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 …title...
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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.(DoctoralDegree) 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。
2015
1
8
11
1. 12
2. 13
3. Multiplex PCR SE/SEls
13
4. Genomic DNA 15
5. Multi locus sequence typing 17
6. SaPI scanning 20
7. 22
8. Sandwich ELISA SEA 23
1. MLST 25
2.
Clonal complex 26
3. SE/SEls
Clonal complex
26
4. SaPI
Clonal complex
27
5. CC81 sea
SEA
29
29
35
42
SaPI
51
1. 52
2. 53
3. 53
4. Genomic DNA 54
5. SaPI scanning 54
6. Southern blott ing LA-PCR SEs
A. pKOB pKOC pKOL 54
B. seb sec sel 56
C. seb sec sel 57
D. Southern blott ing 58
7. SaPI 62
8. Sandwich ELISA SE 62
9. Rep
A. Rep 63
B. Rep 66
C. rRep 69
10.
A. ori s i te 73
B. 74
11. 75
12. 77
1. seb sec sel GI 77
2. seb sec sel SaPI 79
3. SaPI SaPI sel 80
4. SaPI rep-ori site 81
5. SaPI SEs 82
6. rRep ori site 82
7. rRep 84
84
91
105
SE/SEls sed
115
1. 116
2. 117
3. 117
4. 118
5.
119
6. Sandwich ELISA SE/SEl 120
7. sed , selj ser EcoRI
121
8. RNA 122
9. 124
10. 125
11. RT-PCR 125
12. 127
1.
127
2. SE/SEls
128
3. sed EcoRI
129
4. RT-PCR sed
sed
130
130
136
139
144
148
149
at t at tachment
BHI Brain heart infusion
BLAST Basic local al ignment search tool
BSA Bovine serum albumin
CC Clonal complex
CoaT Coagulase type
dsDNA double-stranded DNA DNA
E. coli Escherichia coli
egc enterotoxin gene cluster
EtBr Ethidium bromide
f tsZ Cell division protein gene
GI Genomic island
HRP Horseradish peroxidase
IPTG Isopropyl β -D-1-thiogalactopyranoside
IS Insertion sequence
LA-PCR Long and accurate polymerase chain reaction
LB Luria-Bertani
MHC Major histocompatibil i ty complex
MLST Multi locus sequence typing
mSED Mutant staphylococcal enterotoxin D SED
msed Mutant staphylococcal enterotoxin D gene SED
MSEY Mannitol salt agar with egg yolk
NCBI National Center for Biotechnology Information
ORF Open reading frame
ori s i te Replication origin
PBS Phosphate buffered saline
PBST Phosphate buffered saline with Tween 20 Tween 20
Rep Replication init iator protein
rep Replicat ion ini t iator protein gene
RFLP Restriction fragment length polymorphism
rpoB RNA polymerase beta subunit gene
RT-PCR Reverse transcription polymerase chain reaction
rRep recombinant replication init iator protein
SAG Superantigen
SaPI Staphylococcus aureus pathogenicity island
S. aureus Staphylococcus aureus
SDS-PAGE Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis
-
SE Staphylococcal enterotoxin
SEl Staphylococccal enterotoxin-l ike toxin
slv Single locus variant
sodA Superoxide dismutase gene
ST Sequence type
TBS Tris buffered saline
TBST Tris buffered saline with Tween 20 Tween 20
TET Tris-EDTA-Triton X-100
TSB Tryptic soy broth
1
Staphylococcus aureus S. aureus
Staphylococcal enterotoxins SEs
SEs SEs
SEs SEs
5 67
3
29 73
50
http:/ /www.mhlw.go.jp/stf/seisakunitsuite/bunya/kenkou_iryou/shoku
hin/syokuchu/ 10%
5
http:/ /www.fukushihoken.metro.tokyo.jp/shokuhin/tyuudoku/
2
2000
SEs
13,420 4 S. aureus
S. aureus
17 25 68 69 77 79
S. aureus
S. aureus
68 77 S.
aureus
62 68 Shimizu 68
2001 2 S. aureus 38
S. aureus variable
S. aureus
25 Multi locus sequence typing MLST
S.
aureus 27 37 60 63
83 MLST
3
21
MLST
23 27 37 60 63 S. aureus MLST
27 37 60 63 65
79
1930 Dack 18 S. aureus
1971
5 SEs SEA SEE
6 7 15 16
1994 SEs
SEs SEs
Staphylococcal enterotoxin-l ike toxin SEl
SE/SEls SEA SEE SEG
4
SElX 23
2 28 29 53 55 57 72 80
SE/SEls International Nomenclature Committee
for Staphylococcal Superantigen
”SE”
”SEl”
40
SEs 2
20 24 57
SEs Major
histocompatibil i ty complex MHC class II T cell receptor
T
T
1 34
SEs
SEs
34 58 5
SEs
5 20 SE/SEls
S. aureus
S. aureus
Insertion
5
sequence IS Staphylococcal cassette chromosome the highly variable
genomic region νSaβ Staphylococcus aureus pathogenicity island SaPI
2 42 44 14-17 kb
SaPI 6 8 ′ 9 ′ 18 ′ 19 ′ 44 ′
49 ′ 1
44 51 1998 SaPI
20 SaPI 2 39
41 51 64 SaPI 2 SaPI
att
44
51 SEs
50 51
SaPI
S. aureus 51 75 76 SaPI
3 SaPI Helper
phage DNA SOS
Helper phage SaPI
SaPI
integrase excisionase
SaPI
terminase
SaPI SaPI Helper
phage
6
SaPI SaPI
Replication init iator protein Rep Replication
origin ori s i te 13 51 74 75 76
Rep DNA
51 74 76 ori site 300 bp
rep 3’ 80 bp AT
AT-rich 6 bp
74 Rep
AT-rich
DNA SaPIs
51
SE/SEls
2 28 44 SE/SEls S. aureus
42
44 49
S. aureus
S. aureus
S. aureus
S. aureus
7
S. aureus
1994 2012
S. aureus
SE/SEls
SaPI
4
SaPI
SaPI 1 SaPI
S. aureus SE/SEls
S. aureus SED C SED
SED
1. S. aureus Staphylococcus aureus pathogenicity island SaPI
8
Integration Repression
Excision Replication Encapsulation and packaging
att
SaPI
ori site
2. SaPI
SaPI 51 64
int integrase stl
master repressor str regulatory protein xis exisionase pri primase rep replication
initiator ppi phage interference cp packaging genes ter terminase small subunit
9
3. SaPI
DNA SOS Helper phage SaPI
Helper phage dUTPase SaPI SaPI
integrase excisionase SaPI
terminase SaPI SaPI Helper phage
Novick 51
10
11
S. aureus
17 25 68 69 77 79
S. aureus
S. aureus
62 68 S. aureus
MLST
S. aureus
SaPI
SaPI scanning SaPI 64
S. aureus
64
S. aureus
S. aureus
MLST
MLST SE/SEls
SaPI
Multiplex PCR SaPI scanning
12
S. aureus
1.
1994 2012
83 203 S. aureus
SE/SEls
S. aureus
1 L
Bacto Casitone Becton Dickinson Sparks MD USA 10 g
Bacto agar Becton Dickinson 5 g
NaCl 5 g
pH7.4 Mill iQ 1 L 121 15
S. aureus Mannitol salt
13
agar with egg yolk MSEY Tryptic
soy broth TSB Becton Dickinson Brain heart infusion BHI
broth Becton Dickinson
2.
S. aureus 203
MSEY 2
5% w/v
BHI broth 37
3,000 rpm 30 100 L
- 100 L
37 1 10% 200 L
37
3. Multiplex PCR SE/SEls
S. aureus 203 SE/SEls Omoe 54
Hu 33 Multiplex PCR
MSEY
14
2
5 L Lyse-N-Go PCR Reagent Thermo Fisher Scientific
Wilmington DE USA Genomic DNA
65 30
8 30
65 1 30
97 3
8 1
65 3
97 1
65 1
80
45 L Multiplex PCR
PCR 1
Multiplex PCR QIAGEN Multiplex PCR Kit QIAGEN GmbH
Hilden Germany Multiplex PCR
Multiplex PCR 45 L
2 Multiplex PCR Master Mix 25 L
Primer set set1, set2, set3 set4 5 L
Nuclease-free H2O Thermo Fisher Scientific L
15
95 15
94 30
57 1 30
72 1 30
72 10
4% Agarose S
0.5 μg/mL Ethidium
bromide EtBr Sigma-Aldrich St. Louis MO USA
DNA AE-6933FXES
ATTO
SE/SEls
2 SE/SEls
S. aureus SE/SEls
4. Genomic DNA
Genomic DNA QIAamp DNA Mini kit QIAGEN
5 mL
BHI broth 37
3,000 rpm 10
lysostaphin 5 μg/mL
35
16
Tris-EDTA-Triton X-100 TET 37 1
25 L proteinase K
200 L Buffer AL 56 1
200 L 99.5%
DNeasy Mini Spin Column 8,000 rpm DNeasy Mini
Spin Column 1 500 L Buffer
AW1 8,000 rpm 1
500 L Buffer AW2 13,000 rpm 3
DNeasy Mini Spin Column
13,000 rpm 1 Buffer AW2
DNeasy Mini Spin Column 1.5 mL 200 L Buffer
AE 1 8,000 rpm 1
genomic DNA genomic DNA NanoDrop 2000c
Thermo Fisher Scientific DNA
A260/A280 1.8 4
TET 1 M Tris-HCl pH 8.0
TET 20 mL
1 M Tris-HCl pH 8.0 0.4 mL
0.5 M EDTA pH 8.0 0.08 mL
10% Triton X-100 2.4 mL
pH8.0 Mill iQ 20 mL
17
1 M Tris-HCl pH 8.0 500 mL
60.55 g
6 N HCl 50 mL
Mill iQ
pH8.0 Mill iQ 500 mL
5. Multi locus sequence t yping
83 1 Enright 21
MLST 7
PCR MLST
http:/ /saureus.mlst .net/
1 PCR
4. genomic DNA TaKaRa Ex Taq Hot
Start Version
PCR 50 L
10 Ex Taq Buffer 5 L
dNTP Mixture 2.5 mM 4 L
Forward primer 20 M 0.5 L
Reverse primer 20 M 0.5 L
Ex Taq Hot Start Version 5 units/μL 0.25 L
Template genomic DNA < 1 g 1 L
18
Nuclease-free H2O Thermo Fisher Scientific 38.75 L
95 10
95 30
55 1
72 30
72 10
MonoFas DNA Purification Kit GL
PCR
500 L Buffer A MonoFas
I spin column PCR 50 L 9,000 rpm 1
500 L Buffer B 9,000 rpm 1
MonoFas I spin column
9,000 rpm 1 Buffer B
MonoFas I spin column 1.5 mL 20 L
Buffer C 1 9000 rpm 1
PCR
PCR BigDye Terminator v3.1
Cycle Sequencing Kit Thermo Fisher Scientific
PCR Forward primer
Reverse primer
30
19
10 L
5 Sequencing Buffer 2 L
Template DNA 150-300 ng 1 L
Forward Reverse primer 1 L
Terminator ready reaction mix 1 L
Nuclease-free H2O Thermo Fisher Scientific 5 L
96 1
96 10
50 5
60 4
10 L Nuclease-free H2O Thermo Fisher Scientific
BigDye XTerminator Purification Kit Thermo Fisher Scientific
20 L 96 Well Reaction Plate
Thermo Fisher Scientific 90 L SAM Solution
20 L XTerminator Solution
PCR Foil Seal 96 Well Reaction
Plate 30 1,000 rpm 2
XTerminator Solution
ABI PRISM 3130-Avant Genetic Analyzer
Thermo Fisher Scientific
25
20
50 cm 50 m
POP-7 Thermo Fisher Scientific
10 Capillary Electrophoresis Running Buffer
Sigma-Aldrich
Dye Set Z
Instrument Protocol BDx_FastSeq50_POP7_BDv3
Analysis Protocol 3130KB_POP7_v3
MEGA5 software 70
7
MLST allele number
Sequence t ype ST Clonal
complex CC ST Single locus variant slv
Double locus variant CC MLST
eBURST software
6. SaPI scanning
83 1 83 Sato’o
SaPI scanning 64 SaPI
Long and accurate LA -PCR
4. genomic DNA
TaKaRa LA Taq Hot Start Version
21
LA-PCR Sato’o 64
1 LA-PCR
LA-PCR 50 L
10 LA PCR Buffer II Mg2 +plus 5 L
dNTP Mixture 2.5 mM 8 L
Forward primer 20 M 0.5 L
Reverse primer 20 M 0.5 L
LA Taq Hot Start Version 5 units/μL 0.5 L
Template genomic DNA < 1 g 1 L
Nuclease-free H2O Thermo Fisher Scientific 34.5 L
LA-PCR
96 1
98 10
68 20
68 10
10 kb MonoFas DNA Purification Kit
5.
LA-PCR HindIII
Restrict ion fragment length
polymorphism RFLP
37 1 LA-PCR
33
22
20 L
10 M Buffer 2 L
LA-PCR < 1 g 3 L
HindIII 5 units/μL 1 L
Nuclease-free H2O Thermo Fisher Scientific 14 L
1/10 10 Loading Buffer
DNA 0.7%
SeaKem GTG Agarose gels Lonza Rockland ME USA
0.5 μg/mL EtBr DNA
AE-6933FXES ATTO RFLP
National Center for Biotechnology Information
NCBI http:/ /www.ncbi.nlm.nih.gov/ SaPI
HindIII in si l ico Genetyx software
program, ver. 8 GENETYX Corp.
RFLP SaPI
SaPI
7.
5. MLST
83
61
23
Kimura 2 36
1,000
MEGA5 software 70
8. Sandwich ELISA SEA
CC81 sea S. aureus 10 Sandwich
ELISA SEA Omoe
52
MSEY 2
5 mL 1% w/v Yeast Extract Becton Dickinson
BHI broth 1 37 seed culture
seed culture 0.6 mL 60 mL 1% w/v Yeast
Extract BHI broth 37 48
10,000 rpm 20 0.22 μm
MILLEX GP Merck Mill ipore Darmstadt Germany
20
S. aureus
10,000 rpm 20
A
ImmunoPure Normal Rabbit Serum Thermo Fisher
Scientific 4
5 Can Get Signal Immunoreaction Enhancer Solut ion 1
10 Can Get
24
Signal Immunoreact ion Enhancer Solution 1 10
100,000
100 ng/mL 0.1% w/v Bovine
serum albumin BSA SEA Can Get
Signal Immunoreaction Enhancer Solution 1
Nunc Thermo Fisher Scientific Capture
Rabbit IgG Isotype Control, Purified (UNLB) IgG
100 μL/well 4 Capture
IgG Capture
SEA 1
μg/mL Capture 200
μL/well 1 Phosphate buffered saline 1 PBS
3 300 μL/well Start ingBlock (PBS) Blocking
Buffer Thermo Fisher Scientific 3
StartingBlock (PBS) Blocking Buffer 200 μL/well
1 PBS 3
SEA 100 μL/well 37 1
Atlantis
Biochrom Cambridge UK Tween 20
0.05% w/v 1 PBS 1 PBST
8 Detection 100 μL/well
37 1 Detection
Horseradish peroxidase HRP SEA
Can Get Signal Immunoreaction Enhancer Solution 2
25
20,000 Detection
1 PBST 8
SuperSignal ELISA FemtoMaxmum Sensit ivity Substrate Thermo
Fisher Scientific 100 μL/well SpectraMax Paradigm
Molecular Devices Sunnyvale CA USA
A Rabbit IgG Isotype Control, Purified
(UNLB) IgG
1%
w/v yeast extract BHI broth 100 μL/well
1 PBS
1 PBS 1 L
NaCl 8 g
KCl 0.2 g
Na2HPO4 12H2O 2.9 g
KH2PO4 0.2 g
pH7.4 Mill iQ 1 L
1. MLST
MLST S. aureus 83
17 ST ST1 ST5 ST6 ST8 ST12 ST20 ST30 ST45
26
ST59 ST81 ST96 ST121 ST398 ST508 ST2272 ST45slv
ST81slv ST eBURST
CC 8 CC 4
CC81 ST1 ST81 ST81slv S. aureus
CC81 25.3%
CC8 ST8 CC6 ST6 CC5 ST5 CC508 ST45
ST508 ST45slv CC20 ST20 CC30 ST30 CC59
ST59 S. aureus 8.4% 8
CC ST ST12 ST96 ST121 ST398 ST2272
2. Clonal complex
8 CC S. aureus 5
2 VII CoaT VII
S. aureus 38.5% 2
CC81 CoaT VII
7 CC CC
CC8-CoaT III CC6-CoaT IV CC5-CoaT II CC508-CoaT VII
CC20-CoaT VIII CC30-CoaT IV CC59-CoaT VII
3. SE/SEls Clonal
complex
27
sea CC508 CC20 6 CC
S. aureus CC sea
CC81 76% CC8 100%
CC6 100% sea 5 CC
SE/SEls
SE/SEls
CC
CC
3 CC8 SE
sea CC8
SE sed selj ser
CC8 SaPI SE
seb sec sel sek seq the highly variable
genomic region νSaβ SE seg sei sem
sen seo 7
CC S. aureus 3
4. SaPI Clonal
complex
S. aureus 6 SaPI
49 10 kb
4 5
10 kb SaPI Genomic
28
island GI
CC8 CC5 6 CC S. aureus
CC GI
CC81 9 10 kb
RFLP CC6 9 CC508
9 CC20 9 CC30 8 44
CC59 9 19 GI
RFLP 6 Common insertion RFLP
SaPI 51 64
CC59 19 RFLP SaPI3
CC30 8 44 RFLP
SaPI4 SaPI2 CC81
CC6 9 RFLP MW2 9
GI MW2-like RFLP 64 4
6 Common insertion
CC S. aureus CC RFLP
GI CC81 19%
18 SaPIishikawa11 24% CC81 19
SaPIhhm2 14% 44 SaPIno10
CC20 50% 44 SaPIhirosaki4 CC508
29% 18 SaPI68111
RFLP SaPIs
RFLP 4 6 Frequent
insertion
MLST S.
29
aureus CC
7A S. aureus SaPI
CC
SaPI
CC81 MW2-like RFLP SaPIishikawa11 SaPIhhm2
SaPIno10 3 SaPI
CC CC81
SaPI2 SaPI3 SaPI4 SaPI68111
SaPIhirosaki4 CC
7B 7C
5. CC81 sea SEA
CC81 sea S. aureus
SEA SEA
2 μg/mL SEA 10 7
6 μg/mL SEA 8
SEs S. aureus
17 68 69 79 81
S. aureus
30
4
MLST
S. aureus 8 CC 4
S. aureus MLST
17
79 81 CC CC81 CC8
CC6
S. aureus
CC
S. aureus CC97 CC705
27 CC508 CC15 CC188
CC S. aureus
65 CC81 CC8 CC6 CC
S. aureus
CoaT VII
S. aureus 2
68 S. aureus
CC CoaT
27 CC
CoaT 2 2
S. aureus
31
SE/SEls CC
SE/SEls SE/SEls
CC
CC
3
SE/SEls
SEs S. aureus
MLST S. aureus
CC 7A
SaPI CC5 CC8 6
CC
SaPI CC
CC
SaPIishikawa11 SaPIhhm2 SaPIno10 CC81
CC 7B
CC5 CC8
SaPI GI CC
sed selj ser S. aureus
3 sed selj ser pIB485-like plasmid
pUO-Sa-SED1 pUO-Sa-SED2
32
2 CC5 CC8 SE/SEls
6 CC
DNA DNA
DNA
Waldron Lindsay
78 S. aureus
McCarthy 47 S. aureus
SEA
2 65 68 81 S. aureus
CC CC81 CC8 CC6
sea 5 sea
2 67
genotype
33
2 12 67 sea
CC81
φSa3MW2 14 65
SEA 6 μg/mL 65
sea CC81 6
μg/mL SEA 10 7 70%
8
sea CC81 CC8 CC6
MLST SE/SEls
CC5 CC508 CC20 SEs
CC enterotoxin gene cluster egc
seg sei sem sen seo
5 egc SE
48 56
egc
52 67
SEs
pH
67 egc
egc egc
egc
CC
34
S. aureus
MLST 8 CC
CC SE/SEls SE/SEls
S. aureus
S. aureus
S. aureus
35
1.
5'→
3'
Prim
er se
t‡
Mul
tiple
x PC
R
sea
SEA
3 C
CTT
TGG
AA
AC
GG
TTA
AA
AC
G
set1
SEA
4 TC
TGA
AC
CTT
CC
CAT
CA
AA
AA
C
set1
se
b SE
B5
AA
GG
AC
AC
TAA
GTT
AG
GG
AA
TTA
TGA
se
t1
SEB
6 C
ATC
ATG
TCA
TAC
CA
AA
AG
CTA
TTC
T se
t1
se
c SE
C3
CTC
AA
GA
AC
TAG
AC
ATA
AA
AG
CTA
GG
se
t1
SEC
4 TC
AA
AAT
CG
GAT
TAA
CAT
TATC
C
set1
se
d SE
D3
CTA
GTT
TGG
TAAT
ATC
TCC
TTTA
AA
CG
se
t1
SED
4 TT
AAT
GC
TATA
TCTT
ATA
GG
GTA
AA
CAT
C
set1
se
e SE
E2
TAA
CTT
AC
CG
TGG
AC
CC
TTC
se
t1
SEE3
C
AG
TAC
CTA
TAG
ATA
AA
GTT
AA
AA
CA
AG
C
set1
se
g SE
G1
AA
GTA
GA
CAT
TTTT
GG
CG
TTC
C
set2
SEG
2 A
GA
AC
CAT
CA
AA
CTC
GTA
TAG
C
set2
se
h SE
H1
GTC
TATA
TGG
AG
GTA
CA
AC
AC
T se
t2
SEH
2 G
AC
CTT
TAC
TTAT
TTC
GC
TGTC
se
t2
se
i SE
I1
GG
TGAT
ATTG
GTG
TAG
GTA
AC
se
t2
SEI2
AT
CC
ATAT
TCTT
TGC
CTT
TAC
CA
G
set2
se
lj SE
J1
ATA
GC
ATC
AG
AA
CTG
TTG
TTC
CG
se
t2
SEJ2
C
TTTC
TGA
ATTT
TAC
CA
CC
AA
AG
G
set2
se
k SE
K1
TAG
GTG
TCTC
TAAT
AAT
GC
CA
se
t3
SEK
2 TA
GAT
ATTC
GTT
AG
TAG
CTG
se
t3
se
l SE
L1
TAA
CG
GC
GAT
GTA
GG
TCC
AG
G
set4
SEL2
C
ATC
TATT
TCTT
GTG
CG
GTA
AC
se
t4
se
m
SEM
1 G
GAT
AAT
TCG
AC
AG
TAA
CA
G
set3
36
SEM
2 TC
CTG
CAT
TAA
ATC
CA
GA
AC
se
t3
se
n se
n fo
rwar
d G
CTT
ATG
AG
ATTG
TTC
TAC
ATA
GC
TGC
se
t4
sen
reve
rse
CAT
TAA
CG
CC
TATA
AC
TTTC
TCTT
CAT
C
set4
se
o SE
O1
TGTG
TAA
GA
AG
TCA
AG
TGTA
G
set3
SEO
2 TC
TTTA
GA
AAT
CG
CTG
ATG
A
set3
se
p SE
P3
TGAT
TTAT
TAG
TAG
AC
CTT
GG
se
t2
SEP4
AT
AA
CC
AA
CC
GA
ATC
AC
CA
G
set2
se
q SE
Q1
AAT
CTC
TGG
GTC
AAT
GG
TAA
GC
se
t4
SEQ
2 TT
GTA
TTC
GTT
TTG
TAG
GTA
TTTT
CG
se
t4
se
r SE
R1
GG
ATA
AA
GC
GG
TAAT
AG
CA
G
set4
SER
4 G
TATT
CC
AA
AC
AC
ATC
TAA
C
set4
se
s SE
S-ka
noS
TCG
GA
ATAT
AC
TATG
GG
GC
AA
A
set1
SES-
kano
AS
GG
TCTA
AC
TCTT
GA
ATTG
TAG
GTT
C
set1
se
t SE
T-ka
noS
GG
TTG
GTG
ATTA
TGTA
GAT
GC
TTG
se
t3
SET-
kano
AS
GTA
GG
CTT
GTC
TAA
AG
GG
CTA
TG
set3
fe
mA
fem
A1
AA
AA
AA
GC
AC
ATA
AC
AA
GC
G
set2
and
set3
fem
A2
GAT
AA
AG
AA
GA
AA
CC
AG
CA
G
set2
and
set3
fe
mB
fem
B3
CA
CAT
GG
TTA
CG
AG
CAT
CAT
se
t1 a
nd se
t4
fem
B4
TGTT
TCG
GG
TGTT
TTA
CC
TT
set1
and
set4
MLS
T* ar
cC
arcC
-Up
TTG
ATT
CA
CC
AG
CG
CG
TATT
GTC
arcC
-Dn
AG
GTA
TCTG
CTT
CA
ATC
AG
CG
ar
oE
aroE
-Up
ATC
GG
AA
ATC
CTA
TTTC
AC
ATT
C
aroE
-Dn
GG
TGTT
GTA
TTA
ATA
AC
GA
TATC
gl
pF
glpF
-Up
CTA
GG
AA
CTG
CA
ATC
TTA
ATC
C
glpF
-Dn
TGG
TAA
AA
TCG
CA
TGTC
CA
AT
TC
37
gm
k gm
k-U
p A
TCG
TTTT
ATC
GG
GA
CC
ATC
gmk-
Dn
TCA
TTA
AC
TAC
AA
CG
TAA
TCG
TA
pt
a pt
a-U
p G
TTA
AA
ATC
GTA
TTA
CC
TGA
AG
G
pta-
Dn
GA
CC
CTT
TTG
TTG
AA
AA
GC
TTA
A
tp
i tp
i-Up
TCG
TTC
ATT
CTG
AA
CG
TCG
TGA
A
tpi-D
n TT
TGC
AC
CTT
CTA
AC
AA
TTG
TAC
yq
iL
yqiL
-Up
CA
GC
ATA
CA
GG
AC
AC
CTA
TTG
GC
yqiL
-Dn
CG
TTG
AG
GA
ATC
GA
TAC
TGG
AA
C
SaPI
† scan
ning
8′
site
Sa
PI4S
AT
GG
CA
GG
TGG
AC
CA
AG
AA
GA
GG
CG
GA
CG
TCG
TCG
SaPI
4AS
CAT
TCTA
GTA
GA
CA
CC
TCTG
TCG
TTTT
AA
CAT
AA
G
9′
site
Sa
PIbo
vS
GTT
ATG
GG
AG
AC
TAC
CG
TAC
GTA
TGAT
CA
CA
CA
G
SaPI
bovA
S G
TTG
CTA
TTTA
TGG
TGG
CC
TTTC
AA
GC
AAT
ATTA
G
18
′site
Sa
PIm
w2S
C
ATC
ATG
TTG
GTT
GTT
TATC
AC
TTTT
CAT
GAT
GC
SaPI
mw
2AS
GA
AA
GA
AA
AA
GA
AA
AG
CA
AA
AA
GAT
GA
CG
AC
CA
CA
G
19
′site
Sa
PI3e
xter
nalS
G
CTG
TAG
AA
AG
TGC
GA
AA
GAT
AAT
CC
TTAT
GC
SaPI
3ext
erna
lAS
GC
GA
AAT
AG
ATTC
ATC
ATTA
AC
TTG
AG
GG
AG
44
′site
Sa
PI2S
A
AAT
TAC
CG
CTA
CA
CTA
AA
GG
AC
AA
ATTT
ATC
SaPI
2AS
GG
TATC
GTT
GAT
CC
AA
CTA
AA
GTA
AC
AC
GC
TCA
GC
49
′site
et
dS
CG
TTG
TGA
AA
GAT
AAT
GA
ATC
GG
TTTG
GG
CTG
etdA
S C
TAA
AG
TGG
CA
GTA
AC
AA
ATG
ATTT
TAG
TTTC
*MLS
TM
ulti
locu
s seq
uenc
e ty
ping
†S
aPI
Stap
hylo
cocc
us a
ureu
s pat
hoge
nici
ty is
land
‡P
rimer
set
Mul
tiple
x PC
RPr
imer
38
2.
1994
2012
S. a
ureu
sC
lona
l
com
plex
Coa
T* C
lona
l com
plex
83
%
VII
C
C† 81
CC
508
CC
59
38.5
IV
CC
6C
C30
19
.3
III
CC
8 18
.1
II
CC
5 10
.8
VII
I C
C20
4.
8
*Coa
T:
†CC
: Clo
nal c
ompl
ex
39
3.
S. a
ureu
s SE
/SEl
Stap
hylo
cocc
al
ente
roto
xin/
Stap
hylo
cocc
al e
nter
otox
in-li
ke to
xin
Clo
nal
com
plex
CC
*S.
aur
eus
C
CS.
aur
eus
SE/S
El
† SE
/SEl
*
81
seh
Tran
spos
on
sea,
seb,
sek,
seq
Prop
hage
or S
aPI
8 se
a Pr
opha
ge
sed,
selj,
ser
Plas
mid
6 se
a Pr
opha
ge
seb,
sec,
sel
SaPI
5 se
g, se
i, se
m, s
en, s
eo
νSa
geno
mic
isla
nd c
onta
inin
g
the
ente
roto
xin
gene
clu
ster
se
a, se
d, se
lj, se
r Pr
opha
ge o
r Pla
smid
508
seg,
sei,
sem
, sen
, seo
νS
a ge
nom
ic is
land
con
tain
ing
th
e en
tero
toxi
n ge
ne c
lust
er
sec,
sel,
sek,
seq
Prop
hage
or S
aPI
20
seg,
sei,
sem
, sen
, seo
νS
a ge
nom
ic is
land
con
tain
ing
th
e en
tero
toxi
n ge
ne c
lust
er
seb
SaPI
30
seg,
sei,
sen,
νS
a ge
nom
ic is
land
con
tain
ing
th
e en
tero
toxi
n ge
ne c
lust
er
sea
Prop
hage
59
seb,
sek,
seq
SaPI
se
a Pr
opha
ge
*CC
: Clo
nal c
ompl
ex
†3.
40
4.
S. a
ureu
s C
lona
l com
plex
Gen
omic
isla
nd
Clo
nal
com
plex
SaPI
8'
9'
18'
19'
44'
49'
81
- M
W2-
like
RFL
P (1
00 %
)
SaPI
ishi
kaw
a11
(seb
; 19
%)
SaPI
hhm
2 (N
o SA
G g
enes
; 24
%)
SaPI
no10
(s
eb; 1
4 %
) -
8 8'
-Unk
now
n G
I* s typ
e1
(20
%)
- 18
'-Unk
now
n G
Is ty
pe1
(7 %
) -
- -
6
8'-U
nkno
wn
GIs
type
2 (8
%)
8'
- Unk
now
n G
Is ty
pe3
(8 %
)
MW
2-lik
e R
FLP
(100
%)
18'-U
nkno
wn
GIs
type
2 (1
7 %
)
18'- U
nkno
wn
GIs
type
3 (8
%)
- -
-
5 -
- 18
'-Unk
now
n G
Is ty
pe1
(11
%)
- -
-
508
- 9'
-Unk
now
n G
Is ty
pe1
(100
%)
SaPI
6811
1 (s
ec, s
el, t
st-1‡ ; 2
9 %
)
18'- U
nkno
wn
GIs
type
4 (1
4 %
)
- -
-
41
20
- 9'
-Unk
now
n G
Is ty
pe2
(100
%)
-
- Sa
PIhi
rosa
ki4
(seb
; 50
%)
-
30
SaPI
4 (N
o SA
G† g
enes
; 100
%)
- -
- Sa
PI2
(tst-1
; 100
%)
-
59
- 9'
- Unk
now
n G
Is ty
pe3
(100
%)
-
SaPI
3 (seb
, sek
, seq
; 100
%)
- -
GI
GI
SAG
CC
S. a
ureu
s
GI
%C
CS.
aur
eus
GI
*GI
Gen
omic
isla
nd
†SA
GSu
pera
ntig
en
‡tst
-1to
xic
shoc
k sy
ndro
me
toxi
n-1
gene
4. S. aureus Clonal complex
“Others” ST12 ST96 ST121 ST398 ST2272
42
5. S. aureus SE/SEl
Clonal complex
43
6. S. aureus SaPI
Genomic island Restriction fragment length polymorphism RFLP
HindIII Common insertion CC
S. aureus GI RFLP Frequent insertion
CC S. aureus GI RFLP
GI RFLP
”Unknown GIs type” GI RFLP
M: DNA
44
6.
45
6.
46
7. S. aureus Genomic island
A MLST S. aureus ST
”81slv” ”45slv” ST81 ST45 Single locus variant
CC
47
7.
B C GI GI
Box GI SaPI Box GI
CC GI ”B” ”A”
Clade1 ”C” ”A” Clade2
48
Genomic islands 8’ 9’ 18’ 19’ 44’ 49’
8′-Unknown GIs type1 8′
-insertion
9′
-insertion
SaPIishikawa11 18′-Unknown GIs type1
18′
-insertion
8′-Unknown GIs type2 8′-Unknown GIs type3
9′-Unknown GIs type2 9′-Unknown GIs type4
18′-Unknown GIs type2 18′-Unknown GIs type3 18′-Unknown GIs type5
19′
-insertion
SaPIhhm2 19′-Unknown GIs type1
44′
-insertion
SaPIno10 SaPIhirosaki4
MW2-like RFLP
7.
49
Genomic islands 8’ 9’ 18’ 19’ 44’ 49’
SaPI4 8′
-insertion
9′
-insertion
18′-Unknown GIs type4
9′-Unknown GIs type1 9′-Unknown GIs type3 9′-Unknown GIs type5
SaPI68111 18′
-insertion
18′-Unknown GIs type2
SaPI3 19′
-insertion
SaPI2 44′
-insertion
8. CC81 sea S. aureus Staphylococcal enterotoxin A
3 3
50
51
SaPI
SEs S. aureus
44 51 64
S. aureus
42 44
49
S. aureus
SaPI SEs 44 51
23 SE/SEls seb , sec , sel , sek seq
5 SEs SaPI
SEs SaPI SEs 51
64 67
S. aureus SaPI SEs
5 SaPI SaPI3 SaPIishikawa11 SaPIno10
SaPI68111 SaPIhirosaki4 S. aureus
4 SaPI SEs
S. aureus SaPI
S. aureus SEs
52
SaPI
SaPI
S. aureus SEs
SaPI SaPI S. aureus
SaPI
SaPI
1.
S. aureus 5 S. aureus
Tokyo11212 Tokyo12381 Tokyo12413 Tokyo12571 Tokyo12709
Tokyo12954
SEs
sea seb 2 Tokyo11212 Tokyo12413
sea sec sel 1 Tokyo12381 sec seg sei sel
sem sen seo 2 Tokyo12571 Tokyo12709
seb seh 1 Tokyo12954 Tokyo12709
sec sel SaPI SaPI68111 Tokyo12954
seb SaPI SaPIno10
S. aureus
1.
53
2.
Escherichia col i E. coli 5 K12
DH5α Promega Madison WI USA B
BL21 DE3 Merck Darmstadt Germany
E. coli Luria-Bertani LB
Sigma-Aldrich LB Sigma-Aldrich
Sigma-Aldrich 50 μg/mL
3.
6
7 pKOB
pKOC pKOL
seb sec sel
pGEM-3Zf(+) vector Promega
pGTR212 pGTR413 pGTR381/571 pGTO212 pGTO413
pGTO381/571 pGTN413 7
PCR pGEM-T easy vector Promega
pER212 pER413 pER381/571 pET-15b
vector Merck pGTR212 pGTR413 pGTR381/571
Nde I BamHI
DNA
54
4. Genomic DNA
S. aureus 4 Tokyo11212 Tokyo12381 Tokyo12413
Tokyo12571 Genomic DNA
4.
5. SaPI scanning
S. aureus 4 Tokyo11212 Tokyo12381 Tokyo12413
Tokyo12571 SaPI scanning 6.
6. Southern blott ing LA-PCR SEs
5. LA-PCR
SaPI SEs Southern
blott ing
A. pKOB pKOC pKOL
pKOB pKOC pKOL
E. coli DH5 80
10
55
15 L 100 ng 20
50 μg/mL LB 50 L 2%
5-Bromo-4-chloro-3-indolyi β -D-galactopyranoside
50 L 100 mM Isopropyl -β-D-thiogalactopyranosid IPTG
42 42
90 37
SOC Promega 150 L 37 60
37
LB
37
50 μg/mL 5 mL LB
37 16
QIAprep Spin Miniprep Kit QIAGEN
3,000 rpm 5
250 L Buffer
P1 1.5 mL 250 L
Buffer P2 6 350 L
Buffer N3 13,000 rpm
10 QIAprep spin column
13,000 rpm spin column 1 750
L Buffer PE spin column 13,000 rpm 1
spin column 13,000 rpm 1
Buffer PE QIAprep spin column
56
1.5 mL 50 L Buffer EB 1
13,000 rpm 1
DNA Nanodrop 2000c Thermo fisher
scientific A260/A280 1.8
20
5. SE
7
M13F M13R pKOB
pKOC pKOL
B. seb sec sel
pKOB pKOC pKOL 2
37 1
seb sec sel
EcoRI BamHI Sal I
seb sec 20 L
10 K Buffer 2 L
pKOB pKOC < 1 g 3 L
BamHI 15 units/μL 1 L
EcoRI 15 units/μL 1 L
Nuclease-free H2O Thermo Fisher scientific 13 L
57
sel 20 L
10 T Buffer 3 L
pKOL < 1 g 3 L
BamHI 15 units/μL 1 L
Sal I 15 units/μL 1 L
Nuclease-free H2O Thermo Fisher scientific 12 L
1/10 10 Loading Buffer
0.7% SeaKem GTG Agarose gels Lonza EtBr 0.5
μg/mL DNA
AE-6933FXES ATTO 700-800 bp
MonoFas DNA purification Kit GL
seb sec sel
Buffer A 70
10 MonoFas I spin column
column 9,000 rpm 1
500 L Buffer B 9,000 rpm 1
column 9,000 rpm 1
Buffer B MonoFas I spin
column 1.5 mL 20 L Nuclease-free H2O Thermo
Fisher scientific 1 9,000 rpm 1
seb sec sel
C. seb sec sel
58
seb sec sel ECL Direct Nucleic Acid
Labelling and Detect ion System GE Healthcare Amersham Place Litt le
Chalfont Buckinghamshire HP7 9NA UK
Horseradish peroxidase HRP
100 ng
seb sec sel 10 μL Water
95 10 5
10 μL DNA labell ing reagent Glutaraldehyde solution
37 10 HRP
D. Southern blott ing
5. 250-500 ng LA-PCR
100 units HindIII New England Biolabs Ipswich
MA USA 0.7% SeaKem GTG Agarose gels Lonza
18V 15 EtBr 0.5
μg/mL DNA
AE-6933FXES ATTO LA-PCR
300 mL 0.35 N HCl 10
DNA 300 mL
denaturalization buffer 25 2 DNA
300 mL neutralization buffer 15
2
59
Hybond-N+ GE
Healthcare DNA
UV CL-1000
DNA 120 mj DNA
10 SSC
ECL Direct Nucleic Acid
Labelling and Detect ion System GE Healthcare
DNA
21 mL hybridization buffer
MULTI-SHAKER OVEN HB+S
42 61 rpm 3
900 μL hybridization buffer
HRP seb sec sel
42
61 rpm
300 mL primary wash buffer 20
2 300 mL secondary wash
buffer 5 2 8
mL ECL Detection reagent 1 8 mL ECL Detection reagent 2
1 ATTO CoolSaver
AE-6955 ATTO seb
sec sel 0.35 N HCl
denaturalization buffer neutralization buffer 10 SSC hybridization
60
buffer primary wash buffer secondary wash buffer 5 M NaCl 1
M Tris-HCl pH7.5
0.35 N HCl 1 L
1 N HCl 350 mL
Mill iQ 650 mL
denaturalization buffer 300 mL
5 M NaCl 90 mL
6 g
Mill iQ 210 mL
neutralization buffer 1 L
5 M NaCl 300 mL
Tris-HCl pH 7.5 500 mL
Mill iQ 200 mL
10 SSC 1 L
Mill iQ 500 mL
20 SSC Thermo fisher scientific 500 mL
hybridization buffer 200 mL
Gold hybridization buffer ECL Direct Nucleic Acid
Labelling and Detect ion System
200 mL
Blocking agent ECL Direct Nucleic Acid Labelling
61
and Detection System 10 g
NaCl 3.5 g
primary wash buffer 1 L
Mill iQ
360 g
10% SDS 40 mL
20 SSC Thermo Fisher Scientific 15 mL
Mill iQ 1 L
secondary wash buffer 1 L
Mill iQ 900 mL
20 SSC Thermo Fisher Scientific 100 mL
5 M NaCl 1 L
NaCl 292.2 g
Mill iQ
Mill iQ 1 L
1 M Tris-HCl pH 7.5 500 mL
60.55 g
6 N HCl 67 mL
Mill iQ
pH7.5 Mill iQ 500 mL
62
7. SaPI
DNA
SaPI
5. LA-PCR
5.
MEGA5 software LA-PCR
SaPI Genetyx
software program, ver. 8 SaPI att
SaPI
GeneMark http:/ /exon.gatech.edu/ ORF Finder
http:/ /www.ncbi.nlm.nih.gov/projects/gorf/
SaPI Open reading frame ORF
Basic local al ignment search t ool BLAST
http:/ /blast .ncbi.nlm.nih.gov/Blast .cgi
ORF
SaPI DDBJ/EMBL/GenBank
AB860415 SaPITokyo12413 AB860416 SaPITokyo11212
AB860417 SaPITokyo12571 AB860418 SaPITokyo12381
8. Sandwich ELISA SE
63
SaPI S. aureus 4 SaPI 2
8. Sandwich
ELISA SEB SEC SEL
SEB SEC SEL Capture Detection
Capture
2 μg/mL Anti-SEB 1 μg/mL Anti-SEC 5 μg/mL
Anti-SEL Detection
4,800 Anti-SEB 3,200 Anti-SEC 200 Anti-SEL
100 ng/mL 0.1% w/v BSA SEB SEC
SEL Can Get Signal Immunoreaction Enhancer Solution
1
9. Rep
7. ORF
rep SaPITokyo11212-ORF9
SaPITokyo12413-ORF9 SaPITokyo12381-ORF8 6
A. Rep
5. LA-PCR
SaPITokyo11212-ORF9 SaPITokyo12413-ORF9
64
SaPITokyo12381-ORF8
PCR 7 PCR
TaKaRa LA Taq Hot Start Version
PCR 50 L
10 LA PCR Buffer II Mg2 +plus 5 L
dNTP Mixture 2.5 mM 8 L
Forward primer 20 M 0.5 L
Reverse primer 20 M 0.5 L
LA Taq Hot Start Version 5 units/μL 0.5 L
Template DNA < 1 g 1 L
Nuclease-free H2O Thermo Fisher scientific 34.5 L
95 10
95 30
55 30
72 2 30
72 10
PCR pGEM-T Easy Vector Promega
4 16
18
65
10 L
2 Rapid Ligation Buffer 5 L
PCR 3.5 L
pGEM-T Easy 50 ng/ L 0.5 L
T4 DNA Ligase 3 Unit/ L 1 L
6.
rep
pGTR212 pGTR413 pGTR381/571
7 M13F M13R
rep pET-15b vector
37 1
2
NdeI BamHI
20 L
10 K Buffer 2 L
pGTR212 pGTR413 pGTR381/571 pET-15b
(< 1 g)
3 L
NdeI 10 units/μL 1 L
BamHI 15 units/μL 1 L
66
Nuclease-free H2O Thermo Fisher scientific 13 L
1/10 10 Loading Buffer
6.
DNA Ligation high
rep pET-15b
16 16
8 L
rep 3.5 L
pET-15b 0.5 L
Ligation high 4 L
6.
Rep pER212
pER413 pER381/571
E. coli BL21 DE3 7
T7UP T7TER
B. Rep
Rep rRep QIAGEN The
67
QIAexpressionist
http:/ /kirschner.med.harvard.edu/files/protocols/QIAGEN_QIAexpres
sionist_EN.pdf pER212
pER413 pER381/571 E. coli BL21 DE3
50 μg/mL 100 mL
LB 37 O.D.6 0 0 0.5
0.8 100 mM IPTG 1 mL
20 15 3,000 rpm 20
80
10 mL Lysis buffer
Ultrasonic Homogenizer SMT
14,500 rpm 30
1.5 mL Ni-NTA Agarose QIAGEN
10 mL Lysis buffer 1,000 rpm 10
3 Ni-NTA Agarose
Ni-NTA Agarose 4 90
1,000 rpm 10 Ni-NTA
Agarose 10 mL Wash buffer
1,000 rpm 10 8
Ni-NTA Agarose Ni-NTA Agarose
Wash buffer 2 mL Poly-Prep Chromatography Columns
Bio-rad Hercules CA USA
Elution buffer 1 mL
3 rRep
68
Nanodrop 2000c Thermo Fisher Scient ific 280 nm
rRep rRep
Slide-A-Lyzer G2, NMCO
10K Thermo Fisher Scientific 4
1 L 1 PBS 5
1 PBS Lysis buffer Wash buffer
Elution buffer
Lysis buffer 100 mL
Mill iQ
NaH2PO4 H2O 0.69 g
NaCl 1.754 g
Imidazole 0.068 g
pH8.0 Mill iQ 100 mL
0.5 M Sigma-Aldrich
200 L 100 mg/mL 100 L 2
mg/mL / 1 mL 2-
40 L
Wash buffer 100 mL
Mill iQ
NaH2PO4 H2O 0.69 g
NaCl 1.754 g
Imidazole 0.136 g
pH8.0 Mill iQ 100 mL
69
Elution buffer 100 mL
Mill iQ
NaH2PO4 H2O 0.69 g
NaCl 1.754 g
Imidazole 1.7 g
pH8.0 Mill iQ 100 mL
rRep Quick Start Bradford Protein assay kit Bio-rad
Quick Start Bovine Serum Albumin (BSA) Standard Set Bio-rad
0 125 250 500 750 1000 1500 μg/mL BSA
rRep
10
Nanodrop 2000c Thermo Fisher Scientific
595 nm rRep
C. rRep
rRep SDS-PAGE western blot t ing
SDS-PAGE 500 ng/10 μL rRep
10 μL rRep 10 μL 2 Laemmli sample buffer
70
Bio-rad 100 10 4–20% precast
polyacrylamide gel Bio-rad 40 mA 40
Quick-CBB
100 mL 10
60 mL 30 100 mL
15
Precision Plus Protein Dual Color Standards Bio-rad
3 Bio-rad
SDS-PAGE
10 SDS-PAGE 1 L
Mill iQ
30.3 g
144 g
SDS 10 g
Mill iQ 1 L MilliQ
10
100 mL
50 mL
10 mL
Mill iQ 40 mL
71
60 mL
Quick-CBB A 30 mL
Quick-CBB B 30 mL
100 mL
25 mL
7.5 mL
Mill iQ 67.5 mL
Western blott ing 100 ng/10 μL rRep
SDS-PAGE iBlot Gel Transfer Device Thermo Fisher
Scientific iBlot Transfer Stack, PVDF Mini Thermo Fisher
Scientific
30
10 mL
4
6 His epitope tag antibody Rockland Immunochemicals Inc.
Gilbertsvil le PA USA 2,000
1 Tris Buffered Saline with Tween 20
1 TBST 10 3
10 mL
1 Goat
anti-Rabbit IgG-Horseradish Peroxidase Conjugated Thermo Fisher
Scientific 2,000
72
1 TBST 10 3
ECL Plus Western Blotting Detection
System GE Healthcare 8 mL Solution A
200 μL Solution B 5
Typhoon Trio variable image analyzer GE Healthcare
1 TBST
10 TBS 1 L
Mill iQ
60.6 g
NaCl 80.6 g
KCl 2 g
pH7.4 Mill iQ 1 L
MilliQ 10 0.5
mL Tween 20 1 TBST
100 mL
1 TBST 100 mL
5 g
10.
9. rRep ori s i te
73
Masuda 46
A. ori s i te
5. LA-PCR
SaPITokyo11212 7,765 bp 8,093 bp
SaPITokyo12413 6,016 bp 6,185 bp 7,769 bp 8,340
bp SaPITokyo12381/SaPITokyo12571 5,708 bp 6,007 bp
PCR
7 PCR
9. PCR TA
TA
9. PCR pGEM-T Easy Vector
Promega pGTO212 pGTO413 pGTN413
pGTO381/571
PCR
Forward primer T7UP-Cy5 T7UP Reverse primer
pGTO212 ST212ORI329R pGTO413
ST413ORI171R pGTN413 ST413-8340R pGTO381/571
ST381ORI300R PCR
5. 5’ Cy5
T7UP-Cy5
Fasmac
1/6 6 Loading Buffer
74
6. DNA DNA 5’
Cy5 ori site 5’ Cy5 ori
site
B.
5’ Cy5 ori site 20 ng/μL
1 μL 2 μL Gel Shift Binding 5 Buffer
Promega 15 0 1000 ng
rRep Nuclease-free H2O Thermo Fisher scientific
10 μL 15
4% 120 V 45
1 TBE buffer Thermo
Fisher scientific
Typhoon Trio variable image analyzer GE Healthcare
Cy5 rRep ori site
Cy5 ori site
Gel Shift Binding 5 Buffer Cy5 ori site
10 Cy5 ori site
4%
4% 10 mL
30% Acrylamide/Bis Solution Bio-rad 1.33 mL
75
10 TBE Thermo Fisher scientific 1 mL
10% 0.1 mL
N,N,N',N'- 20 μL
Nuclease-free H2O Thermo Fisher scientific 7.55 mL
11.
9. rRep DNA
7
5’ Cy3 Fluorescent
strand 3’ BHQ2
Quencher strand Fasmac
Boguszewska-Chachulska 9
Tani 71 20 mM
Tris-HCl pH 7.5 Fluorescent st rand Quencher strand 1:2
95 5 2 3
double-stranded DNA dsDNA
14.5 μL 0
75 150 300 500 750 1000 1500 nM rRep
5.5 μL Cy3
ABI 7500 Real Time PCR System Thermo Fisher
Scientific 37 99 1
t F( t)
a
76
C A ko b s C
A ko b s F’(0 )
F( t) = C + A[1-exp(-ko b s t)] , (a)
20 mM Tris-HCl pH 7.5 300 mM Tris-HCl pH
7.5 1.5% Triton X-100
14.5 μL
300 mM Tris-HCl pH 7.5 2 μL
50 mM MgCl2 2 μL
1.5% Triton X-100 1 μL
100 mM ATP 0.2 μL
1 μM dsDNA 0.4 μL
12.5 μM capture strand Fasmac 0.2 μL
Nuclease-free H2O Thermo Fisher Scientific 8.7 μL
20 mM Tris-HCl pH 7.5 100 mL
1 M Tris-HCl pH 7.5 6. 2 mL
Mill iQ
pH7.5 Mill iQ 100 mL
300 mM Tris-HCl pH 7.5 100 mL
1 M Tris-HCl pH 7.5 6. 30 mL
Mill iQ
77
pH7.5 Mill iQ 100 mL
1.5% Triton X-100 100 mL
Triton X-100 1.5 mL
Mill iQ 98.5 mL
12.
S. aureus SEs one way
ANOVA Tukey-Kramer HSD
F
t
p 0.01
JMP software SAS Insti tute Inc. Cary NC USA
1. seb sec sel GI
seb sec sel S. aureus 4 Tokyo12413
Tokyo11212 Tokyo12571 Tokyo12381 SaPI scanning
SaPI SaPI 10 kb
78
LA-PCR Tokyo12413 8 9
Tokyo11212 9 19 Tokyo12571 9 18
Tokyo12381 9 18
9A LA-PCR HindIII
RFLP Tokyo12413 Tokyo11212 Tokyo12381
9 GIs RFLP 6
”MW2-like RFLP” Tokyo12571 9 GI
RFLP 9 -Unknown GIs type1
”MW2-like RFLP” CC81 CC6
GI 9 -Unknown GIs type1 CC508
GI 4 seb sec
sel ”MW2-like RFLP” 9 -Unknown GIs type1
CC81 CC6 CC508
seb sec sel 100%
CC
100% 5 ”MW2-like RFLP” 9
-Unknown GIs type1 seb sec sel
9B 4 LA-PCR
RFLP Tokyo12413 8 Tokyo11212 19
Tokyo12571 18 Tokyo12381 18
SaPI RFLP
Southern blott ing 4 LA-PCR seb
sec sel seb
Tokyo12413 8 Tokyo11212 19
9C sec sel
79
Tokyo12571 Tokyo12381 18
9D 9E
2. seb sec sel SaPI
seb sec sel LA-PCR
14 kb
8 11 10 4
LA-PCR SaPI
att
8 AAAGAAGAACAATAATAT 18
TCCCGCCGTCTCCAT 19 TTATTCCTGCTAAATAA 51
SaPI int pri ter cp
ear
8 11 10 4 14 kb 17 kb
SaPI 10 SaPI
NCBI
SaPI SaPI 50
SaPITokyo12413 Tokyo12413 8 SaPITokyo11212
Tokyo11212 19 SaPITokyo12571 Tokyo12571
18 SaPITokyo12381 Tokyo12381 18
SaPI SaPITokyo12413 SaPITokyo11212
seb SaPITokyo12571 SaPITokyo12381 sec
80
sel 99% 8 11
10 Southern blott ing
SaPI 14,157 bp
SaPITokyo12413 AB860415 ORF 20
ORF SaPI3 SaPI4 SaPIivm10 SaPIivm60
SaPIj50 SaPI ORF
8 10 16,725 bp SaPITokyo11212
AB860416 20 ORF 9 10
SaPITokyo11212 SaPIivm60 92%
SaPITokyo11212 SaPIivm60 SaPITokyo11212
seb 1,342 bp Insertion sequence IS256
9 16,305 bp SaPITokyo12571
AB860417 15,419 bp SaPITokyo12381
AB860418 22 20 ORF
2 SaPI 16 ORF ear
99% SaPI ORF
SaPI3 SaPIno10 SaPImw2 SaPIj11 SaPIj50
ORF 10 11 10
3. SaPI SaPI sel
SaPImw2 SaPITokyo12381 SaPITokyo12571 sel
SaPITokyo12381 sel SaPI
99% A 249
A
81
TAA
11
4. SaPI rep-ori site
SaPI rep ori site
Ubeda 74 rep 3’
ORF ori s i te SaPITokyo11212 rep
ori site SaPIivm60 SaPIbov1
97% 92% 9
SaPITokyo11212 ori site GTACCC 6
GGGTAC 4
SaPIbov1 12A GC
AT-rich
13 SaPITokyo12571 rep ori site
SaPITokyo12381 2 SaPI
rep ori site SaPI3 98%
97% 10 11 SaPITokyo12571
SaPITokyo12381 ori site A/AGACA
6 TGTCTT 3
SaPI3 12B AT-rich
13 SaPITokyo12413-ORF9
SaPIj50 rep 98% 8
SaPITokyo12413-ORF9 3’ ori s i te
90% SaPI
82
bacteriophage PT1028
10 ori s i te 300 bp
170 bp 170 bp
74 5
GGTTA 3
TAACC 2 14
5. SaPI SEs
S. aureus SaPI SEs Sandwich
ELISA seb
SaPI S. aureus 80 μg/mL
SEB Tokyo11212 SEB
12
sec sel SaPI 20 μg/mL
SEC SEL 1 μg/mL
Tokyo12571 SEC SaPI
SaPI68111 Tokyo12709
SEL Tokyo12709
Tokyo12381 SEC
SEL
20 ng/mL 12
6. rRep ori site
83
SaPI Rep
SaPI Rep rRep
SDS-PAGE western blott ing
SaPITokyo12413-rRep 62.7 kDa SaPITokyo11212-rRep 64.8 kDa
SaPITokyo12381-rRep 55.0 kDa 6 rRep
15 rRep
SaPI Rep ori site
SaPITokyo11212-rRep Cy5 SaPITokyo11212-ori
site
5 DNA-
16A DNA- rRep
SaPITokyo12381-rRep Cy5
SaPITokyo12381-ori site
16A SaPI rRep ori s i te
SaPITokyo11212-rRep Cy5
SaPITokyo12381-ori site
SaPITokyo12381-rRep Cy5 SaPITokyo11212-ori site
16B SaPITokyo11212-rRep SaPITokyo12381-rRep
Cy5 ori site DNA
10 Cy5 ori si te
16C SaPITokyo11212-rRep
SaPITokyo12381-rRep 500 ng
SaPITokyo12413-rRep Cy5 ori s i te
170 bp
84
16A SaPITokyo12413-rRep rep
7,769 bp 8,340 bp
16A
7. rRep
rRep DNA
SaPITokyo11212-rRep
SaPITokyo11212-rRep
17A SaPITokyo12381-rRep
17B SaPITokyo12413-rRep 1 μM
17C SaPITokyo11212-rRep
SaPITokyo12413-rRep
SaPITokyo11212-rRep 300 nM
SaPITokyo12413-rRep
17D
S. aureus SaPI
S. aureus
S. aureus SE 4 SaPI
85
SaPI SaPI
10 SaPI Yarwood
82 SaPI DNA
SaPI
SaPITokyo12571 SaPIj50
SaPI3 SaPIj11 SaPImw2 10
SaPITokyo12571 sec sel
SaPI SaPImw2
10 Yarwood
SaPITokyo12571
SaPI SaPITokyo12571
SaPITokyo12571
SaPImw2 SaPI SaPI
SaPITokyo12571
SaPImw2 SaPI
SEs SEA SEB SEC SED SEE
ng ng
10 11 22 35 52 59
SaPI S. aureus SE
SaPI S. aureus
1 mL SEB SEC
SaPI
SE 12 pH
86
S. aureus SE SE
SE
52 67 SaPI S. aureus
SaPI S. aureus
Tokyo11212 SEB
2.5 12 seb
SaPITokyo11212 IS256 seb 172 bp
IS256
IS256
30
45 Tokyo11212 IS256 SEB
SaPITokyo12381 sel
SaPI sel 99% 11 Tokyo12381
SEL 12
11 SaPITokyo12381 sel
SEB SEL
SaPI S. aureus SE
SaPI SE S. aureus
SaPI
87
Rep ori s i te
SaPI Rep ori site
51 74
SaPI rep ori s i te
SaPITokyo11212 rep ori site
SaPI SaPIivm60 SaPIbov1
9 3 SaPI ori s i te
GTACCC AT-rich
12 13 SaPITokyo12381 SaPITokyo12571 SaPI3
rep ori s i te 3 SaPI
ori s i te A/ AGACA AT-rich
10 11 12 13
SaPITokyo11212 SaPIivm60 SaPIbov1 3 SaPI
SaPITokyo12381 SaPITokyo12571 SaPI3 3 SaPI SaPI
Ubeda 74
ori s i te Rep ori s i te
ori site Rep 9
RepA-RepI SaPITokyo11212
Rep RepB SaPITokyo12381
Rep RepA
SaPI Rep SaPI
rRep
SaPITokyo11212-rRep
SaPITokyo12381-rRep SaPI ori s i te
88
SaPI ori s i te 16A 16B
SaPITokyo11212-rRep SaPITokyo12381-rRep
17 SaPI
Rep SaPITokyo11212-Rep SaPITokyo12381-Rep
ori s i te
SaPITokyo12413 rep
ori s i te
SaPITokyo12413-ORF9 SaPIj50 rep 98%
8 ori site ori site 170 bp
rep Ubeda Rep
170 bp
bacteriophage PT1028 14
bacteriophage PT1028 rep ori site
170 bp ori site
170 bp
GGTTA 14
SaPITokyo12413-ORF9 rep
SaPITokyo12413 170 bp ori site
SaPITokyo12413-ORF9
rRep
SaPITokyo12413-rRep ori site 170bp
16A
SaPITokyo12413-rRep
89
17
SaPITokyo12413-Rep
SaPITokyo12413 rep SaPITokyo12413-ORF9
NCBI SaPIj50
rep
ORF9
bacteriophage PT1028 10
bacteriophage PT1028 rep
ori site bacteriophage
PT1028-ORF12
SaPITokyo12413-ORF9 2 ORF
ORF
SaPITokyo12413-ORF9
SaPITokyo12413
rep ori site
S. aureus SEs 4
SaPI
SaPI S. aureus SEs SaPI
SE
SaPI
SaPITokyo11212 SaPITokyo12571
SaPITokyo12381 SaPI
90
SaPITokyo12413
SaPI
SaPITokyo12413
SaPI
S. aureus
91
5.
SE
s
Stap
hylo
cocc
us a
ureu
s
To
kyo1
1212
se
a se
b
T
okyo
1238
1 se
a se
c se
l
To
kyo1
2413
se
a se
b
To
kyo1
2571
se
c se
g se
i sel
sem
sen
seo
T
okyo
1270
9 se
c se
g se
i sel
sem
sen
seo
SaPI
6811
1
To
kyo1
2954
se
b se
h
SaPI
no10
Esch
eric
hia
coli
D
H5α
Prom
ega
B
L21(
DE3
)
Mer
ck
SaPI
SE
92
6.
pGEM
-3Zf
(+)
Apr , c
loni
ng v
ecto
r Pr
omeg
a
pGEM
-T e
asy
Apr , T
A c
loni
ng v
ecto
r Pr
omeg
a
pET-
15b
Apr , H
istid
ine
tag
fusi
on e
xpre
ssio
n ve
ctor
M
erck
pKO
B
Apr , p
GEM
-3Zf
(+) w
ith se
b ‡
pKO
C
Apr , p
GEM
-3Zf
(+) w
ith se
c ‡
pKO
L A
pr , pG
EM-3
Zf(+
) with
sel
‡
pGTR
212
Apr , p
GEM
-T e
asy
with
clo
ned
PCR
pro
duct
con
tain
ing
SaPI
* Toky
o112
12-O
RF† 9
pGTR
413
Apr , p
GEM
-T e
asy
with
clo
ned
PCR
pro
duct
con
tain
ing
SaPI
Toky
o124
13-O
RF9
pGTR
381/
571
Apr , p
GEM
-T e
asy
with
clo
ned
PCR
pro
duct
con
tain
ing
SaPI
Toky
o123
81/S
aPIT
okyo
1257
1-O
RF8
pGTO
212
Apr , p
GEM
-T e
asy
with
clo
ned
PCR
pro
duct
con
tain
ing
SaPI
Toky
o112
12 se
quen
ce (7
765
bp -
8093
bp)
pGTO
413
Apr , p
GEM
-T e
asy
with
clo
ned
PCR
pro
duct
con
tain
ing
SaPI
Toky
o124
13 se
quen
ce (6
016
bp -
6185
bp)
pGTO
381/
571
Apr , p
GEM
-T e
asy
with
clo
ned
PCR
pro
duct
con
tain
ing
SaPI
Toky
o123
81/S
aPIT
okyo
1257
1 se
quen
ce (5
708
bp -
6007
bp)
pGTN
413
Apr , p
GEM
-T e
asy
with
clo
ned
PCR
pro
duct
con
tain
ing
SaPI
Toky
o124
13 se
quen
ce (7
769
bp -
8340
bp)
pER
212
Apr , p
ET-1
5b w
ith S
aPIT
okyo
1121
2-O
RF9
93
pER
413
Apr , p
ET-1
5b w
ith S
aPIT
okyo
1241
3-O
RF9
pER
381/
571
Apr , p
ET-1
5b w
ith S
aPIT
okyo
1238
1/Sa
PITo
kyo1
2571
-OR
F8
*SaP
ISt
aphy
loco
ccus
aur
eus p
atho
geni
city
isla
nd
†OR
FO
pen
read
ing
fram
e
‡
94
7.
5'→
3'
Clo
ning
Sa
PI* To
kyo1
1212
-OR
F† 9 ST
212R
EP1F
TA
CA
TATG
GA
AA
CA
GG
TAA
AA
GTG
ATG
TAC
N
deI
ST21
2REP
1710
R
TAG
GA
TCC
TCA
ATA
ATC
AG
AA
TTA
GG
ATT
A
Bam
HI
Sa
PITo
kyo1
2413
-OR
F9
ST41
3REP
1F
TAC
ATA
TGG
AC
GA
AG
TATC
AC
TTTA
TAA
AA
N
deI
ST41
3REP
1614
R
TAG
GA
TCC
CTA
ATC
ATT
AA
TTA
ATT
CTA
AA
Ba
mH
I
Sa
PITo
kyo1
2381
/SaP
ITok
yo12
571-
OR
F8
ST38
1REP
1F
TAC
ATA
TGA
TTG
ATA
GC
AG
AA
CC
GG
TG
Nde
I
ST38
1REP
1425
R
TAG
GA
TCC
CTA
AA
CTT
TAA
GTT
TCTG
ATA
A
Bam
HI
Sa
PITo
kyo1
1212
sequ
ence
(776
5 bp
- 80
93 b
p)
ST21
2OR
I1F
TAA
GTA
CC
CA
TCG
GTA
CC
CG
TTG
AG
ST21
2OR
I329
R
GTG
TAA
CC
TCC
AG
AA
TTA
ATT
TG
Sa
PITo
kyo1
2413
sequ
ence
(601
6 bp
- 61
85 b
p)
ST41
3OR
I1F
GG
TTA
GA
AA
TAA
CTC
TTG
CTA
A
ST41
3OR
I171
R
TGA
AC
TCC
TCC
TTTT
AA
AA
CTA
A
Sa
PITo
kyo1
2381
/SaP
ITok
yo12
571
sequ
ence
(570
8 bp
- 60
07 b
p)
ST38
1OR
I1F
AC
ATC
TTA
TTTT
TAG
GA
CA
GTA
AG
ST38
1OR
I300
R
TTTC
TTC
CTC
CTA
ATT
TTTG
ATA
GG
TG
Sa
PITo
kyo1
2413
sequ
ence
(776
9 bp
- 83
40 b
p)
ST41
3-77
69F
GTT
GA
GG
GA
TTA
AA
ATT
AG
TAA
C
ST41
3-83
40R
TTC
TAC
AC
CTC
TTTC
TTTT
AA
Inse
rt ch
eck
pUC
/M13
For
war
d se
quen
cing
prim
er b
indi
ng si
te
M13
F C
GC
CA
GG
GTT
TTC
CC
AG
TCA
CG
AC
pU
C/M
13 R
ever
se se
quen
cing
prim
er b
indi
ng si
te
M13
R TC
AC
AC
AG
GA
AA
CA
GC
TATG
AC
95
T7
pro
mot
er si
te
T7U
P TA
ATA
CG
AC
TCA
CTA
TAG
GG
T7
term
inat
or si
te
T7TE
R
GC
TAG
TTA
TTG
CTC
AG
CG
G
Mob
ility
-shi
ft A
ssay
T7
pro
mot
er si
te
T7U
P TA
ATA
CG
AC
TCA
CTA
TAG
GG
T7
pro
mot
er si
te
T7U
P-C
y5
TAA
TAC
GA
CTC
AC
TATA
GG
G
5'
Cy5
-labe
led
Hel
icas
e A
ssay
Fluo
resc
ent s
trand
TA
GTA
CC
GC
CA
CC
CTC
AG
AA
CC
TTTT
TTTT
TTTT
TT
5'
Cy3
-labe
led
Que
nche
r stra
nd
GG
TTC
TGA
GG
GTG
GC
GG
TAC
TA
3’
BH
Q2-
labe
led
Cap
ture
stra
nd
TAG
TAC
CG
CC
AC
CC
TCA
GA
AC
C
*SaP
ISt
aphy
loco
ccus
aur
eus p
atho
geni
city
isla
nd
†OR
FO
pen
read
ing
fram
e
96
8. S
aPIT
okyo
1241
3O
pen
read
ing
fram
e
O
RF
SaPI
OR
F
%
att
OR
F§
Rep
eat
1
18
18
at
tA
AA
GA
AG
AA
CA
ATA
ATAT
SaPI
* Toky
o124
13-O
RF† 1
247
1080
83
4 hy
poth
etic
al
SaPI
4 99
%pa
rtial
‡
sim
ilar t
o Pe
mK
like
pro
tein
99%
, par
tial
SaPI
Toky
o124
13-O
RF2
10
81
2295
12
15
int
SaPI
4 99
%
SaPI
Toky
o124
13-O
RF3
24
65
3079
61
5 st
l Sa
PI4
99%
SaPI
Toky
o124
13-O
RF4
31
74
3392
21
9 st
r Sa
PI4
98%
SaPI
Toky
o124
13-O
RF5
33
93
3665
27
3 xi
s Sa
PI3
86%
SaPI
Toky
o124
13-O
RF6
36
56
3823
16
8 hy
poth
etic
al
SaPI
3 91
%
sim
ilar t
o Sa
PI3O
RF1
6
SaPI
Toky
o124
13-O
RF7
38
16
4025
21
0 hy
poth
etic
al
SaPI
3 99
%
sim
ilar t
o Sa
PI3O
RF1
5
SaPI
Toky
o124
13-O
RF8
40
28
4327
30
0 hy
poth
etic
al
SaPI
ivm
10
95%
parti
al
sim
ilar t
o Sa
PIiv
m10
OR
F10
sim
ilar t
o SA
T013
1_00
383
in T
0131
94%
SaPI
Toky
o124
13-O
RF9
44
02
6015
16
14
hypo
thet
ical
Sa
PIj5
0 98
%
si
mila
r to
repl
icat
ion
initi
ator
(rep
) in
SaPI
j50¶
SaPI
Toky
o124
13-O
RF1
0 61
86
6989
80
4 pr
i
si
mila
r to
bifu
nctio
nal D
NA
prim
ase/
poly
mer
ase
N-te
rmin
al d
omai
n pr
otei
n in
bac
terio
phag
e PT
1028
96%
SaPI
Toky
o124
13-O
RF1
1 69
76
7248
27
3 hy
poth
etic
al
SaPI
j50
98%
si
mila
r to
SaPI
j50O
RF
SaPI
Toky
o124
13-O
RF1
2 72
50
7768
51
9 hy
poth
etic
al
SaPI
j50
99%
parti
al
sim
ilar t
o Sa
PIj5
0OR
F
97
SaPI
Toky
o124
13-O
RF1
3 83
41
8682
34
2 cp
1 Sa
PIiv
m10
99
%
SaPI
Toky
o124
13-O
RF1
4 86
94
9272
57
9 cp
2 Sa
PI2
99%
SaPI
Toky
o124
13-O
RF1
5 92
90
9508
21
9 cp
3 Sa
PIiv
m60
10
0%
SaPI
Toky
o124
13-O
RF1
6 95
59
1008
6 52
8 cp
4 Sa
PIiv
m10
98
%
SaPI
Toky
o124
13-O
RF1
7 10
089
1043
0 34
2 cp
5 Sa
PIj1
1 98
%
SaPI
Toky
o124
13-O
RF1
8 10
427
1099
6 57
0 te
r Sa
PIiv
m10
99
%
SaPI
Toky
o124
13-O
RF1
9 11
304
1210
4 80
1 se
b Sa
PIiv
m10
99
%
SaPI
Toky
o124
13-O
RF2
0 12
182
1273
9 55
8 ea
r Sa
PIiv
m10
94
%
iden
tical
to C
427;
bet
a-la
ctam
ase
100%
Rep
eat
14
140
1415
7 18
att
AA
AG
AA
GA
AC
AAT
AAT
AT
*SaP
ISt
aphy
loco
ccus
aur
eus p
atho
geni
city
isla
nd
†OR
FO
pen
read
ing
fram
e
‡par
tial
OR
F
§O
RF
SaPI
Toky
o124
13O
RF
OR
FSa
PITo
kyo1
2413
OR
F
%pa
rtial
¶sim
ilar t
o re
plic
atio
n in
itiat
or (r
ep) i
n Sa
PIj5
0Sa
PITo
kyo1
2413
-OR
F9Sa
PIj5
0re
p98
%
98
9. S
aPIT
okyo
1121
2O
pen
read
ing
fram
e
O
RF
SaPI
OR
F
%
att
OR
F§
Rep
eat
1
17
17
at
tTT
ATTC
CTG
CTA
AAT
AA
SaPI
* Toky
o112
12-O
RF† 1
160
1404
12
45
int
SaPI
ivm
60
99%
SaPI
Toky
o112
12-O
RF2
13
94
2782
13
89
hypo
thet
ical
Sa
PIiv
m60
10
0%
iden
tical
to S
aPIiv
m60
OR
F2
SaPI
Toky
o112
12-O
RF3
28
09
3381
57
3 st
l Sa
PIiv
m60
10
0%
SaPI
Toky
o112
12-O
RF4
35
53
3774
22
2 hy
poth
etic
al
SaPI
Toky
o112
12-O
RF5
37
75
4047
27
3 hy
poth
etic
al
SaPI
Toky
o112
12-O
RF6
44
04
4787
38
4 hy
poth
etic
al
SaPI
ivm
60
100%
id
entic
al to
SaP
Iivm
60O
RF4
SaPI
Toky
o112
12-O
RF7
47
88
5105
31
8 hy
poth
etic
al
SaPI
ivm
60
100%
id
entic
al to
SaP
Iivm
60O
RF5
SaPI
Toky
o112
12-O
RF8
51
72
6041
87
0 pr
i Sa
PIhh
m2
99%
si
mila
r to
SaPI
hhm
2OR
F6
SaPI
Toky
o112
12-O
RF9
60
55
7764
17
10
rep
SaPI
ivm
60
100%
sim
ilar t
o Sa
PIbo
v1-r
ep97
%¶
ori s
ite‡
77
65
8064
Sa
PIiv
m60
10
0%
si
mila
r to
SaPI
bov1
-ori
site
92%
**
SaPI
Toky
o112
12-O
RF1
0 80
94
8474
38
1 pp
i Sa
PIiv
m60
10
0%
SaPI
Toky
o112
12-O
RF1
1 84
71
9112
64
2 hy
poth
etic
al
SaPI
ivm
60
100%
id
entic
al to
SaP
Iivm
60O
RF9
SaPI
Toky
o112
12-O
RF1
2 96
29
9970
34
2 cp
1 Sa
PIiv
m60
10
0%
SaPI
Toky
o112
12-O
RF1
3 99
82
1056
0 57
9 cp
2 Sa
PIiv
m60
10
0%
99
SaPI
Toky
o112
12-O
RF1
4 10
578
1076
9 21
9 cp
3 Sa
PIiv
m60
10
0%
SaPI
Toky
o112
12-O
RF1
5 10
799
1137
4 57
6 cp
4 Sa
PIiv
m60
10
0%
SaPI
Toky
o112
12-O
RF1
6 11
377
1171
8 34
2 cp
5 Sa
PIiv
m60
10
0%
SaPI
Toky
o112
12-O
RF1
7 11
715
1228
4 57
0 te
r Sa
PIiv
m60
10
0%
SaPI
Toky
o112
12-O
RF1
8 12
479
1365
1 11
73
tnp
trans
posa
se fo
r ins
ertio
n se
quen
ceIS
256
SaPI
Toky
o112
12-O
RF1
9 13
924
1472
4 80
1 se
b Sa
PIiv
m60
10
0%
SaPI
Toky
o112
12-O
RF2
0 14
802
1535
9 55
8 ea
r Sa
PIiv
m60
10
0%
Rep
eat
16
709
1672
5 17
att
TTAT
TCC
TGC
TAA
ATA
A
*SaP
ISt
aphy
loco
ccus
aur
eus p
atho
geni
city
isla
nd
†OR
FO
pen
read
ing
fram
e
‡ori
site
§O
RF
SaPI
Toky
o112
12O
RF
OR
FSa
PITo
kyo1
1212
OR
F
¶sim
ilar t
o Sa
PIbo
v1-r
ep97
%Sa
PITo
kyo1
1212
-OR
F9Sa
PIbo
v1re
p97
%
** si
mila
r to
SaPI
bov1
-ori
site
92%
SaPI
Toky
o112
12or
i site
SaPI
bov1
ori s
ite92
%
100
10.
SaP
ITok
yo12
571
Ope
n re
adin
g fr
ame
O
RF
SaPI
OR
F
%
att
OR
F¶
Rep
eat
1
15
15
at
tTC
CCG
CC
GTC
TCC
AT
SaPI
* Toky
o125
71-O
RF† 1
128
1234
11
07
int
SaPI
j50
97%
SaPI
Toky
o125
71-O
RF2
12
24
2027
80
4 hy
poth
etic
al
iden
tical
to T
1031
; SA
T013
1_00
856
100%
SaPI
Toky
o125
71-O
RF3
21
63
2366
20
4 hy
poth
etic
al
iden
tical
to T
1031
; SA
T013
1_00
857
100%
SaPI
Toky
o125
71-O
RF4
24
02
2620
21
9 st
r Sa
PIis
hika
wa1
1 99
%
iden
tical
to T
1031
; SA
T013
1_00
858
100%
SaPI
Toky
o125
71-O
RF5
27
71
2980
21
0 hy
poth
etic
al
SaPI
3 98
%
sim
ilar t
o Sa
PI3O
RF1
5
SaPI
Toky
o125
71-O
RF6
29
83
3300
31
8 hy
poth
etic
al
SaPI
hiro
saki
4 99
%
sim
ilar t
o Sa
PIhi
rosa
ki4O
RF5
SaPI
Toky
o125
71-O
RF7
33
64
4233
87
0 pr
i Sa
PIno
10
96%
SaPI
Toky
o125
71-O
RF8
42
83
5707
14
25
rep
SaPI
3 98
%
ori s
ite‡
57
08
6007
Sa
PI3
97%
SaPI
Toky
o125
71-O
RF9
60
08
6370
36
3 pp
i Sa
PI3
99%
SaPI
Toky
o125
71-O
RF1
0 63
72
6656
28
5 hy
poth
etic
al
SaPI
no10
99
%
sim
ilar t
o Sa
PIno
10O
RF9
SaPI
Toky
o125
71-O
RF1
1 66
53
7294
64
2 hy
poth
etic
al
SaPI
j11
96%
si
mila
r to
SaPI
j11O
RF1
5
SaPI
Toky
o125
71-O
RF1
2 80
03
8347
34
5 cp
1 Sa
PIj1
1 99
%
SaPI
Toky
o125
71-O
RF1
3 83
82
8960
57
9 cp
2 Sa
PIj1
1 99
%pa
rtial
§
iden
tical
to 2
1305
; SA
2130
5_05
4010
0%
101
SaPI
Toky
o125
71-O
RF1
4 89
60
9136
17
7 cp
3
id
entic
al to
213
05; S
A21
305_
0539
100%
SaPI
Toky
o125
71-O
RF1
5 91
89
9716
52
8 cp
4 Sa
PI3
98%
SaPI
Toky
o125
71-O
RF1
6 97
19
1006
0 34
2 cp
5 Sa
PIj5
0 99
%
SaPI
Toky
o125
71-O
RF1
7 10
057
1042
5 36
9 te
r
id
entic
al to
213
05; S
A21
305_
0536
100%
SaPI
Toky
o125
71-O
RF1
8 10
731
1177
1 10
41
hypo
thet
ical
si
mila
r to
2130
5; S
A21
305_
0534
99%
SaPI
Toky
o125
71-O
RF1
9 12
065
1273
3 66
9 hy
poth
etic
al
iden
tical
to 2
1305
; SA
2130
5_05
3210
0%
sim
ilar t
o re
stric
tion
endo
nucl
ease
100%
SaPI
Toky
o125
71-O
RF2
0 12
954
1350
8 55
5 ea
r Sa
PI4
99%
SaPI
Toky
o125
71-O
RF2
1 14
292
1509
2 80
1 se
c Sa
PIm
w2
99%
SaPI
Toky
o125
71-O
RF2
2 15
259
1598
1 72
3 se
l Sa
PIm
w2
99%
Rep
eat
16
291
1630
5 15
att
TCCC
GC
CG
TCTC
CA
T
*SaP
ISt
aphy
loco
ccus
aur
eus p
atho
geni
city
isla
nd
†OR
FO
pen
read
ing
fram
e
‡ori
site
§par
tial
OR
F
¶O
RF
SaPI
Toky
o125
71O
RF
OR
FSa
PITo
kyo1
2571
OR
F
%
102
11.
SaP
ITok
yo12
381
Ope
n re
adin
g fr
ame
O
RF
SaPI
OR
F
%
att
OR
F¶
Rep
eat
1
15
15
at
tTC
CCG
CC
GTC
TCC
AT
SaPI
* Toky
o123
81-O
RF† 1
128
1234
11
07
int
SaPI
j50
97%
SaPI
Toky
o123
81-O
RF2
12
24
2027
80
4 hy
poth
etic
al
iden
tical
to T
1031
; SA
T013
1_00
856
100%
SaPI
Toky
o123
81-O
RF3
21
63
2366
20
4 hy
poth
etic
al
iden
tical
to T
1031
; SA
T013
1_00
857
100%
SaPI
Toky
o123
81-O
RF4
24
02
2620
21
9 st
r Sa
PIis
hika
wa1
1 99
%
iden
tical
to T
1031
; SA
T013
1_00
858
100%
SaPI
Toky
o123
81-O
RF5
27
71
2980
21
0 hy
poth
etic
al
SaPI
3 98
%
sim
ilar t
o Sa
PI3O
RF1
5
SaPI
Toky
o123
81-O
RF6
29
83
3300
31
8 hy
poth
etic
al
SaPI
hiro
saki
4 99
%
sim
ilar t
o Sa
PIhi
rosa
ki4O
RF5
SaPI
Toky
o123
81-O
RF7
33
64
4233
87
0 pr
i Sa
PIno
10
96%
SaPI
Toky
o123
81-O
RF8
42
83
5707
14
25
rep
SaPI
3 98
%
ori s
ite‡
57
08
6007
Sa
PI3
97%
SaPI
Toky
o123
81-O
RF9
60
08
6370
36
3 pp
i Sa
PI3
99%
SaPI
Toky
o123
81-O
RF1
0 63
72
6656
28
5 hy
poth
etic
al
SaPI
no10
99
%
sim
ilar t
o Sa
PIno
10O
RF9
SaPI
Toky
o123
81-O
RF1
1 66
53
7294
64
2 hy
poth
etic
al
SaPI
j11
96%
si
mila
r to
SaPI
j11O
RF1
5
SaPI
Toky
o123
81-O
RF1
2 80
04
8348
34
5 cp
1 Sa
PIj1
1 99
%
SaPI
Toky
o123
81-O
RF1
3 83
83
8961
57
9 cp
2 Sa
PIj1
1 99
%pa
rtial
§
iden
tical
to 2
1305
; SA
2130
5_05
4010
0%
103
SaPI
Toky
o123
81-O
RF1
4 89
61
9137
17
7 cp
3
id
entic
al to
213
05; S
A21
305_
0539
100%
SaPI
Toky
o123
81-O
RF1
5 91
90
9717
52
8 cp
4 Sa
PI3
98%
SaPI
Toky
o123
81-O
RF1
6 97
20
1006
1 34
2 cp
5 Sa
PIno
10
99%
SaPI
Toky
o123
81-O
RF1
7 10
058
1062
7 57
0 te
r Sa
PIno
10
100%
SaPI
Toky
o123
81-O
RF1
8 10
904
1187
2 96
9 hy
poth
etic
al
SaPI
no10
10
0%
iden
tical
to C
AA
X p
rote
ase
100%
SaPI
Toky
o123
81-O
RF1
9 12
069
1262
3 55
5 ea
r Sa
PI4
99%
SaPI
Toky
o123
81-O
RF2
0 13
362
1420
7 80
1 se
c Sa
PIm
w2
99%
14
374
1509
5 72
2 se
l Sa
PIm
w2
99%
sim
ilar t
o se
l in
SaPI
mw
2
puta
tive
fram
eshi
ft m
utat
ion
beca
use
of d
elet
ion
at 1
4843
th "T
"**
Rep
eat
15
405
1541
9 15
att
TCCC
GC
CG
TCTC
CA
T
*SaP
ISt
aphy
loco
ccus
aur
eus p
atho
geni
city
isla
nd
†OR
FO
pen
read
ing
fram
e
‡ori
site
§par
tial
OR
F
¶O
RF
SaPI
Toky
o123
81O
RF
OR
FSa
PITo
kyo1
2381
OR
F
%
** p
utat
ive
fram
eshi
ft m
utat
ion
beca
use
of d
elet
ion
at 1
4843
th "
T"Sa
PITo
kyo1
2381
1437
4 bp
1509
5 bp
SaPI
mw
2se
l
99%
1484
3T
104
1
2.
SaPI
S. a
ureu
sSt
aphy
loco
ccal
ent
erot
oxin
33
***
***
S. a
ureu
sSE
p<0.
01
† SaPI
Stap
hylo
cocc
us a
ureu
s pat
hoge
nici
ty is
land
Sa
PI†
SEB
SE
C
SEL
μg/m
L
μg/m
L
ng/m
L
Toky
o124
13
SaPI
Toky
o124
13
82.6
± 2
.9*
Toky
o112
12
SaPI
Toky
o112
12
201.
8 ±
52.8
**
Toky
o129
54
SaPI
no10
80
.6 ±
7.8
*
Toky
o123
81
SaPI
Toky
o123
81
52.3
± 1
.6**
N
ot d
etec
ted*
Toky
o125
71
SaPI
Toky
o125
71
23.2
± 2
.2*
56.1
± 8
.7**
Toky
o127
09
SaPI
6811
1 30
.4 ±
2.8
* 14
9.3
± 18
.5**
*
9. Staphylococcal enterotoxin Long and accurate-PCR
A S. aureus 4 Tokyo12413 Tokyo11212 Tokyo12571 Tokyo12381 SaPI
Long and accurate LA PCR
SaPI 10 kb LA-PCR
B LA-PCR RFLP HindIII M DNA
C D E LA-PCR Southern blotting
HindIII “C” seb ”D” sec ”E” sel
105
106
10. SaPI
SaPI SaPITokyo12413 SaPITokyo11212 SaPITokyo12571
SaPITokyo12381 Open reading frame ORF
SaPI 90% SaPI S. aureus
int integrase stl
master repressor str regulatory protein xis exisionase pri primase rep replication
initiator ppi phage interference cp packaging genes ter terminase small subunit seb
staphylococcal enterotoxin B ear penicillin-binding protein tnp transposase sec
staphylococcal enterotoxin C sel staphylococcal enterotoxin L Δsel
staphylococcal enterotoxin L
ori site
ori site
ori site
11. SaPImw2 SaPITokyo12571 SaPITokyo12381 Staphylococcal enterotoxin L
SaPITokyo12381 sel
107
12. SaPI
AT-rich
“A” SaPTokyo11212 SaPIivm60 SaPIbov1 ori site “B”
SaPITokyo12571 SaPITokyo12381 SaPI3 ori site
108
13. SaPI
ori site
ori site GC AT-rich
109
14. SaPITokyo12413 Bacteriophage PT1028
110
15. SaPI
A recombinant replication initiator protein; rRep
SDS-PAGE
Lane 1 500 ng SaPITokyo12413-rRep lane 2 500 ng
SaPITokyo11212-rRep lane 3 500 ng SaPITokyo12381-rRep M
B rRep Western blotting 6 His epitope tag antibody
Lane 1 100 ng SaPITokyo12413-rRep lane 2 100 ng SaPITokyo11212-
rRep lane 3: 100 ng SaPITokyo12381-rRep
111
16. -DNA
A recombinant replication initiator protein rRep
Cy5 DNA SaPITokyo11212-rRep
Cy5 SaPITokyo11212-ori sites SaPITokyo12381-rRep
Cy5 SaPITokyo12381-ori sites SaPITokyo12413-rRep
Cy5 SaPITokyo12413-ori sites SaPITokyo12413-rRep
SaPITokyo12413 7769 bp 8340 bp Cy5
”Shift” rRep-Cy5 DNA ”Probe” rRep
Cy5 DNA
112
113
16.
B rRep ori site SaPI
SaPITokyo11212-rRep Cy5 SaPITokyo12381-ori site
SaPITokyo12381-rRep Cy5 SaPITokyo11212-ori site
C rRep Cy5 DNA
Cy5 DNA
SaPITokyo11212-rRep 100-1000 ng Cy5 SaPITokyo11212-ori site
10 Cy SaPITokyo11212-ori site
SaPITokyo12381-rRep 100-1000 ng Cy5 SaPITokyo12381-ori site
10 Cy5 SaPITokyo12381-ori site DNA
”Shift” rRep-Cy5 DNA
”Probe” rRep Cy5 DNA
17. SaPI DNA
A B C recombinant replication initiator protein
rRep DNA “A” SaPITokyo11212-
rRep ”B” SaPITokyo12381-rRep ”C” SaPITokyo12413-rRep DNA
1 D
rRep SaPITokyo11212-rRep SaPITokyo12413-rRep
3
3 *
p<0.01
114
115
SE/SEls
sed
S. aureus
S aureus
SEs
SaPI S. aureus
23 SE/SEls 5 SE/SEls
sed, selj , ser, ses set
2 53 57 sed , selj ser
pIB485-like plasmid pUO-Sa-SED1 pUO-Sa-SED2
2 53 selj , ser,
ses set pF5 57
SEs SED
SEs
1994 2012
sed
S. aureus 5 SED
sed S. aureus selj ser
116
5
sed selj ser S. aureus
SElJ SER
sed S. aureus SEs
SE/SEls
S. aureus
sed
1.
S. aureus 13 S. aureus
Tokyo10539 Tokyo11726 Tokyo12261 Tokyo12804 Tokyo12902
Tokyo13057 Tokyo13231
SE/SEls sea sed selj ser
5 Tokyo11726 Tokyo12261 Tokyo12804
Tokyo12902 Tokyo13231 sed seg sei selj sem sen seo
ser 2 Tokyo10539 Tokyo13057
S. aureus
117
1.
2.
E. coli 13 K12
DH5α Promega E. coli
2.
3.
14
15 pTokyo10539
pTokyo11726 pTokyo12261 pTokyo12804 pTokyo12902 pTokyo13057
pTokyo13231 S. aureus 7
pG10539F pTokyo10539 4,916 bp EcoRI
pGEM-3Zf(+) vector Promega
pG11726F pG12261F pG12804F pG12902F pG13057F
pG13231F pTokyo11726 pTokyo12261 pTokyo12804
pTokyo12902 pTokyo13057 pTokyo13231 4.9 kbp
EcoRI pGEM-3Zf(+) vector
pGTsed, pGTftsZ, pGTsodA pGTrpoB sed ftsZ sodA rpoB
15
PCR pGEM-T easy vector Promega
118
4.
S. aureus Birnboim Doly 8
MSEY 2
5 mL BHI broth 37
3,000 rpm 10
5 μg/mL lysostaphin
37 1
400 μL SDS
300 μL High salt
10 4 14,500 rpm
15 DNA
Phenol:Chloroform:Isoamyl Alcohol
10 4 14,500 rpm
10 DNA
DNA
1/10 3 M
20 1 4
14,500 rpm 20 1 mL 70%
4 14,500 rpm
10 DNA 10
DNA TE (pH8.0)
DNA NanoDrop 2000c Thermo
Fisher Scientific A260/A280 1.8
119
4
SDS High salt
100 mL
1 M Tris-HCl pH 8.0 4. 2.5 mL
0.5 M EDTA pH 8.0 2 mL
0.9 g
pH8.0 Mill iQ 100 mL
SDS 100 mL
2 N NaOH 10 mL
10% SDS 10 mL
Mill iQ 100 mL
High salt 100 mL
29.5 g
11.5 mL
pH4.8 Mill iQ 100 mL
5.
4. S. aureus
120
EcoRI RFLP
37 1
20 μL
10 M Buffer 2 μL
S. aureus < 1 μg 1 μL
EcoRI 15 units/μL 1 μL
Nuclease-free H2O Thermo Fisher Scientific 16 μL
1/10 10 loading buffer
DNA 0.7%
SeaKem GTG Agarose gels Lonza EtBr
0.5 μg/mL DNA
AE-6933FXES ATTO RFLP
RFLP RFLP 53
6. Sandwich ELISA SE/SEl
S. aureus 7 8.
Sandwich ELISA SEA SED SEG SEI SElJ
SEM SEN SEO SER SED
SEG SEI SElJ SEM SEN SEO SER Capture Detection
121
Capture 2 μg/mL Anti-SED 5 μg/mL
Anti-SEG 2 μg/mL Anti-SEI 2 μg/mL Anti-SElJ 2 μg/mL
Anti-SEM 1 μg/mL Anti-SEN 2 μg/mL Anti-SEO 5 μg/mL
Anti-SER Detection
3,200 Anti-SED 800 Anti-SEG 1,200 Anti-SEI 4,800
Anti-SElJ 800 Anti-SEM 1,000 Anti-SEN 3,500
Anti-SEO 1,200 Anti-SER
100 ng/mL 0.1% w/v
BSA SED SEG SEI SElJ SEM SEN SEO
SER Can Get Signal Immunoreaction Enhancer
Solut ion 1
7. sed , selj ser EcoRI
sed , selj ser EcoRI
DNA
Omoe 53 sed , selj ser pIB485-l ike plasmid
4.5-5.0 kbp EcoRI
5.
4.5-5.0 kbp 18
SeaKem GTG Agarose gel Lonza Monofas DNA
Purification kit GL
7. SE/SEls DNA
37 1
122
pGEM-3Zf(+) vector EcoRI
20 μL
10 M Buffer 2 μL
pGEM-3Zf(+) < 1 μg 1 μL
EcoRI 15 units/μL 1 μL
Nuclease-free H2O Thermo Fisher Scientific 16 μL
EcoRI EcoRI pGEM-3Zf(+) vector
9.
pGEM-3Zf(+) vector EcoRI
5. MEGA5
software EcoRI
GeneMark ORF Finder ORF BLAST
ORF
pGEM-3Zf(+) vector EcoRI ORF
Genetyx software program, ver. 8
8. RNA
S. aureus 7 RNA RiboPure-Bacteria Kit
Thermo Fisher Scientific
123
5 mL 1% w/v yeast extract BHI broth
37 24 5,000
rpm 10 350 μL
RNAwiz 250 μL Zirconia Beads
10
4 14,500 rpm 5
1/5
10 4 14,500 rpm
5 RNA
RNA
Filter Cartridge 4 14,500 rpm
cartridge 1 700 μL Wash
Solution 1 14,500 rpm 1
500 μL Wash Solution 2/3 14,500 rpm 1
2 Filter Cart ridge
14,500 rpm 1 Wash
Solution 2/3 Filter Cartridge
100 25 μL Elution Solution
1 14,500 rpm 1 RNA
genomic DNA RNA 1/9
10 DNase Buffer 4 μL DNase 1 2 U/μL 37
30 1/5 DNase Inactivation Reagent
2 14,500 rpm 1
RNA
RNA NanoDrop 2000c Thermo Fisher
124
Scientific A260/A280 2.0
1% 23S 16S ribosomal RNA
80
9.
2 g RNA High-Capacity cDNA Reverse
Transcription Kits Thermo Fisher Scientific
cDNA
20
50 μL
10 RT Buffer 5 μL
25 dNTP Mix (100 mM) 2 μL
10 RT Random Primers 5 μL
MultiScribe Reverse Transcriptase 2.5 μL
RNase Inhibitor 2.5 μL
RNA 2 μg/25 μL 25 μL
Nuclease-free H2O Thermo Fisher Scientific 8 μL
25 10
37 120
85 5
125
10.
Reverse transcription RT PCR
PCR
pGEM-T easy vector PCR
4. genomic
DNA
5. PCR NCBI
Primer3
(http:/ /frodo.wi.mit.edu/primer3/ )
15
PCR TA
TA 9.
PCR pGEM-T easy vector
pGTsed pGTsodA pGTftsZ pGTrpoB
7 M13F M13R
11. RT-PCR
S. aureus sed sed mutant sed msed
Power SYBR Green PCR Master Mix Thermo Fisher
Scientific ABI7500 Real Time PCR System Thermo Fisher
Scientific RT-PCR
19 3
126
Superoxide dismutase
sodA Cell division protein f tsZ RNA polymerase beta subunit
rpoB
10.
1.0 102 copy/μL 1.0 108 copy/μL
Ct
RNA 40 ng RNA
RT-PCR sodA
ftsZ rpoB sed msed
SYBR green DNA
PCR
DNA SYBR green
DNA
RT-PCR
NCBI
Primer Express Software Version 3.0 Thermo Fisher
Scientific 15
RT-PCR 25 μL
SYBR Green PCR Master Mix 2 12.5 μL
Forward primer 5 μM 1.5 μL
Reverse primer 5 μM 1.5 μL
Template cDNA or Standard plasmid 1 μL
Nuclease-free H2O Thermo Fisher Scientific 8.5 μL
127
50 2
95 10
95 15
60 1
95 15
60 30
95 15
12.
S. aureus 7 SEs
one way ANOVA Tukey-Kramer HSD
p 0.05
Statcel2 software
1.
S. aureus 7 EcoRI RFLP
EcoRI 3 RFLP
40
128
18 RFLP pTokyo11726,
pTokyo12804, pTokyo12902, pTokyo13057 pTokyo13231
pTokyo12261 RFLP pTokyo11726
9.4 kb
pTokyo10539
RFLP pTokyo11726
1
2. SE/SEls
S. aureus 7 SED SElJ SER SED
2 500 ng/mL
SED SED
0.1 ng/mL Tokyo10539 Tokyo12261
Tokyo12902 Tokyo13057 Tokyo11726
Tokyo12804 Tokyo13231 19A SElJ
Tokyo13231 SElJ SER
SElJ 37-135 ng/mL SER 523-2925 ng/mL
19B 19C
SE/SEls
20 SED
0 76% SElJ 0 5%
SER 16 66% sea
129
5 Tokyo11726 Tokyo12261 Tokyo12804 Tokyo12902
Tokyo13231 SEA 71 78%
seg sei sem sen seo 2 Tokyo10539
Tokyo13057 SEs 0.1%
3. sed EcoRI
3 SED sed
sed
S. aureus
4.5-5.0 kbp EcoRI 18
pGEM-3Zf(+) vector sed
4,915 bp 4,916 bp 4,917 bp
4,915 bp pTokyo11726 pTokyo12804
pTokyo13231 EcoRI 4,916 bp
pTokyo10539 pTokyo12261 pTokyo13057 EcoRI
4,917 bp pTokyo12902 EcoRI
EcoRI ORF 3
3 ORF sed , selj ser
pTokyo11726, pTokyo12804 pTokyo13231 sed
A 514 A
TAG
21 DNA sed
130
msed pTokyo10539, pTokyo12261, pTokyo12902
pTokyo13057 sed NCBI
sed KF007920.1
100%
4. RT-PCR sed sed
sed 100% DNA
S. aureus 4 Tokyo10539 Tokyo12261 Tokyo12902
Tokyo13057 sed 22A
sodA sed SED
19A SED Tokyo11726
Tokyo12804 Tokyo13231 msed
sodA
p>0.05 22D
f tsZ rpoB sodA
sed msed 22B 22C
22E 22F
SEs
5 20 SED
SEs
131
3 S. aureus sed
SED
sed pIB485-like
plasmid selj ser sed
SE/SEls 2 53
7 sed, selj ser
S. aureus RFLP
pTokyo11726 pTokyo12804 pTokyo12902 pTokyo13057
pTokyo13231 RFLP Omoe 53 pIB485-like
plasmid p196E p361 RFLP
pTokyo12261 RFLP pIB485like-plasmid
p1151 pI7 pTokyo10539 RFLP
pIB485like-plasmid
18 S. aureus pIB485-like plasmid
sed, selj ser S. aureus
SE/SEls
Tokyo13231 S. aureus 6 SElJ SER
19 SER S. aureus
523 2925 ng/mL 19C SE/SEl
20 SER
SER 57 SElJ
132
SElJ
SEs 31 SEA
64.6% SEE 63.4%
SElJ SEs
Omoe 56
SEs SEs
SEs
Tokyo13057 SER SElJ
SE/SEls 70% SEs
SEs SElJ SER
SE/SEls
S. aureus
sed 2
sed 100%
sed msed pTokyo10539,
pTokyo12261, pTokyo12902 pTokyo13057
sed pTokyo11726, pTokyo12804
pTokyo13231 sed
A 514 A
21 pTokyo11726, pTokyo12804 pTokyo13231
sed
133
mSED msed
SED
C 80
msed NCBI
1963 S.
aureus 502A 502A plasmid
CP007455.1
SEs
SEs
34 43 66 SEA 21-50
81-100
161-180
43 SEA
MHC class II 187
, 225 227
34 66 SED MHC
SEA 34 66
MHC
C
mSED C 80
N
mSED N
134
26 32 mSED
msed S. aureus msed
22 mSED SED
Sandwich ELISA
19A 1 mSED
S. aureus
msed
S. aureus msed
mSED
1 mSED
mSED SED SED
SED
SED
mSED
SED
mSED mSED
mSED
S. aureus
pIB485-like
135
plasmid
SElJ SER
S. aureus SER
SElJ
SED
S. aureus pIB485-like plasmid SED C
80 mSED S.
aureus msed
SED mSED
mSED SE
136
13.
SE
/SEl
s
Stap
hylo
cocc
us a
ureu
s
To
kyo1
0539
sed
seg
sei selj s
em se
n se
o ser
To
kyo1
1726
se
a sed selj ser
To
kyo1
2261
se
a sed selj ser
To
kyo1
2804
se
a sed selj ser
To
kyo1
2902
se
a sed selj ser
To
kyo1
3057
sed
seg
sei selj s
em se
n se
o ser
To
kyo1
3231
se
a sed selj ser
Esch
eric
hia
coli
D
H5α
Prom
ega
SE
/SEl
s
137
14.
pG
EM-3
Zf(+
) A
pr , clo
ning
vec
tor
Prom
ega
pG
EM-T
eas
y A
pr , TA
clo
ning
vec
tor
Prom
ega
pT
okyo
1053
9 S.
aur
eus p
lasm
id p
urifi
ed fr
om st
rain
Tok
yo10
539
pT
okyo
1172
6 S.
aur
eus p
lasm
id p
urifi
ed fr
om st
rain
Tok
yo11
726
pT
okyo
1226
1 S.
aur
eus p
lasm
id p
urifi
ed fr
om st
rain
Tok
yo12
261
pT
okyo
1280
4 S.
aur
eus p
lasm
id p
urifi
ed fr
om st
rain
Tok
yo12
804
pT
okyo
1290
2 S.
aur
eus p
lasm
id p
urifi
ed fr
om st
rain
Tok
yo12
902
pT
okyo
1305
7 S.
aur
eus p
lasm
id p
urifi
ed fr
om st
rain
Tok
yo13
057
pT
okyo
1323
1 S.
aur
eus p
lasm
id p
urifi
ed fr
om st
rain
Tok
yo13
231
pG
1053
9F
Apr , p
GEM
-3Zf
(+) w
ith c
lone
d 49
16 b
p Ec
oRI-f
ragm
ent o
f pTo
kyo1
0539
pG11
726F
A
pr , pG
EM-3
Zf(+
) with
clo
ned
4915
bp
EcoR
I-fra
gmen
t of p
Toky
o117
26
pG
1226
1F
Apr , p
GEM
-3Zf
(+) w
ith c
lone
d 49
16 b
p Ec
oRI-f
ragm
ent o
f pTo
kyo1
2261
pG12
804F
A
pr , pG
EM-3
Zf(+
) with
clo
ned
4915
bp
EcoR
I-fra
gmen
t of p
Toky
o128
04
pG
1290
2F
Apr , p
GEM
-3Zf
(+) w
ith c
lone
d 49
17 b
p Ec
oRI-f
ragm
ent o
f pTo
kyo1
2902
pG13
057F
A
pr , pG
EM-3
Zf(+
) with
clo
ned
4916
bp
EcoR
I-fra
gmen
t of p
Toky
o130
57
pG13
231F
A
pr , pG
EM-3
Zf(+
) with
clo
ned
4915
bp
EcoR
I-fra
gmen
t of p
Toky
o132
31
pGTs
ed
Apr , p
GEM
-T e
asy
with
clo
ned
PCR
pro
duct
con
tain
ing
sed
(mse
d) se
quen
ce (4
1 bp
–425
bp)
pGTs
odA
A
pr , pG
EM-T
eas
y w
ith c
lone
d PC
R p
rodu
ct c
onta
inin
g so
dA se
quen
ce (7
1 bp
–539
bp)
pGTf
tsZ
Apr , p
GEM
-T e
asy
with
clo
ned
PCR
pro
duct
con
tain
ing
ftsZ
sequ
ence
(53
bp–4
48 b
p)
pGTr
poB
Apr , p
GEM
-T e
asy
with
clo
ned
PCR
pro
duct
con
tain
ing
rpoB
sequ
ence
(965
bp–
1576
bp)
138
15.
5'→
3'
TA-C
loni
ng
sed
or m
sed
SED
41F
CTA
GTT
TGG
TAA
TATC
TCC
TTTA
AA
CG
SED
425R
TT
AC
CTT
CG
TGTG
GA
GTG
AC
A
so
dA
SOD
A71
F TG
GA
AA
TTC
AC
CA
TGA
CA
GA
SOD
A53
9R
CC
AA
TGTA
GTC
AG
GG
CG
TTT
fts
Z FT
SZ53
F G
TGTA
GG
TGG
TGG
CG
GTA
AC
FTSZ
448R
C
AG
CA
GC
AG
CTT
GA
GTT
TGA
rp
oB
RPO
B96
5F
TTG
AA
TCA
AA
CG
CA
AA
CA
GC
RPO
B15
76R
A
TCC
AA
TGTT
TGG
TCC
CTC
A
Qua
ntita
tive
RT-
PCR
se
d or
mse
d SE
D29
1F
TTC
AA
AA
GA
AA
TGG
CTC
AA
CA
SED
425R
TT
AC
CTT
CG
TGTG
GA
GTG
AC
A
so
dA
SOD
A37
3F
GG
TTC
AG
GTT
GG
GC
TTG
GT
SOD
A44
0R
TCTT
GG
TTTG
GTG
TAG
TCA
CA
ATT
TC
fts
Z FT
SZ31
5F
GG
GTG
GC
GG
AA
CTG
GTA
CT
FTSZ
376R
C
GC
CC
ATT
TCTT
TTG
CA
ATT
rp
oB
RPO
B14
82F
AC
GTG
AA
CG
TGC
TCA
AA
TGG
RPO
B15
63R
TC
CC
TCA
GG
CG
TTTC
AA
TTG
18. S. aureus 7
Restriction fragment length polymorphism
sed selj ser S. aureus
EcoRI sed selj ser EcoRI
4.9 kbp M DNA
139
19. S. aureus 7
Staphylococcal enterotoxin/Staphylococcal enterotoxin-like toxin
A SED B SElJ C SER
3 3
p<0.05
140
20. S. aureus 7 Staphylococcal
enterotoxin/Staphylococcal enterotoxin-like toxin SE/SEl
SE/SEl
S. aureus SE/SEls Sandwich ELISA
3
”Others” SEG SEI SEM SEN SEO
141
21. S. aureus 7 sed
sed
142
143
22. S. aureus 7 sed
sed
A B C sed S. aureus Tokyo10539 Tokyo12261
Tokyo12902 Tokyo13057 sed ”A”
sodA ”B” ftsZ ”C” rpoB
D E F sed S. aureus Tokyo11726 Tokyo12804 Tokyo13231
sed ”D” sodA ”E”
ftsZ ”F” rpoB 3
3
p<0.05
144
S. aureus SEs
S.
aureus
S. aureus
SE/SEls
S.
aureus
S. aureus
S. aureus
4 S.
aureus S. aureus
MLST 8 CC
CC CC
CC S. aureus
SE/SEls SE/SEls
145
CC SE/SEls
SE/SEls
S. aureus
S. aureus
S. aureus
SEs 4 SaPI SaPITokyo12413
SaPITokyo11212 SaPITokyo12571 SaPITokyo12381
SaPI S. aureus SEs
SaPI SE
SaPI
“ ” Rep
SaPITokyo11212 SaPITokyo12571
SaPITokyo12381 SaPI Rep
SaPITokyo12413
Rep SaPI
SaPITokyo12413
SaPI
S. aureus
S. aureus
146
1
pIB485-like plasmid
SElJ SER
pIB485-like plasmid
SED
S. aureus pIB485-like plasmid SED C
80 mSED
S. aureus msed
SED mSED
mSED
SE
S. aureus
S. aureus
S.
aureus
NCBI
S. aureus
2014
100 SaPI
147
S. aureus
S. aureus
S. aureus
148
149
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