Შ ᆔƷȩǯǿȞȸǼǛஊƢǔ ‷†⁕⁞⁛ Ʒ Ў܇ဃཋܖႎࣱឋǛᚐௌ ٤ ቝ ቁῺ ᄹ⦟⋵┙ක⑼ᄢቇ㒝ዻ∛㒮 ਛጊ ┨ᢥ ᄹ⦟⋵┙ක⑼ᄢቇ㒝ዻ∛㒮㧔 ጘ㒂ක≮⑼ቇᄢቇஜ⑼ቇㇱ⥃ᐥᬌᩏቇ⑼㧕 ⮮ᧄ ⢒ሶ ᄹ⦟⋵┙ක⑼ᄢቇ㒝ዻ∛㒮 㧨⎇ⓥ⋡⊛㧪 ㄭᐕ Extended spectrum--Lactamase㧔ESBL㧕ࠆߪ Metallo--Lactamase㧔MBL㧕ࠍ↥↢ߔࠆ ᭽ࡓࠣߩࡊࠗ࠲ߥޘ㒶ᕈ⩶߇࿖ౝᄖߢჇടߒߡ߅ࠅ㧘ᐢၞ -ࠢ࠲ࡓ⮎߳ߩ⠴ᕈൻ߇ᷓೞ ߥක≮㗴ߣߥߞߡࠆ㧚ᧄቇ㒝ዻ∛㒮ߦߡ㧘ߎߩਔᣇߩǪࠢ࠲ࡑࠍ↥↢ߔࠆ Escherichia coli ߇ᬌߐࠇ㧘ߎࠇࠄ 2 ⒳㘃ߩ⠴ᕈㆮવሶ߇ Plasmid ߦࠦ࠼ߐࠇߡࠆߎߣߩ⏕ࠍⴕ㧘 ߎߩ Plasmid ߩಽᨆࠍⴕ߁ߎߣࠍ⋡⊛ߣߒߚ㧚 㧨᧚ᢱߣᣇᴺ㧪 1㧚 ↪⩶ᩣ ESBL ߣ MBL ࠍ↥↢ߔࠆ E. coli㧔⥃ᐥಽ㔌ᩣ E. coli NM5759㧕 ㆮવሶવ㆐⹜㛎ߩฃኈ⩶ߦ↪ߚ E. coli GN42㧔ࠕࠫൻ࠽࠻࠙ࡓ⠴ᕈᩣ㧕 ᒻ⾰ォ឵ߢ Competent cell ߣߒߡ↪ߚ E. coli JM109㧔TOYOBO㧕 2㧚⩶⒳หቯ߅ࠃ߮⮎ᗵฃᕈ⹜㛎 ฦ⩶ᩣߩ↢ൻቇ⊛ᕈ⁁⹜㛎߅ࠃ߮⮎ᗵฃᕈ⹜㛎ߪ㧘⥄േ᷹ቯᯏེ VITEK 2-XL㧔SYSMEX bioMerieux ␠㧕ࠍ↪ߒ㧘ࡃࠗ࠹࠶ࠢ 2 GN หቯࠞ࠼ߣࡃࠗ࠹࠶ࠢ 2 ࠣࡓ㒶ᕈ⩶ᗵฃᕈࠞ ࠼ AST-N125 ࠍ↪ߡᷝઃᢥᦠߦᓥታᣉߒߚ㧚 3㧚ESBL ߅ࠃ߮ MBL ⏕⹜㛎 ⹜㛎⩶ᩣ߅ࠃ߮ㆮવሶવ㆐⩶ᩣߩ ESBL ߅ࠃ߮ MBL ↥↢ᕈߩ⏕⹜㛎ߪ㧘Double Disk synergy test㧔DDST㧕ߦࠃߞߡⴕߞߚ㧚 4㧚ㆮવሶવ㆐⹜㛎 ฃኈ⩶㧔E. coli GN42㧕߅ࠃ߮ଏਈ⩶㧔E. coli NM5759㧕ࠍᷙวၭ㙃ߒߚ㧚⊒⢒ߒߚࠦࡠ࠾ ࠍ 25g/mL ABPC ߣ 1mM ࠕࠫൻ࠽࠻࠙ࡓࠍ Luria-Bertani㧔LB㧕ኙᄤၭߢၭ㙃ߒߚ㧚 ⊒⢒ߒߚࠦࡠ࠾ߦߟߡ↢ൻቇ⊛ᕈ⁁߅ࠃ߮⮎ᗵฃᕈߩ⏕ࠍⴕߞߚ㧚ߥ߅㧘⹜㛎೨ߩฃ ኈ⩶㧘ଏਈ⩶ࠍߘࠇߙࠇ LB ኙᄤၭ㧔ABPC㧘ࠕࠫൻ࠽࠻࠙ࡓࠍ㧕ߦߡห᧦ઙߢၭ㙃ߒ㧘 ⊒⢒ߒߥߎߣࠍ⏕ߒߚ㧚 5㧚ࡊࠬࡒ࠼ DNA ߩ⺞ᢛ ࡊࠬࡒ࠼♖ࠠ࠶࠻ NucleoBondBAC100 (MACHEREY-NAGEL ␠) ࠍ↪ߡᷝઃߩ⺑ᦠ ߦᓥ㧘ࡊࠬࡒ࠼ DNA ߩ⺞ᢛࠍⴕߞߚ㧚ࡊࠬࡒ࠼ߩ⏕ߪ 0.7㧑ࠕࠟࡠࠬࠥ࡞ߦߡ㔚᳇ ᵒേ㧔100V㧘45 ಽ㧕ߒ㧘GelRed㧔Nucleic Acid Gel Stain, 10,000x in Water㧘Biotium㧕ߢ 20 ಽᨴ 㧙 㧙 ⦡ᓟ㧘࠻ࡦࠬࠗ࡞ࡒࡀ࠲ߢ⏕ߒߚ㧚 6㧚ᒻ⾰ォ឵ታ㛎 E. coli NM5759 ࠃࠅ♖ߒߚࡊࠬࡒ࠼ DNA ࠍޔE. coli Competent Cell Kit㧔Competent high JM109㧘TOYOBO㧕ߦኻߒߡᒻ⾰ォ឵ታ㛎ࠍⴕߞߚ㧚 7㧚PCR ᴺߦࠃࠆ ESBL㧘MBL ↥↢ㆮવሶߩ⏕ ․⇣ࡊࠗࡑࠍ↪ߚ PCR ᴺߦࠃࠆ⮎⠴ᕈㆮવሶ blaCTX-M㧘blaIMP-1 ߩᬌࠍⴕߞߚ㧚 PCR ᔕᶧߩ⚵ᚑߪ 5Green buffer㧧5L㧘 25mM MgCL2㧧2.5L㧘10mM dNTPs㧧0.5L㧘10M CTX-M-F1(IMP1-F1)㧧1L㧘10M CTX-M-R1(IMP1-R1)㧧1L㧘5units/L GoTaq PCR polymerase㧧 0.25L㧘Ṍ⩶ H2O 12.25L㧘㍌ဳ DNA 2.5L ߢో㊂߇ 25L ߦߥࠆࠃ߁ߦ⺞ᢛߒߚ㧚PCR ߩ ᔕ᧦ઙߪ 94͠㧘2 ಽߩࡊࠨࠗࠢ࡞ᓟ㧘94͠ 30 ⑽㧘58͠ 60 ⑽㧘72͠60 ⑽ࠍ 35 ࠨࠗࠢ࡞ⴕ ߥ㧘ᦨᓟߦ 72͠ߢ 3 ಽ㑆ᔕߐߖߚ㧚Ⴧ↥‛ߪ 2㧑ࠕࠟࡠࠬࠥ࡞㧔Invitrogen Ultrapure agarose 1200mg in 60mL 1TAE buffer + 6L SYBR SAFE DNA stain㧘×10000㧕ߢ㔚᳇ᵒേࠍⴕ ⏕ߒߚ㧚 8㧚Ⴎၮ㈩ߩ⸃ᨆ ࡊࠬࡒ࠼ p-NM5759-JM ߩႮၮ㈩ߩ⸃ᨆߦߪ㧘blaCTX-M㧘blaIMP-1 ߩᬌߦ↪ߒߚ PCR ࡊࠗࡑࠍ↪ߡ㧘ESBL ߅ࠃ߮ MBL ㆮવሶࠍὐߣߒߡႮၮ㈩ࠍ⺞ߴߚ㧚Ⴎၮ㈩ߩಽ ᨆߪ㧘Sanger ࠄ 1)ߩᣇᴺߦᓥࠪࠤࡦࠬᔕߦߪ BigDye terminator v3.1 cycle sequencing kit ࠍ↪ߒ㧘ABI prism 310 genetic analyzer㧔 Applied Biosystems)ߦࠃߞߡಽᨆߒߚ㧚߹ߚ㧘ᓧࠄ ࠇߚႮၮ㈩ߩ⸃ᨆߪ㧘Basic Local Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov/) ࠍ↪ߡⴕߞߚ㧚 㧨⚿ᨐ㧪 1㧚E. coli NM5759 ߩ ESBL ߅ࠃ߮ MBL ↥↢ᕈ⏕⹜㛎 E. coli NM5759 ߪ ESBL ߅ࠃ߮ MBL ࠍหᤨߦ↥↢ߒߡࠆߎߣ߇⏕ߐࠇߚ㧚 b a CAZ CPDX ACV CFPM CAZ CTX CFPM SMA IPM MEPM Double Disk synergy test㧔DDST㧕 㧔a㧕 Extended spectrum-Ǫ-lactamase㧔b㧕 Metallo-Ǫ-lactamase 2㧚ㆮવሶવ㆐⹜㛎 ଏਈ⩶ᩣ㧔E. coli NM5759㧕㧘ฃኈ⩶ᩣ㧔E. coli GN42㧕㧘ㆮવሶવ㆐⩶ᩣ㧔E. coli NM5759/GN42㧕 ߩ↢ൻቇ⊛ᕈ⁁㧔 1㧕߅ࠃ߮㧘⮎ᗵฃᕈࡄ࠲ࡦ㧔 2㧕ࠍᲧセߒߚ㧚↢ൻቇ⊛ᕈ⁁ߪ E. coli GN42 ߣ E. coli NM5759/GN42 ߦ߅ߡቢోߦ৻⥌ߒ㧘⮎ᗵฃᕈࡄ࠲ࡦߪᱴߤߩ -ࠢ࠲ࡓ ♽⮎ߦ߅ߡ E. coli GN42 ߦᲧߒߡ㧘E. coli NM5759/GN42 ߪ MIC ࠍߚ㧚 㧙 㧙 1.↢ൻቇ⊛ᕈ⁁ߩᲧセ E. coli NM5759 E. coli GN42 E. coli NM5759/GN42 BGAL䋻Beta-galactosidase ProA䋻L-Proline arylamidase (donor) (recipient) (transconjugant) BGAL 㪂 㪄 㪄 ProA 㪄 㪂 㪂 TyrA 㪄 㪂 㪂 SAC䋻saccharose/sucrose SAC 㪂 㪄 㪄 5KG䋻5-Keto-D-gluconate 5KG 㪄 㪂 㪂 PHOS 㪄 㪂 㪂 ODC 㪂 㪄 㪄 ODC䋻Ornithine Decarboxylase BGUR 㪂 㪄 㪄 BGUR䋻Beta-glucoronidase O129R 㪄 㪂 㪂 ELLM 㪄 㪂 㪂 MOT 㪂 㪄 㪄 TyrA䋻Tyrosine arylamidase PHOS䋻phosphatase O129R䋻O/129 Resistance ELLM䋻Ellman 2.⮎ᗵฃᕈࡄ࠲ࡦߩᲧセ MIC (㱘g/mL䋩 E. coli NM5759 E. coli GN42 E. coli NM5759/GN42 (donor) (recipient) (transconjugant) Ampicillin 㪕㪔㪊㪉 㪓㪔㪉 㪕㪔㪊㪉 Piperacillin 㪕㪔㪈㪉㪏 㪓㪔㪋 㪕㪔㪈㪉㪏 Amoxicillin/ 㪕㪔㪊㪉 㪋 㪈㪍 Cefazolin 㪕㪔㪍㪋 㪓㪔㪋 㪕㪔㪍㪋 Cefmetazole 㪕㪔㪍㪋 㪓㪔㪈 㪊㪉 Cefpodoxime 㪕㪔㪏 㪓㪔㪇㪅㪉㪌 㪕㪔㪏 Cefotaxime 㪕㪔㪍㪋 㪓㪔㪈 㪕㪔㪍㪋 Ceftazidime 㪈㪍 㪓㪔㪈 㪈㪍 Aztreonam 㪕㪔㪍㪋 㪓㪔㪈 㪈㪍 㪋 㪓㪔㪈 㪉 㪓㪔㪈 㪓㪔㪈 㪓㪔㪈 Meropenem 㪈 㪓㪔㪇㪅㪉㪌 㪉 Gentamicin 㪋 㪓㪔㪈 㪓㪔㪈 Levofloxacin 㪕㪔㪏 㪓㪔㪇㪅㪈㪉 㪓㪔㪇㪅㪈㪉 Ciprofloxacin 㪕㪔㪋 㪓㪔㪇㪅㪉㪌 㪓㪔㪇㪅㪉㪌 㪓㪔㪇㪅㪉㪌 clavulanate Cefepim Imipenem 3㧚E. coli NM5759 ߩࡊࠬࡒ࠼ࡄ࠲ࡦ ࠨࠗ࠭ߩ⇣ߥࠆ 3 ⒳㘃ߩࡊࠬࡒ࠼ߦ⋧ᒰߔࠆߣ⠨߃ࠄࠇࠆ DNA ࡃࡦ࠼߇⏕ߐࠇߚ㧚 㧙 㧙 M 1 ( kbp ) Plasmid䉰䉟䉵䈱ᄢ䈐䈇䉅䈱䈎䉌A,B,C䈫䈚䈢 A 19.33 Lane M䋬DNA ladder䋨㱗-Sty I䋩lane 1䋬E. coli NM5759 7.74 6.22 0.7䋦䉝䉧䊨䊷䉴䉭䊦㔚᳇ᵒേ䋨100V䋬45ಽ䋩 4.26 3.47 2.69 B 䊒䊤䉴䊚䊄 DNA䈱⺞ᢛ C NucleoBondBAC100 (MACHEREY-NAGEL) 1.88 4㧚ESBL ߅ࠃ߮ MBL ㆮવሶࠍߔࠆࡊࠬࡒ࠼ DNA ߩන㔌 ᦨ߽ࠨࠗ࠭ߩᄢ߈ p-NM5759-JM ߩߺࠍߒ㧘ESBL ߅ࠃ߮ MBL ㆮવሶߩ PCR ߇㓁ᕈࠍ␜ ߔᒻ⾰ォ឵ᩣ߇ᓧࠄࠇߚ㧚 1 2 3 4 5 M ߩߔࠆplasmidA䈮⋧ᒰ䈜䉎 A: E. coli NM5759ߩ Lane 1䋺p-NM5759-JM1䋬lane 2䋺p-NM5759-JM2 A lane 3䋺p-NM5759-JM3, lane 4䋺p-NM5759-JM4 lane 5䋺p-NM5759䋬lane M䋺DNA ladder䋨2.5Kb䋩 0.9䋦䉝䉧䊨䊷䉴䉭䊦㔚᳇ᵒേ䋨100V䋬45ಽ䋩 5㧚ࡊࠬࡒ࠼ p-NM5759-JM ߩಽᨆ ESBL ㆮવሶࠍὐߣߒߚ㈩ߢߪ blaCTX-M-2 ߩ᭴ㅧㆮવሶߣߘߩᵹߦ ccgAII㧘ccgEIII㧘ardR㧘 ardB㧘ਅᵹߦ tnpA ߩႮၮ㈩߇⏕ߐࠇߚ㧔ਅ࿑-a㧕㧚ߘߒߡ㧘blaIMP-6 ࠍὐߣߒߚ㈩ߢߪ MBL ↥↢ㆮવሶߩ᭴ㅧㆮવሶߣߘߩᵹߦ intI1㧘aacA4㧘ਅᵹߦ aadA2㧘tnp ߩႮၮ㈩߇⏕ ߐࠇߚ㧔ਅ࿑-b㧕㧚 a b ccgAII䋻CcgAII regulatory protein䋬ccgEIII 䋻CcgEII regulatory protein䋬ardR䋻ArdR regulatory protein䋬ardB䋻ArdB antirestriction protein䋬tnpA䋻ISEcp1B transposase䋬intI1䋻Class I integrase䋬aacA4䋻aminoglycoside (6)`-N-acetyltransferase䋬aadA2䋻aminoglycoside adenyltransferase䋬tnp䋻Transposase of IS91 family 㧙 㧙 㧨⠨ኤ㧪 2012 ᐕߦ Shigemoto ࠄ 2)߇ K. pneumoniae ߦ߅ߡ blaCTX-M-2 ߣ blaIMP-6 ࠍࠦ࠼ߔࠆࡊࠬࡒ ࠼ pKPI-6 ࠍႎ๔ߒߡࠆ㧚࿁ E. coli NM5759 ߆ࠄන㔌ߒߚ p-NM5759-JM ߩ blaCTX-M-2 ߣ blaIMP-6 ࿐ߩႮၮ㈩ߪ pKPI-6 ߣߩ⋧หᕈ߇㜞ߊ㧔blaCTX-M-2 ㄝ 4505bpЈ⋧หᕈ 98%㧘 blaIMP-6 ㄝ 4594bpЈ⋧หᕈ 90%㧕pKPI-6 ߣห৻ߩࡊࠬࡒ࠼ߩน⢻ᕈ߇␜ໂߐࠇߚ㧚߹ߚ㧘Yano ࠄ 3)ߪ㧘PCR ᴺߦߡ IMP-1 ㆮવሶ㓁ᕈߢߞߚ E. coli 54 ᩣߩ߁ߜ㧘Ⴧ↥‛ߩႮၮ㈩ࠍ⺞ߴߚ ߣߎࠈ㧘49 ᩣ߇ IMP-6 ࠲ࠗࡊߢࠅ㧘IMP-6 ㆮવሶࠍߔࠆᩣߩߔߴߡ߇ CTX-M ㆮવሶ㓁ᕈ ߢߞߚߣႎ๔ߒߡࠆߎߣ߆ࠄ㧘 CTX-M ߣ IMP-6 ߩㆮવሶࠍࠦ࠼ߔࠆห᭽ߩࡊࠬࡒ࠼߇㧘 ⒳ዻࠍ߃ߡᏒਛߦᢔߒߡࠆน⢻ᕈ߇ෂᗋߐࠇࠆ㧚 㧨߹ߣ㧪 ࿁ᚒߪޘ㧘ESBL ߅ࠃ߮ MBL ࠍหᤨߦ↥↢ߔࠆ E. coli NM5759 ߦߟߡ㧘⠴ᕈㆮવሶߩሽ ㇱ߅ࠃ߮ㆮવ⊛ߥᕈ⾰ߦߟߡ⺞ߴߚ㧚E. coli NM5759 ߪዋߥߊߣ߽㧟⒳㘃ߩࠨࠗ࠭ߩ⇣ߥࠆ ࡊࠬࡒ࠼ࠍߒߡ߅ࠅ㧘ᦨ߽ࠨࠗ࠭ߩᄢ߈ p-NM5759-JM ߦਔᣇߩ⠴ᕈㆮવሶ߇ሽߒߡ ߚ㧚 㧨ෳ⠨ᢥ₂㧪 1) Sanger,F., Nicklen,S.,and Coulson,A.R.: DNA sequencing with chain-terminating inhibitors.Proc. Natl.Acad. Sci. USA.74:5463–5467,1977. 2) Shigemoto,N.,Kuwahara,R.,Kayama,S.,Shimizu,W.,Onodera,M.,Yokozaki,M.,Hisatsune,J.,Kato,F.,Oh ge,H. and Sugai,M.:Emergence in Japan of an imipenem-susceptible, meropenem- resistant Klebsiella pneumoniae carrying blaIMP-6. Diagnostic Microbiology and Infec-tious Disease. 72 :109–112,2012. 3) Yano,H.,Ogawa,M.,Endo,S.,Kakuta,R.,Kanamori,H.,Inomata,S.,Ishibashi,N.,Aoyagi,T.,Hatta,M.,Gu, Y.,Yam-ada,M.,Tokuda,K.,Kunishima,H.,Kitagawa,M.,Hirakata,Y. and Kaku,M.: High frequency o f IMP-6 among clinical isolates of metallo--lactamase-producing Escherichia coli in Japan. Antimicrobial A-gents and Chemotherapy.56:4554-4555, 2012. 㧨⚻⾌ㅜ⚦㧪 ㆮવሶ⸃ᨆᖱႎಣℂ࠰ࡈ࠻࠙ࠚࠕ)'0'6;:8GT ോ▤ℂᚻᢙᢱ ว ⸘ એ 㧙 㧙
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