecyl sulfate FǠɢecFz SE: Staphylococcal enterotoxin qdFǁȥH acMU /qdFǁȥșǰƖ SEA: Staphylococcal enterotoxin serotype A qdFǁȥH acMU A/qdFǁ ȥșǰƖ A SEB: Staphylococcal enterotoxin serotype B qdFǁȥH acMU B/qdFǁ ȥșǰƖ B SEC: Staphylococcal enterotoxin serotype C qdFǁȥH acMU C/qdFǁ ȥșǰƖ C SED: Staphylococcal enterotoxin serotype D qdFǁȥH acMU D/qdF ǁȥșǰƖ D SEE: Staphylococcal enterotoxin serotype E qdFǁȥH acMU E/qdFǁ ȥșǰƖ E. .

(9) SEG: Staphylococcal enterotoxin serotype G qdFǁȥH acMU G/qdF ǁȥșǰƖ G SEH: Staphylococcal enterotoxin serotype H qdFǁȥH acMU H/qdF ǁȥșǰƖ H SEI: Staphylococcal enterotoxin serotype I qdFǁȥH acMU I/qdFǁ ȥșǰƖ I SEK: Staphylococcal enterotoxin serotype K qdFǁȥH acMU K/qdF ǁȥșǰƖ K SEL: Staphylococcal enterotoxin serotype L qdFǁȥH acMU L/qdFǁ ȥșǰƖ L SEM: Staphylococcal enterotoxin serotype M qdFǁȥH acMU M/qdF ǁȥșǰƖ M SEN: Staphylococcal enterotoxin serotype N qdFǁȥH acMU N/qdF ǁȥșǰƖ N SEO: Staphylococcal enterotoxin serotype O qdFǁȥH acMU O/qdF ǁȥșǰƖ O SEP: Staphylococcal enterotoxin serotype P qdFǁȥH acMU P/qdFǁ ȥșǰƖ P SEQ: Staphylococcal enterotoxin serotype Q qdFǁȥH acMU Q/qdF ǁȥșǰƖ Q SER: Staphylococcal enterotoxin serotype R qdFǁȥH acMU R/qdFǁ ȥșǰƖ R. .

(10) SEl: Staphylococcal enterotoxin like toxin qdFǁȥH acMU ƉƖǺ/qdF ǁȥșǰƖƉƖǺ SElJ: Staphylococcal enterotoxin like toxin serotype J qdFǁȥH acMU ƉƖǺ J/qdFǁȥșǰƖƉƖǺ J SElX: Staphylococcal enterotoxin like toxin serotype X qdFǁȥH acMU ƉƖǺ X/qdFǁȥșǰƖƉƖǺ X SFP: Staphylococcal food poisoning qdFǁȥɾƖ SSC: Saline sodium citrate OH ɢȇȭɾĆƙ ST: Sequence type TAE buffer: Tris acetate ethylenediaminetetraacetic acid buffer cW/ɠɢ/EDTA ȇ ȭƦ TBE: Tris-boric acid-EDTA buffer cWuFɢ EDTA ȇȭƦ TET buffer: Tris-EDTA-Triton buffer cW EDTA cEc ȇȭƦ TST-1: Toxic shock syndrome toxin-1 ƖǺńU~ÒǏ²ȋƖǺ UV: Ultraviolet: ǼčȆ VRSA: Vancomycin-resistant Staphylococcus aureus k RxEU ȎńʃȣqdF ǁȥ. .

(11) ȅȹ ʃȣqdFǁȥ.ɖńĔƘń-Pzɱńǁȥ(

(12) Ðƹ9mc-ǕȚ9ɻǕ

(13) ʄðɻ Ǵț+*,įÿ= źȥ.

(14) ÑȜńǍŅ (&

(15) :

(16) ȨīƳ) 9ɾƖ-:+Ɏķ -Ǎǎ;ŦȩǏ9Ł¼țƳ

(17) ĊƓńȐƳ

(18) ƖǺńU~ÒǏ²ȋ+*-ɥǲ(ȠƓǔ +Ǎǎ4(

(19) Ɖ+Ǎǎ-Ɋùȥ(= (24, 48) Í'

(20) ɓĳ

(21) {_U Ȏńʃȣ qdFǁȥ (Methicilline-resistant Staphylococcus aureus

(22) MRSA) 9k RxEU ȎńʃȣqdFǁȥ (Vancomycin-resistant Staphylococcus aureus

(23) VRSA) +*-Ȧ ÉȎńʃȣqdFǁȥ,:=ŉƂǏ

(24) mcÓǐ(õɼ)+%'= (31, 53) 4"

(25) ƾÓėɺĂ,'8

(26) ʃȣqdFǁȥ.Ƹ-ŌƳ9ĞǤ-ɬǱƳ+*-Ɋùȥ) 'ɥȳȶ>'= (23, 91) ʃȣqdFǁȥ,Ɋù=Ǎǎ-$ɾƖ.

(27) ɾñ(źȥqdFǁȥH a cMU (qdFǁȥșǰƖ

(28) Staphylococcal enterotoxins

(29) SEs) @ǅǄ

(30) SEs ,ƛ Ƃ>"ɾñ@ţà=),:<ǑǄ= źɾƖ-Ǐƻ.öé9ņŁ(= (1, 4, 29, 40) SEs .Âĕɧ 22~29kDa -Õǹ] lOɉ(

(31) mc@.7)=ɷɪɽ 9V}RFhXy,öéƣń@Ǣ TSST-1 @ë7" SEs WlpBy.ȎƵ ń@Ǣ))8,crU 9trU )%"ǩ-] lOɉÂȸɡǺ,Ģ'8 ŕœń@Ǣ (4, 7, 42) 4"

(32) SEs .WlœÚƣń@ŷ

(33) MHC classII Âĕ) TCR Âĕ@Ɓƍ

(34) Ďŧ- T ǽȒ@ƣńÑ=. -Ȁƀ

(35) T ǽȒ;-ɚË+SEc. KE ǅǄ@ɀǑ=)(ƖǺńU~ÒǏ²ȋ-ǑǏ,8ɬ= (4, 7, 9, 80) SEs .. -œÚń,:<āÆ>

(36) Classical SEs )' SEA

(37) SEB

(38) SEC

(39) SED

(40). SEE - 5 ǩǝ;>'"

(41) ɓĳ,+%'Ďŧ-Ŭā SE :0Ŭā SEls (qdFǁ ȥșǰƖƉƖǺ

(42) Staphylococcal enterotoxin like toxins) ąì>

(43) ǀÿ4(, 5 ǩ. .

(44) ɽ- classical SEs ,Í

(45) 18 ǩɽ-Ŭā SEs/SEls (SEG~SElX) -Ėÿű;,+ %'=(1, 4, 7, 40, 41, 57, 59, 61, 76, 87) qdFǁȥɾƖ.ǈ(ǑǄ'=ɾñȬǄɥȳ+ɾƖ(= ÙǄÎ¶ Ǜ-ɾƖȁȺ,:=)

(46) ɓĳ

(47) Űź(.ĳɫŧÔ-¬ŧ)ŧǓ-ŅȍǑǄ

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(49) - 20 ĳɫ.Ņȍŧ

(50) ŧ)8,ƌ/,Şǧ'<

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(52) ƺ, MRSA ŉƂǏ(.ÂĕǌėȸſŐȫ-ə Ħ,¡ǎÚńȸſ9SsE WǖA,Ȫ?>'= (16, 43, 53)

(53) ɾƖ,ɬ ɘ=ʃȣqdFǁȥ-ɝ ėǔȑŴ9. -ńƻȸſ.Ź#Ȫ?>'+-ǀƻ(. = ǽȥ-ǌėȸſ-ŎƟ.ĎƉ(=

(54) ǽȥėÂɦ(.

(55) ȦÉŉáńȽʀ9pB VŉáńȽʀ+*â;«ǆ>'= (74) 4"

(56) 1984 ĳ,ǽȥ-ºQiz @ÇɯɡǺ(Ãū'ɶƘơÐl] @ȸſ=lWpDdɶƘơÐƟąì >'ɮ

(57) ǽȥė-ɺĂ(.źŎƟĴǆ;>'= (69) ;,ɓĳ(.

(58) ǌ ėȸſ-,ÂĕǄƹėǔŎƟ@ǿ5ɒA#Âĕǌėȸſ

(59) ǽȥė-Âɦ(ǖA,Ȫ ?>'= (27, 73) -:+Âĕǌėȸſ)'

(60) PCR 9UOH U P,:< ȥQiz-ɞɺĂ@ȸſ=ŎƟ9

(61) ŵɓ(.ƐUOH S@ǆ'ȥQi z@ȃȊǔ,ȸſ=ŎƟǝ;>'<

(62) >;.ʃȣqdFǁȥ,'8ɞŃ ǆ>Ē7'= (14, 66) 2001 ĳ,Å7'ʃȣqdFǁȥ-ºQizɟÄąì>'ɮ (39)

(63) ǈ( Ɖ+Ǎǎ;Âɴ>"ʃȣqdFǁȥ-QizɟÄƜĚ>'= 2013 ĳ 1. .

(64) Ŷǀÿ

(65) 43 Ƅ-ęº+QizɟÄb]sW,ǒɨ>

(66) dpcɟÄ@ë7= )ŧǓƄ,-3=QizɟÄąì>'= -:+ʃȣqdFǁȥ-QiyO Wȸſ,:<

(67) źȥ-Qiz. 3 &-ƈŋȳǺ

(68) +?$RCQiz

(69) RCkCq :0 genomic elements ,:<ƈŋ>=)ű;,+%" (43, 44) RCQiz .

(70) 23º'-Ƅ(»ɖ=źȥ-ǄîȂř9ĄźǔǄÑėǔńƻ@Ļ©=ɝ ĕȋ( = RCkCq.

(71) ʃȣqdFǁȥɳú-$ƺĚ-Pr¯ŷ=ɝ ĕ ȋ-)(= genomic elements .rpBV9 Staphylococcal chromosome cassette mec (SCCmec) +*-čŽńɝ ĕȋ@Ś

(72) źȥč;ȟɌ-Qiz,à <ɒA#ɝ ĕȋ(= ʃȣqdFǁȥ-ǎÚùĕ.

(73) ƖǺ9ERUV

(74) R CPZ+*-RCQiz9RCkCq,Ėÿ=8-8=

(75) Ď-ƖǺ 9ȦÉȎńɝ ĕ. genomic elements ,Ėÿ= (38, 43) -"7

(76) genomic elements Ƅɫ@ǧÐQiz,à<ɒ4>=)

(77) źȥ-ǎÚùĕ-ƿľ,ɬ =)Ȍ;>'= (43, 50) 4"

(78) źȥ-ŉƂǏÂɴƄ-Âĕǌėȸſ,:<

(79) ƺĚ -Qizƈŋ (RCQiz+RCkCq+genomic elements) -ɳú (O ) ƺ Ě-Ǎǎ)ɬɘƧ)ǝ;>'= (18) ƺ,

(80) MRSA -ȸſ(.

(81) RCQi z@ȸſ= multi locus sequence typing (MLST) (20) ) genomic elements -&( = SCCmec āÆ (11, 79) @ªǆ"Âĕǌėȸſ,:=O çĚ). -ȸſǖ. A,Ȫ?>'= MLST .źȥ-jFWMo Pɝ ĕ-ȸſ(<

(82) MRSA ) {_U ŉáńʃȣqdFǁȥ (Methicillin-susceptible S. aureus (MSSA) -*$; (8«ǆãȔ(= (20)

(83) SCCmec .źȥ-{_U Ȏń,ɬ?=ãÐńɝ ùĕɺĂ(< (71)

(84) MSSA (.>@¯ŷ'+ ɚÛ-ąì,'

(85) ɾ ƖÂɴƄ-Ď. MSSA (=)ǝ;>'<

(86) źÂɴƄ@ MRSA )çÂĕǌ. .

(87) ėǔŎƟ,:%'O çĚ@Ȫ).ãȔ(%" (2, 13) -"7 SCCmec āÆ,?=Ŭ"+ȸſŎƟ-ǡǫłȳ(= źė¤Ʉũ(.

(88) +ęº+ŒƑûɵ+qdFǁȥɾƖ,&'

(89) ɾƖ¬ ÂɴʃȣqdFǁȥ-ÂĕǌėȸſźɾƖ-ǑǄŒƑ9SsE W,ŷǆ)Ȍ

(90) Ǯ 1 Ǭ(. SCCmec ,?=Ŭȵ-ɝ ėǔȸſŎƟ-ǡǫ

(91) ǮʉǬ(.Űź, =ɾƖÚńO -ŝǻ)ɝ ėǔƺŀ-ȸű@Ȫ%". .

(92) Ǯ1Ǭ ʃȣqdFǁȥ-Ŭȵɝ ėǔȸſŎƟ-ǡǫ. 1-1. ĶɄ S. aureus pathogenicity islands (SaPIs) . 1998 ĳ, Lindsay ; (46) ,:<Å7' ąì>"ãÐńɝ ùĕ(<

(93) ɾƖ-ÚùƖǺ(= SEs 9-ƖǺ

(94) ȦÉȎń ɝ ĕ

(95) ǜùĕ+*-ɝ ĕĖÿ= (54, 85, 89) SaPIs .ʃȣqdFǁȥ-Qi z-ƺĚɺĂ,Ŝ¹>

(96) >4( 6 ɺĂ- SaPIs Ŝ¹ɺĂ (ʃȣqdFǁȥ-Qi z 8

(97) 9

(98) 18

(99) 19

(100) 44

(101) 49ɺĂ) ąì>'= SaPIs -ºɪ. 13ʋ 17kbp (. -ǭ,.Ŝ¹ɞ¤ƺǋǔ+ȉ<ɔɟÄĖÿ

(102) SaPIs -Qiz1-ǿ. 5ɒ59ȱȰ=.pBVƉǳĕ1-ǿ5ɒ5+* SaPIs ȟɌ-Ȃř9ȥƄɫ-ǧ Ð,ɬ=ɝ ĕ;ƈŋ>'= 4"

(103) MRSA ) MSSA .*$;8 SaPIs @ ¯ŷ="7

(104) SaPIs @Ł)" genomic elemants -ȸſ.

(105) SCCmec āÆ,. ãȔ(=ʃȣqdFǁȥɳúº§@Ģɇ)"ȃȊǔ+ȸſƟ)'ŷÏ)Ȍ;> = źǬ(.ʃȣqdFǁȥ,= SaPIs @Ł)" genomic elemants -ȸſƟ ʅgenomic elements-scanning Ɵʆ-Ŭȵǡǫ@Ȫ))8,

(106). -ŷÏń@Ƈȼ=. "7,Źǝ-ǎÚùĕ@ë6 SaPIs -çĚ@Ƚ5" 4"

(107) ɾƖǇŽƄ-ɝ ėǔƺ ŀ@őš="7,.H acUM ɝ ĕ-¯ŷƻƞ-ºɈ@őš=łȳ= "7

(108) SaPIs č- SEs ɬɘ genomic elements (1, 4, 29, 40)

(109) pBV

(110) Sa - enterotoxin gene cluster (egc)

(111) truncated-c Ww\ -ȸſŎƟ-ǡǫ@Ƚ5 " . .

(112) 1-2. ŻŪ:0ŭƟ 1-2-1. «ǆȥƄ)ăɿż Ȯ 1-1 ,ǢʃȣqdFǁȥ-ƊƯȥƄ 3 Ƅ:0ȞĵÂɴ 10 Ƅ@ǆ" N315 Ƅ ) MW2 Ƅ.Qiz-ºĆĄɟÄąì>'=ƊƯȥƄ (5, 39)(<

(113) 243 .ºQ izɟÄ-ǒɨ.+lWpDdɶƘơÐƟ:< SaPI3 @¯ŷ=)ŞĚ >=ƊƯȥƄ (7) (= ȞĵÂɴƄ)'.

(114) ɾƖǇŽƄ 4 Ƅ

(115) ʄȘǇŽƄ 4 Ƅ

(116) mcŅȍǇŽƄ@ 2 Ƅ-Ⱥ 10 Ƅ@«ǆ" åƄ.. >!>ǋ+=ɾƖ¬

(117). ³įmcʄȘ

(118) Ņȍ:<Âɴ>"Ƅ(= J11 @ɰåȞĵÂɴƄ-ÜȌũƽ.Ȯ 1-1 -ɖ<(<

(119) J11 .đĕƃɿďė:<Â'"#" ʃȣqdFǁȥ-ă ɿ,.º' BHI ĠĐăĀ (Difco) ) BHI broth (Difco) @ǆ

(120) Ƙǚ

(121) 37(ųăɿ @Ȫ%". 1-2-2. Genomic elements ȸſǆ LA-PCR rEx-ȻȺ)Ĉİǅƹ-Ʈ Genomic elements -ɺĂ@Ĉİ="7,

(122) 6 ɺĂ- SaPIs

(123) seh ɬɘc Ww\

(124) egc

(125) Sa3 -Ŝ¹ɺĂɓ´-¯Ėń-ʂɺĂ, LA-PCR rEx@ȻȺ " Genomic elements íɑ-¯Ėń-ʂɺĂ-çĚ,.

(126) CLUSTAL W (77) ) in silico MolecularCloning (in silico biology) @ǆ

(127) b]sW,ǒɨ>'= N315 (BA000018)

(128) MW2 (BA000033)

(129) COL (NC_002951)

(130) USA300 (NC_007793)

(131) RF122 (AJ938182)

(132) MRSA252 (BX571856) :0 MSSA476 (BX571857) Qiz-º ĆĄɟÄ,&'ȸſ" Genomic elements -$

(133) SaPIs

(134) seh ɬɘc Ww\ :0 egc .

(135) Ŝ¹ɺĂɓ´,ȻȺ"rEx1 Y`c(-Ĉİ@Ȫ=: ,

(136) çĚ"¯Ėń-ʂɺĂ,rEx@ȻȺ" Sa3 -ºɪ. 40-50kbp ) SaPIs +*)Ɨɏ=)ɪ"7

(137) rpBV¼-¯ĖńʂɺĂ@ 3 ÂÌ

(138). .

(139) >!>-ɞÂ,ĢŃ= 3 ǩ-rExY`c@ǆň'Ĉİ@Ƚ5" (ü 1-1) Ȯ 1-2 ,źǬ(«ǆ"rExɟÄ@Ǣ" >;-rEx@ǆ"Ĉİǅƹ - in silico ŞĚ,.

(140) in silico MolecularCloning @ǆ". 1-2-3. Genomic DNA -ǵȰ) long-accurate (LA) -PCR 37(ųśǘăɿ"ȥƦ@ɛŁ'ȥ§@øß

(141) TET buffer (10mM Tris-HCl pH7.5

(142) 1mM EDTA

(143) 2.4% TritonX-100

(144) Lysostaphin (Sigma) 5g/ml ƩÍ) (ŊƱ ļ

(145) 37( 30 ÂÍƭ"ǽȒĉǟĊƦ@ǆ'

(146) QIAamp DNA Mini Kit (QIAGEN) ,:= genomic DNA -ǵȰ@Ȫ%" ǵȰ" genomic DNA -ǹķǡȿ.

(147) 1x c W/ɠɢ/EDTA k`pB (TAE buffer) -1% CLWQ) 1xTAE buffer @ǆ" ɶƘơÐƟ,:<Ȫ%" CLWQ,. SeaKem GTG Agarose (Lonza)

(148) 1xTAE buffer -©ŋ,. 50xTAE (Bio-Rad) @ǆ" LA-PCR ,. LA-taq HS (TAKARA) @«ǆ

(149) 0.5M dNTP

(150) 0.5pM årEx

(151) 2.5U LA-taq @Í" 1x LA-taq buffer ʅ49µlʆ) genomic DNAʅ1µlʆ@ƨæ " 50µl ÞŃƦ@©ŋ" PCR ÞŃ. MyCycler l[eSxSEO (Bio-Rad) 8. Veriti SxSEO (Applied biosystems) ,:<ěŮ

(152) 961 Â-ƵČń-ļ

(153) 9810 Ǧ+;0, 6820 Â@ʈSEO)=ĬǨ@ 28 øȉ<ɔ

(154) 6810 Â-ŵǾ¢ɪ@Ȫ%" Ĉİǅƹ.

(155) 1% precast ready gel 64well (Bio-Rad) ) 1xTAE-0.5g/ml Ethidium Bromide (EtBr)-buffer @ǆ'ɶƘ ơÐ,:<ǡȿ" 1xTAE-0.5g/ml EtBr buffer -ɃȰ,.

(156) 50xTAE (Bio-Rad) ) Ethidium Bromide (Bio-Rad) @ǆ" ľ;>"Ĉİǅƹ.

(157) QIAquick PCR Purification Kit (QIAGEN) @ǆ'ǵȰ". .

(158) 1-2-4. LA-PCR Ĉİǅƹ-ȸſ 1-2-4-1. ÇɯɡǺÃūɪĎāȸſ ÇɯɡǺÃūɪĎāȸſ.

(159) 2U - HindIII (New England Biolabs, NEB) 4". HaeIII (NEB) @ǆ'

(160) 3730 Â-ż(ěŮ" ƥÑ" DNA ɩ-ǡȿ.

(161) 1% precast ready gel 20well (Bio-Rad) ) 1xTAE-0.5g/ml EtBr-buffer @ǆ"ɶƘ ơÐ,:<Ȫ%". 1-2-4-2. ST jEqÊZU~ sec rq-©ŋ)ƊɆ,.

(162) sec ɝ ĕūƷ@Of PsO](= pGEM-3zf (+) ,ǿ5ɒA# pKOC3 (Òɤďė ȓžȢÖċ:<Â) @ďșȥ DH5α (TOYOBO) , 42

(163) 45 Ǧɫ-mcU~Ò,:<rWydģ¹" Ɛ(

(164) 100μg/ml C oU (Wako) ƩÍ LB agar (Sigma) (ɜŔļ

(165) ľ;>"ĻɉɍŠ§ @ 100μg/ml C oU ƩÍ LB broth (Sigma) ,'

(166) 37(ųśǘăɿ

(167) QIAprep Spin Miniprep Kit (QIAGEN) @ǆ'rWyd DNA @ǵȰ" ǵȰ " pKOC3 . BamH (NEB) :0 EcoR (NEB) @ǆ'Ãūļ

(168) 1x TAE-0.9%C LWQ@ǆ"ɶƘơÐ@Ȫ%" ;,

(169) Ǚǔ- DNA ūƷ@ë6Q@øß ļ

(170) MinElute Gel Extraction Kit (QIAGEN) @ǆ' sec ƺǋǔ DNA rq@ǵȰ

(171) ECL Direct Nucleic Acid Labeling and Detection System (Amercham Bioscience) ,:<ƊɆ" LA-PCR Ĉİǅƹ-Ǚǔɝ ĕ-ƇÁ-"7,

(172) ǵȰļ-Ĉİǅƹʅ0.5~1μgʆ@ Hind ,:<Ãū

(173) 1x TAE-0.9% agarose gel

(174) 1xTAE buffer

(175) Sub-Cell GT (BioRad)+;0, PowerPac Basic (Bio-Rad) @ǆ' 15V -Ěɶþ(ųɶƘơÐ@Ȫ% ". -ļ

(176) ơÐļ-Q,ɪɩ DNA -ÍƙÂȸÀǂ (350mM HCl10 Âɫ)

(177) 2 ź. .

(178) ɩ DNA - denaturing Àǂ (0.5M NaOH/ 1.5M NaCl25 Âɫ) @Ȫ%"ļ

(179) Q- ïÀǂ (0.5M Tris-HCl/ 1.25M NaCl15 Âɫ) @ 2 øȪ

(180) Ĝƭ(ųɍ¾ļ

(181) UV cross linker ,:< DNA @ýĚ" +

(182) ɍ¾.

(183) k`pB)' 20xSSC (BioRad) @Įɣ" 10x SSC

(184) { q )' Hybond-N+ (Amersham Bioscience) @ «ǆ

(185) DNA ýĚ. TL-2000 Ultraviolet Translinker (UVP) @ǆ

(186) Ơɪ(Ȫ% " DNA ýĚļ-{ q ,Ģ'

(187) 421 Ųɫ-rjEqÊZU ~ ļ

(188) ƊɆ" sec ƺǋǔrq@ǆ'

(189) 42ų-jEqÊZU~ @Ȫ%" jEqÊZU~ k`pB.

(190) ECL gold hybridization buffer (Amersham Bioscience) , 0.1M NaCl (Nacali Tesuque) )ħ- Blocking reagent @ Í 1 Ųɫťŗļ

(191) 42(ÍƵ©ŋ" jEqÊZU~ ļ.

(192) ǮƢƤƦ (6M ĥǺ (Nacalai tesque)

(193) 0.1x SSC (pH=7.0)

(194) 0.4% SDS (Life technology)20 Â) @ǆ' 42( 2 øƢƤ@ȉ<ɔ

(195) Ɛ(ǮƢƤƦ (2x SSC5 Â) @ǆ'Ĝƭ ( 2 øƢƤ@ȉ<ɔ" ŵļ, ECL Direct Nucleic Acid Labeling and Detection System (Amersham Bioscience) ) Chemidoc XRS (Bio-Rad) @ǆ'

(196) Ĉİǅƹ, = sec -ǡȿ@Ȫ%". 1-2-4-3. ĆĄɟÄ-ƜĚ ÇɯɡǺÀǂ,:<Ŭȵ genomic elements -ĖÿǢó>"Ĉİǅƹ,&' .

(197). -ĆĄɟÄ@ƜĚ="7

(198) QIAquick Gel Extraction Kit (QIAGEN) @ǆ'. UOH Wǆa rc-ǵȰ@Ȫ%" ǵȰ"a rc-ǹķǡȿ. 1x TAE-0.9% agarose gel ) 1x TAE buffer ,:=ɶƘơÐ,:<Ȫ

(199) Quant-it dsDNA Assay Kit BR (Invitrogen) @ǆ'Ʋķ@ƮĚ" Ɛ(

(200) ǵȰ DNA

(201) BigDye® Terminator v3.1 Cycle sequencing kit (Applied Biosystems) ) BigDye® Terminator. .

(202) v3.1 5x Cycle sequencing Buffer (Applied Biosystems) @ǆ'

(203) SEOUOH U P (96 1 Â-ļ

(204) 9610 Ǧ

(205) 505 Ǧ

(206) 604 Â-ȪǨ@ 25 SEO) @ Ȫ%" ÞŃǅƹ. 10 Â- 1 ɧ- 3M ɠɢecFz (pH 5.2)

(207) 10 Â- 1 ɧ125mM EDTA (pH7.5)

(208) 2.5 ±ɧ- 100%H]i@ǆ'H]iƝƕ@Ȫ

(209) ƪɰÛļ 3.5 ±ɧ- 70%H]i(ƢƤƶ

(210) -20(¯Ė" ¯ĖS r@ Hi-Di formamide (Applied Biosystems) @ǆ'½ưȸ

(211) 95( 2 ÂɫÍƵ ļ,ƚ( 2 Âɫ¿Ø

(212) JcUOH S (ABI PRISM 3100- Avant Genetic Analyzer (Applied Biosystems)) @ǆ'ĆĄɟÄƜĚ@Ȫ%" ľ;>"ĆĄɟÄ@ ATGC6 software Ver. 10 (GENETYX) @ǆ'ŨÄ

(213) >;Ŭ",ȻȺ"r Ex@ǆ"rExFIM PƟ () ,:=ĆĄɟÄ-ƜĚ@Ȫ%" Open reading frame (ORF) -ŖÁ,. ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) ) in silico MolecularCloning software @ǆ'Ȫ%" 4"

(214) National Center for Biotechnology Information - BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) @ǆ'ǚçńƇǻ,:<

(215) å ORF -ƎȔŞ Ě@Ȫ%". 1-2-5. Accession number źǬ(çĚ>"Ŭȵ genomic elements -ĆĄɟÄ.

(216) DDBJ/EMBL/GenBank , ǒɨ>" Accession numberʊAB704539 (IVM60)

(217) AB704540 (HHMS2)

(218) AB704541 (J11)

(219) AB716349 (IVM10)

(220) AB716350 (Ishikawa11)

(221) AB716351 (No. 10)

(222) AB716352 (Hirosaki4). .

(223) 1-2-6. ěʀŏȿǊä źǞǪ-$

(224) ɝ ĕǿŠ,®=8-.ĩŎďėɝ ĕǿŠǄƹĘºěʀēò -ŏȿ@á'ěŮ" (ŏȿǊä: B201016). 1-3. Ȁƀ 1-3-1. ƊƯȥƄ@ǆ" SaPIs -Ĉİ-ǡȿ ü 1-2 , N315 Ƅ,= LA-PCR ,:= SaPIs ȸſ-Ȁƀ@Ǣ N315 Ƅ, 6 ɺĂ- SaPI Ŝ¹ãȔɺĂ¯Ė>'=

(225). -$ SaPIn1 (Ǹ 15kb) Ŝ¹>. = 44’ɺĂ (39) (. 20Kbp ĺ-k dƇÁ>" (ü 1-2A) ŭ

(226) SaPI Ŝ¹ɟÄ (attC) .Ėÿ= SaPI -Ŝ¹+ 8

(227) 9

(228) 18

(229) 19

(230) 49ɺĂ(. 4kbp Ĉİǅƹľ;>" 44ɺĂ-Ĉİǅƹ- HindIII ƥÑļ-ơÐ·ʅü 1-2Bʆ( .

(231) in silico Ʈ)Ƞ= 3 ǩɽ-SEX- HindIII ƥÑūƷ (8,225bp

(232) 6,473bp

(233) 1,700bp) ǡȿ>" in silico ȸſ:< 6,473bp -ƥÑūƷ,. sec Ėÿ=) Ʈ>

(234) ƺǋǔrq@ǆ"ST jEqÊZU~ ȸſ,:<źūƷ , sec ƇÁ>" (ü 1-2C). 1-3-2. ȞĵÂɴƄ,= SaPI rpBEȸſ Ȯ 1-1 ,Ǣ" 243 Ƅ:0 10 Ƅ-ȞĵÂɴƄ@ǆ

(235) SaPI ǆ-rExY`c ,:= LA-PCR @Ȫ)(

(236) º'-ɺĂ(¨;-Ĉİǅƹľ;>" (ü 1-3) >;-$ SaPI -ĖÿǢó>= 10kbp -Ĉİǅƹ. 22 °Ėÿ'" ;,

(237) Ĉİǅƹ@ HindIII (ƥÑ"Ȁƀ

(238) 8’ɺĂ) 49’ɺĂ(. Ă

(239) 18’ɺĂ:0 44’ɺĂ(.. >!> 1 ǩɽ

(240) 9’ɺ. >!> 2 ǩɽ

(241) 19’ɺĂ(. 4 ǩɽ-ơÐ·@Ǣ". (ü 1-4) >;-$

(242) No.1 Ƅ

(243) No.10 Ƅ

(244) Ishikawa11 Ƅ

(245) Nagasaki Ƅ

(246) IVM60. .

(247) Ƅ

(248) HHMS2 Ƅ

(249) J11 ƄǇŽ- 9ɺĂ-Ĉİǅƹ. MW2 -Qiz 9ɺĂ,Ėÿ =ɸ SaPIs ń genomic elements - HindIII ƥÑūƷ-Ʈ (6,745bp

(250) 2,936bp

(251) 1,940bp

(252) 428bp) (5) )Ƞ"

(253). >č- 6 ɺĂº 9 ǩɽ-Ĉİǅƹ-. HindIII ƥÑūƷ-ơÐ·.b]sW (DDBJ/EMBL/GenBank) ,Ƞ=8Ėÿ+%" (2011 ĳ 9 ŶŲƴ) Ɛ(

(254) Ĉİǅƹ-Ŭȵ SaPIs -ĆĄɟÄ@ ƜĚ="7,

(255) UOH U Pļ

(256) SaPIs -ŖÁ

(257) Open reading frame (ORF) ŖÁ+;0, annotation @Ȫ+

(258) 7 ǩ-Ŭȵ SaPIs @çĚ" ü 1-5 ,Ŭȵ SaPIs -Ƌĸü@Ǣ

(259). -Ⱦǽ@Ȯ 1-3~1-9 ,Ǣ Ŭȵ SaPIs -îè. Novick ;. -şô (55) ,Ľ

(260) SaPI + Ƅè(Ȫ%" (SaPIivm10

(261) SaPIishikawa11

(262) SaPIivm60

(263) SaPIhhms2

(264) SaPIj11

(265) SaPIno10 :0 SaPIhirosaki4) å SaPIs -Ŝ ¹ɺĂ.

(266) SaPIivm10 ( 8'

(267) SaPIishikawa11 ( 18'

(268) SaPIivm60

(269) SaPIhhms2 : 0 SaPIj11 ( 19'

(270) SaPIno10 :0 SaPIhirosaki4 ( 44'(<

(271) >;-º', ' SaPI -ĄźʁƆ(=ǭ-ÊOcoc (attL :0 attR)

(272) intstl. strrep

(273) ter @¯ŷ'" ŭ

(274) SaPIishikawa11 (. pri :0 cp ƏŢ' " ;,

(275) >;- SaPIs (.ĄźʁƆ-ɫ,ǎÚùĕ)' SE ɝ ĕ9ȦÉȎ ńɝ ĕ@¯ŷ'" +?$

(276) SaPIivm10 Ƅ

(277) SaPIishikawa11 Ƅ

(278) SaPIivm60 Ƅ

(279) SaPIno10 Ƅ:0 SaPIhirosaki4 Ƅ(. SE ɝ ĕ)' seb @

(280) SaPIj11 Ƅ( . sek ) seq @¯ŷ'" 4"

(281) SaPIivm10 Ƅ

(282) SaPIishikawa11 Ƅ

(283) SaPIivm60 Ƅ

(284) SaPIhhms2 Ƅ

(285) SaPIno10 Ƅ:0 SaPIhirosaki4 Ƅ(.ȦÉȎńɝ ĕ) '

(286) C oU Ȏńɝ ĕ-u|P(= ear @¯ŷ'". 1-3-3. H acMU ɝ ĕɬɘ genomic elements -ȸſ. .

(287) ƊƯȥƄ(= N315 Ƅ:0 MW2 Ƅ¯ŷ= SEs ɬɘ genomic elements @Ĉ İ

(288) ÇɯɡǺÀǂ@Ȫ%"Ȁƀ@ü 1-6 ,Ǣ 4" in silico (-ūƷɪƮ@Ȯ 110 ,Ǣ MW2 Ƅ¯ŷ= seh ɬɘc Ww\ @Ĉİ HindIII (Ãū= )

(289) 5,000bp ɓ,k dľ;>" (ü 1-6A) -Ȁƀ. in silico (-ūƷɪƮ ʅ5,000bp ɓ, 2 &- DNA ūƷʆ)Ƞ" N315 Ƅ¯ŷ= egc ,&'8 ĈÊǅƹ-ÇɯɡǺÀǂ@Ȫ) 2,500bp ɓ) 750bp ɓ,k dľ;> (ü 16B)

(290) in silico (-ūƷɪƮʅǸ 2,500bp , 3 &- DNA ūƷ)Ǹ 750bp ,ʈ&DNA ūƷʆ)Ƞ'" N315 Ƅ) MW2 Ƅ¯ŷ=Sa3 ɺĂ,Ėÿ=pB V- 3 ɺĂ-ȸſɺĂ (Left

(291) Center

(292) Right) - HaeIII ,:=ÇɯɡǺƥÑl] .,ǋ+%'< (ü 1-6C~E)

(293) -Ȁƀ8 in silico (Ʈ>" DNA ūƷl ] -Ʈ)Ƞ'" c Ww\ -ȸſ,'

(294) ɾƖǇŽ 4 Ƅº')ŉƂǏǇŽ 2 Ƅº'. MW2 Ƅ¯ŷ=8-)ç-ơÐl] @ǢĈİǅƹľ;>" (ü 1-7A, ü 1-8A) --Ƅ(.

(295) c Ww\ @ë6)Ȍ;>=Ĉİǅƹľ;>+%" çƉ ,

(296) ʄȘǇŽ- 3 Ƅ( N315 )çā- egc @ë6)Ȍ;>=Ĉİǅƹǡȿ>" (ü 1-7B, ü 1-8B). -- 7 Ƅ(.Ĉİǡȿ>+%" ;,Sa3 ɺĂ-r. pBV@ȸſ=)

(297) ɾƖǇŽ 3 Ƅ)ŉƂǏǇŽ 2 Ƅ(Ĉİǡȿ>" ɾ ƖǇŽƄ 3 Ƅ) HHMS2 ƄǇŽ-Ĉİǅƹ. MW2 ǇŽ-Ĉİǅƹ)ơÐ·Ƞ" (ü 1-7C~E, ü 1-8C~E) Ɣ<-ŉƂǏǇŽƄ (J11 Ƅ) -¯ŷ=pBV- 3 ɺĂDNA ÃūūƷ-ơÐl] .b]sW,Ƞ=8-Ėÿ+%" (2012 ĳ 4 ŶŲƴ) ŭ

(298) - 6 Ƅ(.pBV-Ŝ¹ǡȿ>+%". .

(299) ȞĵÂɴƄ- SaPI )H acMU ɬɘ genomic elements -¯ŷƻƞ.Ȯ 1-11 -ɖ<(<

(300) ø«ǆ"º'-ȞĵÂɴƄ(. -rpBEȸſ)Ŭȵ genomic. elements -çĚãȔ(%". 1-4. Ȍġ ʃȣqdFǁȥ-Qiz@ƈŋ= 3 ǩɽ-ƈŋȳǺ (RCQiz

(301) RCkCq

(302) genomic elements) -$

(303) źȥ-ǎÚùĕ-Ď. genomic elements ,Ėÿ '<

(304) ɾƖ-ÚùƖǺ(= SEs -ɝ ĕ. SElX @ɰº' genomic elements ,Ėÿ=)ǝ;>'= (1, 4, 29, 40, 87) >; SEs ɝ ĕɬɘ genomic elements -ƿľ.

(305) źȥ SEs @ǅǄɾƖ@ĹɊ"7,łɹ-ɚ Ǩ(<

(306) SEs ɬɘ genomic elements -¯ŷƻƞ@ȸſ=).ɾƖÂɴƄ-ɝ ėǔȑŴ@ǂȸ=(ɥȳ(= ɖį- PCR (ƒǡ, 10kbp @ɋ=ɪɩ DNA -Ĉİǅƹ@ľ= PCR ÞŃƦ9S xSEO-rcR,ɬ=Ňą.ɯ;>'= Í'

(307) øȻȺ"r ExY`c.

(308) MW2 Ƅ9 MSSA476 Ƅ ()8, MLST ,:< sequence type 1

(309) ST1)

(310) N315 Ƅ (ç ST5)

(311) USA300 Ƅ (ç ST8)

(312) MRSA252 Ƅ (ç ST36)

(313) RF122 Ƅ (ç ST151)

(314) COL Ƅ (ç ST250) +*-ǋ+=Ƕȁ-ȥƄ (5, 19, 26, 30, 32, 39) -ĆĄ ɟÄ@8),ȻȺ"

(315) ʃȣqdFǁȥ-Qiz-¯Ėń-ʂɺĂ(8Ć ĄĎāĖÿ="7

(316) ƊƯȥƄ)çƉ,

(317) ȞĵÂɴƄ,'8Ĉİ@ǡȿ=łȳ %" Qiz-ºĆĄɟÄȸɂ>

(318) 44’, SaPIn1 @¯ŷ=)ǝ;>' =ƊƯȥƄ(= N315 Ƅ@ǆ'

(319) ƒǡ+ SaPI ºɪ-Ĉİ@ǡȿ" øȻȺ " 6 ǩ- SaPIs ǆrExY`c) PCR ɡǺʅLA-taqʆ

(320) ;, 2 Wa`r PCR Ɵ @ǆ=)(

(321) 20kbp ĺ- SaPIn1 @ë6Ĉİǅƹľ;>" -Ĉİǅƹ@. .

(322) HindIII ƥÑ"ōȴ. in silico (-ūƷɪƮȀƀ)Ƞ

(323) ST jEqÊZ U~ (8 SaPIn1 ¯ŷ= sec ǡȿ>" (ü 1-2) 3 ǩ-ɸ SaPI ń genomic elements ,'8çƉ, N315 Ƅ) MW2 Ƅ¯ŷ= genomic elements @ƒǡ, ȸſ(=)ǡȿ> (ü 1-6)

(324) ź LA-PCR ƊƯȥƄ-ȸſ,ŃǆãȔ(=) Ȍ;>" ;,

(325) QizɟÄű+ 10 Ƅ-ȞĵÂɴƄ(8

(326) ƊƯƄ)çƉ, SaPIs @ë7" genomic elements -ƒǡ+Ĉİǡȿ> (ü 1-3)

(327) Í'

(328) ĽŽąì +%"Ŭȵ- SaPIs 8 7 ǩɽçĚ>" (ü 1-5) ;,

(329) SEs ɬɘɸ genomic elements -$Sa3 ,Ėÿ=Ŭȵ-pBV)Ʈ>=Ŭȵ genomie element -çĚ8ãȔ(%" (ü 1-8) źǞǪ(Ŭ",çĚ>"º'- SaPIs ,.

(330) SaPI -Ąźƈɗ¯Ė>'" SaPI -ǭ,.ÊOcocĖÿ'<

(331). -ɟÄ.>4(ąì>'. =ɟÄ)çɽ£=8-(<

(332) Ŝ¹ɺĂ8ůą)Ƞ'" (Ȯ 1-3~1-9ʆ ;,

(333) SaPI -Qiz1-Ŝ¹,ɬ?=ɺĂƺǋǔE aPW@Rd= int . att )ĢŃ=)ǝ;>'<

(334) źǞǪ(8ɚÛ-ąì (54) ɖ<ĢŃȴ;>" -

(335) SaPI -ɝ ĕÇĿ,ɬ?= stl ) str

(336) ȟɌ-Qiz;-Ã<Á,ɬ?=. xis

(337) ȟɌ-ȱȰ,ɬ?= rep

(338) pBVıƫ,ɬ?= pif

(339) pBVƉǳĕ1-Qiz Ŝ¹,ɬ?= ter (51, 54, 63, 81-83) @º'-Ƅ¯ŷ'" (Ȯ 1-3~Ȯ 1-9) 4"

(340) SaPIishikawa11 Ƅč- SaPI (.rExæŋ,ɬ?= pri )pBVǆǳĕ-Ļ ŋ,ɬ?= cp ȱæ§@¯ŷ'" >; 2 ɝ ĕ. MW2 Ƅ¯ŷ= SaPImw2 (.Ə'=)ąì>'<

(341) SaPIs ,łɹ-ɝ ĕ(.+)ŞĚ>= (5, 54) SaPIs -ĄźʁƆ@©=ɝ ĕȋ.

(342) SaPIs ȟɌȥƄɫ@ Ť

(343) Qiz,Ŝ¹ >=ɲ,łȳ+ɝ ĕȋ(=

(344) -ĄźʁƆ¯Ė>'=);

(345) źǞǪ. .

(346) (çĚ" SaPIs .ƙĲ ŤʃȣqdFǁȥ- SEs ɝ ĕƿľ,ɬ?%'=)Ȍ ;>" ʃȣqdFǁȥ¯ŷ= genomic elements ,.ĎƉń=)ǝ;>' = ¬/

(347) SEs ɬɘ genomic elements @¬,=) sec . SaPIs ,Ėÿ=

(348) ƸŌƳǇŽƄ (RF122 Ƅ) (>/Qiz- 9',Ŝ¹>= SaPIbov1 @

(349) USA400 Ƅ (ĭƿľā MRSA) O (= MW2 (>/ 18'ɺĂ,Ėÿ= SaPImw2 @

(350) N315 Ƅ9 Mu50 +*-ÓǐŮȻɬɘā MRSA O -ǩ(>/ SaPIn1/m1 @¯ŷ=)ǝ;>'<

(351) çɝ ĕ(8ǇŽ),ǋ+= SaPIs ǋ+=ɺĂ,Ėÿ'= (5, 30, 39) >4(,. seb ɬɘ SaPIs )' 19’ɺĂ ,Ŝ¹>= SaPI3 -5ąì>'" (26, 90) źǞǪ(.

(352) seb ɬɘ SaPIs ) 'Ŭ", 8’ɺĂ,Ŝ¹>= SaPIivm10

(353) 18’ɺĂ,Ŝ¹>= SaPIishikawa11

(354) 19’ ɺĂ,Ŝ¹>= SaPIivm60 :0 44’ɺĂ,Ŝ¹>= SaPIno10 ) SaPIhirosaki4 - 5 ǩ@çĚ" sec ɬɘ SaPIs )çƉ, seb ɬɘ SaPIs 8Qiz-ĎƉ+ɺĂ, Ėÿ=)ű;,+%" 4"

(355) SaPI3 . seb -, sek

(356) seq ɝ ĕ@ SaPI ,¯ŷ=

(357) øçĚ"º'- seb ɬɘ SaPI . SEs ɝ ĕ)' seb @ÕƼ(¯ ŷ'" ;,

(358) źǞǪ(. sek/seq ɬɘ SaPI

(359) SaPIj11 @çĚ" sek/seq ɬ ɘ SaPIs )'

(360) Èɕ- SaPI3 -, SaPI1 ) SaPI5 ąì>'< (19, 46)

(361) ;,>;- 2 &-ɝ ĕ. SaPIs #(.+Sa3mw2 +*-pBV,Ė ÿ=)ąì>'= (5, 32, 49) SaPIs #(.+

(362) - SEs ɝ ĕɬɘ genomic elements ,ɬ'8ĎƉń =)ǝ;>'= McCathey ;-ʃȣqdFǁȥpBV-ȸſ,:=)ȱŧ-. sea ɬɘpBVąì>'= (49) çƉ, egc .. -]Er,:< 1ʋ4 ā4(ā. Æ(=)ǝ;>'= (12) seh ɬɘc Ww\ ,ɬ=ĎƉń.

(363) ǀÿ4. .

(364) (,ąì>'+ źǞǪ(.

(365) ɾƖǇŽ 3 Ƅ)ŉƂǏǇŽ 1 Ƅ sea

(366) sek

(367). seq ɬɘpBV(=Sa3mw2 @¯ŷ'<

(368) ʄȘǇŽ 3 Ƅ N315 Ƅā- egc (1 ā) @¯ŷ'" ŭ

(369) J11 Ƅ(.Źǝ-pBV-ĖÿŞĚ>

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(372) 58 58 58 58 58 58 33. 標準菌株食中毒食中毒. IVM60. Hirosaki11 鼻腔ヒト感染症. IVM10. Hirosaki4 鼻腔ヒト感染症. No.10. Ishikawa11食中毒鼻腔. No.1 Nagasaki 食中毒鼻腔. 243. HHMS2 J11. . 58. 標準菌株. N315. 本研究. 58. 7. 39. 5. 標準菌株. MW2. 引用文献. 由来. 株. 表1-1 供試菌株.

(373) 表1-2. 使用プライマープライマー SaPI4S SaPI4AS. 塩基配列 (5’ to 3’) ATGGCAGGTGGACCAAGAAGAGGCGGACGTCGTCG CATTCTAGTAGACACCTCTGTCGTTTTAACATAAG. 増幅領域 8’SaPI 8’SaPI. Priming位置 (株) 412747-412781 (N315) 416278-416312 (N315). SaPIbovS SaPIbovAS. GTTATGGGAGACTACCGTACGTATGATCACACAG GTTGCTATTTATGGTGGCCTTTCAAGCAATATTAG. 9’SaPI 9’SaPI. 435843-435876 (N315) 439465-439499 (N315). SaPImw2S SaPImw2AS. CATCATGTTGGTTGTTTATCACTTTTCATGATGC GAAAGAAAAAGAAAAGCAAAAAGATGACGACCACAG. 18’SaPI 18’SaPI. 844043-844076 (N315) 845724-845759 (N315). SaPI3externalS SaPI3externalAS. GCTGTAGAAAGTGCGAAAGATAATCCTTATGC GCGAAATAGATTCATCATTAACTTGAGGGAG. 19’SaPI 19’SaPI. 877986-878017 (N315) 878357-878387 (N315). SaPI2S SaPI2AS. AAATTACCGCTACACTAAAGGACAAATTTATC GGTATCGTTGATCCAACTAAAGTAACACGCTCAGC. 44’SaPI 44’SaPI. 2056106-2056137 (N315) 2072459-2072493 (N315). etdS etdAS. CGTTGTGAAAGATAATGAATCGGTTTGGGCTG CTAAAGTGGCAGTAACAAATGATTTTAGTTTC. 49’SaPI 49’SaPI. 2275743-2275774 (N315) 2278259-2278290 (N315). SEH exS SEH exAS. ACTAATGTCTCCATTGGTTGTTCTCTATTAAAAGCGCGAT Transposon ATATCGTCTTTCGACACGTTGTAAGTGAAGCAGCTAGA Transposon. 60574-60614 (MW2) 70511-70548 (MW2). egcS egcAS. TCTTAGAAGAGGATGGCTTCGAAACATTTACAGCG egc GGTGGTATTGCGATCCCATTAAAAGATATTGAAGATTTAGA egc. 1883029-1883063 (N315) 1874827-1874867 (N315). Sa3RS Sa3RAS. GTGAGTTTCGTGATTTCAAAGGTTGGACTAAGATG GACAATAGTGCCAAAGCCGAATCTAAGAAAGATGAT. 2018126-2018160 (N315) 2005236-2005271 (N315). Sa3CS Sa3CAS. ATCTATCCACTCTTTCTCATCCATATCAATAGGTTTACG φSa3 center TACGCGTTTAAGCGAGTACAGAGAAAAGAAAACAATAGA φSa3 center. 2017613-2017651 (N315) 2035055-2035093 (N315). Sa3LS Sa3LAS. GAATCTTCAGATTGTGTATGTGTACCGATAACGT AACCCGTTGTATCCTTTAGTTTTAACTACTTCATCAAG. 2049941-2049974 (N315) 2032829-2032866 (N315). . φSa3 left φSa3 left. φSa3 right φSa3 right.

(374) 表1-3. SaPIivm10のORFマップ ORF No. attL 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 attR. Gene. Annotation or function. aaagaagaacaataatatataatttattgtcaaaccccgtagg Hypothetical protein Hypothetical protein int Integrase stl Helix-turn-helix XRE family protein str Transcriptional regulator xis excisionase Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein pri Primase rep Replication initiator pif Interference with phage growth Hypothetical protein Hypothetical protein cp1 Genome packaging cp2 Genome packaging cp3 Genome packaging cp4 Genome packaging ter Terminase small subunit seb Enterotoxin type B ear Penicillin binding protein aaagaagaacaataatatataatttattgtcaaaccccgtagg. Location (bp). length (bps). 1-43 43 197-1027 (complement) 831 1046-1513 (complement) 468 1510-2727 (complement) 1218 2753-3616 (complement) 864 4022-4234 213 4235-4507 273 4519-4692 174 4659-4862 204 4864-5247 384 5248-5568 321 5634-6503 870 6520-7989 1470 8268-8630 363 8632-8895 264 8897-9538 642 10074-10415 342 10446-11099 654 11152-11679 528 11682-12023 342 12020-12589 570 12897-13697 801 13468-14025 (complement) 558 15735-15751 17. . Comments. No similar protein on database No similar protein on database Similar to SAA6008_00346 (S. aureus JKD6008) No similar protein on database Similar to SAT0131_00379 (S. aureus T0131) Similar to SAA6008_00349 (S. aureus JKD6008) Similar to SAT0131_00381 (S. aureus T0131) Similar to SAT0131_00859 (S. aureus T0131) Similar to SAT0131_00382 (S. aureus T0131) Similar to SAV0789 (S. aureus Mu50) Similar to SAV0790 (S. aureus Mu50) Similar to SAV0791 (S. aureus Mu50) Similar to SAV0792 (S. aureus Mu50) Similar to SAA6008_00357 (S. aureus JKD6008) Similar to SAV2019 (S. aureus Mu50) Similar to SAV0795 (S. aureus Mu50) Similar to SAT0131_00867 (S. aureus T0131) Similar to SAV0798 (S. aureus Mu50) Similar to SAT0131_00392 (S. aureus T0131) Similar to SAV0796 (S. aureus Mu50) Similar to SA21266_1666 (S. aureus 21266) Similar to SA21266_1665 (S. aureus 21266).

(375) 表1-4. SaPIishikawa11のORFマップ ORF. Gene. Annotation or function. Location (bp). length (bps). Comments. No. attL. tcccgccgtctccat. 1-15. 15. 1. int. Integrase. 348-1463 (complement). 1116. Similar to MW0745 (S. aureus MW2). 2. stl. Helix-turn-helix XRE family protein. 1469-2206 (complement). 738. Similar to MW0746 (S. aureus MW2). 3. str. Transcriptional regulator. 2358-2576. 219. Similar to MW0747 (S. aureus MW2). 4. xis. excisionase. 2588-2734. 147. Similar to MW0748 (S. aureus MW2). 5. Hypothetical protein. 2727-2951. 225. Similar to MW0749 (S. aureus MW2). 6. Hypothetical protein. 2952-3251. 300. Similar to MW0750 (S. aureus MW2). 7. rep. Replication initiator. 3344-5716. 2373. Similar to MW0751 (S. aureus MW2). 8. pif. Interference with phage growth. 6404-6748. 345. Similar to MW0752 (S. aureus MW2). 9. Hypothetical protein. 6973-7179. 207. Similar to MW0753 (S. aureus MW2). 10. Hypothetical protein. 7166-8224. 1059. Similar to MW0754 (S. aureus MW2). Terminase small subunit. 8402-8893. 492. Similar to MW0755 (S. aureus MW2). 12. Hypothetical protein. 9322-10290. 969. Similar to SAV0801 (S. aureus Mu50). 13. Hypothetical protein. 10515-10709. 195. Similar to SAV0802 (S. aureus Mu50). 11. ter. 14. seb. Enterotoxin type B. 11447-112247. 801. Similar to SA21266_1666 (S. aureus 21266). 15. ear. Penicillin binding protein. 12325-12882 (complement) 558. Similar to SA21266_1665 (S. aureus 21266). attR. tcccgccgtctccat. 13463-13447. 15. .

(376) 表1-5. SaPIivm60のORFマップ ORF. Gene. Annotation or function. Location (bp). length (bps). Comments. 1-17. 17. Integrase. 160-1404 (complement). 1245. Similar to SA21266_1686 (S. aureus 21266). SAP domain protein. 1394-2782 (complement). 1389. Similar to SA21266_1685 (S. aureus 21266). Helix-turn-helix XRE family protein. 2809-3381 (complement). 573. Similar to SA21266_1683/1684 (S. aureus 21266). 4. Hypothetical protein. 4404-4787. 384. Similar to SA21266_1678 (S. aureus 21266). 5. Hypothetical protein. 4788-5105. 318. Similar to SA21266_1677 (S. aureus 21266). No. attL 1. ttattcctgctaaataa int. 2 3. stl. 6. pri. Primase. 5172-5981. 810. Similar to SA21266_1676 (S. aureus 21266). 7. rep. Replication initiator. 6054-7763. 1710. Similar to SA21266_1675 (S. aureus 21266). 8. pif. Interference with phage growth. 8093-8473. 381. Similar to SA21266_1674 (S. aureus 21266). Hypothetical protein. 8470-9111. 642. Similar to SA21266_1673 (S. aureus 21266). 9 10. cp1. Genome packaging. 9627-9968. 342. Similar to SA21266_1672 (S. aureus 21266). 11. cp2. Genome packaging. 9980-10558. 579. Similar to SA21266_1671 (S. aureus 21266). 12. cp3. Genome packaging. 10576-10794. 219. Similar to SA21266_1670 (S. aureus 21266). 13. Genome packaging. 10797-11372. 576. Similar to SA21266_1669 (S. aureus 21266). 14. Genome packaging. 11375-11716. 342. Similar to SA21266_1668 (S. aureus 21266). 15. ter. Terminase small subunit. 11713-12282. 570. Similar to SA21266_1667 (S. aureus 21266). 16. seb. Enterotoxin type B. 12590-13390. 801. Similar to SA21266_1666 (S. aureus 21266). 17. ear. Penicillin binding protein. 13468-14025 (complement) 558. Similar to SA21266_1665 (S. aureus 21266). attR. ttattcctgctaaataa. 15375-15391. 17. .

(377) 表1-6. SaPIhhms2のORFマップ ORF. Gene. Annotation or function. Location (bp). length (bps). Comments. No. attL. ttattcctgctaaataa. 1-17. 17. 1. int. Integrase. 160-1404 (complement). 1245. Similar to SA21266_1686 (S. aureus 21266). SAP domain protein. 1394-2782 (complement). 1389. Similar to SA21266_1685 (S. aureus 21266). Helix-turn-helix XRE family protein. 2809-3381 (complement). 573. Similar to SA21266_1683/1684 (S. aureus 21266). 4. Hypothetical protein. 4404-4787. 384. Similar to SA21266_1678 (S. aureus 21266). 5. Hypothetical protein. 4788-5105. 318. Similar to SA21266_1677 (S. aureus 21266). 2 3. stl. 6. pri. Primase. 5172-6041. 870. Similar to SA21266_1676 (S. aureus 21266). 7. rep. Replication initiator. 6055-7764. 1710. Similar to SA21266_1675 (S. aureus 21266). 8. pif. Interference with phage growth. 8103-8483. 381. Similar to SA21266_1674 (S. aureus 21266). Hypothetical protein. 8480-9121. 642. Similar to SA21266_1673 (S. aureus 21266). 9 10. cp1. Genome packaging. 9638-9979. 342. Similar to SA21266_1672 (S. aureus 21266). 11. cp2. Genome packaging. 9991-10569. 579. Similar to SA21266_1671 (S. aureus 21266). 12. cp3. Genome packaging. 10587-10805. 219. Similar to SA21266_1670 (S. aureus 21266). 13. Genome packaging. 10808-11383. 576. Similar to SA21266_1669 (S. aureus 21266). 14. Genome packaging. 11386-11727. 342. Similar to SA21266_1668 (S. aureus 21266). Terminase small subunit. 11724-12293. 570. Similar to SA21266_1667 (S. aureus 21266). Phage abortive infection. 12685-13896. 1212. Low homology with Tepidanaerobacter sp. Abi family protein. Penicillin binding protein. 14111-14665 (complement). 558. Similar to SAUSA300_0815 (S. aureus USA300_FRI3757). 16017-16033. 17. 15. ter. 16 17. ear. attR. ttattcctgctaaataa. .

(378) 表1-7. SaPIj11のORFマップ ORF No. attL 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21. 22 attR. Gene. Annotation or function. Location (bp). length (bps). 1-17 160-1380 (complement) 1468-2196 (complement) 2220-2954 (complement) 2966-3424 (complement) 3436-3768 (complement) 3982-4224 4217-4489 4845-5213 5214-5534 5601-6470 6487-7956 8209-8604 8597-8890 8887-9528 10238-10582 10617-11012 11423-11950 11953-12294 12291-12860 12993-13814. 17 1221 729 735 459 318 243 273 369 321 870 1437 396 294 642 345 396 528 342 570 774. Hypothetical protein. 14082-14639. 558. Hypothetical protein ttattcctgctaaataa. 14990-15505 15735-15751. 516 17. ttattcctgctaaataa int Integrase sek Enterotoxin-like type K seq Enterotoxin-like type Q Toxin-antitoxin system stl Cro/CI family transcriptional regulator str Transcriptional regulator xis excisionase Hypothetical protein Hypothetical protein pri Primase rep Replication initiator pif Interference with phage growth Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein cp2 Genome packaging cp3 Genome packaging ter Terminase small subunit Hypothetical protein. . Comments. Similar to SAUSA300_0799 (S. aureus USA300_FPR3757) Similar to SAUSA300_0800 (S. aureus USA300_FPR3757) Similar to SAUSA300_0801 (S. aureus USA300_FPR3757) Similar to SAUSA300_0802 (S. aureus USA300_FPR3757) Similar to SAUSA300_0803 (S. aureus USA300_FPR3757) Similar to SAUSA300_0804 (S. aureus USA300_FPR3757) Similar to SAUSA300_0805 (S. aureus USA300_FPR3757) Similar to SAAV_0792 (S. aureus ED98) Similar to SAAV_0795 (S. aureus ED98) Similar to SAV0790 (S. aureus Mu50) Similar to SAV0791 (S. aureus Mu50) Similar to SAV0792 (S. aureus Mu50) Similar to SAV0793 (S. aureus Mu50) Similar to SAV2019/2020 (S. aureus Mu50) Similar to SA21200_0582 (S. aureus 21200) Similar to SA21200_0581 (S. aureus 21200) Similar to HMPREF0769_10096 (S. aureus MN8) Similar to SAB1895c (S. aureus RF122) Similar to SA21193_1849 (S. aureus 2119) Low homology with Bacillus cereus hypothetical protein (B. cereus 03BB102 strain, BCA_4773, 35% of maximum identity) Low homology with Penibacillus curdlanolyticus hypothetical protein (P. curdlanolyticus YK9 strain, PaecuDRAFT _3564, 29% of maximum identity) Similar to SAAV_0810 (S. aureus ED98).

(379) 表1-8. SaPIno10のORFマップ ORF No. attL 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 attR. Gene. Annotation or function. ttttacatcattcc int Integrase stl Helix-turn-helix XRE family protein str Transcriptional regulator xis excisionase Hypothetical protein pri Primase rep Replication initiator pif Interference with phage growth Hypothetical protein Hypothetical protein cp1 Genome packaging cp2 Genome packaging cp3 Genome packaging cp4 Genome packaging cp5 Genome packaging ter Terminase small subunit Hypothetical protein Hypothetical protein seb Enterotoxin type B ear Penicillin binding protein ttttacatcattcc. Location (bp). length (bps). 1-14 14 81-1253 (complement) 1173 1267-1974 (complement) 708 2160-2348 189 2352-2669 318 3017-3334 318 3401-4270 870 4287-5756 1470 5958-6404 447 6406-6690 285 6687-7328 642 7866-8207 342 8220-8798 579 8816-9034 219 9085-9612 528 9615-9956 342 9953-10522 570 10799-11767 969 11992-12186 195 12925-13725 801 13803-14360 (complement) 558 15735-15751 17. . Comments. Similar to SAB1912 (S. aureus RF122) Similar to SAB1911 (S. aureus RF122) Similar to SAB1910c (S. aureus RF122) Similar to SAB1909c (S. aureus RF122) Similar to SAV0789 (S. aureus Mu50) Similar to SAV0790 (S. aureus Mu50) Similar to SAV0791 (S. aureus Mu50) Similar to SAV0792 (S. aureus Mu50) Similar to SAV0793 (S. aureus Mu50) Similar to SAV0794 (S. aureus Mu50) Similar to SAV0795 (S. aureus Mu50) Similar to SAV0796 (S. aureus Mu50) Similar to SAV0797 (S. aureus Mu50) Similar to SAV0798 (S. aureus Mu50) Similar to SAV0799 (S. aureus Mu50) Similar to SAV0800 (S. aureus Mu50) Similar to SAV0801 (S. aureus Mu50) Similar to SAV0802 (S. aureus Mu50) Similar to SA21266_1666 (S. aureus 21266) Similar to SA21266_1665 (S. aureus 21266).

(380) 表1-9. SaPIhirosaki4のORFマップ ORF No. attR 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 attR. Gene. Annotation or function. ttttacatcattccggcat int Integrase stl Helix-turn-helix XRE family protein str Transcriptional regulator xis excisionase Hypothetical protein pri Primase rep Replication initiator pif Interference with phage growth Hypothetical protein cp1 Genome packaging cp2 Genome packaging cp3 Genome packaging Genome packaging Genome packaging ter Terminase small subunit Hypothetical protein Hypothetical protein seb Enterotoxin type B ear Penicillin binding protein ttttacatcattccggcat. Location (bp). length (bps). 1-19 19 86-1258 (complement) 1173 1272-1946 (complement) 675 2119-2337 219 2342-2659 318 3007-3324 318 3388-4257 870 4271-5971 1701 6301-6681 381 6678-7319 642 7881-8222 342 8234-8812 579 8830-9048 219 9099-9626 528 9629-9970 342 9967-10536 570 10813-11781 969 12006-12200 195 12938-13738 801 13816-14373 (complement) 558 15115-15134 19. . Comments. Similar to SAT0131_02174 (S. aureus T0131) Similar to SAT0131_02173 (S. aureus T0131) Similar to SAT0131_02172 (S. aureus T0131) Similar to SAV2025 (S. aureus Mu50) Similar to SaPI2 ORF7 (S. aureus RN3984) Similar to SaPI2 ORF8 (S. aureus RN3984) Similar to SaPI2 ORF9 (S. aureus RN3984) Similar to SaPI2 ORF10 (S. aureus RN3984) Similar to SaPI2 ORF11 (S. aureus RN3984) Similar to SAV0795 (S. aureus Mu50) Similar to SAV0796 (S. aureus Mu50) Similar to SAV0797 (S. aureus Mu50) Similar to SAV0798 (S. aureus Mu50) Similar to SaPI2 ORF17 (S. aureus RN3984) Similar to SAV0800 (S. aureus Mu50) Similar to SAV0801 (S. aureus Mu50) Similar to SAV0802 (S. aureus Mu50) Similar to SA21266_1666 (S. aureus 21266) Similar to SA21266_1665 (S. aureus 21266).

(381) 表1-10. N315株およびN315株における各増幅genomic elementsの消化DNA断片長 (bp). 増幅産物 (株). 制限酵素消化断片1. 消化断片2. 消化断片3. 消化断片4. 消化断片5. 消化断片6. 消化断片7. 消化断片8. 消化断片9. 消化断片10. 消化断片11. HindIII. 5,032 (bp). 4,634. 243. 79. -. -. -. -. -. -. -. egc (N315). HindIII. 2,594. 2,561. 2,349. 747. -. -. -. -. -. -. -. Sa3L* (MW2). HaeIII. 5,237. 3,351. 1,587. 1,049. 847. -. -. -. -. -. -. Sa3L* (N315). HaeIII. 8,730. 3,351. 842. -. -. -. -. -. -. -. -. Sa3C** (MW2). HaeIII. 4,039. 3,036. 2,380. 2,059. 1,571. 1,193. 854. 845. 722. 707. 137. Sa3C** (N315). HaeIII. 13,858. 3,645. -. -. -. -. -. -. -. -. -. Sa3R*** (MW2). HaeIII. 7,310. 4,419. 1,792. 1,545. 232. -. -. -. -. -. -. Sa3R*** (N315). HaeIII. 14,592. 3,645. -. -. -. -. -. -. -. -. -. seh-トランスポゾン (MW2). *Sa3L: Sa3 Left,**Sa3C: Sa3 Center, ***Sa3R: Sa3 Right. .

(382) 表1-11. 本章で使用した株のgenomic elementsプロファイリング. 株. 由来. 8’. 9’. 18’. 19’. 44’. 49’. トランスポゾン. egc. φSa3. No. 1. 食中毒. -. GE TypeA (MW2). SaPIishikawa11. -. -. -. ○ (MW2). -. -. No. 10. 食中毒. -. GE TypeA. -. -. SaPIno10. -. ○. -. φSa3mw2. Ishikawa11. 食中毒. -. GE TypeA. SaPIishikawa11. -. -. -. ○. -. φSa3mw2. Nagasaki. 食中毒. -. GE TypeA. SaPIishikawa11. -. -. -. ○. -. φSa3mw2. IVM10. 鼻腔. SaPIivm10. -. -. -. -. GE. -. ○ (N315). -. IVM60. 鼻腔. -. GE TypeA. -. SaPIivm60. -. -. -. -. -. Hirosaki4. 鼻腔. -. GE TypeB. -. -. SaPIhirosaki4. -. -. ○. -. Hirosaki11. 鼻腔. SaPIivm10. GE TypeB. -. -. -. GE. -. ○. -. HHMS2. 感染症. -. GE TypeA. -. SaPIhhms2. -. -. ○. -. φSa3mw2. J11. 感染症. -. GE TypeA. -. SaPIj11. -. -. ○. -. unknown phage. GE: genomic elements. .

(383) 図1-1. Sa3 Left. φSa3n (from N315). Sa3 Center. Sa3 Right 10Kbp. 図1-1. φSa3領域ファージのLA-PCRでの増幅領域。Left、Center、Rightは各プライマーセットでの増幅領域を示す。図の作成には、データベース上のN315株のゲノム配列 (BA000018) より、φSa3nの配列を抽出し、 In Silico MolecularCloning を使用してファージマップを作成した。 .

(384) 図1-2 A M. B 8’. 9’ 18’ 19’ 44’ 49’ M. C M. 44’. 20 9.6 6.6 4.4 kbp. 1 2. 4.4 2.3 2.0 kbp. N315. 3. 図1-2. N315株におけるSaPIs解析。A: 全6カ所のSaPIs挿入領域の解析。8’、9’、18’、19’、44’、49’はそれぞれ SaPIs挿入領域の8’、9’、18’、19’、44’、49’を示す。M: マーカー (λ-HindIII digest (Takara))。 B. 44’領域増幅産物のHindIII消化断片の泳動像。矢頭1-3は消化断片1-3を示す。M: マーカー (λ-HindIII digest (Takara))。C. sec 特異的DNAプローブを用いたSouthern hybridization。矢頭2は消化断片2を示す。. . 2.

(385) 図1-3. M 1 2 3 4 5 6 7 8 9 10 11 N M. M 1 2 3 4 5 6 7 8 9 10 11 12 M. 8’. 20 4.4 kbp. 9’. 20 4.4 kbp. 20 4.4 44’kbp. 20 4.4 18’ kbp. 20 4.4 49’kbp. 20 4.4 19’kbp. 図1-3. 臨床分離株10株のSaPIs解析。M: 分子量マーカー (λ-HindIII digest (Takara))、1: 243. 2: No. 1、3: No. 10、4: Nagasaki、5: Ishikawa11、6: IVM10、7: IVM60、8: Hirosaki4、9: Hirosaki11、10: HHMS2、11: J11、N: No DNA (超純水) 矢頭: SaPIsが含まれていると考えられる増幅産物。 .

(386) 図1-4 20 9.4 6.4 4.4. 8’. 9’. 20 9.4 6.4 4.4. M 6 9 M. 19’. 20 9.4 6.4 4.4. 2.3 2.0. 2.3 2.0. kbp. kbp. M 1 7 10 11 M. M 2 3 4 5 7 8 9 10 11 M. M 3 8 M. 44’. 2.3 2.0 kbp. 20 9.4 6.4 4.4 2.3 2.0 kbp. M 6 9 M. M 2 4 5 9M. 18’. 20 9.4 6.4 4.4. 49’. 2.3 2.0. 20 9.4 6.4 4.4 2.3 2.0 kbp. kbp. 図1-4. SaPIsが存在すると考えられる増幅産物のHindIII処理。M: 分子量マーカー (λ-HindIII digest (Takara))、1: 243. 2: No. 1、3: No. 10、4: Nagasaki、5: Ishikawa11、6: IVM10、7: IVM60、8: Hirosaki4、 9: Hirosaki11、10: HHMS2、11: J11。500bp以下のDNA断片は確認困難 .

(387) 図1-5 SaPIivm10 seb ear SaPIishikawa11 seb ear SaPIivm60 seb ear SaPIhhms2 ear SaPIj11 sek seq SaPIno10 seb ear SaPIhirosaki4 Lysogen. Replication Phage Packaging interference. seb ear. 1Kbp 図1-5. 本研究で同定した7種の新規SaPIs。青矢印: 溶原化、遺伝子制御に関わる遺伝子。緑矢印: 複製に関わる遺伝子、橙矢印: ファージ干渉に関わる遺伝子。灰矢印: ファージ様粒子へのパッケージングに関わる遺伝子、赤矢印: スーパー抗原遺伝子、黄矢印: 薬剤耐性遺伝子. .

(388) A. 図1-6. B Length M (kbp). 1.5’ sehTransposon HindIII. MW2. 5.0 4.0 3.0. egc HindIII. 3.0 2.5 2.0. M. N315. 0.75. C. M. MW2 N315. D. 3.0. 1.5. MW2 N315. E. φSa3 Center HaeIII. M. MW2 N315. 10.0. 10.0 5.0. 8.0 6.0 5.0. φSa3 Left HaeIII. M. 5.0. φSa3 Right HaeIII. 2.0 1.0. 2.0 1.0. :不明瞭なバンド図1-6. N315株およびN315株における3種のgenomic elements挿入領域における増幅産物の制限酵素処理。A. トランスポゾン (MW2)、B. egc (N315)、C. φSa3 Left (MW2とN315)、D. φSa3 Center (MW2とN315)、E. φSa3 Right (MW2とN315)。 M: 1kbp Ladder。白矢頭: 不明瞭なDNA断片。500bp以下のDNA断片は確認困難。 .

(389) 図1-7 A. B. C. D. E. Length (kbp) M 10.0. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. M. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. M. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. M. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. M. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 1.5’ sehTransposon egc. 10.0. φSa3 Left. 10.0. φSa3 Center10.0. φSa3 Right. 10.0. 図1-7. 臨床分離株のSEs関連genomic elementsの解析。 M: 1kbp Ladder。1: No. 1、2: No. 10、3: Nagasaki、4: Ishikawa11、5: IVM10、6: IVM60、7: Hirosaki4、8: Hirosaki11、9: HHMS2、10: J11。白矢頭: 不明瞭なPCR産物。 .

(390) 図1-8. Length (kbp) M. A 1.5’ sehTransposon HindIII. 2. 3. 4. M. 10. 9. B. 5.0 4.0 3.0. M. C. 1. egc HindIII. 2. 3. 4. 9. 10. 10.0 5.0 φSa3 3.0. M. D. 5. 7. 8. 3.0 2.0 1.0. 2. 3. 4. 9. 10. 5.0 4.0 φSa3 3.0 Center 2.0. Left HaeIII. HaeIII 1.0. 1.0. E. M. 2. 3. 4. 9. 10. 10.0 φSa3 5.0. :不明瞭なバンド. Right HaeIII 2.0 1.0. 図1-8. 臨床分離株のSEs関連genomic elementsの解析。A. トランスポゾン、B. egc、C. φSa3 Left、D. φSa3 Center、E. φSa3 Right 。 M: 1kbp Ladder。1: No. 1、2: No. 10、3: Nagasaki、4: Ishikawa11、5: IVM10、6: IVM60、7: Hirosaki4、8: Hirosaki11、9: HHMS2、10: J11。白矢頭: 不明瞭なDNA断片。500bp以下のDNA断片は確認困難。. .

(391) ƿ 2 ƾ Ņŏ,=ȩŦÍĥN-Øā. 2-1. Ėǹ ȮǡocFƏǢ-»ýƛÿǮœ(. ŏǢ--ǮœĈǻ,ƫƨ=)ú mobile genetic elements )RCQgw@Ǯœ=ĬŰ,»Ȩ=)(=

(392) Ãƾ(genomic elements-scanning Ű. SCCmec îÀ)ØŚ, genomic elements Ǯœ, ě!=

(393) - RCQgw@Ǯœ=ĬŰ)' MLST 9RCO{Y (Coa) îÀ+ *Ƭ;>'=

(394) MLST .ŏǢ¥Ō=ƒßǑĳ,ÔŞ+ 7 %-hFVLm OȎý-÷ƚj\;Ǣ-ǆǏǮœ@ǥŃŰ( < (20) Ǝë MRSA ĩŕƠ ǀ-ÖƹĩŕƠƕŒŗ-Ǯœ,ūƔ>'=

(395) Coa îÀ. ŏǢƓƒ= Coa )Þ />=nDo|¸ê,ȗ=\jN-İÍĥ,ñ&»ȨŰ( = (92)

(396) %' .Ö Coa î,Ĉ=İ,:=ĮÁǴȬǥ?>'! ȃĕ :<šƱĥ-ȭ PCR (-»ȨŰƱƽ>! (68)

(397) Coa îÀ.Ņŏ,'ȩŦ»ȟŗ-ŘŖ,ūƔ >'< ȩŦƕŒŗ.Ɖā-ǤŻî (II î/III î/IV î/VII î) ,Ȟƺ=) Ƭ;>'= (36, 56, 68, 75)

(398) ȮǡocFƏǢ-# MRSA ,:=ĩŕƠ(. MLST ) SCCmec îÀ@ǌ5×? !ǮœƥA,ǥ?> ǩĽ-Ȏÿƣ,ȃǓ+NòÝ>'=

(399) Ó´ƣ + MRSA N)' ST250 (CC30)/SCCmecI Ƭ;>'= (18, 21)

(400) ğ ùŚ+NòÝ> Êơńǲȗȉî- MRSA N)'Ņŏ9ÈǄ (. CC5/SCCmecII - New York/Japan N z_j(. CC5/SCCmecI - EMRSA16/pediatric N-ƟȚµ(-ĩŕäȧ)+$'= (18)

(401) Ń 1990 ĕș Ņŏ@Û7!Ɨ( Êơńǲø(- MRSA ĩŕ (ďĩŕî MRSA) äȧǭ>'= (16)

(402) Ɖ,ÈǄ(. ST8/SCCmecVI - USA300 N -þëŸÂ+äȧ,+$'< ȃĕ(.Êơńǲ,'8»ȟòÝ>' .

(403) = (62)

(404) -:,ĩŕƠ;-»ȟŗ(.-ǵǊ+ȎÿƣǚŊǮœ> Ǎň ƣ%ìƐƣ+SqE{V,:$' ĩŕƠĮŠ-!7-ñƲħòƱƽ>' =

(405) ŏƾ(. ȩŦƢƒĮŠ-!7-ƶÿƣñƦ-ĜĪ@ƨƣ)' Ŏ"òÝ-+ ȩŦÍĥN-Øā)ȎÿƣƉġ-Ǯœ@Ǵ5!

(406). 2-2. Őŀ:0ŃŰ 2-2-1. ǴǢŗ ðȫő+;0, DNA ıº ŏƾ(.Ņŏ(»ȟ>! 506 ŗ-ȮǡocFƏǢ@Ɣ!

(407) -# 42 ŗ. 1990-2010 ĕ,Ƣƒ!ȩŦ»ȟŗ( < ²'ƚ+=ȩŦ;»ȟ> !

(408) 329 ŗ. 2005-2010 ĕ,'ƚ+=©ĒǙ-ȯǜ;-»ȟŗ( =

(409) 85 ŗ. 1990 ĕș,Ƣƒ!ÖƹȮǡocFƏǢƝĦ (ļǤƠ ŕĥǟƞƜ ocFƏǢĥ ƤǞƅ«ŚƠ§Ǘ CbmĥƤǞƃ+*) -ĦǙ;-»ȟŗ( =

(410) 4! Ţ<. 2007 ĕ,úȘ-VjuP_b-Ǽƈ;»ȟ>!Ƒô»ȟŗ 50 ŗ (52) @Ɣ!

(411) Ö»ȟŗ-»ȟìï@é 2-1 ,Ƴ

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(413) 4!ŏƮƻ(Ɣ!ŗ,. ȟč( »ȟ>!8-.þë+

(414) + ȮǡocFƏǢ-ðȫ) DNA ıº.ƿ 1 ƾ,Ǳ Ȃ!ŃŰ)ØŚ,ǥ$!

(415). 2-2-2. ȮǡocFƏǢ- genotyping ŏƮƻ(. Coa îÀ SE/SEl î:0 MLST @ genotyping Ű)'ǥ$!

(416) Ɣ!p{EuX_b@ǧ 2-1 ,Ƴ

(417) Coa îÀ. Sakai ; (68) òÝ!p{E uX_b@Ɣ'ǥ$!

(418) ŏŰ(. 2 ƹȨ-p{EuX_b < X_b A )X _b B ,.>. > CoaIII CoaIV CoaVII CoaVIII ) CoaI CoaII CoaV. .

(419) CoaVI @Ɖƚƣ,õē(=p{EuÛ4>'=

(420) PCR ÏĤ. QIAGEN Multiplex PCR Kit (QIAGEN) @Ɣ' ÏĤŷ.xJ-pbR},ſĲ MyCycler™ jZd}Su}SEN{ (Bio-Rad) @Ɣ'ǥ$!

(421) ÏĤő. 9515 »-ğ 35 æ-õē (9430 Ʒ 5730 Ʒ 7290 Ʒ) @ǥ ŋ ğ, 7210 »(ŋǋĚ@ǥ$!

(422) õēƓƈ. 3% NuSieve 3:1 Agarose Gel (Cambrex Bio Science Rockland) ) 0.5 × Tris-boric acid-EDTA (TBE) buffer @Ɣ' ȠŨűÆ@ǥ 0.5μg/ml EtBr ) UV b{VE}vf\@Ɣ'ÔǭÇ ƱǶ !

(423) 0.5×TBE . 10x b|V/sFȑ/EDTA (TBE) (Bio-Rad) @đȒ'Ī!

(424) SE/SEl -ȎýîÀ. Omoe ; (58) òÝ!p{EuX_b@Ɣ'ǥ$ !

(425) SEA-SER Ȏý) TSST-1 Ȏý@Ɖƚƣ,õē=(= 4 ƹ-p{Eu X_b (X_b 1: seasebsecsedseefemB, X_b 2: segsehseiselj. sep femA, X_b 3: seksemseotst-1 femA, X_b 4: selsenser seq femB) @Ɣ'õē!