mlst for epidemiological study - mut
TRANSCRIPT
MLST FOR EPIDEMIOLOGICAL STUDY
1
Presented by Watsawan Prapasawat
Department of Clinic, Faculty of Veterinary Medicine
Mahanakorn University of Technology
• Multilocus sequence typing (MLST) is a procedure for characterising isolates of bacterial species using the sequences of internal fragments of (usually) seven house-keeping genes.
• MLST is based on PCR amplification and sequencing of internal fragments of a number (usually 6 or 7) of essential or housekeeping genes spread around the bacterial chromosome.
• The differences between the sequences are used to define sequence types (STs).
2
Principle of MLST
• It is provide a portable, accurate, and highly discriminating typing system that can be used for most bacteria and some other organisms.
• If the same genes were used in different studies, then the typing information of the isolates from these different studies may be compared and analyzed together.
3
The aim of MLST
• MLST schemes have been developed for the most important bacterial pathogens, including
Streptococcus pyogenes
Haemophilus influenza
Staphylococcus aureus
Campylobacter jejuni
Enterococcus faecium
Escherichia coli Salmonella spp.
4
Multilocus sequence typing (MLST)
5
DNA EXTRACTION
PCR FOR 7
HOUSEKEEPING GENES
DNA SEQUENCING
EDITING DNA SEQUENCE
MATCH ALLELE; IN DATANASE
ANALYSIS OF MLST
Diagrammatic representative for procedure used for MLST
6
Gene Primer Anealing Amplicon
(Temp) size (bp)
adk ATTCTGCTTGGCGCTCCGGG (F)
CCGTCAACTTTCGCGTATTT (R)
54 583
fumC TCACAGGTCGCCAGCGCTTC (F)
GTACGCAGCGAAAAAGATTC (R)
54 806
gyrB TCGGCGACACGGATGACGGC (F)
ATCAGGCCTTCACGCGCATC (R)
60 911
icd ATGGAAAGTAAAGTAGTTGTTCCGGCACA (F)
GGACGCAGCAGGATCTGTT (R)
54 878
mdh ATGAAAGTCGCAGTCCTCGGCGCTGCTGGCGG(F)
TTAACGAACTCCTGCCCCAGAGCGATATCTTTCTT (R)
60 932
purA CGCGCTGATGAAAGAGATGA (F)
CATACGGTAAGCCACGCAGA (R)
54 816
recA CGCATTCGCTTTACCCTGACC (F)
TCGTCGAAATCTACGGACCGGA (R)
58 780
PCR primers of housekeeping genes of E. coli for MLST
(Aanensen and Spratt, 2005)
7
Demonstrate for MLST
Prove sequence by Bioedit program
Allele number for adk gene
Submit Forward and Reward sequence
for generating new allele type
Analysis of MLST
• Allele-based method • Nucleotide-based method
20
Nucleotide-based method
Algorithm Functionality Link MAFFT Sequence alignment http://mafft.cbrc.jp/alignment/software/
MAUVE Sequence alignment http://asap.ahabs.wisc.edu/mauve/index.php
JModeltest2 Selection of models of nucleotide substitution https://code.google.com/p/jmodeltest2/
RAxML ML inference of evolutionary relationships http://www.exelixis-lab.org/
GARLI ML inference of evolutionary relationships http://code.google.com/p/garil/
PHYML ML inference of evolutionary relationships http://code.google.com/p/phyml/
MrBayes Bayesian inference of evolutionary relationships http://mrbayes.sourceforge.net/
BEAST Bayesian inference of evolutionary relationships http://beast.bio.ed.ac.uk/Main_Page
ClonalFrame Bayesian inference of clonal relationships considering recombination
http://www.xavierdidelot.xtreemhost.host/clonalframe.htm
Mega5 http://www.megasoftware.net/mega5/
Nucleotide-based method
Nucleotide-based method
24
Application of MLST
• Molecular epidemiology and public health • Population structure and dynamics • Other applications
Virulence gene profiles, antimicrobial resistance
patterns and phylogenetic relationship of
Escherichia coli isolated from diarrheal and
healthy weaning piglets and their environments in swine farm
27
28
Introduction: Main causes according to pig’s age
AT BIRTH-SUCKLING 1-4 WEEKS
WEANING 5-9 WEEKS
GROWING PIG – SLAUGHTER
10 WEEKS -6 MONTHS
DISEASES:
CLAUSE:
EFFECTS:
ECO- LOSS:
Iron deficiency, Arthritis,
Aujeszky disease, Clostridia,
Coccidiosis, Diarrhea,
Colibacilosis, Epidemic diarrhea,
PRRS, Salmonellosis,
Swine dysentery, TGE
Trauma, Vitamin deficiency
Viral infection,
Bacterial infection
Morbility, Mortality, Stunt growth
Depend on the diseases
Atrophic Rhinitis, Aujeszky disease, Clostridia, Coccidiosis, Post weaning
diarrheal, Colibacilosis, Epidemic diarrhea, PRRS, FMD, Swine dysentery, TGE,
Glasser disease, Swine influenza, Parvovirus, Classcical swine fever etc.
Trauma, Vitamin deficiency, Viral infection, Bacterial infection
Morbility, Mortality, Stunted and grow slowly
Depend on the diseases
POST WEANING DIARRHEA
• To analyze the phenotype and genotype of diarrheagenic
E. coli isolated from weaning piglets with and without
diarrhea and their environments in a swine farm
29
General Objective
30
Schematic process
31
Sampling source
Total No. of positive
E. coli
No. (%)
Pathogenic E. coli
Non pathogenic E. coli
Diarrheal piglet pens 182 68 (37.4) 114 (62.6)
Rectal swab 130 61 (46.9) 69 (53.1)
Feed from feeder 20 3 (15.0) 17 (85.0)
Water from nipple 12 0 12 (100.0)
Waste swab 16 2 (12.5) 14 (87.5)
Worker’s hand 4 2 (50.0) 2 (50.0)
Healthy piglet pens 170 47 (27.6) 123 (72.4)
Rectal swab 116 35 (30.2) 81 (69.8)
Feed from feeder 20 4 (20.0) 16 (80.0)
Water from nipple 9 1 (11.1) 8 (88.9)
Waste swab 21 7 (33.3) 14 (66.7)
Worker’s hand 4 0 4 (100.0)
Total 352 115 (32.7) 237 (67.3)
Table 5.3 Detection rate of Escherichia coli positive for virulence genes detected in diarrheal and healthy weaning piglets and environmental samples in
swine farm at Central region, Thailand
32
Table 5.6 Detection rate of antimicrobial resistance from diarrheal and healthy weaning piglets and environmental samples in swine farm at Central region, Thailand
• E. coli isolates from diarrheal and healthy weaning piglets and
their environmental samples showed the low resistance rate to CT in 7.1% (13/182) from diarrheal pens and 7.6% (13/170) from healthy
pens.
33
0
20
40
60
80
100
120
140
160
180
200
AMP AMC CTX CAZ KAN STR GEN TET DO OTC ENR SXT CT
No. of
anti
mic
robia
l re
sist
ance
Antimicrobial agents
Antimicrobial resiatance
Diarrhea Healthy
Fig. 5.5 Comparison of individual antimicrobial resistance against E. coli isolates between diarrheal and healthy pens in swine farm at Central region, Thailand.
• Detection rates of E. coli isolates resistant to 13 tested antimicrobial
agents were not different from both diarrheal and healthy piglet pens in this study .
34
Sampling source
Total no. of
tested
E. coli
isolates
Total no. of integron
positive
isolates
No. (%) of E. coli isolate positive for
Class1 Class2
Class1
and 2
Diarrheal piglet pens 182 122 (67.0) 120 (98.4) 1 (0.8) 1 (0.8)
Rectal swab 130 93 (71.5)a 92 (98.9) 0 1 (1.1)
Feed from feeder 20 10 (50.0) 10 (100.0) 0 0
Water from nipple 12 5 (41.7) 4 (80.0) 1 (20.0) 0
Waste swab 16 11 (68.8) 11 (100.0) 0 0
Worker’s hand 4 3 (75.0) 3 (100.0) 0 0
Healthy piglet pens 170 101 (59.4) 95 (94.0) 4 (4.0) 2 (2.0)
Rectal swab 116 64 (55.2)a 61 (95.3) 3 (4.7) 0
Feed from feeder 20 14 (70.0) 14 (100.0) 0 0
Water from nipple 9 8 (88.9) 8 (100.0) 0 0
Waste swab 21 13 (61.9) 10 (76.9) 1 (7.7) 2 (15.4)
Worker’s hand 4 2 (50.0) 2 (100.0) 0 0
Total 352 223 (63.4) 215 (96.4)b 5 (2.2) b 3 (1.4)
Table 5.9 Detection rates of Class 1, 2 and 3 integrons of Escherichia coli from diarrheal and healthy weaning piglets and environmental samples in swine farm
• Class 1 integron of E. coli was more commonly detected in all tested samples in both diarrheal and healthy pens.
35
DNA EXTRACTION
PCR FOR 7
HOUSEKEEPING GENES
DNA SEQUENCING
FORWARD AND REWARD TRIMIMG
MATCH ALLELE; IN DATANASE
GENERATE CLONAL
COMPLEX & PHYLYGENETIC TREE
Precipitation and
bind in column
E. coli cultured in LB
Lysis, Buffer ATL,
proteinase K
Washing :
Buffer AW1
Washing :
Buffer AW2
Elution:
Buffer AE S
T1
00
:EC
58
-Rec
tal
swab
:Th
aila
nd
ST
100:E
C132-F
eed
:Thail
and
26
ST
100:E
C29-R
ecta
l sw
ab:T
hai
land
62
ST
5125:J
apan
87
ST
5707:E
C283-R
ecta
l sw
ab:T
hai
land
ST
5707:E
C338-W
aste
:Thai
land
ST
5707:E
C339-W
aste
:Thai
land
23
62
11
ST
1114:E
C8-R
ecta
l sw
ab:T
hai
land
2
ST
1114:E
C253-R
ecta
l sw
ab:T
hai
land
ST
1114
:EC
304-
Fee
d:T
hailan
d
1
1
ST
1114
:EC
241-
Rec
tal s
wab
:Tha
ilan
d
3
ST
1114
:EC
163-
Was
te:T
hailan
d
48
ST36
57:C
hina
ST42
41:C
hinaST
165:
EC13
-Rec
tal s
wab
:Tha
iland
ST3899
:Chi
na
223654
61
ST5709
:EC11
3-Rec
tal s
wab
:Tha
iland
ST5709:EC172-W
aste:
Thailan
d
81
59
ST1112:EC183-R
ectal
swab
:Thail
and
ST4704:Lao
s
ST1695:ChinaST34:EC216-R
ectal swab:Thaila
nd
ST3484:China
476327620
ST4429:EC343-Waste:Thailand
ST3298:China
80 ST3656:China
20ST1731:China
14
ST3661:China
ST5714:EC325-Water:Thailand
ST10:EC310-Feed:Thailand
ST3658:China4
ST10:EC83-Rectal swab:ThailandST10:EC184-Rectal swab:ThailandST10:EC215-Rectal swab:Thailand
ST10:EC203-Rectal swab:Thailand
ST5713:EC179-Hand:ThailandST3628:China
123
22
4
13
116
3
2
ST1702:ChinaST3659:China
29
9
ST218:EC156-Water:Thailand
ST3744:China
82ST4545:Japan
ST3940:China
ST4242China
ST542:EC78-Rectal swab:Thailand
ST3725:China
64
69
70
31
12
3
ST5069:China
ST47:Indonesia
ST5222:C
hina
ST
3402:China
ST
2962:China
ST
1421:EC
44-Rectal sw
ab:Thailand
ST
3660:China
6795
9645
5542
2
ST
48:E
C142-F
eed:T
hailan
d
ST
48:E
C315-F
eed:T
hailan
d
34 S
T48:E
C305-F
eed:T
hailan
d
84
ST
1688:C
hin
a
34
ST
1700:C
hin
a
ST
3404:C
hin
a
ST
4543:C
hin
a
15
ST
1690:C
hin
a
ST
3630:C
hin
a
ST
3631:C
hin
aS
T3634:C
hin
a
98 5
9 40
15
38
43
22
ST
5708:E
C43-R
ecta
l sw
ab:T
hai
land
ST
1691:C
hin
a 36
ST
5176:E
C219-R
ecta
l sw
ab:T
hai
land
ST
3629:C
hin
a
ST
206:E
C134-F
eed:T
hai
land
ST
206:
EC
275-
Rec
tal sw
ab:T
hail
and
72 64 43 6
12
ST
156:
EC
133-
Fee
d:T
hailan
d
ST
156:
EC
299-
Fee
d:T
hailan
d
93
ST57
12:E
C15
2-W
ater
:Tha
ilan
d
ST36
75:C
hina 26
12
ST52
18:E
C1-
Rec
tal s
wab
:Tha
iland
ST5218
:EC68
-Rec
tal s
wab
:Tha
iland
82
ST5541
:EC18
-Rec
tal s
wab
:Tha
iland
98
ST67 :Indones
ia
ST4547:Japan 69
26ST1820:Korea
11ST3939:C
hina
ST29:EC226-Rectal sw
ab:Thailand
ST1693:China
ST410:EC131-Feed:Thailand
ST2273:Japan
48ST88:EC98-Rectal swab:Thailand
ST88:EC65-Rectal swab:Thailand
ST5706:EC9-Rectal swab:ThailandST3870:China 54
3582
48
5939
94
42
14
ST710:ChinaST1701:China
ST4544:Japan
99
ST1689:China
ST2292:Japan
ST5710:EC136-Feed:Thailand
ST101:EC187-Rectal swab:Thailand
ST5229:EC239-Rectal swab:Thailand
879979
59
75
72
18
ST5711:EC143-Feed:ThailandST3485:China
ST641:EC104-Rectal swab:Thailand
ST641:EC210-Rectal swab:Thailand
895099
22
ST1699:China
30
ST117 :Laos
ST1698:China
100
38
ST1694:China
ST1687:China
ST1697:China
8998
38
ST2291:Japan
ST4546:Japan
ST5171:C
hina
6743
45
ST
3057:EC
5-Rectal sw
ab:Thailand
ST
3057:China
96
ST
3058:China
ST
3277:C
hin
a
38100
ST
5084:C
hin
a
MLST
10
0
80
60
40
20
0
MLST
adk
fum
C
gyrB
icd
mdh
purA
recA
6 7 5 1 8 18 2
6 7 5 1 8 18 2
427 636 188 1 8 18 6
6 4 12 1 20 12 7
6 4 12 1 20 12 7
6 4 12 1 20 18 7
6 4 12 1 411 12 7
6 4 12 16 9 7 7
6 29 32 16 11 8 44
6 29 32 16 11 8 44
6 4 33 16 11 7 6
6 19 3 16 9 8 341
6 19 3 16 9 8 341
6 19 3 16 9 9 341
9 6 33 131 24 8 7
9 6 33 131 24 8 7
9 175 4 131 24 8 7
10 11 4 12 8 8 2
10 11 4 426 8 8 2
10 11 4 1 8 8 2
10 11 4 8 8 8 2
10 11 4 8 8 8 2
10 11 4 8 8 8 2
10 11 4 8 8 8 2
10 11 4 8 8 8 2
6 11 4 8 8 8 2
6 11 4 8 8 8 2
6 11 4 8 8 8 2
10 11 4 8 179 8 6
10 4 5 8 8 8 2
8 7 1 8 8 8 2
112 11 5 8 8 8 86
112 11 5 12 8 8 86
10 11 5 8 12 1 2
10 11 5 8 12 1 2
10 11 5 10 8 1 2
10 27 5 10 12 9 2
10 27 5 10 12 9 2
10 27 5 10 12 9 2
10 27 5 10 12 8 2
10 736 5 10 12 1 2
10 736 5 10 12 1 2
10 736 5 10 12 1 2
10 27 5 10 12 1 2
10 27 5 10 12 1 2
10 27 5 10 12 1 2
10 27 5 10 12 1 2
10 27 5 10 12 1 2
43 41 15 18 11 7 6
43 41 15 18 11 7 44
43 41 15 90 50 8 6
290 54 55 324 35 40 223
EC-E134
EC-R275
EC-R219
EC-R65
EC-R98
EC-E131
EC-R9
EC-R226
EC-E133
EC-E299
EC-E152
EC-R1
EC-R68
EC-R18
EC-R104
EC-R210
EC-E143
EC-E156
EC-E179
EC-R216
EC-E310
EC-R184
EC-R203
EC-R215
EC-R83
EC-E142
EC-E305
EC-E315
EC-E325
EC-R43
EC-R44
EC-E343
EC-R78
EC-E172
EC-R113
EC-R183
EC-E132
EC-R29
EC-R58
EC-R13
EC-E338
EC-E339
EC-R283
EC-E163
EC-E304
EC-R241
EC-R253
EC-R8
EC-R187
EC-R239
EC-E136
EC-R5
ST-206
ST-206
ST-5176
ST-88
ST-88
ST-410
ST-5706
ST-29
ST-156
ST-156
ST-5712
ST-5218
ST-5218
ST-5541
ST-641
ST-641
ST-5711
ST-218
ST-5713
ST-34
ST-10
ST-10
ST-10
ST-10
ST-10
ST-48
ST-48
ST-48
ST-5714
ST-5708
ST-1421
ST-4429
ST-542
ST-5709
ST-5709
ST-1112
ST-100
ST-100
ST-100
ST-165
ST-5707
ST-5707
ST-5707
ST-1114
ST-1114
ST-1114
ST-1114
ST-1114
ST-101
ST-5229
ST-5710
ST-3057
D
H
H
D
D
D
D
H
D
H
D
D
D
D
D
H
D
D
D
H
H
H
H
H
D
D
H
H
H
D
D
H
D
D
D
H
D
D
D
D
H
H
H
D
H
H
H
D
H
H
D
D
E
P
P
P
P
E
P
P
E
E
E
P
P
P
P
P
E
E
E
P
E
P
P
P
P
E
E
E
E
P
P
E
P
E
P
P
E
P
P
P
E
E
P
E
E
P
P
P
P
P
E
P
PEN-CEP-AMINO-TET-QUIN-SULF
PEN-CEP-AMINO-TET-QUIN-SULF
PEN-AMINO-TET-QUIN-SULF
PEN-CEP-AMINO-QUIN
PEN-BETA-CEP-AMINO-TET-SULF
PEN-CEP-AMINO-TET-QUIN
PEN-CEP-AMINO-TET-QUIN
PEN-CEP-AMINO-TET-QUIN-SULF
PEN-CEP-AMINO-TET-QUIN-SULF
PEN-CEP-AMINO-TET-QUIN-SULF
PEN-CEP-AMINO-TET-QUIN
PEN-CEP-AMINO-TET-QUIN-SULF
PEN-CEP-AMINO-TET-QUIN-SULF
PEN-CEP-AMINO-TET-QUIN-SULF-POLY
PEN-AMINO-TET-SULF
PEN-CEP-AMINO-QUIN
QUIN
PEN-AMINO-TET-QUIN-SULF
PEN-CEP-AMINO-TET-QUIN-SULF
PEN-BETA-CEP-AMINO-TET-QUIN
PEN-CEP-AMINO-TET-QUIN-SULF
PEN-CEP-AMINO-TET-QUIN-SULF-POLY
PEN-CEP-AMINO-TET-QUIN-SULF
PEN-CEP-AMINO-TET-QUIN
PEN-TET-QUIN
PEN- CEP-AMINO-TET-QUIN-SULF
PEN-CEP-AMINO-TET-QUIN
PEN-CEP-AMINO-TET-QUIN
PEN-CEP-AMINO-TET-QUIN-SULF-POLY
PEN-AMINO-TET-QUIN-SULF
PEN-AMINO-TET-QUIN
PEN-BETA-CEP-AMINO-TET-QUIN-SULF
PEN-AMINO-TET-QUIN-SULF
PEN-CEP-AMINO-TET-QUIN-SULF
PEN-CEP-AMINO-TET-QUIN-SULF
PEN-CEP-AMINO-TET-QUIN-SULF
PEN-BETA-CEP-AMINO-TET-QUIN-SULF
PEN-BETA-CEP-AMINO-TET-QUIN-SULF
PEN-BETA-CEP-AMINO-TET-QUIN-SULF
PEN-BETA-CEP-AMINO-TET-QUIN
PEN-BETA-CEP-AMINO-TET-QUIN-SULF
PEN-AMINO-TET-QUIN-SULF
PEN-CEP-AMINO-QUIN-SULF
PEN-CEP-AMINO-TET-QUIN-SULF-POLY
PEN-CEP-AMINO-QUIN-SULF
PEN-CEP-AMINO-TET-QUIN-SULF
PEN-CEP-TET
PEN-CEP-AMINO-TET-QUIN
PEN-AMINO-TET-QUIN-POLY
PEN-CEP-AMINO-TET-QUIN-POLY
PEN-BETA-AMINO-TET-QUIN-SULF
PEN-CEP-AMINO-TET
Sampling source No. of isolate No. of STs Sequence types (STs)
Diarrheal piglet pens 29 26
Rectal swab 17 14 ST10, ST88, ST100, ST165, ST542, ST641, ST1114, ST1421, ST3057,
ST5218, ST5541, ST5706, ST5708, ST5709
Feed from feeder 7 7 ST48, ST100, ST156, ST206, ST410, ST5710, ST5711
Water from nipple 2 2 ST218, ST5712
Waste swab 2 2 ST1114, ST5709
Worker’s hand 1 1 ST5713
Healthy piglet pens 23 18
Rectal swab 14 11 ST10, ST29, ST34, ST101, ST206, ST641, ST1112, ST1114, ST5176,
ST5229, ST5707
Feed from feeder 5 4 ST10, ST48, ST156, ST1114
Water from nipple 1 1 ST5714
Waste swab 3 2 ST4429, ST5707
Worker’s hand 0 0 -
36
Table 5.14 Frequency of sequence types from 52 representative isolates from diarrheal and healthy weaning piglets and environmental samples
• The 52 E. coli isolates were assigned to 32 distinct STs with 11 new STs. • Among 11 novel STs
• 9 STs were detected from diarrheal piglet pens. • 2 STs were detected from healthy piglet pens.
• Majority of isolates were assigned to ST10 and ST1114.
37
Fig. 5.15 Clonal relationship of 32 STs among 52 Escherichia coli isolates identified by goeBURST. SLV are connected via black lines. The size of the circles is related to the number of isolates in STs.
• 18 out of 32 identified STs were grouped into 6 different
CCs • 14 STs were singleton
• Isolates from different sources were closely related especially CC10 and CC1114.
• CC10 were recovered from rectal swab, feed, water, and hand samples from both diarrheal and healthy piglet pens.
• CC1114 were also found in rectal swab, feed, waste samples from diarrheal and healthy piglet pens.
38
Fig. 5.16 Population structure identified by fullMST (minimum spanning tree) using goeBURST analysis based on STs and among 296 STs including STs of this study strains and those deposit in the database
• 30 CCs were generated in this study.
• 10 CCs belonging to CC10, CC88, CC101, CC156, CC542, CC877, CC1114, CC1421, CC2951, and CC5218 were associated with STs in this study .
• The CC10 was the large CC that related to isolates collected from Hungary, Italy, UK, and Germany in 1988, 1993, 1999, and 2004, respectively.
• The CC88 was related to isolates collected from Germany in 2004.
• The CC1114 was related to isolates collected from UK, Germany, and Canada in 1990, 1997, and 1998, respectively.
39
Table 5.16 Genetic variation among single locus variant members of novel sequence types (STs) and clonal complex of Escherichia coli isolates in this study
40
EC58-Rectal swab:ST100
EC132-Feed:ST100
EC29-Rectal swab:ST100
EC163-Waste:ST1114
EC8-Rectal swab:ST1114
EC13-Rectal swab:ST165
EC113-Rectal swab:ST5709
EC172-Waste:ST5709
EC83-Rectal swab:ST10
EC179-Hand:ST5713
EC78-Rectal swab:ST542
EC156-Water:ST218
EC142-Feed:ST48
EC43-Rectal swab:ST5708
EC44-Rectal swab:ST1421
EC134-Feed:ST206
EC136-Feed:ST5710
EC152-Water:ST5712
EC1-Rectal swab:ST5218
EC68-Rectal swab:ST5218
EC18-Rectal swab:ST5541
EC133-Feed :ST156
EC131-Feed:ST410
EC65-Rectal swab:ST88
EC9-Rectal swab:ST5706
EC98-Rectal swab:ST88
EC104-Rectal swab:ST641
EC143-Feed:ST5711
E.coli ATCC25922
EC5-Rectal swab:ST3057
100
90
99
9952
31
18
9
45
90
34
11
63
82
92
96
87
74
0.005
Fig. 5.21
• 29 E. coli isolates from diarrheal weaning piglets and environmental samples were
analyzed by ML tree based on the concatenated sequences of seven housekeeping genes loci (3,423 bp).
• Same STs of E. coli isolates in diarrheal piglet pens were isolated from different sources.
• ST100 represented isolates recovered from rectal swab and feed samples.
• ST1114 and ST5709 represented isolates recovered from rectal and waste swab samples.
Intra-relationship analysis
• These results indicated that E. coli isolates from rectal swab and environmental samples might be originated from the same clone.
41
EC338-Waste:ST5707
EC339-Waste:ST5707
EC283-Rectal swab:ST5707
EC304-Feed:ST1114
EC253-Rectal swab:ST1114
EC241-Rectal swab:ST1114
EC183-Rectal swab:ST1112
EC216-Rectal swab:ST34
EC325-Water:ST5714
EC184-Rectal swab:ST10
EC215-Rectal swab:ST10
EC203-Rectal swab:ST10
EC310-Feed:ST10
EC343-Waste:ST4429
EC305-Feed:ST48
EC315-Feed:ST48
EC275-Rectal swab:ST206
EC219-Rectal swab:ST5176
EC210-Rectal swab:ST641
EC187-Rectalswab:ST101
EC239-Rectal swab:ST5229
EC226-Rectal swab:ST29
EC299-Feed:ST156
E.coli ATCC25922
100
64
63
35
17
55
79
98
76
66
95
89
56
84
0.002
Fig. 5.22
• 23 E. coli isolates from weaning piglets
without diarrhea and environmental samples were also analyzed by ML
• The same STs were isolated from different sources.
• ST10 and ST1114 represented isolates obtained from rectal swab and feed samples.
• ST5707 represented isolates obtained from rectal and waste swab samples.
Intra-relationship analysis
• These results indicated that E. coli from
rectal swab and environmental samples might be originated from same origin
42
EC58-Rectal swab:ST100 EC132-Feed:ST100 EC29-Rectal swab:ST100
EC283-Rectal swab:ST5707 EC338-Waste:ST5707 EC339-Waste:ST5707
EC304-Feed:ST1114 EC253-Rectal swab:ST1114 EC241-Rectal swab:ST1114 EC163-Waste:ST1114 EC8-Rectal swab:ST1114 EC13-Rectal swab:ST165
EC113-Rectal swab:ST5709 EC172-Waste:ST5709
EC183-Rectal swab:ST1112 EC216-Rectal swab:ST34
EC83-Rectal swab:ST10 EC184-Rectal swab:ST10 EC215-Rectal swab:ST10 EC203-Rectal swab:ST10 EC179-Hand:ST5713
EC310-Feed:ST10 EC325-Water:ST5714
EC156-Water:ST218 EC78-Rectal swab:ST542
EC343-Waste:ST4429 EC142-Feed:ST48 EC305-Feed:ST48 EC315-Feed:ST48
EC43-Rectal swab:ST5708 EC143-Feed:ST5711 EC104-Rectal swab:ST641 EC210-Rectal swab:ST641
EC44-Rectal swab:ST1421 EC219-Rectal swab:ST5176
EC134-Feed:ST206 EC275-Rectal swab:ST206
EC133-Feed :ST156 EC299-Feed:ST156
EC187-Rectalswab:ST101 EC239-Rectal swab:ST5229
EC136-Feed:ST5710 EC152-Water:ST5712
EC1-Rectal swab:ST5218 EC68-Rectal swab:ST5218
EC18-Rectal swab:ST5541 EC226-Rectal swab:ST29
EC131-Feed:ST410 EC65-Rectal swab:ST88 EC9-Rectal swab:ST5706
EC98-Rectal swab:ST88 EC5-Rectal swab:ST3057
100
9399
8689
99
83
98
46
27
89
23
69
21
11
12
31
60
98
76
89
85
33
6266
75
63
77
40
0.005
Fig. 5.23
Inter-relationship analysis
• 52 E. coli isolates from weaning piglets with and without diarrheal were analyzed phylogenetic relationship by using ML.
• ST10, ST48, ST156, ST206, ST641 and ST1114 were isolated from both diarrheal and healthy piglet pens.
• ST3057 isolated from rectal swab sample in diarrheal piglet pen was demonstrated different from other isolates that presenting high degree of genetic diversity in ML
• Circle: E. coli from diarrheal piglet pens. • Bold circles: E. coli from health piglet pens
43 adk fumC gyrB icd mdh purA recA Virulence profile
Rectal swab 10 27 5 10 12 9 2 ST-100 lt/F4/astA
Rectal swab 10 27 5 10 12 9 2 ST-100 lt/F18/astA
Feed 10 27 5 10 12 9 2 ST-100 lt/F4/astA
Waste 10 736 5 10 12 1 2 ST-5707 astA
Rectal swab 10 736 5 10 12 1 2 ST-5707 -
Waste 10 736 5 10 12 1 2 ST-5707 astA
Rectal swab 10 27 5 10 12 1 2 ST-1114 astA
Waste 10 27 5 10 12 1 2 ST-1114 astA
Rectal swab 10 27 5 10 12 1 2 ST-1114 astA
Rectal swab 10 27 5 10 12 1 2 ST-1114 astA
Feed 10 27 5 10 12 1 2 ST-1114 -
Rectal swab 10 27 5 10 12 8 2 ST-165 lt/F4/astA
Rectal swab 10 11 5 10 8 1 2 ST-1112 -
Rectal swab 10 11 5 8 12 1 2 ST-5709 -
Waste 10 11 5 8 12 1 2 ST-5709 -
Feed 10 11 4 8 8 8 2 ST-10 -
Rectal swab 10 11 4 8 8 8 2 ST-10 -
Rectal swab 10 11 4 8 8 8 2 ST-10 -
Rectal swab 10 11 4 8 8 8 2 ST-10 -
Rectal swab 10 11 4 8 8 8 2 ST-10 -
Worker 10 11 4 426 8 8 2 ST-5713 astA
Water 10 11 4 8 179 8 6 ST-5714 -
Water 10 11 4 12 8 8 2 ST-218 -
Rectal swab 10 11 4 1 8 8 2 ST-34 -
Rectal swab 112 11 5 12 8 8 86 ST-542 astA
Waste 112 11 5 8 8 8 86 ST-4429 -
Feed 6 11 4 8 8 8 2 ST-48 -
Feed 6 11 4 8 8 8 2 ST-48 -
Feed 6 11 4 8 8 8 2 ST-48 -
Rectal swab 10 4 5 8 8 8 2 ST-5708 F41/astA
Rectal swab 8 7 1 8 8 8 2 ST-1421 -
Rectal swab 427 636 188 1 8 18 6 ST-5176 -
Feed 6 7 5 1 8 18 2 ST-206 astA
Rectal swab 6 7 5 1 8 18 2 ST-206 -
Feed 6 29 32 16 11 8 44 ST-156 -
Feed 6 29 32 16 11 8 44 ST-156 astA
Rectal swab 43 41 15 18 11 7 6 ST-101 -
Rectal swab 43 41 15 18 11 7 44 ST-5229 -
Feed 43 41 15 90 50 8 6 ST-5710 F4
Water 6 4 33 16 11 7 6 ST-5712 -
Rectal swab 6 19 3 16 9 8 341 ST-5218 F18/astA
Rectal swab 6 19 3 16 9 8 341 ST-5218 F18/astA
Rectal swab 6 19 3 16 9 9 341 ST-5541 astA
Rectal swab 6 4 12 16 9 7 7 ST-29 -
Feed 6 4 12 1 20 18 7 ST-410 -
Rectal swab 6 4 12 1 20 12 7 ST-88 lt
Rectal swab 6 4 12 1 411 12 7 ST-5706 F18
Rectal swab 6 4 12 1 20 12 7 ST-88 lt
Feed 9 175 4 131 24 8 7 ST-5711 -
Rectal swab 9 6 33 131 24 8 7 ST-641 -
Rectal swab 9 6 33 131 24 8 7 ST-641 -
Rectal swab 290 54 55 324 35 40 223 ST-3057 -
Source ST type
Allele profile
EC29-Rectal swab:ST100
EC58-Rectal swab:ST100
EC132-Feed:ST100
EC338-Waste:ST5707
EC283-Rectal swab:ST5707
EC339-Waste:ST5707
EC241-Rectal swab:ST1114
EC163-Waste:ST1114
EC8-Rectal swab:ST1114
EC253-Rectal swab:ST1114
EC304-Feed:ST1114
EC13-Rectal swab:ST165
EC183-Rectal swab:ST1112
EC113-Rectal swab:ST5709
EC172-Waste:ST5709
EC310-Feed:ST10
EC203-Rectal swab:ST10
EC83-Rectal swab:ST10
EC215-Rectal swab:ST10
EC184-Rectal swab:ST10
EC179-Hand:ST5713
EC325-Water:ST5714
EC156-Water:ST218
EC216-Rectal swab:ST34
EC78-Rectal swab:ST542
EC343-Waste:ST4429
EC305-Feed:ST48
EC142-Feed:ST48
EC315-Feed:ST48
EC43-Rectal swab:ST5708
EC44-Rectal swab:ST1421
EC219-Rectal swab:ST5176
EC134-Feed:ST206
EC275-Rectal swab:ST206
EC133-Feed :ST156
EC299-Feed:ST156
EC187-Rectalswab:ST101
EC239-Rectal swab:ST5229
EC136-Feed:ST5710
EC152-Water:ST5712
EC1-Rectal swab:ST5218
EC68-Rectal swab:ST5218
EC18-Rectal swab:ST5541
EC226-Rectal swab:ST29
EC131-Feed:ST410
EC65-Rectal swab:ST88
EC9-Rectal swab:ST5706
EC98-Rectal swab:ST88
EC143-Feed:ST5711
EC104-Rectal swab:ST641
EC210-Rectal swab:ST641
EC5-Rectal swab:ST3057
98
9399
89
76
75
69100
6999
46
4186
89
83
3498
33
32
3085
28
27
23
69
32
22
28
60
89
2162
21
16
49
6
4
4
3
5
12
57
66
77
12
78
9
3
4
Fig. 5.24 The association between ST and virulence profiles of 52 E. coli isolates
• Association between ST and virulence profiles were observed in 6 STs, ST10, ST48, ST88, ST641, ST5709 and ST5218.
• 4 STs were non pathogenic E. coli.
• ST88 (lt+) • ST5218 (F18+/astA+)
• 5 STs; ST100, ST5707, ST1114, ST206, and ST156 showed different virulence profiles within same STs.
Association between ST and virulence profiles
44
ABO resistance profile Integron
Rectal swab ST-100 AMP-AMC-CTX-CAZ-KAN-TET-DO-OTC-ENR-SXT Int1
Rectal swab ST-100 AMP-AMC-CTX-CAZ-KAN-STR-GEN-TET-DO-OTC-ENR-SXT Int1
Feed ST-100 AMP-AMC-CTX-CAZ-KAN-GEN-TET-DO-OTC-ENR-SXT -
Waste ST-5707 AMP-AMC-CTX-CAZ-KAN-STR-GEN-TET-DO-OTC-ENR-SXT Int1- Int2
Rectal swab ST-5707 AMP-CTX-CAZ-KAN-GEN-ENR-SXT Int2
Waste ST-5707 AMP-STR-GEN-TET-DO-OTC-ENR-SXT Int2
Rectal swab ST-1114 AMP-CTX-CAZ-KAN-STR-GEN-TET-DO-OTC-ENR-SXT Int1
Waste ST-1114 AMP-CTX-CAZ-STR-GEN-TET-DO-OTC-ENR-SXT-CT -
Rectal swab ST-1114 AMP-CTX-KAN-GEN-TET-DO-OTC-ENR -
Rectal swab ST-1114 AMP-CTX-TET-DO-OTC -
Feed ST-1114 AMP-CTX-CAZ-KAN-STR-GEN-ENR-SXT Int1
Rectal swab ST-165 AMP-AMC-CTX-CAZ-KAN-GEN-TET-DO-OTC-ENR Int1
Rectal swab ST-1112 AMP-CTX-STR-GEN-TET-DO-OTC-ENR-SXT Int1
Rectal swab ST-5709 AMP-CTX-STR-GEN-TET-DO-OTC-ENR-SXT -
Waste ST-5709 AMP-CTX-STR-GEN-TET-DO-OTC-ENR--SXT Int1
Feed ST-10 AMP-CTX-KAN-STR-GEN-TET-DO-OTC-ENR-SXT Int1
Rectal swab ST-10 AMP-CTX-KAN-STR-GEN-TET-DO-OTC-ENR-SXT Int1
Rectal swab ST-10 AMP-TET-DO-OTC-ENR Int1
Rectal swab ST-10 AMP-CTX-GEN-TET-DO-OTC-ENR -
Rectal swab ST-10 AMP-CTX-STR-GEN-TET-DO-OTC-ENR-SXT-CT Int1
Worker ST-5713 AMP-CTX-STR-GEN-TET-DO-OTC-ENR-SXT Int1
Water ST-5714 AMP-CTX-CAZ-STR-GEN-TET-DO-OTC-ENR-SXT-CT Int1
Water ST-218 AMP-STR-TET-DO-OTC-ENR-SXT Int2
Rectal swab ST-34 AMP-AMC-CTX-CAZ-STR-GEN-TET-DO-OTC-ENR -
Rectal swab ST-542 AMP-STR-TET-DO-OTC-ENR-SXT Int1-Int2
Waste ST-4429 AMP-AMC-CTX-CAZ-KAN-STR-GEN-TET-DO-OTC-ENR-SXT Int1- Int2
Feed ST-48 AMP-CTX-CAZ-KAN-STR-GEN-TET-DO-OTC-ENR -
Feed ST-48 AMP-CTX-KAN-STR-GEN-TET-DO-OTC-ENR-SXT Int1
Feed ST-48 AMP-CTX-CAZ-STR-GEN-TET-DO-OTC-ENR Int1
Rectal swab ST-5708 AMP-KAN-STR-GEN-TET-DO-OTC-ENR-SXT Int1
Rectal swab ST-1421 AMP-KAN-GEN-TET-DO-OTC-ENR -
Rectal swab ST-5176 AMP-KAN-GEN-TET-DO-OTC-ENR-SXT Int1
Feed ST-206 AMP-CTX-CAZ-KAN-STR-GEN-TET-DO-OTC-ENR-SXT Int1
Rectal swab ST-206 AMP-CTX-CAZ-KAN-STR-GEN-TET-DO-OTC-ENR-SXT Int1
Feed ST-156 AMP-CTX-KAN-STR-GEN-TET-DO-OTC-ENR-SXT Int1
Feed ST-156 AMP-CTX-KAN-STR-GEN-TET-DO-OTC-ENR-SXT -
Rectal swab ST-101 AMP-STR-TET-DO-OTC-ENR-CT -
Rectal swab ST-5229 AMP-CTX-CAZ-GEN-TET-DO-OTC-ENR-CT -
Feed ST-5710 AMP-AMC-STR-GEN-TET-DO-OTC-ENR-SXT Int1
Water ST-5712 AMP-CTX-KAN-GEN-TET-DO-OTC-ENR -
Rectal swab ST-5218 AMP-CTX-CAZ-STR-GEN-TET-OTC-ENR-SXT Int1
Rectal swab ST-5218 AMP-CTX-CAZ-KAN-STR-GEN-TET-DO-OTC-ENR-SXT -
Rectal swab ST-5541 AMP-CTX-STR-GEN-TET-DO-OTC-ENR-SXT Int1
Rectal swab ST-29 AMP-CTX-STR-GEN-TET-DO-OTC-ENR-SXT Int1
Feed ST-410 AMP-CTX-STR-GEN-TET-DO-OTC-ENR -
Rectal swab ST-88 AMP-CTX-KAN-ENR -
Rectal swab ST-5706 AMP-CTX-CAZ-KAN-STR-GEN-TET-OTC-ENR -
Rectal swab ST-88 AMP-AMC-CTX-CAZ-STR-GEN-TET-DO-OTC-SXT Int1
Feed ST-5711 ENR -
Rectal swab ST-641 AMP-STR-GEN-TET-DO-OTC-SXT Int1
Rectal swab ST-641 AMP-CTX-CAZ-STR-GEN-ENR -
Rectal swab ST-3057 AMP-CTX-CAZ-GEN-TET-OTC -
Source ST type
EC29-Rectal swab:ST100
EC58-Rectal swab:ST100
EC132-Feed:ST100
EC338-Waste:ST5707
EC283-Rectal swab:ST5707
EC339-Waste:ST5707
EC241-Rectal swab:ST1114
EC163-Waste:ST1114
EC8-Rectal swab:ST1114
EC253-Rectal swab:ST1114
EC304-Feed:ST1114
EC13-Rectal swab:ST165
EC183-Rectal swab:ST1112
EC113-Rectal swab:ST5709
EC172-Waste:ST5709
EC310-Feed:ST10
EC203-Rectal swab:ST10
EC83-Rectal swab:ST10
EC215-Rectal swab:ST10
EC184-Rectal swab:ST10
EC179-Hand:ST5713
EC325-Water:ST5714
EC156-Water:ST218
EC216-Rectal swab:ST34
EC78-Rectal swab:ST542
EC343-Waste:ST4429
EC305-Feed:ST48
EC142-Feed:ST48
EC315-Feed:ST48
EC43-Rectal swab:ST5708
EC44-Rectal swab:ST1421
EC219-Rectal swab:ST5176
EC134-Feed:ST206
EC275-Rectal swab:ST206
EC133-Feed :ST156
EC299-Feed:ST156
EC187-Rectalswab:ST101
EC239-Rectal swab:ST5229
EC136-Feed:ST5710
EC152-Water:ST5712
EC1-Rectal swab:ST5218
EC68-Rectal swab:ST5218
EC18-Rectal swab:ST5541
EC226-Rectal swab:ST29
EC131-Feed:ST410
EC65-Rectal swab:ST88
EC9-Rectal swab:ST5706
EC98-Rectal swab:ST88
EC143-Feed:ST5711
EC104-Rectal swab:ST641
EC210-Rectal swab:ST641
EC5-Rectal swab:ST3057
98
9399
89
76
75
69100
6999
46
4186
89
83
3498
33
32
3085
28
27
23
69
32
22
28
60
89
2162
21
16
49
6
4
4
3
5
12
57
66
77
12
78
9
3
4
Fig. 5.25 The correlation between MLST and virulence profiles of 52 E. coli isolates
• Most of STs from MLST analysis and profiles of antimicrobial resistance phenotypes and integrons were not correlated.
• The same STs showed highly variable patterns of multi-antimicrobial
resistance including ST100, ST5707, ST1114, ST10, ST48, ST5218, ST88, and ST641.
• 3 STs including ST156, ST206, and ST5709 revealed correlation between STs and antimicrobial resistance profiles.
• Interestingly, ST5709 were detected from rectal swab and waste swab samples in diarrheal piglet pens that showed correlation with virulence and antimicrobial resistance profiles
Association between ST and antimicrobial resistance patterns
45
Fig. 5.26 Genetic relationship of Escherichia coli isolates. The ML tree was constructed based on the concatenated sequences of seven housekeeping genes loci (3,423 bp) of 95 STs from 115 isolates (32 STs from 52 isolates from this study) obtained from swine sources in Asia from database.
• The result demonstrated that the 32 STs of this study showed diverse genetic background.
• ST3057 that showed high genetic diversity for this study but it was identical with E. coli isolates from China.
ST
10
0:E
C5
8-R
ecta
l sw
ab:T
hai
lan
d
ST
100:E
C132-F
eed
:Thail
and
26
ST
100:E
C29-R
ecta
l sw
ab:T
hai
land
62
ST
5125:J
apan
87
ST
5707:E
C283-R
ecta
l sw
ab:T
hai
land
ST
5707:E
C338-W
aste
:Thai
land
ST
5707:E
C339-W
aste
:Thai
land
23
62
11
ST
1114:E
C8-R
ecta
l sw
ab:T
hai
land
2
ST
1114:E
C253-R
ecta
l sw
ab:T
hai
land
ST
1114
:EC
304-
Fee
d:T
hailan
d
1
1
ST
1114
:EC
241-
Rec
tal s
wab
:Tha
ilan
d
3
ST
1114
:EC
163-
Was
te:T
hailan
d
48
ST36
57:C
hina
ST42
41:C
hinaST
165:
EC13
-Rec
tal s
wab
:Tha
iland
ST3899
:Chi
na
223654
61
ST5709
:EC11
3-Rec
tal s
wab
:Tha
iland
ST5709:EC172-W
aste:
Thailan
d
81
59
ST1112:EC183-R
ectal
swab
:Thail
and
ST4704:Lao
s
ST1695:ChinaST34:EC216-R
ectal swab:Thaila
nd
ST3484:China
476327620
ST4429:EC343-Waste:Thailand
ST3298:China
80 ST3656:China
20ST1731:China
14
ST3661:China
ST5714:EC325-Water:Thailand
ST10:EC310-Feed:Thailand
ST3658:China4
ST10:EC83-Rectal swab:ThailandST10:EC184-Rectal swab:ThailandST10:EC215-Rectal swab:Thailand
ST10:EC203-Rectal swab:Thailand
ST5713:EC179-Hand:ThailandST3628:China
123
22
4
13
116
3
2
ST1702:ChinaST3659:China
29
9
ST218:EC156-Water:Thailand
ST3744:China
82ST4545:Japan
ST3940:China
ST4242China
ST542:EC78-Rectal swab:Thailand
ST3725:China
64
69
70
31
12
3
ST5069:China
ST47:Indonesia
ST5222:C
hina
ST
3402:China
ST
2962:China
ST
1421:EC
44-Rectal sw
ab:Thailand
ST
3660:China
6795
9645
5542
2
ST
48:E
C142-F
eed:T
hailan
d
ST
48:E
C315-F
eed:T
hailan
d
34 S
T48:E
C305-F
eed:T
hailan
d
84
ST
1688:C
hin
a
34
ST
1700:C
hin
a
ST
3404:C
hin
a
ST
4543:C
hin
a
15
ST
1690:C
hin
a
ST
3630:C
hin
a
ST
3631:C
hin
aS
T3634:C
hin
a
98 5
9 40
15
38
43
22
ST
5708:E
C43-R
ecta
l sw
ab:T
hai
land
ST
1691:C
hin
a 36
ST
5176:E
C219-R
ecta
l sw
ab:T
hai
land
ST
3629:C
hin
a
ST
206:E
C134-F
eed:T
hai
land
ST
206:
EC
275-
Rec
tal sw
ab:T
hail
and
72 64 43 6
12
ST
156:
EC
133-
Fee
d:T
hailan
d
ST
156:
EC
299-
Fee
d:T
hailan
d
93
ST57
12:E
C15
2-W
ater
:Tha
ilan
d
ST36
75:C
hina 26
12
ST52
18:E
C1-
Rec
tal s
wab
:Tha
iland
ST5218
:EC68
-Rec
tal s
wab
:Tha
iland
82
ST5541
:EC18
-Rec
tal s
wab
:Tha
iland
98
ST67 :Indones
ia
ST4547:Japan 69
26ST1820:Korea
11ST3939:C
hina
ST29:EC226-Rectal sw
ab:Thailand
ST1693:China
ST410:EC131-Feed:Thailand
ST2273:Japan
48ST88:EC98-Rectal swab:Thailand
ST88:EC65-Rectal swab:Thailand
ST5706:EC9-Rectal swab:ThailandST3870:China 54
3582
48
5939
94
42
14
ST710:ChinaST1701:China
ST4544:Japan
99
ST1689:China
ST2292:Japan
ST5710:EC136-Feed:Thailand
ST101:EC187-Rectal swab:Thailand
ST5229:EC239-Rectal swab:Thailand
879979
59
75
72
18
ST5711:EC143-Feed:ThailandST3485:China
ST641:EC104-Rectal swab:Thailand
ST641:EC210-Rectal swab:Thailand
895099
22
ST1699:China
30
ST117 :Laos
ST1698:China
100
38
ST1694:China
ST1687:China
ST1697:China
8998
38
ST2291:Japan
ST4546:Japan
ST5171:C
hina
6743
45
ST
3057:EC
5-Rectal sw
ab:Thailand
ST
3057:China
96
ST
3058:China
ST
3277:C
hin
a
38100
ST
5084:C
hin
a
Genetic relationship of E. coli isolated from swine sources in Asia
• 52 E. coli isolates were generated 32 distinct STs with 11 new STs from
indicating that they were genetically diverse.
• ST10 and ST1114 were the common ST circulating in both diarrheal and healthy weaning piglets and their environments.
• Phylogenetic analysis revealed that the same STs in various sources among diarrheal and healthy piglet pens were found indicating the same clone contaminated in these sources causing the infection.
46
Conclusion and Recommendation
• ST10, ST48, ST156, ST206, ST641 and ST1114 were isolated from both diarrheal and healthy piglet pens.
• Notably, it was demonstrated that E. coli from either diarrheal or
healthy weaning piglets could be a source of transmission through their environments, or vice versa.
• Moreover, the STs of E. coli isolates and virulence/ antimicrobial resistance/integrons were not correlated in this study.
47
Conclusion and Recommendation
THANK YOU FOR ATTENTION
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