Title 黄色ブドウ球菌食中毒における分子疫学解析と可動性遺伝因子の特性に関する研究( 本文(Fulltext) ) Author(s) 鈴木, 康規 Report No.(Doctoral Degree) 博士(獣医学) 甲第444号 Issue Date 2015-09-24 Type 博士論文 Version ETD URL http://hdl.handle.net/20.500.12099/53639 ※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。
㯤 Ⰽࣈ ࢻ࢘⌫ ⳦㣗 ୰ẘ ࠾ࡅ ࡿ
ศ Ꮚ Ꮫゎᯒ ྍ ືᛶ㑇 ఏᅉ Ꮚࡢ
≉ ᛶ 㛵ࡍࡿ ◊✲
2015 ᖺ
ᒱ 㜧 ᏛᏛ 㝔㐃 ྜ⋇་ Ꮫ◊ ✲⛉
㸦 ᒾᡭ Ꮫ㸧
㕥 ᮌ ᗣつ
┠ ḟ ⥴ ゝ 1 ᅗ 8 ➨ ୍ ❶ ᮾ ி 㒔 ࡛ Ⓨ ⏕ ࡋ ࡓ 㣗 ୰ ẘ ⏤ ᮶ 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ศ 㞳 ᰴ ࡢ ศ Ꮚ Ꮫ ゎ ᯒ ᗎ ㄽ 11 ᮦ ᩱ ཬ ࡧ ᪉ ἲ 1. 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ᰴ ⏝ ᇵ ᆅ 12 2. ࢥ ࢢ ࣛ ࣮ ࢮ ᆺ ู 13 3. Multiplex PCR ἲ ࢆ ⏝ ࠸ ࡓ SE/SEls 㑇 ఏ Ꮚ ࡢ ᆺ ู ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡢ ࢱ ࣉ ࡢ ᥎ 13 4. 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ Genomic DNA ࡢ ⢭ 〇 15
5. Multi locus sequence typing 17
6. SaPI scanning 20
7. ศ Ꮚ ⣔ ⤫ ゎ ᯒ 22
8. Sandwich ELISA ࢆ ⏝ ࠸ ࡓ SEA ࢱ ࣥ ࣃ ࢡ ㉁ ࡢ ᐃ 㔞 23
⤖ ᯝ
1. 㣗 ୰ ẘ ⏤ ᮶ 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ᰴ ࡢ MLST ゎ ᯒ 25
Clonal complex ࡢ 㛵 ಀ ᛶ 26 3. 㣗 ୰ ẘ ⏤ ᮶ 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ᰴ ࡢ SE/SEls 㑇 ఏ Ꮚ ᆺ Clonal complex ࡢ 㛵 ಀ ᛶ 26 4. 㣗 ୰ ẘ ⏤ ᮶ 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ᰴ ࡀ ಖ ᭷ ࡍ ࡿ SaPI ࡢ ྠ ᐃ Clonal complex ࡢ 㛵 ಀ ᛶ 27 5. CC81 ᒓ ࡍ ࡿ sea 㑇 ఏ Ꮚ 㝧 ᛶ 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ᰴ ࡢ SEA ⏘ ⏕ 㔞 29 ⪃ ᐹ 29 ⾲ 35 ᅗ 42 ➨ ❶ ࣈ ࢻ ࢘ ⌫ ⳦ ࢚ ࣥ ࢸ ࣟ ࢺ ࢟ ࢩ ࣥ ࢆ ಖ ᭷ ࡍ ࡿ ᪂ つ SaPI ࡢ ྠ ᐃ ࡑ ࡢ ≉ ᛶ ᗎ ㄽ 51 ᮦ ᩱ ཬ ࡧ ᪉ ἲ 1. 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ᰴ ⏝ ᇵ ᆅ 52 2. ⭠ ⳦ ᰴ ⏝ ᇵ ᆅ 53 3. ⏝ ࡋ ࡓ ࣉ ࣛ ࢫ ࣑ ࢻ ࡢ ᴫ せ 53 4. 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ Genomic DNA ࡢ ⢭ 〇 54 5. SaPI scanning 54
6. Southern blotting ࢆ ⏝ ࠸ ࡓ LA-PCR ቑ ᖜ ⏘ ≀ ୰ ࡢ SEs 㑇 ఏ Ꮚ ࡢ ᳨ ฟ
A. pKOB㸪 pKOC ཬ ࡧ pKOL ࣉ ࣛ ࢫ ࣑ ࢻ ࡢ 㔞 ⢭ 〇 54
B. seb㸪 sec㸪 sel 㑇 ఏ Ꮚ ᩿ ∦ ࡢ ⢭ 〇 56
C. seb㸪 sec㸪 sel ࣉ ࣟ ࣮ ࣈ ࡢ ᶆ ㆑ 57
D. Southern blotting 58 7. ᪂ つ SaPI 㓄 ิ ࡢ ྠ ᐃ 62 8. Sandwich ELISA ࢆ ⏝ ࠸ ࡓ SE ࢱ ࣥ ࣃ ࢡ ㉁ ࡢ ᐃ 㔞 62 9. Rep ࡢ Ⓨ ⌧ ⢭ 〇 A. Rep Ⓨ ⌧ ⏝ ࣉ ࣛ ࢫ ࣑ ࢻ ࡢ ᵓ ⠏ 63 B. ⤌ ࠼ Rep ࡢ Ⓨ ⌧ ཬ ࡧ ⢭ 〇 66 C. rRep ࡢ ⣧ ᗘ ࡢ ☜ ㄆ 69 10. ࢤ ࣝ ࢩ ࣇ ࢺ ࢵ ࢭ A. ori site 㓄 ิ ࣉ ࣟ ࣮ ࣈ ࡢ స 〇 73 B. ࢤ ࣝ ࢩ ࣇ ࢺ ࢵ ࢭ 74 11. ⺯ ග ࣊ ࣜ ࢝ ࣮ ࢮ ࢵ ࢭ 75 12. ⤫ ィ Ꮫ ⓗ ゎ ᯒ 77 ⤖ ᯝ
1. seb㸪 sec㸪 sel 㑇 ఏ Ꮚ ࡀ Ꮡ ᅾ ࡍ ࡿ GI ࡢ ྠ ᐃ 77
2. seb㸪 sec㸪 sel 㑇 ఏ Ꮚ ಖ ᭷ ᪂ つ SaPI 㓄 ิ ࡢ ྠ ᐃ 79
3. ᪂ つ SaPI ᪤ ▱ SaPI ࡀ ಖ ᭷ ࡍ ࡿ sel 㓄 ิ ࡢ ẚ ㍑ 80
4. ᪂ つ SaPI ࠾ ࡅ ࡿ rep-ori site 㡿 ᇦ ࡢ ẚ ㍑ 81
5. ᪂ つ SaPI ࢆ ಖ ᭷ ࡍ ࡿ 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ᰴ ࡢ SEs ⏘ ⏕ 㔞 82
7. rRep ࡢ ࣊ ࣜ ࢝ ࣮ ࢮ ά ᛶ 84 ⪃ ᐹ 84 ⾲ 91 ᅗ 105 ➨ ୕ ❶ SE/SEls 㛵 㐃 ࣉ ࣛ ࢫ ࣑ ࢻ ࡢ 㣗 ୰ ẘ ࡢ 㛵 ኚ ␗ ᆺ sed 㑇 ఏ Ꮚ ࡢ ྠ ᐃ ᗎ ㄽ 115 ᮦ ᩱ ཬ ࡧ ᪉ ἲ 1. 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ᰴ ⏝ ᇵ ᆅ 116 2. ⭠ ⳦ ᰴ ⏝ ᇵ ᆅ 117 3. ⏝ ࡋ ࡓ ࣉ ࣛ ࢫ ࣑ ࢻ ࡢ ᴫ せ 117 4. 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ࣉ ࣛ ࢫ ࣑ ࢻ ࡢ ⢭ 〇 118 5. 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ࣉ ࣛ ࢫ ࣑ ࢻ ࡢ ไ 㝈 㓝 ⣲ ᩿ ∦ 㛗 ከ ᆺ ゎ ᯒ 119
6. Sandwich ELISA ࢆ ⏝ ࠸ ࡓ SE/SEl ࢱ ࣥ ࣃ ࢡ ㉁ ࡢ ᐃ 㔞 120
7. sed, selj ཬ ࡧ ser 㑇 ఏ Ꮚ ࢆ ྵ ࡴ EcoRI ᩿ ∦ ࡢ ࢩ ࣮ ࢡ
࢚ ࣥ ࢫ ゎ ᯒ 121
8. 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ࡽ ࡢ ⥲ RNA ࡢ ⢭ 〇 122
10. ࢫ ࢱ ࣥ ࢲ ࣮ ࢻ ࣉ ࣛ ࢫ ࣑ ࢻ ࡢ స 〇 125 11. ᐃ 㔞 ⓗ ࣜ ࣝ ࢱ ࣒ RT-PCR 125 12. ⤫ ィ Ꮫ ⓗ ゎ ᯒ 127 ⤖ ᯝ 1. 㣗 ୰ ẘ ⏤ ᮶ 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ᰴ ࡀ ಖ ᭷ ࡍ ࡿ ࣉ ࣛ ࢫ ࣑ ࢻ ࡢ ྠ ᐃ 127 2. 㣗 ୰ ẘ ⏤ ᮶ 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ᰴ ࠾ ࡅ ࡿ SE/SEls ࡢ ⏘ ⏕ 㔞 128 3. sed 㓄 ิ ࡀ Ꮡ ᅾ ࡍ ࡿ EcoRI ᩿ ∦ ࡢ ࢡ ࣟ ࣮ ࢽ ࣥ ࢢ ࢩ ࣮ ࢡ ࢚ ࣥ ࢫ ゎ ᯒ 129 4. ᐃ 㔞 ⓗ ࣜ ࣝ ࢱ ࣒ RT-PCR ࡼ ࡿ sed ཬ ࡧ ኚ ␗ ᆺ sed 㑇 ఏ Ꮚ Ⓨ ⌧ ࡢ ᳨ ฟ 130 ⪃ ᐹ 130 ⾲ 136 ᅗ 139 ⥲ ᣓ 144 ㅰ ㎡ 148 ཧ ⪃ ᩥ ⊩ 149
␎ ㄒ ୍ ぴ
att 㓄 ิ 㸸 attachment 㓄 ิ BHI㸸 Brain heart infusion
BLAST㸸 Basic local alignment search tool
BSA㸸 Bovine serum albumin㸦 ࢘ ࢩ ⾑ Ύ ࣝ ࣈ ࣑ ࣥ 㸧 CC㸸 Clonal complex
CoaT㸸 Coagulase type㸦 ࢥ ࢢ ࣛ ࣮ ࢮ ᆺ 㸧 dsDNA㸸 double-stranded DNA 㸦 㔜 㙐 DNA㸧
E. coli㸸 Escherichia coli㸦 ⭠ ⳦ 㸧 egc㸸 enterotoxin gene cluster
EtBr㸸 Ethidium bromide㸦 ⮯ ࢚ ࢳ ࢪ ࢘ ࣒ 㸧
ftsZ㸸 Cell division protein gene
GI㸸 Genomic island
HRP㸸 Horseradish peroxidase㸦 す ὒ ࣡ ࢧ ࣅ ࣌ ࣝ ࢜ ࢟ ࢩ ࢲ ࣮ ࢮ 㸧
IPTG㸸 Isopropyl β -D-1-thiogalactopyranoside
IS㸸 Insertion sequence
LA-PCR㸸 Long and accurate polymerase chain reaction LB㸸 Luria-Bertani
MHC㸸Major histocompatibility c omplex㸦 せ ⤌ ⧊ 㐺 ྜ 㑇 ఏ Ꮚ 」 ྜ య 㸧 MLST㸸 Multi locus sequence t yping
mSED㸸 Mutant staphylococcal enterotoxin D㸦 ኚ ␗ ᆺ SED㸧
msed㸸Mutant staphylococcal e nterotoxin D gene㸦 ኚ ␗ ᆺ SED 㑇 ఏ Ꮚ 㸧 MSEY㸸 Mannitol salt agar with egg yolk㸦 ༸ 㯤 ຍ ࣐ ࣥ ࢽ ࢵ ࢺ 㣗 ሷ 㸧 NCBI㸸 National Center for Biotechnology Information㸦 ᅜ ❧ ⏕ ≀ ᕤ Ꮫ
ሗ ࢭ ࣥ ࢱ ࣮ 㸧
ORF㸸 Open reading frame
ori site㸸 Replication origin㸦 」 〇 㛤 ጞ Ⅼ 㸧
PBS㸸 Phosphate buffered saline 㸦 ࣜ ࣥ 㓟 ⦆ ⾪ ⏕ ⌮ 㣗 ሷ Ỉ 㸧
PBST㸸 Phosphate buffered saline with Tween 20㸦 Tween 20 ῧ ຍ ࣜ ࣥ 㓟 ⦆ ⾪ ⏕ ⌮ 㣗 ሷ Ỉ 㸧
Rep㸸 Replication initiator protein 㸦 」 〇 㛤 ጞ ࢱ ࣥ ࣃ ࢡ ㉁ 㸧
rep㸸 Replication initiator protein gene 㸦 」 〇 㛤 ጞ ࢱ ࣥ ࣃ ࢡ ㉁ 㑇 ఏ Ꮚ 㸧
RFLP㸸 Restriction fragment length pol ymorphism㸦 ไ 㝈 㓝 ⣲ ᩿ ∦ 㛗 ከ ᆺ 㸧
rpoB㸸 RNA pol ymerase beta subunit gene
RT-PCR㸸 Reverse transcription pol ymerase chain reaction
rRep㸸 recombinant replication initiator protein 㸦 ⤌ ࠼ 」 〇 㛤 ጞ ࢱ ࣥ ࣃ ࢡ ㉁ 㸧
SAG㸸 Superantigen 㸦 ࢫ ࣮ ࣃ ࣮ ᢠ ཎ ᛶ ẘ ⣲ 㸧 SaPI㸸 Staphylococcus aureus pathogenicity island
S. aureus㸸 Staphylococcus aureus㸦 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ 㸧
SDS-PAGE㸸Sodium dodecyl sulfate-pol y-acrylamide gel electrophoresis
㸦 ࢻ ࢹ ࢩ ࣝ ◲ 㓟 ࢼ ࢺ ࣜ ࢘ ࣒-࣏ ࣜ ࢡ ࣜ ࣝ ࣑ ࢻ ࢤ ࣝ 㟁 Ẽ Ὃ ື 㸧
SE㸸 Staphylococcal enterotoxin㸦 ࣈ ࢻ ࢘ ⌫ ⳦ ࢚ ࣥ ࢸ ࣟ ࢺ ࢟ ࢩ ࣥ 㸧 SEl㸸Staphylococccal enterotoxin-like toxin㸦 ࣈ ࢻ ࢘ ⌫ ⳦ ࢚ ࣥ ࢸ ࣟ ࢺ ࢟ ࢩ ࣥ ᵝ ẘ ⣲ 㸧
slv㸸 Single locus variant
sodA㸸 Superoxide dismutase gene
TBS㸸 Tris buffered saline㸦 ࢺ ࣜ ࢫ ⦆ ⾪ ⏕ ⌮ 㣗 ሷ Ỉ 㸧
TBST㸸 Tris buffered saline with Tween 20㸦 Tween 20 ῧ ຍ ࢺ ࣜ ࢫ ⦆ ⾪ ⏕ ⌮ 㣗 ሷ Ỉ 㸧
TET ࣂ ࢵ ࣇ ࣮ 㸸 Tris-EDTA-Triton X-100 ࣂ ࢵ ࣇ ࣮ TSB㸸 Tryptic soy broth
1 ⥴ ゝ
㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ 㸦Staphylococcus aureus㸹 S. aureus㸧 ࡣ ့ ங ື ≀ ࡸ
㫽 㢮 ࡞ ࡢ ⾑ ື ≀ ᗈ ࡃ ศ ᕸ ࡍ ࡿ ᖖ ᅾ ⳦ ࡛ ࠶ ࡿ ྠ 㸪㣗 ୰ ẘ 㸪 ẘ ⣲ ᛶ ࢩ ࣙ ࢵ ࢡ ೃ ⩌ 㸪 ࣈ ࢻ ࢘ ⌫ ⳦ ᛶ ⓶ ⬺ ೃ ⩌ ࠸ ࡗ ࡓ ẘ ⣲ ཎ ᛶ ᝈ ࢆ ᘬ ࡁ ㉳ ࡇ ࡍ ཎ ⳦ ࡋ ࡚ 㔜 せ ど ࡉ ࢀ ࡚ ࠸ ࡿ ࠋ ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ࡣ 㸪 ᮏ ⳦ ࡢ ⏘ ⏕ ࡍ ࡿ ࣈ ࢻ ࢘ ⌫ ⳦ ࢚ ࣥ ࢸ ࣟ ࢺ ࢟ ࢩ ࣥ
㸦Staphylococcal enterotoxins㸹 SEs㸧ࡢ ᦤ ྲྀ ࡼ ࡾ ᘬ ࡁ ㉳ ࡇ ࡉ ࢀ ࡿ ẘ
⣲ ᆺ 㣗 ୰ ẘ ࡢ ୍ ࡘ ࡛ ࠶ ࡿ ࠋSEs ࡣ ⪏ ⇕ ᛶ ࡀ 㧗 ࡃ 㸪 ᪤ 㣗 ရ ୰ SEs ࡀ ⏘ ⏕ ࡉ ࢀ ࡚ ࠸ ࡿ ሙ ྜ 㸪 ẅ ⳦ ฎ ⌮ ࡸ ຍ ⇕ ㄪ ⌮ ࡛ ⳦ ࢆ Ṛ ⁛ ࡉ ࡏ ࡚ ࡶ SEs ࡣ ṧ Ꮡ ࡋ 㣗 ୰ ẘ ࡢ ཎ ᅉ ࡞ ࡾ ᚓ ࡿ ࠋ ࡲ ࡓ 㸪 SEs ࡣ ࢺ ࣜ ࣉ ࢩ ࣥ ࡸ ࣌ ࣉ ࢩ ࣥ ࠸ ࡗ ࡓ ✀ ࠎ ࡢ ࢱ ࣥ ࣃ ࢡ ㉁ ศ ゎ 㓝 ⣲ ᑐ ࡋ ࡚ ᢠ ᛶ ࢆ ♧ ࡍ ࡓ ࡵ 㸪 య ෆ ࡢ ᾘ 㓝 ⣲ ࡛ ศ ゎ ࡉ ࢀ ࡃ ࠸ ≉ ᛶ ࢆ ᣢ ࡘ 㹙5㸪 67㹛ࠋ ᮏ 㣗 ୰ ẘ ࡣ 㸪 ᖹ ᆒ 3 㛫 ࡢ ₯ అ 㛫 ࡢ ᚋ Ⓨ ࡍ ࡿ ࠋ ≧ ࡣ ᝏ ᚰ ཬ ࡧ ྤ ࡛ ࠶ ࡾ 㸪ሙ ྜ ࡼ ࡾ ୗ ⑩ ࢆ క ࠺㹙29㸪73㹛ࠋ㏻ ᖖ ࡑ ࡢ ≧ ࡣ ୍ 㐣 ᛶ ᾘ ኻ ࡋ ண ᚋ ࡣ Ⰻ ዲ ࡉ ࢀ ࡿ ࡀ 㸪 ሙ ྜ ࡼ ࡗ ࡚ ࡣ ⬺ Ỉ ≧ ࡸ ⾑ ᅽ ࡢ ప ୗ 㸪 ⬦ ᢿ ᚤ ᙅ ࡞ ࢆ క ࡗ ࡚ ࢩ ࣙ ࢵ ࢡ ࡸ ⬺ 㝗 ࡿ ࡇ ࡶ ࠶ ࡿ ࡓ ࡵ 㸪 ᩆ ᛴ ᦙ ㏦ ࢆ ᚲ せ ࡍ ࡿ ࡞ 㐺 ษ ࡞ ⒪ ࡀ ᚲ せ ࡛ ࠶ ࡿ ࠋ ཌ ⏕ ປ ാ ┬ ࡢ 㣗 ୰ ẘ ⤫ ィ ࡼ ࡿ 㸪᪥ ᮏ ࡛ ࡣ ᖺ 㛫 ⣙ 50 ௳ ࡢ ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ࡀ Ⓨ ⏕ ࡋ ࡚ ࠸ ࡿ 㸦 http://www.mhlw.go.jp/stf/seisakunitsuite/bunya/kenkou_iryou/shoku hin/syokuchu/㸧ࠋࡑ ࡢ ࠺ ࡕ 㸪ᮾ ி 㒔 ࠾ ࡅ ࡿ ࡣ య ࡢ ࠾ ࡼ ࡑ 10% ࢆ ༨ ࡵ 㸪 ᖺ 㛫 5 ௳ ๓ ᚋ ࡢ ሗ ࿌ ࡀ ࡞ ࡉ ࢀ ࡚ ࠸ ࡿ 㸦http://www.fukushihoken.metro.tokyo.jp/shokuhin/tyuudoku/ 㸧ࠋ ᮏ 㣗 ୰ ẘ ࡣ 㸪 㣗 ရ 〇 㐀 ࣭ ㈍ ẁ 㝵 ࡛ ࡢ 㣗 ရ ࡢ ⾨ ⏕ ⓗ ࡞ ྲྀ ᢅ ࠸ ཬ ࡧ 㐺
2 ษ ࡞ ಖ Ꮡ ⟶ ⌮ ࡼ ࡾ ㏆ ᖺ ῶ ᑡ ഴ ྥ ࠶ ࡿ ࡀ 㸪2000 ᖺ 㛵 す ᆅ ᪉ ࠾ ࠸ ࡚ ຍ ᕤ ங ΰ ᅾ ࡋ ࡓ SEs ࢆ ཎ ᅉ ࡍ ࡿ つ ᶍ 㣗 ୰ ẘ 㸦 ᝈ ⪅ ᩘ 13,420 ྡ ௨ ୖ 㸧 ࡀ Ⓨ ⏕ ࡍ ࡿ 㹙 4㹛 ࡞ 㸪 S. aureus ࡣ බ ⾗ ⾨ ⏕ ୖ ᖖ ὀ ព ࢆ ᡶ ࢃ ࡞ ࡅ ࢀ ࡤ ࡞ ࡽ ࡞ ࠸ 㣗 ୰ ẘ ㉳ ᅉ ⳦ ࡢ ୍ ࡘ ࡛ ࠶ ࡾ 㸪 ྠ ⳦ ࡢ ไ ᚚ 㸪 ≉ ࡑ ࡢ ẘ ⣲ ⏘ ⏕ ࡢ ไ ᚚ ࡣ 㸪 㣗 ࡢ Ᏻ ࢆ ☜ ಖ ࡍ ࡿ ୖ ࡛ 㔜 せ ど ࡉ ࢀ ࡿ ࠋ ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ࡣ ᗈ ࡃ ୡ ⏺ ୰ ࡛ Ⓨ ⏕ ࡋ ࡚ ࠾ ࡾ 㸪 ࡇ ࢀ ࡲ ࡛ ᵝ ࠎ ࡞ ᡭ ἲ ࡼ ࡗ ࡚ 㸪ᮏ 㣗 ୰ ẘ 㛵 ࡍ ࡿ S. aureus ᰴ ࡢ Ꮫ ⓗ ≉ ᛶ ࡀ ㄪ ࡽ ࢀ ࡚ ࡁ ࡓ 㹙17㸪 25㸪 68㸪 69㸪 77㸪 79㹛ࠋ ࢥ ࢢ ࣛ ࣮ ࢮ ᆺ ู ἲ ࡣ 㸪 ᪥ ᮏ ࠾ ࡅ ࡿ S. aureus ᰴ ࡢ Ꮫ ゎ ᯒ ࠾ ࠸ ࡚ ࡼ ࡃ ⏝ ࠸ ࡽ ࢀ ࡿ ᡭ ἲ ࡛ ࠶ ࡿ ࠋᮏ ᆺ ู ἲ ࡣ S. aureus ࡀ ⏘ ⏕ ࡍ ࡿ ࢥ ࢢ ࣛ ࣮ ࢮ ࡢ ᢠ ཎ ᛶ ࡢ ᕪ ␗ ᇶ ࡙ ࡃ ᡭ ἲ ࡛ ࠶ ࡾ 㹙68㸪 77㹛㸪 ≉ ᐃ ࡢ ࢥ ࢢ ࣛ ࣮ ࢮ ᆺ ࢆ ᣢ ࡘ S. aureus ᰴ ࡀ 㣗 ୰ ẘ ࡽ ඃ ໃ ศ 㞳 ࡉ ࢀ ࡿ ࡇ ࡀ ▱ ࡽ ࢀ ࡚ ࠸ ࡿ 㹙62㸪 68㹛ࠋ Shimizu ࡽ 㹙 68㹛 ࡣ 㸪 ࢥ ࢢ ࣛ ࣮ ࢮ ᆺ ู ࡣ ཎ ᅉ ⳦ ࡢ ≉ ᐃ ࡸ ఏ ࡞ ࢆ ᥎ ᐃ ࡍ ࡿ Ꮫ ゎ ᯒ ࢆ ⾜ ࠺ ୖ ࡛ ᭷ ⏝ ࡛ ࠶ ࡿ ሗ ࿌ ࡋ ࡚ ࠸ ࡿ ࠋ 2001 ᖺ 2 ✀ 㢮 ࡢ S. aureus ᰴ ࡢ ࢤ ࣀ ࣒ ሷ ᇶ 㓄 ิ ࡀ Ỵ ᐃ ࡉ ࢀ㹙 38㹛㸪 S. aureus ࢤ ࣀ ࣒ ࡣ ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡢ ᤄ ධ ࡸ variable 㑇 ఏ Ꮚ ࡢ Ꮡ ᅾ ➼ 㸪 ከ ࡃ ࡢ ྍ ኚ 㡿 ᇦ ࢆ ྵ ࡴ ࡇ ࡀ ᫂ ࡽ ࡞ ࡗ ࡓ ࠋ ࡑ ࡋ ࡚ 㸪 ࡑ ࢀ ࡽ ࡢ ྍ ኚ 㡿 ᇦ ࢆ ᑐ ㇟ ࡋ ࡓ ศ Ꮚ ⏕ ≀ Ꮫ ⓗ ࡞ ᡭ ἲ ࡀ S. aureus ࡢ Ꮫ ゎ
ᯒ ᛂ ⏝ ࡉ ࢀ ࡚ ࠸ ࡿ 㹙25㹛ࠋ Multi locus sequence typing㸦 MLST㸧 ἲ
ࡣ ศ Ꮚ Ꮫ ゎ ᯒ ᡭ ἲ ࡢ ୍ ࡘ ࡛ ࠶ ࡾ 㸪 ᮏ ἲ ᇶ ࡙ ࡃ ࢡ ࣟ ࣮ ࣥ ゎ ᯒ ࡀ S.
aureus ࢆ ྵ ࡵ ࡓ ᵝ ࠎ ࡞ ⳦ ✀ ࡛ ᗈ ࡃ ⏝ ࠸ ࡽ ࢀ ࡚ ࠸ ࡿ㹙 27㸪37㸪60㸪63㸪
83㹛ࠋMLST ἲ ࡛ ࡣ 㸪᭱ ึ 」 ᩘ ࡢ ࣁ ࢘ ࢫ ࢟ ࣮ ࣆ ࣥ ࢢ 㑇 ఏ Ꮚ ࢆ ᑐ ㇟ ࡋ 㸪 ࡑ ࡢ ሷ ᇶ 㓄 ิ ࡢ ᕪ ␗ ࡢ ゎ ᯒ ࢆ ⾜ ࠺ ࠋ ࡑ ࡢ ᚋ 㸪 ࡑ ࢀ ࡒ ࢀ ࡢ ሷ ᇶ 㓄
3 ิ ࢆ ᪤ Ⓩ 㘓 ࡉ ࢀ ࡚ ࠸ ࡿ ࢹ ࣮ ࢱ ࣋ ࣮ ࢫ ẚ ㍑ ࡋ ࣃ ࢱ ࣮ ࣥ ࡍ ࡿ ࡇ ࡛ ࢱ ࣆ ࣥ ࢢ ࢆ ⾜ ࠺ ࡇ ࡀ ࡛ ࡁ ࡿ㹙21㹛ࠋࣁ ࢘ ࢫ ࢟ ࣮ ࣆ ࣥ ࢢ 㑇 ఏ Ꮚ ࡣ ⣽ ⳦ ࡢ ⏕ Ꮡ 㛵 ࢃ ࡿ 㑇 ఏ Ꮚ ࡛ ࠶ ࡿ ࠋ ᮏ 㑇 ఏ Ꮚ ኚ ␗ ࡀ ㉳ ࡁ ࡓ ⣽ ⳦ ࡢ ከ ࡃ ࡣ 㸪 Ṛ ⁛ ࡍ ࡿ 㞟 ᅋ ࡢ ୰ ࡛ Ὲ ử ࡉ ࢀ ࡚ ࡋ ࡲ ࠺ ࠋ ࡑ ࡢ ࡓ ࡵ ᮏ 㑇 ఏ Ꮚ ࡢ 㐍 ㏿ ᗘ 㸦 ୍ ᐃ 㛫 ࠶ ࡓ ࡾ ࡢ ኚ ␗ Ⓨ ⏕ 㢖 ᗘ 㸧 ࡣ 㸪 ཎ 㑇 ఏ Ꮚ ࡞ ኚ ␗ ࡢ ㉳ ࡇ ࡾ ࡸ ࡍ ࠸ 㑇 ఏ Ꮚ ẚ ㍑ ࡋ ࡚ ⦆ ࡸ ࡛ ࠶ ࡿ ࠋࡇ ࡢ ࡼ ࠺ 㸪 ࣁ ࢘ ࢫ ࢟ ࣮ ࣆ ࣥ ࢢ 㑇 ఏ Ꮚ ࡣ 㛗 ࠸ 㛫 ࢆ ࡅ ࡚ ኚ ␗ ࡢ ✚ ࡳ 㔜 ࡡ ࡀ ㉳ ࡇ ࡾ ከ ᵝ ᛶ ࢆ ⏕ ࡌ ࡿ ࡇ ࡽ 㸪 ࡑ ࡢ ሷ ᇶ 㓄 ิ ࡢ ᕪ ␗ ࡀ ⣽ ⳦ ࡢ 㐍 㛫 ࢆ ᫎ ࡋ ࡚ ࠸ ࡿ ⪃ ࠼ ࡽ ࢀ ࡿ ࡓ ࡵ 㸪MLST ἲ ࡛ ᚓ ࡽ ࢀ ࡓ ࢹ ࣮ ࢱ ࡣ ⳦ ᰴ ࡢ ศ Ꮚ Ꮫ ゎ ᯒ ࡢ ࡳ ࡞ ࡽ ࡎ ศ Ꮚ ⣔ ⤫ Ꮫ ゎ ᯒ ࠾ ࠸ ࡚ ࡶ ⏝ ࡉ ࢀ ࡿ 㹙23㸪 27㸪 37㸪 60㸪 63㹛ࠋ S. aureus ᰴ ࢆ ᑐ ㇟ ࡋ ࡓ MLST ゎ ᯒ ࡣ 㸪 ࣓ ࢳ ࢩ ࣜ ࣥ ⪏ ᛶ 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ឤ ᰁ ศ 㞳 ᰴ ࡸ ⎔ ቃ ⏤ ᮶ ศ 㞳 ᰴ ࡛ ᗈ ࡃ ᐇ ࡉ ࢀ ࡚ ࠸ ࡿ 㹙27㸪37㸪60㸪63㸪65㹛ࠋࡋ ࡋ 㸪ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ศ 㞳 ᰴ ࡢ ሗ ࿌ ࡣ 㝈 ᐃ ⓗ ࡛ ࠶ ࡾ 㸪 ࡑ ࡢ ศ Ꮚ Ꮫ ⓗ ≉ ᛶ ࡸ ศ Ꮚ ⣔ ⤫ Ꮫ ⓗ ࡞ ⫼ ᬒ 㛵 ࡋ ࡚ ࡣ ᮍ ࡔ ᫂ ࡞ Ⅼ ࡀ ከ ࠸ 㹙79㹛ࠋ 1930 ᖺ 㸪 Dack ࡽ 㹙 18㹛 ࡼ ࡾ 㸪 ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ࡣ S. aureus ࡢ ⏘ ⏕ ࡍ ࡿ ⳦ య እ ẘ ⣲ ࡼ ࡾ ᘬ ࡁ ㉳ ࡇ ࡉ ࢀ ࡿ ࡇ ࡀ ド ᫂ ࡉ ࢀ 㸪ࠕ ࢚ ࣥ ࢸ ࣟ ࢺ ࢟ ࢩ ࣥ ࠖ ྡ ࡉ ࢀ ࡓ ᮏ ẘ ⣲ ࡢ ◊ ✲ ࡀ ጞ ࡲ ࡗ ࡓ ࠋ1971 ᖺ ࡲ ࡛ 㸪
ᢠ ཎ ᛶ ࡢ ␗ ࡞ ࡿ 5 ✀ 㢮 ࡢ ᆺ 㸦 ྂ ⓗ SEs㸹 SEA㹼 SEE㸧 ࡀ Ꮡ ᅾ ࡍ ࡿ
ࡇ ࡀ ሗ ࿌ ࡉ ࢀ㹙6㸪7㸪15㸪16㹛㸪㣗 ୰ ẘ ᳨ ᰝ ࡢ ศ 㔝 ࠾ ࠸ ࡚ ࡣ 㸪㛗
ࡽ ࡃ ⾑ Ύ Ꮫ ⓗ ᡭ ἲ ࢆ ⏝ ࠸ ࡚ ࡇ ࢀ ࡽ ࡢ ẘ ⣲ ࡢ ศ 㢮 ࡀ ࡞ ࡉ ࢀ ࡚ ࡁ ࡓ ࠋ ࡋ
ࡋ 㸪 ศ Ꮚ ⏕ ≀ Ꮫ ⓗ ゎ ᯒ ᡭ ἲ ࡢ 㐍 ᒎ ࡼ ࡾ 㸪1994 ᖺ ௨ 㝆 ྂ ⓗ SEs
௨ እ ࡶ ྤ ά ᛶ ࢆ ᣢ ࡘ ᪂ ᆺ SEs㸪 ࠾ ࡼ ࡧ SEs ᵓ 㐀 ࡀ 㢮 ఝ ࡍ ࡿ ࢚
ࣥ ࢸ ࣟ ࢺ ࢟ ࢩ ࣥ ᵝ ẘ ⣲ 㸦Staphylococcal enterotoxin -like toxin㸹 SEl㸧
4
㹼SElX ࡢ 23 ✀ 㢮 ࡀ ྠ ᐃ ࡉ ࢀ 㸪 ከ ✀ ࡢ ศ Ꮚ ✀ ࡼ ࡾ ᵓ ᡂ ࡉ ࢀ ࡿ ẘ ⣲ ⩌
࡛ ࠶ ࡿ ࡇ ࡀ ᫂ ࡽ ࡞ ࡗ ࡚ ࠸ ࡿ㹙2㸪28㸪29㸪53㸪55㸪57㸪72㸪80㹛ࠋ
࡞ ࠾ 㸪ࡇ ࢀ ࡽ ࡢ ᪂ ᆺ SE/SEls ࡣ 㸪International Nomenclature Committee
for Staphylococcal Superantigen ࡢ ྡ つ ⣙ ࡼ ࡾ 㸪 㟋 㛗 㢮 ື ≀ ࡢ
⤒ ཱྀ ᢞ ᐇ 㦂 ࡼ ࡾ ྤ ࡀ ㄆ ࡵ ࡽ ࢀ ࡓ ࡶ ࡢ ࢆ”SE”㸪 ྤ ᐇ 㦂 ᮍ ᐇ ࡶ ࡋ ࡃ ࡣ ྤ ά ᛶ 㝜 ᛶ ࡢ ࡶ ࡢ ࢆ”SEl” ⾲ グ ࡍ ࡿ ᐃ ࡵ ࡽ ࢀ ࡚ ࠸ ࡿ 㹙40㹛ࠋ ከ ࡃ ࡢ SEs ࡣ 㸪 ྤ ά ᛶ ຍ ࠼ ࢫ ࣮ ࣃ ࣮ ᢠ ཎ ά ᛶ ࢆ ᭷ ࡍ ࡿ 㹙 2㸪 20㸪24㸪57㹛ࠋࢫ ࣮ ࣃ ࣮ ᢠ ཎ ά ᛶ ࡼ ࡿ ẘ ⣲ ᛶ ࢩ ࣙ ࢵ ࢡ ೃ ⩌ ࡢ Ⓨ ࣓ ࢝ ࢽ ࢬ ࣒ ࡣ ࡼ ࡃ ゎ ᯒ ࡉ ࢀ ࡚ ࠸ ࡿ ࠋ ࡍ ࡞ ࢃ ࡕ 㸪 SEs ࡀ Major
histocompatibility complex㸦 MHC㸧class II T cell receptor ྠ
⤖ ྜ 㸦 ᯫ ᶫ 㸧 ࡍ ࡿ ࡇ ࡛ 㸪 ከ ᩘ ࡢ T ⣽ ⬊ ࢆ ά ᛶ ࡍ ࡿ ࠋ ࡑ ࡢ ⤖ ᯝ 㸪 ᢠ ཎ 㠀 ౫ Ꮡ ⓗ T ⣽ ⬊ ࡽ ࡢ 㐣 ᗘ ࡢ ࢧ ࢺ ࢝ ࣥ ᨺ ฟ ࢆ ㄏ ㉳ ࡋ 㸪Ⓨ ⑈ 㸪 ⾑ ᅽ ప ୗ 㸪 ✀ ᛶ ⾑ ⟶ ෆ จ ᅛ 㸪 ከ ⮚ ჾ ࡞ ࡢ ⮴ ⓗ ࡞ ែ ࢆ ᘬ ࡁ ㉳ ࡇ ࡍ ẘ ⣲ ᛶ ࢩ ࣙ ࢵ ࢡ ೃ ⩌ ࢆ Ⓨ ࡍ ࡿ 㹙1㸪 34㹛ࠋ ୍ ᪉ ࡛ 㸪 SEs ࡢ ࣄ ࢺ ࠾ ࡅ ࡿ ྤ Ⓨ ࣓ ࢝ ࢽ ࢬ ࣒ ࡢ ศ Ꮚ ᇶ ┙ ࡣ ༑ ศ ᫂ ࡽ ࡉ ࢀ ࡚ ࠸ ࡞ ࠸ ࠋSEs ࡣ ᾘ ⟶ ࡢ ⢓ ⭷ ୗ ⤌ ⧊ Ꮡ ᅾ ࡍ ࡿ ⫧ ‶ ⣽ ⬊ ࡢ ࢭ ࣟ ࢺ ࢽ ࣥ ⬺ 㢛 ⢏ ࢆ ㄏ ㉳ ࡋ 㸪 ୖ ⾜ ᛶ ࡢ ⭡ ⭍ ឤ ⚄ ⤒ ࠾ ࡼ ࡧ ㏞ ㉮ ⚄ ⤒ ࢆ ่ ⃭ ࡍ ࡿ ࡇ ࡛ ྤ ࢆ ᘬ ࡁ ㉳ ࡇ ࡍ ⪃ ࠼ ࡽ ࢀ ࡚ ࠸ ࡿ 㹙34㸪 58㹛ࠋ 5 ✀ 㢮 ࡢ ྂ ⓗ SEs ࡀ ఱ ࢀ ࡶ ࣄ ࢺ ࡢ 㣗 ୰ ẘ ࡢ ཎ ᅉ ࡞ ࡿ ࡇ ࡣ ᫂ ࡽ ࡛ ࠶ ࡿ ࡀ 㹙5㸪 20㹛㸪 ᪂ ᆺ SE/SEls ࡣ 㸪 ࡑ ࡢ ྤ ά ᛶ ࡢ ᭷ ↓ ࡸ ᙉ ᙅ 㸪 S. aureus ࡼ ࡿ ⏘ ⏕ ࡢ ᭷ ↓ ࡸ ⏘ ⏕ 㔞 ࡢ ከ ᐻ ࠸ ࡗ ࡓ Ⅼ ࡛ ◊ ✲ ẁ 㝵 ࡛ ࠶ ࡿ ࡶ ࡢ ࡶ ከ ࡃ 㸪 㣗 ୰ ẘ ࡢ 㛵 ࡣ ᮍ ࡔ ᫂ ࡞ Ⅼ ࡀ ከ ࠸ ࠋ S. aureus ࡣ ᵝ ࠎ ࡞ ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡸ ࡑ ࡢ ᩿ ∦ ࢆ ᣢ ࡘ ࡇ ࡀ ሗ ࿌ ࡉ ࢀ ࡚ ࠾ ࡾ 㸪 ࡑ ࡢ ✀ 㢮 ࡋ ࡚ 㸪 ࣉ ࣟ ࣇ ࣮ ࢪ 㸪 ࣉ ࣛ ࢫ ࣑ ࢻ 㸪Insertion
5
sequence㸦 IS㸧㸪Staphylococcal cassette chromosome 㸪the highly variable genomic region νSaβ 㸪Staphylococcus aureus pathogenicit y island㸦 SaPI㸧
࡞ ࡀ ▱ ࡽ ࢀ ࡚ ࠸ ࡿ 㹙2㸪 42㸪 44㹛ࠋ 14-17 kb ࡢ ࡁ ࡉ ࡢ 㓄 ิ ࢆ ᣢ ࡘ SaPI ࡣ 㸪 ࢤ ࣀ ࣒ ୖ ࡢ 6 ᡤ ࡢ ᤄ ධ ྍ ⬟ 㡿 ᇦ 㸦 8′㸪 9′㸪 18′㸪 19′㸪 44′ ཬ ࡧ 49′㡿 ᇦ 㸹 ᅗ 1㸧 ࡢ ࠸ ࡎ ࢀ ᤄ ධ ࡉ ࢀ ࡚ ࠸ ࡿ ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡛ ࠶ ࡾ 㸪 ࣉ ࣟ ࣇ ࣮ ࢪ ࡢ ᵝ ᐟ ࡢ ࢡ ࣟ ࣔ ࢰ ࣮ ࣒ ෆ ࡛ Ᏻ ᐃ ⓗ Ꮡ ᅾ 㸦 ⁐ ཎ 㸧 ࡋ ࡚ ࠸ ࡿ 㹙44㸪 51㹛ࠋ 1998 ᖺ ึ ࡵ ࡚ SaPI ࡢ ሗ ࿌ ࡀ ࡞ ࡉ ࢀ ࡚ ࡽ 㸪 ⌧ ᅾ ࡲ ࡛ 20 ✀ 㢮 ௨ ୖ ࡢ SaPI ࡀ ྠ ᐃ ࡉ ࢀ ࡚ ࠸ ࡿ 㹙 2㸪 39㸪 41㸪51㸪64㹛ࠋSaPI ࡢ ᣢ ࡘ ୍ ⯡ ⓗ ࡞ 㑇 ఏ Ꮚ ᵓ 㐀 ࢆ ᅗ 2 ♧ ࡋ ࡓ ࠋSaPI ࡢ ୧ ➃ ࡣ ᤄ ධ 㒊 ≉ ␗ ⓗ ࡞ ⧞ ࡾ ㏉ ࡋ 㓄 ิ㸦att 㓄 ิ 㸧ࡀ Ꮡ ᅾ ࡋ 㸪⮬ ㌟ ࡢ ఏ 㛵 ࡍ ࡿ 」 ᩘ ࡢ 㑇 ఏ Ꮚ ࡀ ಖ Ꮡ ࡉ ࢀ ࡓ ᇶ ᮏ ᵓ 㐀 ࢆ ᣢ ࡘ㹙44㸪 51㹛ࠋࡲ ࡓ 㸪 ཎ 㑇 ఏ Ꮚ㸦 SEs 㑇 ఏ Ꮚ ➼ 㸧ࡸ ⸆ ⪏ ᛶ 㑇 ఏ Ꮚ ࡀ ࢡ ࢭ ࢧ ࣜ ࣮ 㑇 ఏ Ꮚ ࡋ ࡚ ᇶ ᮏ 㑇 ఏ Ꮚ ࡢ 㛫 Ꮡ ᅾ ࡍ ࡿ ሙ ྜ ࡀ ࠶ ࡿ㹙50㸪51㹛ࠋ ఏ ࢆ ᢸ ࠺ 㑇 ఏ Ꮚ ࡀ ᶵ ⬟ ࡍ ࡿ ࡇ ࡼ ࡾ ⁐ ཎ ࡋ ࡚ ࠸ ࡿ SaPI ࡢ ࡢ S. aureus ᰴ ࡢ ఏ ࡀ ຠ ⋡ ⓗ ⾜ ࢃ ࢀ ࡿ 㹙 51㸪 75㸪 76㹛ࠋ SaPI ఏ ᵝ ᘧ ࡢ ᴫ ␎ ࢆ ᅗ 3 ♧ ࡋ ࡓ ࠋSaPI ఏ ࡢ ㄏ ᑟ ࡣ ≉ ᐃ ࡢ Helper
phage ࡀ 㛵 ࡍ ࡿ ࠋ ࠼ ࡤ 㸪DNA ᦆ യ క ࠺ SOS ᛂ ⟅ ࡼ ࡾ ᐟ ࡢ
ࢤ ࣀ ࣒ ෆ ࡛ ⁐ ཎ ࡋ ࡚ ࠸ ࡿ Helper phage ࡀ ㄏ ᑟ ࡉ ࢀ ࡿ ࡇ ࡀ 㸪 SaPI
ఏ ࢆ 㛤 ጞ ࡉ ࡏ ࡿ ୍ ᅉ ࡞ ࡿ ࠋᐟ ࡢ ࢤ ࣀ ࣒ ෆ Ꮡ ᅾ ࡍ ࡿ SaPI 㓄 ิ
ࡣ 㸪integrase ࡸ excisionase ࡢ ാ ࡁ ࡼ ࡾ ษ ࡾ ฟ ࡉ ࢀ ࡿ ࠋษ ࡾ ฟ ࡉ ࢀ
ࡓ SaPI 㓄 ิ ࡣ 」 〇 ࡉ ࢀ ᩘ ⓒ ࢥ ࣆ ࣮ ࡀ ┤ 㙐 ≧ 㐃 ࡞ ࡗ ࡓ 㙐 ≧ య㸦 ࢥ ࣥ
࢝ ࢸ ࣐ ࣮ 㸧ࢆ ᙧ ᡂ ࡍ ࡿ ࠋࡇ ࡢ ࢥ ࣥ ࢝ ࢸ ࣐ ࣮ ࡣ terminase 」 ྜ య ࡼ ࡾ
ಶ ࠎ ࡢ SaPI 㓄 ิ ษ ᩿ ࡉ ࢀ ࡿ ࠋ᭱ ⤊ ⓗ 㸪ಶ ࠎ ࡢ SaPI 㓄 ิ ࡀ Helper
phage ࢆ ᵓ ᡂ ࡍ ࡿ እ Ẇ ࢱ ࣥ ࣃ ࢡ ㉁ ྲྀ ࡾ ㎸ ࡲ ࢀ 㸪 ࣇ ࣮ ࢪ ᵝ ⢏ Ꮚ ࡋ ࡚ ᐟ እ ᨺ ฟ ࡉ ࢀ ࡿ ࠋ
6
͆ 」 〇 ͇ࡣ 㸪SaPI ࡢ ఏ ࠾ ࡅ ࡿ 㔜 せ ࡞ 㐣 ⛬ ࡢ ࡦ ࡘ ࡛ ࠶ ࡿ ࠋSaPI
ࡣ ୍ ࡘ ࡢ 」 〇 ༢ ࡀ Ꮡ ᅾ ࡋ 㸪 」 〇 ༢ ≉ ␗ ⓗ ࡞ 」 〇 㛤 ጞ ࢱ ࣥ ࣃ ࢡ
㉁ 㸦Replication initiator protein 㸹 Rep 㸧 」 〇 㛤 ጞ Ⅼ 㸦 Replication
origin㸹ori site㸧ࢆ ᣢ ࡘ ࡇ ࡀ ▱ ࡽ ࢀ ࡚ ࠸ ࡿ㹙 13㸪51㸪74㸪75㸪76㹛ࠋ Rep ࡣ 㔜 㙐 DNA ࢆ ୍ ᮏ 㙐 ኚ ࡉ ࡏ ࡿ ࣊ ࣜ ࢝ ࣮ ࢮ ά ᛶ ࢆ ᣢ ࡘ ࢱ ࣥ ࣃ ࢡ ㉁ ࡛ ࠶ ࡿ㹙51㸪74㸪76㹛ࠋori site ࡣ 300 bp ⛬ ᗘ ࡢ 㓄 ิ ࡛ ࠶ ࡾ 㸪 ㏻ ᖖ rep 㑇 ఏ Ꮚ ࡢ 3’ഃ ㏆ ഐ ⨨ ࡍ ࡿ ࠋྠ 㒊 ࡣ 80 bp ⛬ ᗘ ࡢ AT ᐩ ࢇ ࡔ 㓄 ิ㸦AT-rich 㡿 ᇦ 㸧ࢆ 㸪6 bp ๓ ᚋ ࡢ ྠ ᪉ ྥ 㓄 ิ ㏫ ᪉ ྥ 㓄 ิ ࡽ ࡞ ࡿ ࢸ ࣟ ࣥ 㓄 ิ ࡀ ᣳ ࡴ ᵓ 㐀 ࢆ ᣢ ࡘ㹙74㹛ࠋRep ࢱ ࣥ ࣃ ࢡ ㉁ ࡀ ࢸ ࣟ ࣥ 㓄 ิ ⤖ ྜ ࡋ 㸪 ࣊ ࣜ ࢝ ࣮ ࢮ ά ᛶ ࢆ ㏻ ࡋ ࡚ AT-rich 㡿 ᇦ ࡢ DNA 㔜 㙐 ࢆ ୍ ᮏ 㙐 ࡍ ࡿ ࡇ ࡛ SaPIs ࡢ 」 〇 ࡀ 㛤 ጞ ࡉ ࢀ ࡿ ⪃ ࠼ ࡽ ࢀ ࡚ ࠸ ࡿ 㹙51㹛ࠋ ከ ࡃ ࡢ SE/SEls 㑇 ఏ Ꮚ ࡣ 㸪 ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ୖ Ꮡ ᅾ ࡍ ࡿ ࡇ ࡀ ሗ ࿌ ࡉ ࢀ ࡚ ࠸ ࡿ㹙2㸪28㸪44㹛ࠋࡇ ࡢ ࡇ ࡣ 㸪SE/SEls 㑇 ఏ Ꮚ ࡀ S. aureus ᰴ 㛫 ࡛ Ỉ ᖹ ఏ ࡋ ᚓ ࡿ ࡇ ࢆ ♧ ࡋ ࡚ ࠸ ࡿ ࠋ ࡑ ࢀ ࡺ ࠼ 㸪 ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡢ ఏ ࡀ 㣗 ୰ ẘ ཎ ᛶ ࡢ ⋓ ᚓ ῝ ࡃ 㛵 ࡍ ࡿ ࡇ ࡀ ♧ ၀ ࡉ ࢀ 㹙42㸪 44㸪49㹛㸪ᵝ ࠎ ࡞ ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࢆ ྠ ᐃ ࡋ ࡑ ࡢ ≉ ᛶ ࢆ ゎ ᯒ ࡍ ࡿ ࡇ ࡀ 㸪 S. aureus ࡢ ཎ య ࡋ ࡚ ࡢ ㉳ ᛶ ศ Ꮚ 㑇 ఏ Ꮫ ⓗ ≉ ᛶ ࢆ ⪃ ᐹ ࡍ ࡿ ୖ ࡛ 㠀 ᖖ ᭷ ┈ ࡛ ࠶ ࡿ ⪃ ࠼ ࡽ ࢀ ࡿ ࠋࡍ ࡞ ࢃ ࡕ 㸪 ཎ ᛶ S. aureus ࡢ ศ Ꮚ 㐍 ࢆ ⪃ ᐹ ࡍ ࡿ ࡇ ࡀ ྍ ⬟ ࡞ ࡿ ࠋ ᮏ ◊ ✲ ࡛ ࡣ 㸪 㣗 ࡢ Ᏻ ࢆ ☜ ಖ ࡍ ࡿ ୖ ࡛ 㔜 せ ど ࡉ ࢀ ࡿ ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ࡘ ࠸ ࡚ 㸪ࡑ ࡢ ศ 㞳 S. aureus ᰴ ࡢ Ꮫ ゎ ᯒ ࡼ ࡿ ሗ ࡀ ᮏ 㣗 ୰ ẘ ࡢ Ⓨ ⏕ ≧ ἣ ㄪ ᰝ ࡸ Ⓨ ⏕ 㜵 Ṇ ᭷ ⏝ ࡛ ࠶ ࡿ ⪃ ࠼ 㸪 ྛ ✀ ࡢ Ꮫ ᡭ ἲ ࢆ ⤌ ࡳ ྜ ࢃ ࡏ ࡚ ⏝ ࠸ ࡿ ࡇ ࡼ ࡾ ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ࢆ ㄏ Ⓨ ࡋ ࡸ ࡍ ࠸ S. aureus ࡢ 㑇 ఏ Ꮫ ⓗ ≉ ᛶ ࡢ ゎ ᫂ ࡍ ࡿ ࡇ ࢆ ヨ ࡳ ࡓ ࠋ ࡲ ࡓ 㸪 ྍ ື ᛶ 㑇
7 ఏ ᅉ Ꮚ ࡢ ྠ ᐃ ࠾ ࡼ ࡧ ࡑ ࡢ ≉ ᛶ 㛵 ࡍ ࡿ ሗ ࡀ S. aureus ࡢ 㣗 ୰ ẘ ཎ య ࡋ ࡚ ࡢ ㉳ ᛶ ࢆ ⪃ ᐹ ࡍ ࡿ ୖ ࡛ ᭷ ┈ ࡛ ࠶ ࡿ ⪃ ࠼ 㸪 ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ Ⓨ ⏕ 㛵 ࡍ ࡿ ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡢ ศ Ꮚ 㑇 ఏ Ꮫ ⓗ ≉ ᛶ ࢆ ᳨ ウ ࡋ ࡓ ࠋ ➨ ୍ ❶ ࡛ ࡣ 㸪1994 ᖺ ࡽ 2012 ᖺ ᮾ ி 㒔 ࡛ Ⓨ ⏕ ࡋ ࡓ ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ⏤ ᮶ S. aureus ᰴ ࡢ ศ Ꮚ Ꮫ ୖ ࡢ ≉ ᛶ ࢆ ᫂ ࡽ ࡋ 㸪ࡑ ࡢ ≉ ᛶ ࢥ ࢢ ࣛ ࣮ ࢮ ᆺ 㸪SE/SEls 㑇 ఏ Ꮚ ᆺ ཬ ࡧ ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡢ ಖ ᭷ ࣃ ࢱ ࣮ ࣥ ࡢ 㛫 ⥭ ᐦ ࡞ 㛵 ಀ ࡀ ࠶ ࡿ ࡇ ࢆ ᫂ ࡽ ࡋ ࡓ ࠋ ➨ ❶ ࡛ ࡣ 㸪➨ ୍ ❶ ࡢ ⤖ ᯝ ࡽ ࡑ ࡢ Ꮡ ᅾ ࡀ ᥎ ࡉ ࢀ ࡓ 㸪᪂ つ SaPI 㓄 ิ ࢆ 4 ✀ 㢮 ྠ ᐃ ࡋ ࡓ ࠋ ࡲ ࡓ 㸪 ࡑ ࢀ ࡽ ࡢ 㣗 ୰ ẘ ㉳ ᛶ ཬ ࡧ ఏ ⬟ ຊ 㛵 ࡍ ࡿ ≉ ᛶ ࢆ ゎ ᯒ ࡋ 㸪 ࡚ ࡢ ᪂ つ SaPI ࡀ 㣗 ୰ ẘ ㉳ ᛶ ࢆ ಖ ᣢ ࡍ ࡿ ୍ ᪉ ࡛ 㸪 ᪂ つ SaPI ࡢ 1 ࡘ ࡀ 㸪 SaPI ఏ ࡢ ୍ 㐣 ⛬ ࡛ ࠶ ࡿ ͆ 」 〇 ͇ 㛵 ࡍ ࡿ ⬟ ຊ ࢆ Ḟ ዴ ࡋ ࡚ ࠸ ࡿ ࡇ ࢆ ᫂ ࡽ ࡋ ࡓ ࠋ ➨ ୕ ❶ ࡛ ࡣ 㸪ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ⏤ ᮶ S. aureus ᰴ ࡀ ᣢ ࡘ SE/SEls ಖ ᭷ ࣉ ࣛ ࢫ ࣑ ࢻ ࡢ 㣗 ୰ ẘ ࡢ 㛵 ࢆ ᳨ ウ ࡋ ࡓ ࠋ ࡲ ࡓ 㸪 ゎ ᯒ ᑐ ㇟ ࡋ ࡓ
୍ 㒊 ࡢ S. aureus ᰴ ࡀ 㸪SED 㓄 ิ ࡢ C ᮎ ➃ 㒊 ศ ࢆ Ḟ ኻ ࡍ ࡿ ኚ ␗ ᆺ SED
ࢆ ಖ ᭷ ࡍ ࡿ ࡇ ࢆ ᫂ ࡽ ࡍ ࡿ ࡶ 㸪ྠ ኚ ␗ ᆺ SED ࡢ 㣗 ୰ ẘ ཎ
ᅗ 1. S. aureusࢤࣀ࣒ୖ࠾ࡅࡿStaphylococcus aureus pathogenicity island㸦SaPI㸧ࡢᤄධྍ ⬟㡿ᇦࡢ⨨
ŶőŃƄƘƕƈƘƖ।ঀ
ع0ES
b6D3,V
b6D3,V
b6D3,V
b6D3,V
b6D3,V
b
b6D3,V
8Integration
Repression
Excision
Replication
Encapsulation
and packaging
⧞ࡾ㏉ࡋ㓄ิ㸦att㓄ิ㸧
SaPIࡢఏ㛵ࡍࡿ㑇ఏᏊ 㸦ᇶᮏ㑇ఏᏊ㸧
ཎ㑇ఏᏊࡸᶵ⬟᫂㑇ఏᏊࡀᏑᅾࡍࡿྍ⬟ᛶࡢ࠶ࡿ㡿ᇦ
」〇㛤ጞⅬ㸦
ori site㸧
ᅗ 2. SaPIࡢᇶᮏᵓ㐀 ཎ㑇ఏᏊࡸᶵ⬟᫂㑇ఏᏊࡀᏑᅾࡍࡿྍ⬟ᛶࡢ࠶ࡿ㡿ᇦࡣ㸪᪤ሗSaPI㹙51㸪64㹛ࡀᣢ ࡘ㑇ఏᏊࡢᵓ㐀ࡽ᥎ ࡋࡓࠋྛ㑇ఏᏊࡢ␎ㄒࡣ௨ୗࡢ㏻ࡾ࡛࠶ࡿࠋint㸸integrase㸹stl㸸master repressor㸹str㸸regulatory protein㸹xis㸸exisionase㸹pri㸸primase㸹rep㸸replication initiator㸹ppi㸸phage interference㸹cp㸸packaging genes㸹ter㸸terminase small subunit
ᅗ 3. SaPIఏᵝᘧࡢᴫ␎ DNAᦆയక࠺SOSᛂ⟅ࡼࡗ࡚⁐ཎࡋ࡚࠸ࡿHelper phageࡢㄏᑟࡀ㉳ࡇࡾ㸪SaPIఏ ࢆ㛤ጞࡉࡏࡿࠋHelper phage⏤᮶ࡢdUTPaseࡼࡗ࡚㸪SaPIㄏᑟࡢᢚไࡀゎ㝖ࡉࢀ㸪SaPI 㓄ิࡣintegraseࡸexcisionaseࡢാࡁࡼࡾษࡾฟࡉࢀࡿࠋษࡾฟࡉࢀࡓSaPI㓄ิࡣ」〇ࡉࢀ ᩘⓒࢥࣆ࣮ࡀ┤㙐≧㐃࡞ࡗࡓ㙐≧య㸦ࢥࣥ࢝ࢸ࣐࣮㸧ࢆᙧᡂࡍࡿࠋࡇࡢࢥࣥ࢝ࢸ࣐࣮ࡣ terminase」ྜయࡼࡾಶࠎࡢSaPI㓄ิษ᩿ࡉࢀࡿࠋಶࠎࡢSaPI㓄ิࡀHelper phageࢆᵓᡂ ࡍࡿࣇ࣮ࢪእẆࢱࣥࣃࢡ㉁ྲྀࡾ㎸ࡲࢀ㸪ࣇ࣮ࢪᵝ⢏Ꮚࡋ࡚ᐟእᨺฟࡉࢀࡿࠋ Novickࡽ㹙51㹛ࡢᅗࢆᘬ⏝ࡋ୍㒊ᨵኚࡋࡓࠋ 10
11 ➨ ୍ ❶ ᮾ ி 㒔 ࡛ Ⓨ ⏕ ࡋ ࡓ 㣗 ୰ ẘ ⏤ ᮶ 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ศ 㞳 ᰴ ࡢ ศ Ꮚ Ꮫ ゎ ᯒ ᗎ ㄽ ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ࡣ ᗈ ࡃ ୡ ⏺ ୰ ࡛ Ⓨ ⏕ ࡋ ࡚ ࠾ ࡾ 㸪 ࡇ ࢀ ࡲ ࡛ ᵝ ࠎ ࡞ ᡭ ἲ ࡼ ࡗ ࡚ 㸪ᮏ 㣗 ୰ ẘ 㛵 ࡍ ࡿ S. aureus ᰴ ࡢ Ꮫ ⓗ ≉ ᛶ ࡀ ㄪ ࡽ ࢀ ࡚ ࡁ ࡓ 㹙17㸪25㸪68㸪69㸪77㸪79㹛ࠋ᪥ ᮏ ࡛ ࡣ 㸪 ࢥ ࢢ ࣛ ࣮ ࢮ ᆺ ู ἲ ࡀ S. aureus ᰴ ࡢ Ꮫ ゎ ᯒ ࠾ ࠸ ࡚ ỗ ⏝ ࡉ ࢀ ࡚ ࠾ ࡾ 㸪≉ ᐃ ࡢ ࢥ ࢢ ࣛ ࣮ ࢮ ᆺ ࢆ ᣢ ࡘ S. aureus ᰴ ࡀ 㣗 ୰ ẘ ࡽ ඃ ໃ ศ 㞳 ࡉ ࢀ ࡿ ࡇ ࡀ ▱ ࡽ ࢀ ࡚ ࠸ ࡿ 㹙62㸪 68㹛ࠋ ࡋ ࡋ 㸪 㣗 ୰ ẘ ⏤ ᮶ S. aureus ᰴ ࢆ ᑐ ㇟ ࡋ 㸪MLST ἲ ࡞ ࡢ ศ Ꮚ Ꮫ ゎ ᯒ ࢆ ⾜ ࡗ ࡓ ሗ ࿌ ࡣ 㝈 ᐃ ⓗ ࡛ ࠶ ࡾ 㸪S. aureus ᰴ ࠾ ࡅ ࡿ ศ Ꮚ Ꮫ ୖ ࡢ ≉ ᛶ 㣗 ୰ ẘ Ⓨ ⏕ ࡢ 㛵 㐃 ᛶ ࡣ ᫂ ࡽ ࡛ ࡞ ࠸ ࠋ ㏆ ᖺ 㸪ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡢ ୍ ࡘ ࡛ ࠶ ࡿ SaPI ࢆ ຠ ⋡ ⓗ ࡘ ヲ ⣽ ゎ ᯒ
ࡍ ࡿ SaPI scanning ἲ ࡀ 㛤 Ⓨ ࡉ ࢀ 㸪SaPI ࡢ ྠ ᐃ ⏝ ࡉ ࢀ ࡚ ࠸ ࡿ㹙 64㹛ࠋ
ᮏ ἲ ࡣ 㸪 ẚ ㍑ ⓗ ᗈ ᇦ ࡢ ࢤ ࣀ ࣒ 㓄 ิ ࢆ ゎ ᯒ ࡢ ᑐ ㇟ ࡍ ࡿ ࡓ ࡵ 㸪 ྛ ⳦ ᰴ ࣞ ࣋ ࣝ ࡛ ࡢ ࢱ ࣆ ࣥ ࢢ ࡶ ⏝ ࠸ ࡿ ࡇ ࡀ ࡛ ࡁ 㸪S. aureus ᰴ ࡢ ศ Ꮚ Ꮫ ゎ ᯒ ࡢ ᛂ ⏝ ࡀ ᮇ ᚅ ࡉ ࢀ ࡚ ࠸ ࡿ 㹙64㹛ࠋ ᮏ ❶ ࡛ ࡣ 㸪ᮾ ி 㒔 ࡛ Ⓨ ⏕ ࡋ ࡓ ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ⏤ ᮶ S. aureus ᰴ ࢆ ⏝ ࠸ 㸪ࡑ ࢀ ࡽ ࡢ Ꮫ ⓗ ≉ ᛶ ࢆ ⥙ ⨶ ⓗ ゎ ᯒ ࡋ ࡓ ࠋྛ ⏤ ᮶ S. aureus ᰴ ࡢ ศ Ꮚ Ꮫ ⓗ ≉ ᛶ 㛵 ࡋ ࡚ MLST ἲ ࢆ ⏝ ࠸ ࡚ ᫂ ࡽ ࡋ ࡓ ࠋ⥆ ࠸ ࡚ 㸪MLST ἲ ࢆ ᇶ ࡋ ࡓ ศ Ꮚ ⣔ ⤫ ࢥ ࢢ ࣛ ࣮ ࢮ ᆺ 㸪 ಖ ᭷ SE/SEls 㑇 ఏ Ꮚ ᆺ ཬ ࡧ ಖ ᭷ SaPI ࠾ ࡅ ࡿ 㛵 ಀ ᛶ ࢆ 㸪ᢠ ࢥ ࢢ ࣛ ࣮ ࢮ ⾑ Ύ ࢆ ⏝
12 ࡿ ࡇ ࡛ ᫂ ࡽ ࡋ 㸪㣗 ୰ ẘ ࢆ ㄏ Ⓨ ࡋ ࡸ ࡍ ࠸ S. aureus ࡢ 㑇 ఏ Ꮫ ⓗ ≉ ᛶ ࢆ ⌮ ゎ ࡍ ࡿ ࡇ ࢆ ┠ ⓗ ࡋ ࡓ ࠋ ᮦ ᩱ ཬ ࡧ ᪉ ἲ 1. 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ᰴ ⏝ ᇵ ᆅ ᮏ ◊ ✲ ࡛ ࡣ 㸪1994 ᖺ ࡽ 2012 ᖺ ᮾ ி 㒔 ࡛ Ⓨ ⏕ ࡋ ࡓ ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ 83 ࡽ ศ 㞳 ࡉ ࢀ ࡓ 203 ᰴ ࡢ S. aureus ࢆ ⏝ ࡋ ࡓ ࠋ ࡇ ࢀ ࡽ ࡢ ᰴ ࡣ 㸪ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ࡢ ᝈ ⪅ ⣅ ౽ ⏤ ᮶ 㸪ᝈ ⪅ ྤ ≀ ⏤ ᮶ 㸪 ᥎ ᐃ ཎ ᅉ 㣗 ရ ⏤ ᮶ ཬ ࡧ タ ࡢ ㄪ ⌮ ჾ ල ࡶ ࡋ ࡃ ࡣ ㄪ ⌮ ᚑ ᴗ ဨ ࡢ ᡭ ᣦ ࡢ ᣔ ࡁ ྲྀ ࡾ ᳨ య ⏤ ᮶ ࡛ ࠶ ࡿ ࠋ ᚋ ㏙ ࡍ ࡿ SE/SEls 㑇 ఏ Ꮚ ᆺ ࢥ ࢢ ࣛ ࣮ ࢮ ᆺ ࡀ 㸪 ᝈ ⪅ ᳨ య ⏤ ᮶ ศ 㞳 ᰴ ᥎ ᐃ ཎ ᅉ 㣗 ရ ⏤ ᮶ 㸦 ࡶ ࡋ ࡃ ࡣ ᣔ ࡁ ྲྀ ࡾ ᳨ య ⏤ ᮶ 㸧 ศ 㞳 ᰴ ࡛ ୍ ⮴ ࡋ ࡓ ࡶ ࡢ ࢆ 㸪 ྛ ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ࡢ ཎ ᅉ ⳦ ᰴ ࡋ ࡓ ࠋ S. aureus ᰴ ࡢ ಖ Ꮡ ࡣ ࢝ ࢪ ࢺ ࣥ ༙ ὶ ື ᇵ ᆅ ࢆ ⏝ ࠸ ࡓ ࠋ ⤌ ᡂ ࡣ ௨ ୗ ࡢ ㏻ ࡾ ࡛ ࠶ ࡿ ࠋ ࢝ ࢪ ࢺ ࣥ ༙ ὶ ື ᇵ ᆅ 㸦1 L ࢫ ࢣ ࣮ ࣝ 㸧
ձ Bacto Casitone㸦 Becton Dickinson㸪 Sparks㸪 MD㸪 USA㸧 10 g
ղ Bacto agar㸦 Becton Dickinson㸧 5 g
ճ NaCl㸦 ග ⣧ ⸆ 㸪 㜰 㸪 ᪥ ᮏ 㸧 5 g
pH7.4 ㄪ ᩚ ᚋ 㸪MilliQ Ỉ ࡛ 1 L ࣓ ࢫ ࢵ ࣉ ࡋ 㸪121Υ 15 ศ 㛫 ࢜ ࣮ ࢺ ࢡ ࣞ ࣮ ࣈ ࡋ ࡓ ࠋ
13
agar with egg yolk㸸MSEY㸧ᐮ ኳ ᇵ ᆅ㸦 ᰤ ◊ Ꮫ 㸪ᮾ ி 㸪᪥ ᮏ 㸧㸪Tryptic soy broth㸦 TSB㸸 Becton Dickinson㸧 ཬ ࡧ Brain heart infusion㸦 BHI㸧 broth㸦 Becton Dickinson㸧ࢆ ⏝ ࠸ ࡓ ࠋྛ ᇵ ᆅ ࡢ ㄪ 〇 ࡣ ῧ ࡢ ㄝ ᫂ ᭩ ᚑ ࡗ ࡚ ⾜ ࡗ ࡓ ࠋ 2. ࢥ ࢢ ࣛ ࣮ ࢮ ᆺ ู ౪ ヨ S. aureus 203 ᰴ ࡢ ࢥ ࢢ ࣛ ࣮ ࢮ ᆺ ู ࡣ 㸪 ࣈ ࢻ ࢘ ⌫ ⳦ ࢥ ࢢ ࣛ ࣮ ࢮ ᆺ ู ⏝ ච ⾑ Ύ ࢟ ࢵ ࢺ 㸦 ࢹ ࣥ ࢝ ⏕ ◊ 㸪 ᮾ ி 㸪 ᪥ ᮏ 㸧 ࢆ ⏝ ࠸ 㸪 ῧ ࡢ ㄝ ᫂ ᭩ ࢆ ୍ 㒊 ᨵ ኚ ࡋ ࡚ ⾜ ࡗ ࡓ ࠋ ࡍ ࡞ ࢃ ࡕ 㸪 ࢝ ࢪ ࢺ ࣥ ༙ ὶ ື ᇵ ᆅ ಖ Ꮡ ࡉ ࢀ ࡚ ࠸ ࡿ ⳦ ᰴ ࢆ 㸪MSEY ᐮ ኳ ᇵ ᆅ ࢆ ⏝ ࠸ ࡚ 2 ᅇ ⥅ ௦ ࡋ ࡓ ࠋ ṇ ᖖ ࢘ ࢧ ࢠ ⾑ ₢ 㸦 ࢹ ࣥ ࢝ ⏕ ◊ 㸧 ࢆ 5%㸦 w/v㸧 ࡞ ࡿ ࡼ ࠺ ῧ ຍ ࡋ ࡓ BHI broth ༢ ୍ ࢥ ࣟ ࢽ ࣮ ࢆ ᥋ ✀ ࡋ 㸪37Υ ࡛ ୍ ᬌ 㟼 ⨨ ᇵ 㣴 ࡋ ࡓ ࠋᇵ 㣴 ᾮ ࢆ 3,000 rpm㸪 30 ศ 㐲 ᚰ ศ 㞳 ࡋ ࡚ ୖ Ύ ࢆ ᥇ ྲྀ ࡋ 㸪 100 PL ࡎ ࡘ ヨ 㦂 ⟶ ศ ὀ ࡋ ࡓ ࠋ ྛ ච ⾑ Ύ 㸦 Ϩ ᆺ-ϯ ᆺ 㸧 ࢆ 100 PL ࡎ ࡘ ຍ ࠼ 㸪 ᧠ ᢾ ᚋ 37Υ ࡛ 1 㛫 ᇵ 㣴 ࡋ ࡓ ࠋ⥆ ࠸ ࡚ 10% ṇ ᖖ ࢘ ࢧ ࢠ ⾑ ₢ ⁐ ᾮ ࢆ 200 PL ࡎ ࡘ ຍ ࠼ 㸪᧠ ᢾ ᚋ 37Υ ࡛ ಖ ࡋ ࡓ ࠋྛ ヨ 㦂 ⟶ ෆ ࡢ ⾑ ₢ ࡢ จ ᅛ ࡢ ᭷ ↓ ࢆ ⥅ ⓗ ほ ᐹ ࡋ 㸪จ ᅛ ࢆ 㜼 Ṇ ࡋ ࡓ ⾑ Ύ ᆺ ࢆ ࢥ ࢢ ࣛ ࣮ ࢮ ᆺ ࡋ ࡓ ࠋ 3. Multiplex PCR ἲ ࢆ ⏝ ࠸ ࡓ SE/SEls 㑇 ఏ Ꮚ ࡢ ᆺ ู ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡢ ࢱ ࣉ ࡢ ᥎
౪ ヨ S. aureus 203 ᰴ ࡢ ಖ ᭷ SE/SEls 㑇 ఏ Ꮚ ᆺ ࡣ Omoe ࡽ 㹙 54㹛 ཬ
ࡧ Hu ࡽ㹙 33㹛ࡀ ሗ ࿌ ࡋ ࡓ Multiplex PCR ἲ ࢆ ୍ 㒊 ᨵ ኚ ࡋ ࡚ Ỵ ᐃ ࡉ ࢀ
14
ᐮ ኳ ᇵ ᆅ ࢆ ⏝ ࠸ ࡚ 2 ᅇ ⥅ ௦ ࡋ ࡓ ࠋ ⁛ ⳦ ∎ ᯞ ࢆ ⏝ ࠸ ࡚ ༢ ୍ ࢥ ࣟ ࢽ ࣮
ࢆ 㔮 ⳦ ࡋ 㸪5 PL ࡢ Lyse-N-Go PCR Reagent㸦 Thermo Fisher Scientific㸪
Wilmington㸪DE㸪 USA㸧 ᠱ ⃮ ࡋ 㸪 ୗ グ ࡢ ฎ ⌮ ᮲ ௳ ࡛ Genomic DNA ࢆ ⁐ ฟ ࡉ ࡏ ࡓ ࠋ ձ 65Υ 30 ⛊ ղ 8Υ 30 ⛊ ճ 65Υ 1 ศ 30 ⛊ մ 97Υ 3 ศ յ 8Υ 1 ศ ն 65Υ 3 ศ շ 97Υ 1 ศ ո 65Υ 1 ศ չ 80Υ ಖ ฎ ⌮ ⤊ ᚋ 㸪45 PL ࡢ Multiplex PCR ᛂ ⁐ ᾮ ࢆ ┤ ᥋ ࢳ ࣗ ࣮ ࣈ ຍ ࠼ 㸪PCR ᛂ ࢆ ⾜ ࡗ ࡓ ࠋ ⏝ ࡋ ࡓ ࣉ ࣛ ࣐ ࣮ ࢭ ࢵ ࢺ ࢆ ⾲ 1 ♧ ࡍ ࠋ
Multiplex PCR ࡣ QIAGEN Multiplex PCR Kit 㸦 QIAGEN GmbH㸪 Hilden㸪 Germany㸧 ࢆ ⏝ ࡋ ࡓ ࠋ Multiplex PCR ᛂ ⁐ ᾮ ཬ ࡧ ᛂ ᮲ ௳ ࡣ ௨ ୗ ࡢ ㏻ ࡾ ࡛ ࠶ ࡿ ࠋ
Multiplex PCR ᛂ ᾮ ⤌ ᡂ 㸦 45 PL ࢫ ࢣ ࣮ ࣝ 㸧
ձ 2Multiplex PCR Master Mix 25 PL
ղ Primer set 㸦 set1, set2, set3 ࡶ ࡋ ࡃ ࡣ set4㸧 5 PL
15 ᛂ ᮲ ௳ ձ 95Υ 15 ศ ղ 94Υ 30 ⛊ ճ 57Υ 1 ศ 30 ⛊ մ 72Υ 1 ศ 30 ⛊ յ 72Υ 10 ศ ᛂ ≀ ࢆ 㸪4% ࢞ ࣟ ࣮ ࢫ ࢤ ࣝ 㸦 Agarose S㸹 ࢽ ࢵ ࣏ ࣥ ࢪ ࣮ ࣥ 㸪 ᮾ ி 㸪 ᪥ ᮏ 㸧 ࡛ 㟁 Ẽ Ὃ ື ࡋ ࡓ ࠋ0.5 μg/m L ⮯ ࢚ ࢳ ࢪ ࢘ ࣒ 㸦 Ethidium
bromide㸹 EtBr㸧⁐ ᾮ㸦 Sigma-Aldrich㸪St. Louis㸪MO㸪 USA㸧 ࢞
ࣟ ࣮ ࢫ ࢤ ࣝ ࢆ ᾐ ₕ ࡋ DNA ࢆ ᰁ Ⰽ ࡋ ࡓ ࠋ ࢤ ࣝ ᙳ ⨨ AE-6933FXES 㸦ATTO㸪 ᮾ ி 㸪 ᪥ ᮏ 㸧 ࢆ ⏝ ࠸ ࡚ ⣸ እ ⥺ ↷ ᑕ ࡋ 㸪 ࣂ ࣥ ࢻ ࢆ ᳨ ฟ ࡋ ࡓ ࠋ ྛ SE/SEls 㑇 ఏ Ꮚ ࡀ Ꮡ ᅾ ࡍ ࡿ ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡢ ࢱ ࣉ 㛵 ࡍ ࡿ ሗ ࿌ 㹙2㹛 ᇶ ࡙ ࡁ 㸪 ᮏ ᐇ 㦂 ࡛ ᚓ ࡽ ࢀ ࡓ SE/SEls 㑇 ఏ Ꮚ ᆺ ࡢ ⤖ ᯝ ࡽ 㸪 ౪ ヨ S. aureus ᰴ ࡀ ᣢ ࡘ SE/SEls 㑇 ఏ Ꮚ ࡀ Ꮡ ᅾ ࡍ ࡿ ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡢ ࢱ ࣉ ࢆ ᥎ ࡋ ࡓ ࠋ 4. 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ Genomic DNA ࡢ ⢭ 〇
⳦ ᰴ ࡢ Genomic DNA ࡢ ⢭ 〇 ࢆ QIAamp DNA Mini kit㸦 QIAGEN㸧
ࢆ ⏝ ࠸ 㸪 ῧ ࡢ ㄝ ᫂ ᭩ ࢆ ୍ 㒊 ᨵ ኚ ࡋ ࡚ ⾜ ࡗ ࡓ ࠋ ࡍ ࡞ ࢃ ࡕ 㸪5 mL ࡢ
BHI broth ༢ ୍ ࡢ ࢥ ࣟ ࢽ ࣮ ࢆ ᥋ ✀ ࡋ 㸪37Υ ࡛ ୍ ᬌ ┞ ᇵ 㣴 ࡋ ࡓ ࠋᇵ
㣴 ᾮ ࢆ 3,000 rpm㸪 10 ศ 㐲 ᚰ ศ 㞳 ࡋ ୖ Ύ ࢆ ྲྀ ࡾ 㝖 ࠸ ࡓ ࠋ ᅇ ࡋ ࡓ ⳦
య ࢆ 㸪 lysostaphin 㸦 ග ⣧ ⸆ 㸧 ࢆ ⤊ ⃰ ᗘ 5 μg/mL ῧ ຍ ࡋ ࡓ
16
Tris-EDTA-Triton X-100㸦 TET㸧 ࣂ ࢵ ࣇ ࣮ ࡛ ᠱ ⃮ ࡋ 㸪 37Υ ࡛ 1 㛫
ຍ ࡋ ⣽ ⬊ ቨ ࢆ ◚ ቯ ࡋ ࡓ ࠋ ⣽ ⬊ ቨ ◚ ቯ ⳦ ᾮ 25 PL ࡢ proteinase K
200 PL ࡢ Buffer AL ࢆ ຍ ࠼ ࡼ ࡃ ᠱ ⃮ ࡋ 㸪 56Υ ࡛ 1 㛫 ຍ ࡋ ࡓ ࠋ
ḟ 200 PL ࡢ 99.5%࢚ ࢱ ࣀ ࣮ ࣝ㸦 ග ⣧ ⸆ 㸧ࢆ ຍ ࠼ ࡼ ࡃ ᠱ ⃮ ࡋ 㸪ΰ
ྜ ᾮ ࢆ DNeasy Mini Spin Column ⛣ ࡋ ࡓ ࠋ8,000 rpm ࡛ DNeasy Mini
Spin Column ࢆ 1 ศ 㛫 㐲 ᚰ ศ 㞳 ࡋ 㸪ℐ 㐣 ᾮ ࢆ ᤞ ࡚ ࡓ ࠋ500 PL ࡢ Buffer AW1 ࢆ ຍ ࠼ 㸪8,000 rpm ࡛ 1 ศ 㛫 ࡢ 㐲 ᚰ ศ 㞳 ࢆ ⾜ ࠸ 㸪ℐ 㐣 ᾮ ࢆ ᤞ ࡚ 㸪
⥆ ࠸ ࡚ 500 PL ࡢ Buffer AW2 ࢆ ຍ ࠼ 㸪 13,000 rpm ࡛ 3 ศ 㛫 ࡢ 㐲 ᚰ ศ
㞳 ࢆ ࡋ 㸪DNeasy Mini Spin Column ࢆ Ὑ ί ࡋ ࡓ ࠋℐ 㐣 ᾮ ࢆ ᤞ ࡚ 㸪 ࡉ ࡽ
13,000 rpm ࡛ 1 ศ 㛫 㐲 ᚰ ศ 㞳 ࡋ 㸪Buffer AW2 ࢆ ྲྀ ࡾ 㝖 ࠸ ࡓ ࠋ
DNeasy Mini Spin Column ࢆ 1.5 mL ࢳ ࣗ ࣮ ࣈ ⛣ ࡋ 㸪200 PL ࡢ Buffer AE ࢆ ຍ ࠼ ࡚ 1 ศ 㛫 ࡢ 㟼 ⨨ ᚋ 㸪 8,000 rpm ࡛ 1 ศ 㛫 㐲 ᚰ ศ 㞳 ࡋ 㸪 ⢭ 〇 genomic DNA ⁐ ᾮ ࢆ ᚓ ࡓ ࠋ⢭ 〇 ࡋ ࡓ genomic DNA ࡣ 㸪NanoDrop 2000c
㸦Thermo Fisher Scientific㸧 ࢆ ⏝ ࠸ ࡚ DNA ⃰ ᗘ ࢆ ᐃ ࡋ ࡓ ࠋ ࡲ ࡓ 㸪
A260/A280 ࡀ 1.8 ௨ ୖ ࡛ ࠶ ࡿ ࡇ ࢆ ☜ ㄆ ࡋ 㸪ゎ ᯒ ⏝ ࠸ ࡿ ࡲ ࡛ 4Υ ࡛ ಖ Ꮡ ࡋ ࡓ ࠋ࡞ ࠾ 㸪 ⏝ ࡋ ࡓ TET ࣂ ࢵ ࣇ ࣮ 1 M Tris-HCl㸦 pH 8.0㸧 ࡢ ⤌ ᡂ ࡣ ௨ ୗ ࡢ ㏻ ࡾ ࡛ ࠶ ࡿ ࠋ TET ࣂ ࢵ ࣇ ࣮ ⤌ ᡂ 㸦 20 mL ࢫ ࢣ ࣮ ࣝ 㸧 ձ 1 M Tris-HCl㸦 pH 8.0㸧 㸦 ୗ グ ㄪ 〇 㸧 0.4 mL ղ 0.5 M EDTA㸦 pH 8.0㸧 㸦 ྠ ோ Ꮫ 㸪 ⇃ ᮏ 㸪 ᪥ ᮏ 㸧 0.08 mL ճ 10% Triton X-100㸦 ග ⣧ ⸆ 㸧 2.4 mL pH8.0 ㄪ ᩚ ᚋ 㸪࢜ ࣮ ࢺ ࢡ ࣞ ࣮ ࣈ ῭ ࡳ MilliQ Ỉ ࡛ 20 mL ࣓ ࢫ ࢵ ࣉ ࡋ ࡓ ࠋ
17 1 M Tris-HCl㸦 pH 8.0㸧 ⤌ ᡂ 㸦 500 mL ࢫ ࢣ ࣮ ࣝ 㸧 ձ ࢺ ࣜ ࢫ ࣄ ࢻ ࣟ ࢟ ࢩ ࣓ ࢳ ࣝ ࣑ ࣀ ࣓ ࢱ ࣥ 㸦 ග ⣧ ⸆ 㸧 60.55 g ղ 6 N HCl㸦 ග ⣧ ⸆ 㸧 50 mL ճ MilliQ Ỉ 㐺 㔞 pH8.0 ㄪ ᩚ ᚋ 㸪MilliQ Ỉ ࡛ 500 mL ࣓ ࢫ ࢵ ࣉ ࡋ 㸪࢜ ࣮ ࢺ ࢡ ࣞ ࣮ ࣈ ࡋ ࡓ ࠋ
5. Multi locus sequence t yping
ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ 83 ࡢ ௦ ⾲ 1 ᰴ ࡘ ࠸ ࡚ 㸪 Enright ࡽ 㹙 21㹛
ࡀ ሗ ࿌ ࡋ ࡓ ᪉ ἲ ᚑ ࠸ MLST ゎ ᯒ ࢆ ⾜ ࡗ ࡓ ࠋゎ ᯒ ᑐ ㇟ ࡞ ࡿ 7 ࡘ ࡢ
㑇 ఏ Ꮚ ᗙ 㸪ࣉ ࣛ ࣐ ࣮ 㓄 ิ ཬ ࡧ PCR ᛂ ᮲ ௳ ࡣ 㸪MLST ࢹ ࣮ ࢱ ࣋ ࣮
ࢫ 㸦http://saureus.mlst.net/ 㸧 බ 㛤 ࡉ ࢀ ࡚ ࠸ ࡿ ࡶ ࡢ ᚑ ࡗ ࡓ ࠋ ྛ ࣉ
ࣛ ࣐ ࣮ ࡢ 㓄 ิ ࡣ ⾲ 1 ࡢ ㏻ ࡾ ࡛ ࠶ ࡿ ࠋ PCR ࡣ ๓ 㡯 㸦 ➨ ୍ ❶ ᮦ ᩱ ཬ
ࡧ ᪉ ἲ 4.㸧࡛ ⢭ 〇 ࡋ ࡓ genomic DNA ࢆ 㗪 ᆺ ࡋ ࡚ 㸪TaKaRa Ex Taq Hot
Start Version㸦 ࢱ ࢝ ࣛ ࣂ ࢜ 㸪 ㈡ 㸪᪥ ᮏ 㸧ࢆ ⏝ ࡋ ⾜ ࡗ ࡓ ࠋ ᛂ ⁐ ᾮ ཬ ࡧ ᛂ ᮲ ௳ ࡣ ௨ ୗ ࡢ ㏻ ࡾ ࡛ ࠶ ࡿ ࠋ
PCR ᛂ ᾮ ⤌ ᡂ 㸦 50 PL ࢫ ࢣ ࣮ ࣝ 㸧
ձ 10Ex Taq Buffer 5 PL
ղ dNTP Mixture㸦 ࡑ ࢀ ࡒ ࢀ 2.5 mM㸧 4 PL
ճ Forward primer㸦 20 PM㸧 0.5 PL
մ Reverse primer㸦 20 PM㸧 0.5 PL
յ Ex Taq Hot Start Version㸦 5 units/μ L㸧 0.25 PL
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շ Nuclease-free H2O㸦 Thermo Fisher Scientific㸧 38.75 PL
ᛂ ᮲ ௳ ձ 95Υ 10 ศ ղ 95Υ 30 ⛊ ճ 55Υ 1 ศ մ 72Υ 30 ⛊ յ 72Υ 10 ศ
ᛂ ⤊ ᚋ 㸪MonoFas DNA Purification Kit㸦 GL ࢧ ࢚ ࣥ ࢫ 㸪ᮾ ி 㸪
᪥ ᮏ 㸧ࢆ ⏝ ࠸ ࡚ PCR ቑ ᖜ ⏘ ≀ ࡢ ⢭ 〇 ࢆ ⾜ ࡗ ࡓ ࠋ᪉ ἲ ࡣ ࢟ ࢵ ࢺ ῧ
ࡢ ㄝ ᫂ ᭩ ᚑ ࡗ ࡓ ࠋࡍ ࡞ ࢃ ࡕ 㸪500 PL ࡢ Buffer A ࢆ ຍ ࠼ ࡓ MonoFas
I spin column PCR ᛂ ᾮ 50 PL ࢆ ຍ ࠼ 9,000 rpm ࡛ 1 ศ 㛫 㐲 ᚰ ศ
㞳 ࡋ 㸪 ℐ 㐣 ᾮ ࢆ ᤞ ࡚ ࡓ ࠋ500 PL ࡢ Buffer B ࢆ ຍ ࠼ 㸪 9,000 rpm ࡛ 1
ศ 㛫 㐲 ᚰ ศ 㞳 ࡋ 㸪MonoFas I spin column ࢆ Ὑ ί ࡋ ࡓ ࠋℐ 㐣 ᾮ ࢆ ᤞ ࡚ 㸪
ࡉ ࡽ 9,000 rpm ࡛ 1 ศ 㛫 ࡢ 㐲 ᚰ ศ 㞳 ࢆ ⾜ ࠸ Buffer B ࢆ ྲྀ ࡾ
㝖 ࠸ ࡓ ࠋMonoFas I spin column ࢆ 1.5 mL ࢳ ࣗ ࣮ ࣈ ⛣ ࡋ 㸪 20 PL ࡢ
Buffer C ࢆ ຍ ࠼ ࡚ 1 ศ 㛫 ࡢ 㟼 ⨨ ᚋ 㸪 9000 rpm ࡛ 1 ศ 㛫 ࡢ 㐲 ᚰ ศ 㞳
ࡼ ࡗ ࡚ ⢭ 〇 PCR ቑ ᖜ ⏘ ≀ ࢆ ᚓ ࡓ ࠋ
ୖ グ ࡢ ⢭ 〇 PCR ቑ ᖜ ⏘ ≀ ࢆ 㗪 ᆺ ࡋ ࡚ 㸪 BigDye Terminator v3.1
Cycle Sequencing Kit㸦 Thermo Fisher Scientific㸧ࢆ ⏝ ࠸ ࡚ ࢧ ࢡ ࣝ ࢩ
࣮ ࢡ ࢚ ࣥ ࢫ ᛂ ࢆ ⾜ ࡗ ࡓ ࠋ ࡞ ࠾ 㸪PCR ᛂ ࢆ ⾜ ࡗ ࡓ Forward primer
ཬ ࡧ Reverse primer ࢆ ࡑ ࢀ ࡒ ࢀ ࢩ ࣮ ࢡ ࢚ ࣥ ࢫ ⏝ ࣉ ࣛ ࣐ ࣮ ࡋ ࡚
⏝ ࡋ ࡓ ࠋ ᛂ ⁐ ᾮ ཬ ࡧ ᛂ ᮲ ௳ ࡣ ௨ ୗ ࡢ ㏻ ࡾ ࡛ ࠶ ࡿ ࠋ 30 ࢧ ࢡ ࣝ
19
ࢩ ࣮ ࢡ ࢚ ࣥ ࢫ ᛂ ᾮ ⤌ ᡂ 㸦10 PL ࢫ ࢣ ࣮ ࣝ 㸧
ձ 5Sequencing Buffer 2 PL
ղ Template DNA㸦 150-300 ng㸧 1 PL
ճ Forward ࡶ ࡋ ࡃ ࡣ Reverse primer 1 PL
մ Terminator ready reaction mix 1 PL
յ Nuclease-free H2O㸦 Thermo Fisher Scientific㸧 5 PL
ᛂ ᮲ ௳
ձ 96Υ 1 ศ
ղ 96Υ 10 ⛊
ճ 50Υ 5 ⛊
մ 60Υ 4 ศ
ᛂ ᾮ 10 PL ࡢ Nuclease-free H2O㸦 Thermo Fisher Scientific㸧 ࢆ
ຍ ࠼ 㸪BigDye XTerminator Purification Kit㸦 Thermo Fisher Scientific㸧
ࢆ ⏝ ࠸ ࡚ ⢭ 〇 ࡋ ࡓ ࠋ᪉ ἲ ࡣ ヨ ⸆ ῧ ࡢ ㄝ ᫂ ᭩ ᚑ ࡗ ࡓ ࠋࡍ ࡞ ࢃ ࡕ 㸪
20 PL ࡢ ࢧ ࢡ ࣝ ࢩ ࣮ ࢡ ࢚ ࣥ ࢫ ᛂ ᾮ ࢆ 㸪 96 Well Reaction Plate
㸦Thermo Fisher Scientific㸧 ࡢ ࢘ ࢙ ࣝ ⛣ ࡋ 㸪 90 PL ࡢ SAM Solution
20 PL ࡢ XTerminator Solution ࢆ ࡑ ࢀ ࡒ ࢀ ࡢ ࢘ ࢙ ࣝ ῧ ຍ ࡋ ࡓ ࠋ
PCR Foil Seal㸦 ᪥ ᮏ ࢪ ࢙ ࢿ ࢸ ࢡ ࢫ 㸪ᮾ ி 㸪᪥ ᮏ 㸧࡛ 96 Well Reaction Plate ࢆ ᐦ ᑒ ࡋ 㸪30 ศ 㛫 ᨩ ᢾ ࡋ ࡓ ᚋ 1,000 rpm ࡛ 2 ศ 㛫 ࡢ 㐲 ᚰ ศ 㞳 ࢆ
⾜ ࠸ 㸪XTerminator Solution ࢆ ỿ ⃦ ࡉ ࡏ 㸪ୖ Ύ ࢆ ࢟ ࣕ ࣆ ࣛ ࣜ ࣮ 㟁 Ẽ Ὃ
ື ⏝ ࠸ ࡓ ࠋ
ሷ ᇶ 㓄 ิ ࡢ Ỵ ᐃ ࡣ 㸪ABI PR ISM 3130-Avant Genetic Anal yzer
㸦Thermo Fisher Scientific㸧ࢆ ⏝ ࡋ 㸪௨ ୗ ࡢ ᮲ ௳ ࡛ ࢟ ࣕ ࣆ ࣛ ࣜ ࣮ 㟁
20 Ẽ Ὃ ື ࢆ ⾜ ࡗ ࡓ ࠋ
ձ ࢟ ࣕ ࣆ ࣛ ࣜ ࣮ 㸹 50 cm50 Pm
ղ ࣏ ࣜ ࣐ ࣮ 㸹 POP-7㸦 Thermo Fisher Scientific㸧
ճ Ὃ ື ⏝ ࣂ ࢵ ࣇ ࣮㸹10Capillary Electrophoresis Running Buffer
㸦Sigma-Aldrich㸧
մ Dye Set㸹 Z
յ Instrument Protocol 㸹 BDx_FastSeq50_POP7_BDv3
ն Analysis Protocol 㸹 3130KB_POP7_v3
ᚓ ࡽ ࢀ ࡓ ሷ ᇶ 㓄 ิ ࢆ MEGA5 software㹙 70㹛ࢆ ⏝ ࠸ ࡚ ᩚ ิ ࡉ ࡏ 㸪ゎ
ᯒ ᑐ ㇟ ࡞ ࡿ 7 ࡘ ࡢ 㑇 ఏ Ꮚ ᗙ ࡢ 㓄 ิ ࢆ Ỵ ᐃ ࡋ ࡓ ࠋ Ỵ ᐃ ࡋ ࡓ 㓄 ิ ࢆ ୖ
グ ࡢ MLST ࢹ ࣮ ࢱ ࣋ ࣮ ࢫ ↷ ྜ ࡋ 㸪ྛ 㑇 ఏ Ꮚ ᗙ ࡢ allele number ཬ ࡧ
ࡑ ࡢ ⤌ ࡳ ྜ ࢃ ࡏ ࡽ ᑟ ࢀ ࡿ Sequence t ype 㸦 ST 㸧 ࢆ ᚓ ࡓ ࠋ Clonal
complex㸦 CC㸧 ゎ ᯒ ࡣ ࡑ ࢀ ࡒ ࢀ ࡢ ST ࡢ Single locus variant㸦 slv㸧 Double locus variant ࢆ ྠ ୍ ࡢ CC ᆺ ᐃ ⩏ ࡋ 㸪 ๓ ㏙ ࡢ MLST ࢹ ࣮ ࢱ
࣋ ࣮ ࢫ ୖ බ 㛤 ࡉ ࢀ ࡚ ࠸ ࡿ eBURST software ࢆ ⏝ ࠸ ࡚ ⾜ ࡗ ࡓ ࠋ
6. SaPI scanning
ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ 83 ࡢ ௦ ⾲ ྛ 1 ᰴ㸦 ィ 83 ᰴ 㸧 ࡘ ࠸ ࡚ 㸪Sato’o
ࡽ ࡀ ሗ ࿌ ࡋ ࡓ SaPI scanning ἲ 㹙 64㹛 ࢆ ᐇ ࡋ 㸪 ࢤ ࣀ ࣒ ୖ ࡢ SaPI ࡢ
ᤄ ධ ࡢ ᭷ ↓ ࢆ ⥙ ⨶ ⓗ ゎ ᯒ ࡋ ࡓ ࠋLong and accurate㸦 LA㸧-PCR ࡼ
ࡿ ゎ ᯒ ࡣ 㸪๓ 㡯㸦 ➨ ୍ ❶ ᮦ ᩱ ཬ ࡧ ᪉ ἲ 4.㸧࡛ ⢭ 〇 ࡋ ࡓ genomic DNA
21
⏝ ࡋ ⾜ ࡗ ࡓ ࠋLA-PCR ࡢ ࣉ ࣛ ࣐ ࣮ ࡣ Sato’o ࡽ ࡀ ⏝ ࠸ ࡓ 㓄 ิ㹙 64㹛
ࢆ ⏝ ࡋ ࡓ ࠋ ྛ ࣉ ࣛ ࣐ ࣮ ࡢ 㓄 ิ ࡣ ⾲ 1 ࡢ ㏻ ࡾ ࡛ ࠶ ࡿ ࠋ LA-PCR
ᛂ ⁐ ᾮ ཬ ࡧ ᛂ ᮲ ௳ ࡣ ௨ ୗ ࡢ ㏻ ࡾ ࡛ ࠶ ࡿ ࠋ
LA-PCR ᛂ ᾮ ⤌ ᡂ 㸦 50 PL ࢫ ࢣ ࣮ ࣝ 㸧
ձ 10LA PCR Buffer II 㸦 Mg2 +plus㸧 5 PL
ղ dNTP Mixture㸦 ࡑ ࢀ ࡒ ࢀ 2.5 mM㸧 8 PL
ճ Forward primer㸦 20 PM㸧 0.5 PL
մ Reverse primer㸦 20 PM㸧 0.5 PL
յ LA Taq Hot Start Version㸦 5 units/μ L㸧 0.5 PL
ն Template genomic DNA 㸦 < 1 Pg㸧 1 PL
շ Nuclease-free H2O㸦 Thermo Fisher Scientific㸧 34.5 PL
LA-PCR ᛂ ᮲ ௳
ձ 96Υ 1 ศ
ղ 98Υ 10 ⛊
ճ 68Υ 20 ศ
մ 68Υ 10 ศ
10 kb ௨ ୖ ࡢ ቑ ᖜ ⏘ ≀ ࢆ 㸪 MonoFas DNA Purification Kit ࢆ ⏝ ࠸ ࡚
⢭ 〇 ࡋ ࡓ ࠋ ᪉ ἲ ࡣ ๓ 㡯 㸦 ➨ ୍ ❶ ᮦ ᩱ ཬ ࡧ ᪉ ἲ 5.㸧 ᚑ ࡗ ࡓ ࠋ ᚓ ࡽ
ࢀ ࡓ ⢭ 〇 ῭ ࡳ LA-PCR ቑ ᖜ ⏘ ≀ ࡘ ࠸ ࡚ 㸪 HindIII㸦 ࢱ ࢝ ࣛ ࣂ ࢜ 㸧
ࢆ ⏝ ࠸ ࡓ ไ 㝈 㓝 ⣲ ᩿ ∦ 㛗 ከ ᆺ 㸦 Restriction fragment length
polymorphism㸸 RFLP㸧 ゎ ᯒ ࢆ ⾜ ࡗ ࡓ ࠋ ࡍ ࡞ ࢃ ࡕ 㸪 ௨ ୗ ࡢ ᛂ ᾮ ࢆ ㄪ
〇 ᚋ 㸪37Υ ࡛ 1 㛫 㟼 ⨨ ࡋ LA-PCR ቑ ᖜ ⏘ ≀ ࢆ ษ ᩿ ࡋ ࡓ ࠋ
22
ᛂ ᾮ ⤌ ᡂ 㸦20 PL ࢫ ࢣ ࣮ ࣝ 㸧
ձ 10M Buffer 2 PL
ղ ⢭ 〇 ῭ ࡳ LA-PCR ቑ ᖜ ⏘ ≀ 㸦 < 1 Pg㸧 3 PL
ճ HindIII 㸦 5 units/μL㸧 1 PL
մ Nuclease-free H2O㸦 Thermo Fisher Scientific㸧 14 PL
ᛂ ᾮ ᛂ ᾮ 㔞 ࡢ 1/10 㔞 ௨ ୖ ࡢ 10Loading Buffer 㸦 ࢱ ࢝ ࣛ ࣂ
࢜ 㸧ࢆ ῧ ຍ ࡋ 㸪ไ 㝈 㓝 ⣲ ᛂ ࢆ Ṇ ࡵ ࡓ ࠋษ ᩿ ࡉ ࢀ ࡓ DNA 㙐 ࢆ 0.7%
SeaKem GTG Agarose gels 㸦 Lonza㸪 Rockland㸪 ME㸪 USA㸧 ࢆ ⏝ ࠸ ࡚
㟁 Ẽ Ὃ ື ࡋ ࡓ ࠋ0.5 μg/mL EtBr ⁐ ᾮ ࢞ ࣟ ࣮ ࢫ ࢤ ࣝ ࢆ ᾐ ₕ ࡋ DNA
ᰁ Ⰽ ࡋ 㸪ࢤ ࣝ ᙳ ⨨ AE-6933FXES㸦 ATTO㸧ࢆ ⏝ ࠸ ࡚ 㸪RFLP ࣃ ࢱ
࣮ ࣥ ࢆ ☜ ㄆ ࡋ ࡓ ࠋࡲ ࡓ 㸪National Center for Biotechnology Information
㸦NCBI㸹 http://www.ncbi.nlm.nih.gov/ 㸧 Ⓩ 㘓 ࡉ ࢀ ࡚ ࠸ ࡿ ᪤ ▱ SaPI
ࡢ HindIII ண ษ ᩿ ࣃ ࢱ ࣮ ࣥ ࡢ in silico ゎ ᯒ ࢆ Genetyx software
program, ver. 8㸦 GENETYX Corp.㸪ᮾ ி 㸪᪥ ᮏ 㸧ࢆ ⏝ ࠸ ࡚ ⾜ ࡗ ࡓ ࠋᚓ
ࡽ ࢀ ࡓ RFLP ษ ᩿ ࣃ ࢱ ࣮ ࣥ ᪤ ▱ SaPI ࡢ ண ษ ᩿ ࣃ ࢱ ࣮ ࣥ ࢆ ẚ ㍑ ࡋ SaPI ࢆ ྠ ᐃ ࡋ ࡓ ࠋ 7. ศ Ꮚ ⣔ ⤫ ゎ ᯒ ๓ 㡯 㸦 ➨ ୍ ❶ ᐇ 㦂 ᮦ ᩱ ཬ ࡧ ᐇ 㦂 ᪉ ἲ 5.㸧 MLST ゎ ᯒ ࡛ ᚓ ࡽ ࢀ ࡓ ྛ 㑇 ఏ Ꮚ ᗙ ࡢ 㓄 ิ ࢆ ⧅ ࡂ ྜ ࢃ ࡏ ࡓ ࡶ ࡢ 㸦 ⤖ ྜ 㓄 ิ 㸧 ࢆ ⏝ ࠸ ศ Ꮚ ⣔ ⤫ ゎ ᯒ ࢆ ⾜ ࡗ ࡓ ࠋ౪ ヨ ௦ ⾲ ᰴ ྜ ィ 83 ᰴ ࡢ ⤖ ྜ 㓄 ิ ࡘ ࠸ ࡚ ࣐ ࣝ ࢳ ࣉ ࣝ ࣛ ࣓ ࣥ ࢺ ࢆ ⾜ ࠸ 㸪 ㏆ 㞄 ⤖ ྜ ἲ 㹙61㹛 ࡼ ࡾ ⣔ ⤫ ᶞ ࢆ స 〇 ࡋ
23
ࡓ ࠋࡲ ࡓ 㸪ࡇ ࡢ 㝿 ࡢ ሷ ᇶ ⨨ ࣔ ࢹ ࣝ ࡋ ࡚ Kimura ࡢ 2 ኚ ᩘ ἲ㹙 36㹛
ࢆ ⏝ ࡋ ࡓ ࠋ ᚓ ࡽ ࢀ ࡓ ⣔ ⤫ ᶞ ࡘ ࠸ ࡚ 㸪1,000 ᅇ ࣛ ࣥ ࢲ ࣒ ᢳ ฟ ࡼ
ࡿ ࣈ ࣮ ࢺ ࢫ ࢺ ࣛ ࢵ ࣉ ᳨ ᐃ ࢆ ⾜ ࠸ 㸪 ᶞ ᙧ ࡢ ಙ 㢗 ᛶ ホ ౯ ࢆ ⾜ ࡗ ࡓ ࠋ ⣔ ⤫
ᶞ ࡢ స 〇 ཬ ࡧ ಙ 㢗 ᛶ ホ ౯ ࡣ MEGA5 software 㹙 70㹛 ࢆ ⏝ ࠸ ࡚ ⾜ ࡗ ࡓ ࠋ
8. Sandwich ELISA ࢆ ⏝ ࠸ ࡓ SEA ࢱ ࣥ ࣃ ࢡ ㉁ ࡢ ᐃ 㔞
CC81 ᒓ ࡍ ࡿ sea 㑇 ఏ Ꮚ 㝧 ᛶ S. aureus ᰴ 10 ᰴ ࡘ ࠸ ࡚ 㸪Sandwich ELISA ࡼ ࡿ SEA ࢱ ࣥ ࣃ ࢡ ㉁ Ⓨ ⌧ 㔞 ࢆ ᐃ ࡋ ࡓ ࠋᮏ ᐃ 㔞 ࡣ Omoe ࡽ
㹙52㹛 ࡢ ᪉ ἲ ࢆ ୍ 㒊 ᨵ ኚ ࡋ ࡚ ⾜ ࡗ ࡓ ࠋ ࡍ ࡞ ࢃ ࡕ 㸪 ࢝ ࢪ ࢺ ࣥ ༙ ὶ ື ᇵ
ᆅ ಖ Ꮡ ࡉ ࢀ ࡚ ࠸ ࡿ ⳦ ᰴ ࢆ 㸪MSEY ᐮ ኳ ᇵ ᆅ ࢆ ⏝ ࠸ ࡚ 2 ᅇ ⥅ ௦ ࡋ ࡓ
ᚋ 㸪5 mL ࡢ 1%㸦 w/v㸧 Yeast Extract㸦 Becton Dickinson㸧 ࢆ ῧ ຍ ࡋ ࡓ
BHI broth 1 ࢥ ࣟ ࢽ ࣮ ᥋ ✀ ࡋ 㸪 37Υ ࡛ ୍ ᬌ ┞ ᇵ 㣴 ࡋ seed culture
ᾮ ࡋ ࡓ ࠋ⥆ ࠸ ࡚ 㸪seed culture ᾮ 0.6 mL ࢆ 60 mL ࡢ 1%㸦 w/v㸧Yeast
Extract ῧ ຍ BHI broth ᥋ ✀ ࡋ 㸪 37Υ ࡛ 48 㛫 ┞ ᇵ 㣴 ࡋ ࡓ ࠋ ᇵ
㣴 ᾮ ࢆ 10,000 rpm㸪 20 ศ 㛫 㐲 ᚰ ศ 㞳 ࡋ 㸪 ࡑ ࡢ ୖ Ύ ࢆ 0.22 μm ࡢ ࣇ
ࣝ ࢱ ࣮ 㸦MILLEX GP㸹 Merck Millipore㸪 Darmstadt㸪 Germany㸧 ࡛ ℐ
㐣 ࡋ 㸪 ℐ 㐣 ᾮ ࢆ ゎ ᯒ ⏝ ࠸ ࡿ ࡲ ࡛ 㸫20Υ ࡛ ಖ Ꮡ ࡋ ࡓ ࠋ
ୖ グ ࡛ ㄪ 〇 ࡋ ⤖ ಖ Ꮡ ࡋ ࡓ S. aureus ᇵ 㣴 ୖ Ύ ࢧ ࣥ ࣉ ࣝ ᾮ ࢆ ⼥ ゎ
ᚋ 㸪10,000 rpm㸪 20 ศ 㛫 㐲 ᚰ ศ 㞳 ࡋ ୖ Ύ ࢆ ᅇ ࡋ ᐃ ࢧ ࣥ ࣉ ࣝ ࡋ
ࡓ ࠋ㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ࡀ ⏘ ⏕ ࡍ ࡿ ࣉ ࣟ ࢸ ࣥ A ࢆ ୖ Ύ ࢧ ࣥ ࣉ ࣝ ᾮ ୰
ࡽ 㝖 ཤ ࡍ ࡿ ࡓ ࡵ 㸪ImmunoPure Normal Rabbit Serum㸦 Thermo Fisher
Scientific 㸧 ࢆ ᐃ ࢧ ࣥ ࣉ ࣝ ➼ 㔞 ຍ ࠼ 㸪 4Υ ࡛ ୍ ᬌ 㟼 ⨨ ࡋ ࡓ ࠋ ࡑ ࡢ
ᚋ 㸪5 ಸ 㔞 ࡢ Can Get Signal Immunoreaction Enhancer Solution 1 㸦 ᮾ
24
Signal Immunoreaction Enhancer Solution 1 ࢆ ⏝ ࠸ ࡚ 㐺 ᐅ 10 ಸ ࡽ 100,000 ಸ ࡲ ࡛ ࡢ ᕼ 㔘 ᐃ ࢧ ࣥ ࣉ ࣝ ᾮ ࢆ స 〇 ࡋ ࡓ ࠋ ࡲ ࡓ 㸪 ᒾ ᡭ Ꮫ
ࡢ 㔜 ⱱ ඞ ᙪ ᩍ ᤵ ࡼ ࡾ ศ ࡉ ࢀ ࡓ 100 ng/mL ࡢ 0.1% 㸦 w/v 㸧 Bovine
serum albumin㸦 BSA㸧ῧ ຍ ࣜ ࢥ ࣥ ࣅ ࢼ ࣥ ࢺ SEA ࢱ ࣥ ࣃ ࢡ ㉁ ࢆ 㸪Can Get Signal Immunoreaction Enhancer Solution 1 ࢆ ⏝ ࠸ ࡚ 㝵 ẁ ᕼ 㔘 ࡋ 㸪ᶆ ‽ ᭤ ⥺ ࡢ స 〇 ⏝ ࠸ ࡓ ࠋ
Nunc ࣝ ࣑ ࢾ ࣥ ࢡ ࣉ ࣞ ࣮ ࢺ㸦 Thermo Fisher Scientific㸧 Capture
ᢠ య ཬ ࡧ Rabbit IgG Isot ype Control, Purified (UNLB) IgG 㸦 ࢥ ࢫ ࣔ ࣂ
࢜ 㸪ᮾ ி 㸪᪥ ᮏ 㸧ࢆ 100 μL/well ຍ ࠼ 㸪4Υ ࡛ ୍ ᬌ 㟼 ⨨ ࡋ 㸪Capture
ᢠ య ཬ ࡧ ṇ ᖖ ࢘ ࢧ ࢠ IgG ࢆ ᅛ ┦ ࡋ ࡓ ࠋ Capture ᢠ య ࡣ 㸪 ᒾ ᡭ
Ꮫ ࡢ 㔜 ⱱ ඞ ᙪ ᩍ ᤵ ࡼ ࡾ ศ ࡉ ࢀ ࡓ SEA ᑐ ࡍ ࡿ ࢘ ࢧ ࢠ ᢠ య ࢆ 㸪 1
μg/mL ᕼ 㔘 ࡋ ࡚ ⏝ ࡋ ࡓ ࠋ ᅛ ┦ ᚋ 㸪 Capture ᢠ య ࢆ ᤞ ࡚ 200 μL/well ࡢ 1Phosphate buffered saline㸦 㸫 㸧㹙 1PBS㸦 㸫 㸧㹛 ࡛ ࢘ ࢙
ࣝ ࢆ 3 ᅇ Ὑ ί ࡋ ࡓ ࠋḟ 㸪300 μL/well ࡢ StartingBlock (PBS) Blocking
Buffer㸦 Thermo Fisher Scientific㸧 ࢆ ຍ ࠼ 3 ศ 㛫 㟼 ⨨ ࡋ 㸪 ࣈ ࣟ ࢵ ࢟ ࣥ
ࢢ ࢆ ⾜ ࡗ ࡓ ࠋStartingBlock (PBS) Blocking Buffer ࢆ ᤞ ࡚ 㸪200 μL/well
ࡢ 1PBS㸦 㸫 㸧࡛ ࢘ ࢙ ࣝ ࢆ 3 ᅇ Ὑ ί ᚋ 㸪 ᐃ ࢧ ࣥ ࣉ ࣝ ᾮ ཬ ࡧ ᶆ ‽ ᭤
⥺ స 〇 ⏝ ࣜ ࢥ ࣥ ࣅ ࢼ ࣥ ࢺ SEA ᕼ 㔘 ᾮ ࢆ 100 μL/well ຍ ࠼ 㸪 37Υ ࡛ 1
㛫 㟼 ⨨ ࡋ ࡓ ࠋ ࢧ ࣥ ࣉ ࣝ ᾮ ࢆ ᤞ ࡚ 㸪Atlantis ࣐ ࢡ ࣟ ࣉ ࣞ ࣮ ࢺ ࢘ ࢛
ࢵ ࢩ ࣕ ࣮ 㸦Biochrom㸪 Cambridge㸪 UK㸧 ࢆ ⏝ ࠸ ࡚ 㸪 Tween 20㸦 ග
⣧ ⸆ 㸧ࢆ 0.05%㸦 w/v㸧 ࡞ ࡿ ࡼ ࠺ ῧ ຍ ࡋ ࡓ 1PBS㸦 1PBST㸧࡛
࢘ ࢙ ࣝ ࢆ 8 ᅇ Ὑ ί ࡋ ࡓ ࠋὙ ί ᚋ 㸪Detection ᢠ య ࢆ 100 μL/well ຍ ࠼ 㸪
37Υ ࡛ 1 㛫 㟼 ⨨ ࡋ ࡓ ࠋDetection ᢠ య ࡣ 㸪ᒾ ᡭ Ꮫ ࡢ 㔜 ⱱ ඞ ᙪ ᩍ
ᤵ ࡼ ࡾ ศ ࡉ ࢀ ࡓ Horseradish peroxidase㸦 HRP㸧 ᶆ ㆑ ᢠ SEA ࢘ ࢧ ࢠ
25
ࢆ ⏝ ࠸ ࡚ 㸪20,000 ಸ ᕼ 㔘 ࡋ ࡚ ⏝ ࡋ ࡓ ࠋ ᛂ ᚋ 㸪Detection ᢠ య ࢆ
ᤞ ࡚ 㸪1 PBST ࡛ ࢘ ࢙ ࣝ ࢆ 8 ᅇ Ὑ ί ࡋ ࡓ ࠋ Ὑ ί ᚋ 㸪 ᇶ ㉁ ⁐ ᾮ
㸦SuperSignal ELISA FemtoMaxmum Sensitivity Substrate 㸹 Thermo
Fisher Scientific 㸧 ࢆ 100 μL/well ຍ ࠼ 㸪 SpectraMax Paradigm
㸦Molecular Devices㸪 Sunnyvale㸪 CA㸪 USA㸧 ࡛ Ⓨ ග ᐃ ࢆ ⾜ ࡗ ࡓ ࠋ
ࣉ ࣟ ࢸ ࣥ A ࡢ ᙳ 㡪 ࡢ ᭷ ↓ ࡣ 㸪 Rabbit IgG Isot ype Control, Purified
(UNLB) IgG ࢆ ᅛ ┦ ࡋ ࡓ ࢘ ࢙ ࣝ ࡛ ྠ ୍ ࡢ ࢧ ࣥ ࣉ ࣝ ࢆ ᐃ ࡋ 㸪㠀 ≉ ␗
ᛂ ࡀ ࣈ ࣛ ࣥ ࢡ ྠ ➼ ࡞ ࡿ ࡇ ࡼ ࡾ ☜ ㄆ ࡋ ࡓ ࠋࣈ ࣛ ࣥ ࢡ ࡣ 1%
㸦w/v㸧yeast extract ࢆ ῧ ຍ ࡋ ࡓ BHI broth㸦 100 μL/well㸧ࢆ ⏝ ࡋ ࡓ ࠋ
⏝ ࡋ ࡓ 1PBS㸦 㸫 㸧 ࡢ ⤌ ᡂ ࡣ ௨ ୗ ࡢ ㏻ ࡾ ࡛ ࠶ ࡿ ࠋ 1PBS㸦 㸫 㸧 ⤌ ᡂ 㸦 1 L ࢫ ࢣ ࣮ ࣝ 㸧 ձ NaCl㸦 ග ⣧ ⸆ 㸧 8 g ղ KCl㸦 ග ⣧ ⸆ 㸧 0.2 g ճ Na2HPO4࣭12H2O㸦 ග ⣧ ⸆ 㸧 2.9 g մ KH2PO4㸦 ග ⣧ ⸆ 㸧 0.2 g pH7.4 ㄪ ᩚ ᚋ 㸪 ࢜ ࣮ ࢺ ࢡ ࣞ ࣮ ࣈ ῭ ࡳ MilliQ Ỉ ࡛ 1 L ࣓ ࢫ ࢵ ࣉ ࡋ 㸪 ࢜ ࣮ ࢺ ࢡ ࣞ ࣮ ࣈ ࡋ ࡓ ࠋ ⤖ ᯝ 1. 㣗 ୰ ẘ ⏤ ᮶ 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ᰴ ࡢ MLST ゎ ᯒ MLST ゎ ᯒ ࡢ ⤖ ᯝ 㸪 ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ⏤ ᮶ S. aureus ྜ ィ 83 ᰴ ࡣ 17 ✀ 㢮 ࡢ ST㸦 ST1㸪 ST5㸪 ST6㸪 ST8㸪 ST12㸪 ST20㸪 ST30㸪 ST45㸪
26 ST59㸪 ST81㸪 ST96㸪 ST121㸪 ST398㸪 ST508㸪 ST2272㸪 ST45slv ཬ ࡧ ST81slv㸧 ᆺ ู ࡉ ࢀ ࡓ ࠋ ࡲ ࡓ ࡇ ࢀ ࡽ ࡢ ST ᆺ ࡘ ࠸ ࡚ 㸪 eBURST ࢆ ⏝ ࠸ ࡓ CC ゎ ᯒ ౪ ࡍ ࡿ 㸪8 ✀ 㢮 ࡢ せ ࡞ CC ศ 㢮 ࡉ ࢀ ࡓ㸦 ᅗ 4㸧ࠋ ࡇ ࡢ ࠺ ࡕ CC81㸦 ST1㸪ST81 ཬ ࡧ ST81slv ࢆ ྵ ࡴ 㸧 ᒓ ࡍ ࡿ S. aureus ᰴ 㸦 ௨ ᚋ CC81 ᰴ ⾲ グ ࡍ ࡿ 㸧 ࡀ ᭱ ࡶ ከ ࡃ 㸪 య ࡢ 25.3%ࢆ ༨ ࡵ 㸪 ḟ ࠸ ࡛ CC8㸦 ST8㸧ᰴ 㸪CC6㸦 ST6㸧ᰴ 㸪CC5㸦 ST5㸧ᰴ 㸪CC508㸦 ST45㸪 ST508 ཬ ࡧ ST45slv㸧ᰴ 㸪CC20 ᰴ㸦 ST20㸧㸪CC30㸦 ST30㸧ᰴ 㸪CC59 㸦ST59㸧 ᰴ ࡢ 㡰 ࡛ ࠶ ࡗ ࡓ ࠋ ࡲ ࡓ ౪ ヨ S. aureus ᰴ ࡢ 8.4%ࡀ 㸪 ୖ グ 8 ✀ 㢮 ࡢ CC ᒓ ࡉ ࡞ ࠸ ST ᆺ㸦 ST12㸪ST96㸪ST121㸪ST398 ཬ ࡧ ST2272㸧 ࡛ ࠶ ࡗ ࡓ ࠋ 2. 㣗 ୰ ẘ ⏤ ᮶ 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ᰴ ࡢ ࢥ ࢢ ࣛ ࣮ ࢮ ᆺ Clonal complex ࡢ 㛵 ಀ ᛶ ୖ グ 8 ✀ 㢮 ࡢ CC ᒓ ࡍ ࡿ S. aureus ᰴ ࡣ 㸪 5 ✀ 㢮 ࡢ ࢥ ࢢ ࣛ ࣮ ࢮ
ᆺ ศ 㢮 ࡉ ࢀ ࡓ㸦 ⾲ 2㸧ࠋࡇ ࢀ ࡽ ࡢ ࠺ ࡕ ࢥ ࢢ ࣛ ࣮ ࢮ VII ᆺ㸦 CoaT VII㸧
ᒓ ࡍ ࡿ S. aureus ᰴ ࡀ ᭱ ࡶ ከ ࡃ య ࡢ 38.5%ࢆ ༨ ࡵ ࡚ ࠸ ࡓ ࠋ ⾲ 2
♧ ࡍ ࡼ ࠺ 㸪CC81 ᰴ ࡣ CoaT VII ࢆ ♧ ࡋ ࡓ ࠋ ྠ ᵝ ࡢ 㛵 ಀ ᛶ ࡀ ࡢ
7 ✀ 㢮 ࡢ CC ᆺ ࢥ ࢢ ࣛ ࣮ ࢮ ᆺ ࡶ ㄆ ࡵ ࡽ ࢀ 㸪≉ ᐃ ࡢ CC ᰴ ࡣ ࡑ ࢀ ࡒ ࢀ ≉ ᐃ ࡢ ࢥ ࢢ ࣛ ࣮ ࢮ ᆺ ࢆ ಖ ᭷ ࡍ ࡿ ࠸ ࠺ 㛵 ಀ ᛶ ࡀ Ꮡ ᅾ ࡋ ࡓ ࠋ ิ
ᣲ ࡍ ࡿ CC8-CoaT III㸪CC6-CoaT IV㸪CC5-CoaT II㸪CC508-CoaT VII㸪
CC20-CoaT VIII㸪 CC30-CoaT IV ཬ ࡧ CC59-CoaT VII ࡛ ࠶ ࡗ ࡓ ࠋ
3. 㣗 ୰ ẘ ⏤ ᮶ 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ᰴ ࡢ SE/SEls 㑇 ఏ Ꮚ ᆺ Clonal complex ࡢ 㛵 ಀ ᛶ
27 sea 㑇 ఏ Ꮚ 㝧 ᛶ ᰴ ࡣ 㸪CC508 CC20 ࢆ 㝖 ࡃ 6 ✀ 㢮 ࡢ CC ᒓ ࡍ ࡿ S. aureus ᰴ ࡛ ㄆ ࡵ ࡽ ࢀ ࡓ ࠋ ≉ 㸪 ศ 㞳 ẚ ⋡ ࡀ 㧗 ࠸ CC ᰴ ࡛ sea 㑇 ఏ Ꮚ ࡢ 㧗 ࠸ ಖ ᭷ ⋡ ࢆ ㄆ ࡵ ࡓ ࠋࡍ ࡞ ࢃ ࡕ 㸪CC81 ᰴ ࡢ 76%㸪CC8 ᰴ ࡢ 100%㸪 CC6 ᰴ ࡢ 100%ࡀ sea 㑇 ఏ Ꮚ ࢆ ಖ ᭷ ࡋ ࡚ ࠸ ࡓ 㸦 ᅗ 5㸧 ࠋ ≉ ᐃ ࡢ CC ᰴ ࡣ 㸪 ࡑ ࢀ ࡒ ࢀ ≉ ᐃ ࡢ SE/SEls 㑇 ఏ Ꮚ ࢆ ಖ ᭷ ࡍ ࡿ ࠸ ࠺ 㛵 ಀ ᛶ ࡀ Ꮡ ᅾ ࡋ ࡓ ࠋ ࡑ ࡋ ࡚ 㸪 ࡑ ࢀ ࡽ SE/SEls ࡀ Ꮡ ᅾ ࡍ ࡿ ᥎ ࡉ ࢀ ࡿ ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡢ ಖ ᭷ 㛵 ࡋ ࡚ ࡶ 㸪 ࡑ ࢀ ࡒ ࢀ ࡢ CC ᰴ ࡀ ᚲ ࡎ ಖ ᭷ ࡍ ࡿ ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡑ ࡢ ୰ ࡢ ୍ 㒊 ࡢ CC ᰴ ࡀ ಖ ᭷ ࡍ ࡿ ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡀ Ꮡ ᅾ ࡋ ࡓ㸦 ⾲ 3㸧ࠋ ࠼ ࡤ 㸪 ࡚ ࡢ CC8 ᰴ ࡣ ࣉ ࣟ ࣇ ࣮ ࢪ ୖ Ꮡ ᅾ ࡍ ࡿ SE 㑇 ఏ Ꮚ ࡛ ࠶ ࡿ sea 㑇 ఏ Ꮚ ࢆ ಖ ᭷ ࡋ ࡚ ࠸ ࡓ ࠋࡲ ࡓ 㸪୍ 㒊 ࡢ CC8 ᰴ ࡀ ࣉ
ࣛ ࢫ ࣑ ࢻ ୖ Ꮡ ᅾ ࡍ ࡿ SE 㑇 ఏ Ꮚ ࡛ ࠶ ࡿ sed㸪 selj ཬ ࡧ ser 㑇 ఏ Ꮚ ࢆ
ಖ ᭷ ࡋ ࡚ ࠸ ࡓ ࠋ ୍ ᪉ ࡛ 㸪 ࡚ ࡢ CC8 ᰴ ࡣ 㸪 SaPI ୖ Ꮡ ᅾ ࡍ ࡿ SE 㑇
ఏ Ꮚ ࡛ ࠶ ࡿ seb㸪sec㸪sel㸪sek ཬ ࡧ seq 㑇 ఏ Ꮚ ࡸ 㸪the highly variable
genomic region νSaβ ୖ Ꮡ ᅾ ࡍ ࡿ SE 㑇 ఏ Ꮚ ࡛ ࠶ ࡿ seg㸪 sei㸪 sem㸪
sen ཬ ࡧ seo 㑇 ఏ Ꮚ ࢆ ಖ ᭷ ࡋ ࡚ ࠸ ࡞ ࡗ ࡓ ࠋ ྠ ᵝ ࡢ 㛵 ಀ ᛶ ࡀ ࡢ 7 ✀ 㢮 ࡢ CC ᒓ ࡍ ࡿ S. aureus ᰴ ࠾ ࠸ ࡚ ࡶ ㄆ ࡵ ࡽ ࢀ ࡓ 㸦 ⾲ 3㸧 ࠋ 4. 㣗 ୰ ẘ ⏤ ᮶ 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ᰴ ࡀ ಖ ᭷ ࡍ ࡿ SaPI ࡢ ྠ ᐃ Clonal complex ࡢ 㛵 ಀ ᛶ ྛ ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ⏤ ᮶ S. aureus ᰴ ࡢ 6 ᡤ ࡢ SaPI ᤄ ධ ྍ ⬟ 㡿 ᇦ ࢆ ゎ ᯒ ࡋ ࡓ ࠋ ࠸ ࡎ ࢀ ࡢ ᰴ ࡶ 49͛ 㡿 ᇦ ࡣ 10 kb ௨ ୖ ࡢ ቑ ᖜ ⏘ ≀ ࡣ ㄆ ࡵ ࡽ ࢀ ࡞ ࡗ ࡓ㸦 ⾲ 4㸧ࠋࡑ ࡢ 5 㡿 ᇦ ࡣ 㸪ゎ ᯒ ࡋ ࡓ ఱ ࢀ ࡢ ᰴ ࠾ ࠸ ࡚ 10 kb ௨ ୖ ࡢ ቑ ᖜ ⏘ ≀ ࡀ ㄆ ࡵ ࡽ ࢀ 㸪 SaPI ࢆ ྵ ࡴ Genomic
28 island㸦 GI㸧 ࡀ Ꮡ ᅾ ࡋ ࡓ ࠋ CC8 ཬ ࡧ CC5 ࢆ 㝖 ࡃ 6 ✀ 㢮 ࡢ CC ᒓ ࡍ ࡿ S. aureus ᰴ ࡣ 㸪ࡑ ࢀ ࡒ ࢀ ≉ ᐃ ࡢ 㡿 ᇦ ྛ CC ෆ ࡛ ඹ ㏻ ࡋ ࡓ GI ࡀ Ꮡ ᅾ ࡋ ࡓ ࠋ ࠼ ࡤ 㸪 CC81 ᰴ ࡢ 9͛ 㡿 ᇦ ࡣ 㸪 10 kb ௨ ୖ ࡢ ቑ ᖜ ⏘ ≀ ࡀ ㄆ ࡵ ࡽ ࢀ 㸪 ࡑ ࢀ ࡽ ࡣ ྠ ୍ ࡢ RFLP ࣃ ࢱ ࣮ ࣥ ࢆ ♧ ࡋ ࡓ ࠋྠ ᵝ 㸪CC6 ᰴ ࡢ 9͛ 㡿 ᇦ 㸪CC508 ᰴ ࡢ 9͛ 㡿 ᇦ 㸪CC20 ᰴ ࡢ 9͛ 㡿 ᇦ 㸪CC30 ᰴ ࡢ 8͛ 㡿 ᇦ 44͛ 㡿 ᇦ 㸪 CC59 ᰴ ࡢ 9͛ 㡿 ᇦ 19͛ 㡿 ᇦ Ꮡ ᅾ ࡍ ࡿ GI ࡣ 㸪 ࡑ ࢀ ࡒ ࢀ ྠ ୍ ࡢ RFLP ࣃ ࢱ ࣮ ࣥ ࢆ ♧ ࡋ ࡓ㸦 ᅗ 6 ࡢ͆ Common insertion͇㸧ࠋࡇ ࢀ ࡽ RFLP ࣃ ࢱ ࣮ ࣥ ᪤ ሗ ࡢ SaPI 㓄 ิ㹙 51㸪64㹛ࢹ ࣮ ࢱ ࢆ ẚ ㍑ ࡋ 㸪ྛ 㡿 ᇦ ࡢ ᤄ ධ 㓄 ิ ࢆ ྠ ᐃ ࡋ ࡓ ࠋCC59 ᰴ ࡢ 19͛ 㡿 ᇦ ࡢ RFLP ࣃ ࢱ ࣮ ࣥ ࡣ SaPI3 ࡢ ࡑ ࢀ ୍ ⮴ ࡋ ࡓ ࠋCC30 ᰴ ࡢ 8͛ 㡿 ᇦ 44͛ 㡿 ᇦ ࡢ RFLP ࣃ ࢱ ࣮ ࣥ ࡣ ࡑ ࢀ ࡒ ࢀ SaPI4 ཬ ࡧ SaPI2 ࡢ ࡑ ࢀ ࡽ ୍ ⮴ ࡋ ࡓ ࠋࡲ ࡓ 㸪CC81 ᰴ ཬ ࡧ CC6 ᰴ ࡢ 9͛ 㡿 ᇦ ࡢ RFLP ࣃ ࢱ ࣮ ࣥ ࡣ 㸪MW2 ᰴ ࡀ 9͛ 㡿 ᇦ ᣢ ࡘ ᮍ ゎ ᫂ ࡢ GI ࡢ ࡑ ࢀ ୍ ⮴ ࡋ ࡓ㸦 MW2-like RFLP㸧㹙 64㹛㸦 ⾲ 4 ཬ ࡧ ᅗ 6 ࡢ ͆ Common insertion͇㸧ࠋ ୍ ᪉ 㸪ྛ CC ᒓ ࡍ ࡿ ୍ 㒊 ࡢ S. aureus ᰴ ࡣ 㸪ࡑ ࡢ CC ≉ ᭷ ࡢ RFLP ࣃ ࢱ ࣮ ࣥ ࢆ ᣢ ࡘ GI ࡢ ᤄ ධ ࢆ ㄆ ࡵ ࡓ ࠋ ࡍ ࡞ ࢃ ࡕ 㸪 CC81 ᰴ ࡢ 19%ࡣ 㸪 ࡑ ࡢ 18͛ 㡿 ᇦ SaPIishikawa11 ࢆ ಖ ᭷ ࡋ 㸪 24%ࡢ CC81 ᰴ ࡀ 19͛ 㡿 ᇦ SaPIhhm2 ࢆ 㸪14%ࡀ 44͛ 㡿 ᇦ SaPIno10 ࢆ ࡑ ࢀ ࡒ ࢀ ಖ ᭷ ࡋ ࡚ ࠸ ࡓ ࠋࡲ ࡓ 㸪CC20 ᰴ ࡢ 50%ࡣ 㸪44͛ 㡿 ᇦ SaPIhirosaki4 ࢆ 㸪CC508 ᰴ ࡢ 29%ࡣ 㸪 18͛ 㡿 ᇦ SaPI68111 ࢆ ࡑ ࢀ ࡒ ࢀ ಖ ᭷ ࡋ ࡚ ࠸ ࡓ ࠋ ࡑ ࡢ ࡢ ᤄ ධ 㓄 ิ ࡀ Ꮡ ᅾ ࡋ ࡓ ቑ ᖜ ⏘ ≀ ࡢ RFLP ࣃ ࢱ ࣮ ࣥ ࡣ ᪤ ▱ ࡢ SaPIs ࡢ RFLP ࣃ ࢱ ࣮ ࣥ ࡣ ୍ ⮴ ࡋ ࡞ ࡗ ࡓ 㸦 ⾲ 4 ཬ ࡧ ᅗ 6 ࡢ ͆ Frequent insertion͇㸧ࠋ MLST ゎ ᯒ ࠾ ࡅ ࡿ 㓄 ิ ࢆ ᇶ 㸪 ྛ ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ⏤ ᮶ S.
29
aureus ᰴ ࡢ ศ Ꮚ ⣔ ⤫ ᶞ ࢆ స ᡂ ࡋ ࡓ ⤖ ᯝ 㸪 ྛ CC ᆺ ᑐ ᛂ ࡍ ࡿ ศ Ꮚ ⣔
⤫ ࡀ Ꮡ ᅾ ࡋ ࡓ 㸦 ᅗ 7A㸧ࠋ ࡲ ࡓ 㸪 ౪ ヨ S. aureus ᰴ ࡢ ศ Ꮚ ⣔ ⤫ SaPI
ࡢ ಖ ᭷ ≧ ἣ ࡢ 㛵 ಀ ᛶ ࢆ ᳨ ウ ࡋ ࡓ ⤖ ᯝ 㸪ࡑ ࢀ ࡒ ࢀ ࡢ ⣔ ⤫ ࡍ ࡞ ࢃ ࡕ CC
࡛ SaPI ࡢ ಖ ᭷ ศ ᕸ ࡀ ␗ ࡞ ࡿ ࠸ ࠺ 㛵 ಀ ᛶ ࡀ ᫂ ࡽ ࡞ ࡗ ࡓ ࠋ ࠼ ࡤ 㸪
CC81 ᰴ ࡣ MW2-like RFLP ࡢ 㸪 SaPIishikawa11 㸪 SaPIhhm2 㸪 SaPIno10 ࢆ ಖ ᭷ ࡋ ࡚ ࠸ ࡓ ࠋ ࡇ ࢀ ࡽ 3 ✀ ࡢ SaPI ࡣ 㸪 ศ Ꮚ ⣔ ⤫ ⓗ ㏆
࠸ ࡢ CC ᰴ ࠾ ࠸ ࡚ ࡶ ࡑ ࡢ ศ ᕸ ࡀ ㄆ ࡵ ࡽ ࢀ ࡞ ࠸ 㸪CC81 ᰴ ≉ ᭷ ࡢ
ࡶ ࡢ ࡛ ࠶ ࡗ ࡓ ࠋ ྠ ᵝ 㸪SaPI2 㸪 SaPI3 㸪 SaPI4 㸪 SaPI68111 ཬ ࡧ
SaPIhirosaki4 ࡶ ≉ ᐃ ࡢ ୍ ࡘ ࡢ CC ᰴ ࡢ ࡳ 㸪 ࡑ ࡢ ศ ᕸ ࡀ ㄆ ࡵ ࡽ ࢀ ࡓ 㸦 ᅗ 7B ཬ ࡧ 7C㸧ࠋ 5. CC81 ᒓ ࡍ ࡿ sea 㑇 ఏ Ꮚ 㝧 ᛶ 㯤 Ⰽ ࣈ ࢻ ࢘ ⌫ ⳦ ᰴ ࡢ SEA ⏘ ⏕ 㔞 CC81 ᒓ ࡍ ࡿ sea 㑇 ఏ Ꮚ 㝧 ᛶ S. aureus ᰴ ࡘ ࠸ ࡚ 㸪 ᇵ 㣴 ᾮ ୰ ࡢ SEA ⏘ ⏕ 㔞 ࢆ ᐃ ࡋ ࡓ ࠋSEA ⏘ ⏕ 㔞 ࡣ ࡑ ࢀ ࡒ ࢀ ࡢ ᰴ ࡛ ␗ ࡞ ࡗ ࡚ ࠸ ࡓ ࡀ 㸪 ࡚ ࡢ ᰴ ࡛ 2 μg/mL ௨ ୖ ࡢ SEA ࡢ ⏘ ⏕ ࡀ ㄆ ࡵ ࡽ ࢀ 㸪 10 ᰴ ୰ 7 ᰴ ࡀ 6 μg/mL ௨ ୖ ࡢ SEA ࡢ ⏘ ⏕ ࢆ ㄆ ࡵ ࡓ 㸦 ᅗ 8㸧 ࠋ ⪃ ᐹ SEs ࢆ ⏘ ⏕ ࡍ ࡿ S. aureus ᰴ ࢆ ཎ ᅉ ࡍ ࡿ ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ࡣ 㸪ୡ ⏺ ୰ ࡛ Ⓨ ⏕ ࡍ ࡿ ᭱ ࡶ せ ࡞ 㣗 ရ ፹ ᛶ ᝈ ࡢ ୍ ࡘ ࡛ ࠶ ࡿ ࠋ ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ⏤ ᮶ ᰴ ࡢ Ꮫ ゎ ᯒ ࡣ 㸪 Ⓨ ⏕ ࡋ ࡓ ᆅ ᇦ ࡈ ู ࠎ ࡢ ᡭ ἲ ࢆ ⏝ ࠸ ࡚ ࡞ ࡉ ࢀ ࡚ ࡁ ࡓ 㹙17㸪68㸪69㸪79㸪81㹛ࠋᮏ ❶ ࡛ ࡣ 㸪ᮾ ி 㒔 ࡛ Ⓨ ⏕ ࡋ ࡓ ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ⏤ ᮶ S. aureus ᰴ ࡘ ࠸ ࡚ 㸪ศ Ꮚ ⏕ ≀ Ꮫ ⓗ ᡭ ἲ
30 ࢆ ྵ ࡵ ࡓ 4 ✀ 㢮 ࡢ Ꮫ ゎ ᯒ ࢆ ᐇ ࡋ ࡓ ࠋ ࡉ ࡽ 㸪 ᰴ ࡢ 㑇 ఏ Ꮫ ⓗ ≉ ᛶ ࢆ ᳨ ウ ࡋ ࡓ ࠋ MLST ἲ ࢆ ⏝ ࠸ ࡓ ศ Ꮚ Ꮫ ⓗ ゎ ᯒ ࡢ ⤖ ᯝ 㸪 ᮾ ி 㒔 ࡛ Ⓨ ⏕ ࡋ ࡓ ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ⏤ ᮶ S. aureus ᰴ ࡣ 8 ✀ 㢮 ࡢ CC ศ 㢮 ࡉ ࢀ ࡓ 㸦 ᅗ 4㸧ࠋ 㣗 ୰ ẘ 㛵 ࡍ ࡿ S. aureus ᰴ ࡢ MLST ゎ ᯒ ࡣ ࢻ ࢶ 㸪ࢫ ࢫ 㸪㡑 ᅜ 㸪 ୰ ᅜ ➼ 㸪୍ 㒊 ࡢ ᅜ ࡛ ᐇ ࡉ ࢀ ࡚ ࠸ ࡿ ࡢ ࡳ ࡛ 㸪ࡑ ࡢ ሗ ࿌ ᩘ ࡣ ᑡ ࡞ ࠸㹙17㸪 79㸪 81㹛ࠋ ᮾ ி 㒔 ࠾ ࡅ ࡿ ศ 㞳 ẚ ⋡ ࡀ 㧗 ࠸ CC ᰴ ࡛ ࠶ ࡿ CC81㸪 CC8 ཬ ࡧ CC6 ➼ ࡣ 㸪ࡇ ࢀ ࡽ ࡢ ᅜ ࡢ 㣗 ୰ ẘ ࠾ ࠸ ࡚ ࡶ 㸪 ࡚ ࡛ ࡣ ࡞ ࠸ ࡀ ඃ ඛ ⓗ ศ 㞳 ࡉ ࢀ ࡿ ࡇ ࡀ ሗ ࿌ ࡉ ࢀ ࡚ ࠸ ࡿ ࠋ ୍ ᪉ 㸪 ᪥ ᮏ ᅜ ෆ ࠾ ࠸ ࡚ 㸪 㣗 ୰ ẘ ௨ እ ࡢ ࡸ ࣄ ࢺ ࡢ ᖖ ᅾ ⳦ ࡋ ࡚ ศ 㞳 ࡉ ࢀ ࡿ S. aureus ᰴ ࡢ CC ࡣ 㸪 ᅇ ࡢ ⤖ ᯝ ࡣ ᫂ ࡽ ␗ ࡞ ࡿ ࠋ ࠼ ࡤ 㸪 ங ᡣ ⅖ ࢆ ⨯ ᝈ ࡋ ࡓ ࢘ ࢩ ࡢ ங ⏤ ᮶ ࡢ S. aureus ᰴ ࡣ 㸪 CC97 CC705 ࡛ ࠶ ࡾ 㹙27㹛㸪 ࡲ ࡓ 㸪 ᖖ ே ࡢ 㰯 ⭍ ࡽ ࡣ 㸪 CC508㸪 CC15 ཬ ࡧ CC188 ศ 㢮 ࡉ ࢀ ࡿ CC ࢆ ᣢ ࡘ S. aureus ࡀ ศ 㞳 ࡉ ࢀ ࡿ ሗ ࿌ ࡉ ࢀ ࡚ ࠸ ࡿ 㹙65㹛ࠋ௨ ୖ ࡢ ⤖ ᯝ ࡽ 㸪CC81㸪CC8㸪CC6 ࡞ ࡢ ≉ ᐃ ࡢ CC ࢆ ᣢ ࡘ S. aureus ࡣ 㸪 㣗 ୰ ẘ ࡽ ศ 㞳 ࡉ ࢀ ࡸ ࡍ ࠸ ᰴ ࡛ ࠶ ࡿ ࡇ ࡀ ♧ ၀ ࡉ ࢀ ࡓ ࠋ ࢥ ࢢ ࣛ ࣮ ࢮ ᆺ ู ࡢ ⤖ ᯝ ࡽ 㸪CoaT VII ࡀ ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ⏤ ᮶ S. aureus ᰴ ࠾ ࠸ ࡚ ᭱ ࡶ ከ ࡃ ศ 㞳 ࡉ ࢀ ࡓ㸦 ⾲ 2㸧ࠋࡇ ࡢ ᐇ ࡣ 㸪㐣 ཤ ᪥ ᮏ ࡛ ⾜ ࢃ ࢀ ࡓ ሗ ࿌ ୍ ⮴ ࡋ ࡚ ࠸ ࡓ 㹙68㹛ࠋ ࡲ ࡓ 㸪 S. aureus ᰴ ࠾ ࡅ ࡿ CC CoaT ࡣ ᐦ ᥋ ࡞ 㛵 ಀ ᛶ ࡀ Ꮡ ᅾ ࡍ ࡿ ࡇ ࡀ ௨ ๓ ࡼ ࡾ ሗ ࿌ ࡉ ࢀ ࡚ ࠾ ࡾ㹙27㹛㸪ᮏ ◊ ✲ ࠾ ࠸ ࡚ ࡶ ≉ ᐃ ࡢ CC ᰴ ࡀ እ ࡞ ࡃ ༢ ୍ ࡢ CoaT ࢆ ᣢ ࡘ ࡇ ࡀ ᫂ ࡽ ࡞ ࡗ ࡓ 㸦 ⾲ 2㸧ࠋ ࡇ ࢀ ࡽ 2 ࡘ ࡢ ᆺ ู ἲ ࢆ ⤌ ࡳ ྜ ࢃ ࡏ ࡿ ࡇ ࡛ 㸪ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ࢆ ᘬ ࡁ ㉳ ࡇ ࡍ S. aureus ࡢ Ꮫ ⓗ ≉ ᛶ 㛵 ࡍ ࡿ ᭷ ┈ ࡞ ሗ ࢆ ᚓ ࡿ ࡇ ࡀ ࡛ ࡁ ࡿ ⪃ ࠼ ࡽ ࢀ ࡿ ࠋ
31 SE/SEls 㑇 ఏ Ꮚ ᆺ ู ࡢ ⤖ ᯝ ࡽ 㸪 ྛ CC ᰴ ࠾ ࠸ ࡚ ᚲ ࡎ ಖ ᭷ ࡍ ࡿ SE/SEls 㑇 ఏ Ꮚ ࡑ ࡢ ୍ 㒊 ࡢ ᰴ ࡀ ಖ ᭷ ࡍ ࡿ SE/SEls 㑇 ఏ Ꮚ ࡢ ≉ ␗ ⓗ ࡞ ⤌ ࡳ ྜ ࢃ ࡏ ࡀ Ꮡ ᅾ ࡍ ࡿ ࡇ ࡀ ᫂ ࡽ ࡞ ࡗ ࡓ ࠋ ࡲ ࡓ 㸪 ྛ CC ᰴ ࠾ ࠸ ࡚ 㸪 ᚲ ࡎ ಖ ᭷ ࡉ ࢀ ࡿ ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡀ ࠶ ࡿ 㸪 㒊 ศ ⓗ ࡞ ᰴ ⩌ ࡢ ࡳ ಖ ᭷ ࡀ ㄆ ࡵ ࡽ ࢀ ࡿ ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡀ ࠶ ࡾ 㸪CC ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ 㛫 ୍ ᐃ ࡢ 㛵 ಀ ᛶ ࡀ ࠶ ࡿ ࡇ ࡀ ᥎ ࡉ ࢀ ࡓ 㸦 ⾲ 3㸧 ࠋ ࡇ ࢀ ࡽ ࡢ ࡇ ࡣ 㸪 ࣈ ࢻ ࢘ ⌫ ⳦ ࢤ ࣀ ࣒ ୖ Ꮡ ᅾ ࡍ ࡿ SE/SEls 㑇 ఏ Ꮚ ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡢ ࢱ ࣉ 㛫 ≉ ␗ ⓗ ࡞ ⤌ ࡳ ྜ ࢃ ࡏ ࡀ Ꮡ ᅾ ࡍ ࡿ ࡇ ࢆ ♧ ࡋ ࡚ ࠸ ࡿ ࠋ SEs ࡣ 㣗 ୰ ẘ ㉳ ᅉ ẘ ⣲ ࡛ ࠶ ࡿ ࡇ ࡽ 㸪 㣗 ୰ ẘ ࢆ ㄏ Ⓨ ࡍ ࡿ S. aureus ࡣ ≉ ᐃ ࡢ ࢤ ࣀ ࣒ ᵓ 㐀 ࢆ ᣢ ࡘ ࡇ ࢆ ♧ ၀ ࡋ ࡚ ࠸ ࡿ ࠋ MLST ᇶ ࡙ ࡃ ศ Ꮚ ⣔ ⤫ ᶞ ゎ ᯒ ࢆ ⾜ ࡗ ࡓ ⤖ ᯝ 㸪౪ ヨ S. aureus ᰴ ࡣ ྛ CC ᆺ ᑐ ᛂ ࡍ ࡿ ศ Ꮚ ⣔ ⤫ ࡀ Ꮡ ᅾ ࡋ ࡓ 㸦 ᅗ 7A㸧ࠋ ࡇ ࢀ ࡽ ࡢ ศ Ꮚ ⣔ ⤫ SaPI ಖ ᭷ ศ ᕸ ࢆ ゎ ᯒ ࡋ ࡓ ⤖ ᯝ 㸪 CC5 ཬ ࡧ CC8 ࢆ 㝖 ࡃ 6 ✀ 㢮 ࡢ CC ᰴ ࡣ 㸪 ࡇ ࡢ ୧ ⪅ ࡢ 㛫 ≉ ᚩ ⓗ ࡞ 㛵 ಀ ᛶ ࡀ Ꮡ ᅾ ࡍ ࡿ ࡇ ࡀ ᫂ ࡽ ࡞ ࡗ ࡓ ࠋࡍ ࡞ ࢃ ࡕ 㸪ྛ SaPI ࡣ ศ Ꮚ ⣔ ⤫ ⓗ ༢ ୍ ࡢ CC ᰴ ࠾ ࠸ ࡚ ࡢ ࡳ 㸪ࡑ ࡢ ศ ᕸ㸦 ಖ ᭷ 㸧ࡀ ㄆ ࡵ ࡽ ࢀ 㸪ศ Ꮚ ⣔ ⤫ ⓗ ㏆ ࠸ ࡢ CC ᰴ ࡛ ࡉ ࠼ ࡑ ࡢ ศ ᕸ 㸦 ಖ ᭷ 㸧 ࡀ ㄆ ࡵ ࡽ ࢀ ࡞ ࡗ ࡓ ࠋ ࠼ ࡤ 㸪 SaPIishikawa11 㸪 SaPIhhm2㸪 SaPIno10 ࡣ CC81 ᰴ ࡢ ࡳ ㄆ ࡵ ࡽ ࢀ 㸪
ࡢ CC ᰴ ࡣ ಖ ᭷ ࡋ ࡚ ࠸ ࡞ ࡗ ࡓ 㸦 ᅗ 7B㸧ࠋ
୍ ᪉ ࡛ 㸪 ከ ࡃ ࡢ CC5 ᰴ ཬ ࡧ CC8 ᰴ ࡣ 㸪 ࡢ ᤄ ධ ྍ ⬟ 㡿 ᇦ ࠾ ࠸
࡚ ࡶ SaPI ࢆ ྵ ࡵ ࡓ GI ࡢ ᤄ ධ ࡀ ㄆ ࡵ ࡽ ࢀ ࡞ ࡗ ࡓ ࡀ 㸪 ࡇ ࢀ ࡽ ࡢ CC
ᰴ ࠾ ࠸ ࡚ ࡢ ࡳ sed㸪selj ཬ ࡧ ser 㑇 ఏ Ꮚ ࢆ ಖ ᭷ ࡍ ࡿ S. aureus ᰴ ࡀ Ꮡ
ᅾ ࡋ ࡓ 㸦 ⾲ 3㸧ࠋ sed㸪 selj ཬ ࡧ ser 㑇 ఏ Ꮚ ࡣ pIB485-like plasmid㸪
pUO-Sa-SED1㸪pUO-Sa-SED2 ࡞ ࡢ ࣉ ࣛ ࢫ ࣑ ࢻ ୖ Ꮡ ᅾ ࡍ ࡿ ࡇ ࡀ ሗ ࿌ ࡉ ࢀ ࡚ ࠾ ࡾ 㸪 ࡇ ࢀ ࡽ ࡢ ࣉ ࣛ ࢫ ࣑ ࢻ ࢆ ㏻ ࡌ ࡚ ఏ ࡍ ࡿ ࡇ ࡀ ▱ ࡽ
32 ࢀ ࡚ ࠸ ࡿ 㹙2㹛ࠋ ࡇ ࡢ ࡇ ࡽ 㸪 CC5 ᰴ ཬ ࡧ CC8 ᰴ ࡣ 㸪 SE/SEls ࢆ ࢥ ࣮ ࢻ ࡍ ࡿ ࣉ ࣛ ࢫ ࣑ ࢻ ࢆ ಖ ᭷ ࡋ ࡸ ࡍ ࡃ 㸪 ࡑ ࡢ ࡢ 6 ✀ 㢮 ࡢ CC ᰴ ࡣ ࡇ ࡢ ࣉ ࣛ ࢫ ࣑ ࢻ ࢆ ಖ ᭷ ࡍ ࡿ ྍ ⬟ ᛶ ࡀ ప ࠸ ࡇ ࢆ ♧ ၀ ࡋ ࡚ ࠸ ࡿ ࠋ ࡍ ࡞ ࢃ ࡕ 㸪 ࡑ ࢀ ࡒ ࢀ ࡢ ⣔ ⤫ ࡀ ཷ ࡅ ධ ࢀ ࡸ ࡍ ࠸ ≉ ᐃ ࡢ ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡢ ࢱ ࣉ ࡀ Ꮡ ᅾ ࡍ ࡿ ࡇ ࡀ ᥎ ࡉ ࢀ ࡓ ࠋ ࡇ ࡢ ࡼ ࠺ ࡞ ྛ ⣔ ⤫ ࠾ ࡅ ࡿ ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡢ ཷ ࡅ ධ ࢀ ࡸ ࡍ ࡉ ࡢ 㐪 ࠸ ࡣ 㸪 ྛ ⣔ ⤫ ࡀ ಖ ᭷ ࡍ ࡿ ไ 㝈 ಟ 㣭 ⣔ ࡢ 㐪 ࠸ ࡼ ࡿ ࡶ ࡢ ࡛ ࠶ ࡿ ྍ ⬟ ᛶ ࡀ ⪃ ࠼ ࡽ ࢀ ࡿ ࠋ ไ 㝈 ಟ 㣭 ⣔ ࡣ 㸪 ⮬ ㌟ ࡢ DNA ࢆ ࣓ ࢳ ࣝ ࡞ ࡛ ಟ 㣭 ࡍ ࡿ ಟ 㣭 㓝 ⣲ DNA ࡢ ≉ ᐃ 㓄 ิ ࢆ ㄆ ㆑ ษ ᩿ ࡍ ࡿ ࡀ ಟ 㣭 ࢆ ཷ ࡅ ࡓ 㒊 ࡣ ษ ᩿ ࡋ ࡞ ࠸ ไ 㝈 㓝 ⣲ ࡢ ⤌ ࡳ ྜ ࢃ ࡏ ࡽ ᡂ ࡾ 㸪 እ 㒊 ࡽ ධ ࡋ ࡓ DNA ࢆ 㑅 ᢥ ⓗ ษ ᩿ ศ ゎ ࡍ ࡿ ാ ࡁ ࢆ ᣢ ࡘ ࠋ ࡍ ࡞ ࢃ ࡕ 㸪 ࣇ ࣮ ࢪ ឤ ᰁ ࡞ እ ᮶ ᛶ 㑇 ఏ Ꮚ ࡢ Ỉ ᖹ ఏ ࡽ ⮬ ㌟ ࡢ ࢤ ࣀ ࣒ ࢆ Ᏺ ࡿ 㸪⣽ ⳦ ࡢ ⏕ య 㜵 ᚚ ᶵ ᵓ ࡛ ࠶ ࡿ ࠋWaldron Lindsay 㹙78㹛 ࡣ 㸪 S. aureus ࡢ ≉ ᐃ ࡢ ⣔ ⤫ ࡀ ಖ ᭷ ࡍ ࡿ ไ 㝈 ಟ 㣭 ⣔ ࡼ ࡾ ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡢ Ỉ ᖹ ఏ ࡀ ไ 㝈 ࡉ ࢀ ࡿ ሗ ࿌ ࡋ ࡚ ࠸ ࡿ ࠋ ࡲ ࡓ 㸪 McCarthy ࡽ㹙 47㹛ࡣ 㸪S. aureus ࡣ ྛ ⮬ ␗ ࡞ ࡗ ࡓ ไ 㝈 ಟ 㣭 ⣔ ࢆ ⋓ ᚓ ࡋ ࡓ ࡓ ࡵ ⊂ ❧ ࡋ ࡓ 㐍 ࢆ 㐙 ࡆ ࡓ ⣔ ⤫ ࡀ Ⓨ ⏕ ࡋ 㸪 ࡑ ࢀ ࡽ ࡢ ␗ ࡞ ࡗ ࡓ ⣔ ⤫ ࡀ ␗ ࡞ ࡿ 㢖 ᗘ ࡛ 㸪 ྍ ື ᛶ 㑇 ఏ ᅉ Ꮚ ࡢ ୍ ࡘ ࡛ ࠶ ࡿ ࣉ ࣟ ࣇ ࣮ ࢪ ࢆ ಖ ᭷ ࡍ ࡿ ሗ ࿌ ࡋ ࡚ ࠸ ࡿ ࠋ ࡇ ࢀ ࡽ ࡢ ሗ ࿌ ࡣ 㸪 ᅇ ࡢ ⪃ ᐹ ࢆ ᨭ ᣢ ࡍ ࡿ ࡶ ࡢ ⪃ ࠼ ࡽ ࢀ ࡿ ࠋ SEA ࡣ ࣈ ࢻ ࢘ ⌫ ⳦ 㣗 ୰ ẘ ࡽ ᭱ ࡶ ከ ࡃ ᳨ ฟ ࡉ ࢀ ࡿ ࡇ ࡀ ▱ ࡽ ࢀ ࡚ ࠸ ࡿ 㹙2㸪 65㸪 68㸪 81㹛 ࠋ ᮏ ◊ ✲ ࠾ ࠸ ࡚ ࡶ 㸪 ౪ ヨ S. aureus ᰴ ࠾ ࠸ ࡚ ศ 㞳 ẚ ⋡ ࡀ 㧗 ࠸ CC ᰴ ࡛ ࠶ ࡿ CC81 ᰴ 㸪 CC8 ᰴ ཬ ࡧ CC6 ᰴ ࡢ ከ ࡃ ࡣ sea 㑇 ఏ Ꮚ ࡀ 㝧 ᛶ ࡛ ࠶ ࡗ ࡓ㸦 ᅗ 5㸧ࠋsea 㑇 ఏ Ꮚ ࡣ ࣉ ࣟ ࣇ ࣮ ࢪ ୖ Ꮡ ᅾ ࡍ ࡿ ࡇ ࡀ ▱ ࡽ ࢀ ࡚ ࠸ ࡿ 㹙2㸪 67㹛ࠋ ࡑ ࡢ ⏘ ⏕ 㔞 ࡣ 㸪 ࣉ ࣟ ࣇ ࣮ ࢪ ࡢ genotype 㛵 㐃 ࡋ 㸪㧗 ⏘ ⏕ ᆺ ࡢ ࣉ ࣟ ࣇ ࣮ ࢪ ప ⏘ ⏕
