MLST FOR EPIDEMIOLOGICAL STUDY - MUT

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)

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

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

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


ST3744:China

82ST4545:Japan

ST3940:China

ST4242China


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


ST2273:Japan

48ST88:EC98-Rectal swab:Thailand


ST5706:EC9-Rectal swab:ThailandST3870:China 54

3582

48

5939

94

42

14


ST4544:Japan

99

ST1689:China

ST2292:Japan




879979

59

75

72

18

ST5711:EC143-Feed:ThailandST3485:China



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-AMINO-TET-QUIN-SULF

PEN-CEP-AMINO-QUIN

PEN-BETA-CEP-AMINO-TET-SULF

PEN-CEP-AMINO-TET-QUIN








PEN-CEP-AMINO-TET-QUIN-SULF-POLY

PEN-AMINO-TET-SULF

PEN-CEP-AMINO-QUIN

QUIN



PEN-BETA-CEP-AMINO-TET-QUIN





PEN-TET-QUIN

PEN- CEP-AMINO-TET-QUIN-SULF





PEN-AMINO-TET-QUIN

PEN-BETA-CEP-AMINO-TET-QUIN-SULF








PEN-BETA-CEP-AMINO-TET-QUIN



PEN-CEP-AMINO-QUIN-SULF


PEN-CEP-AMINO-QUIN-SULF


PEN-CEP-TET


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


EC163-Waste:ST1114




EC172-Waste:ST5709


EC179-Hand:ST5713


EC156-Water:ST218

EC142-Feed:ST48



EC134-Feed:ST206

EC136-Feed:ST5710

EC152-Water:ST5712




EC133-Feed :ST156

EC131-Feed:ST410





EC143-Feed:ST5711

E.coli ATCC25922


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


EC304-Feed:ST1114





EC325-Water:ST5714




EC310-Feed:ST10

EC343-Waste:ST4429

EC305-Feed:ST48

EC315-Feed:ST48




EC187-Rectalswab:ST101



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


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



EC131-Feed:ST410 EC65-Rectal swab:ST88 EC9-Rectal swab:ST5706


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


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



Feed 10 27 5 10 12 1 2 ST-1114 -


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 -


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 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



EC132-Feed:ST100

EC338-Waste:ST5707


EC339-Waste:ST5707


EC163-Waste:ST1114



EC304-Feed:ST1114




EC172-Waste:ST5709

EC310-Feed:ST10





EC179-Hand:ST5713

EC325-Water:ST5714

EC156-Water:ST218



EC343-Waste:ST4429

EC305-Feed:ST48

EC142-Feed:ST48

EC315-Feed:ST48




EC134-Feed:ST206


EC133-Feed :ST156

EC299-Feed:ST156



EC136-Feed:ST5710

EC152-Water:ST5712





EC131-Feed:ST410




EC143-Feed:ST5711




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-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 -

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 -



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



EC132-Feed:ST100

EC338-Waste:ST5707


EC339-Waste:ST5707


EC163-Waste:ST1114



EC304-Feed:ST1114




EC172-Waste:ST5709

EC310-Feed:ST10





EC179-Hand:ST5713

EC325-Water:ST5714

EC156-Water:ST218



EC343-Waste:ST4429

EC305-Feed:ST48

EC142-Feed:ST48

EC315-Feed:ST48




EC134-Feed:ST206


EC133-Feed :ST156

EC299-Feed:ST156



EC136-Feed:ST5710

EC152-Water:ST5712





EC131-Feed:ST410




EC143-Feed:ST5711




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



ST3658:China4

ST10:EC83-Rectal swab:ThailandST10:EC184-Rectal swab:ThailandST10:EC215-Rectal swab:Thailand


ST5713:EC179-Hand:ThailandST3628:China

123

22

4

13

116

3

2


29

9


ST3744:China

82ST4545:Japan

ST3940:China

ST4242China


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


ST2273:Japan

48ST88:EC98-Rectal swab:Thailand


ST5706:EC9-Rectal swab:ThailandST3870:China 54

3582

48

5939

94

42

14


ST4544:Japan

99

ST1689:China

ST2292:Japan




879979

59

75

72

18

ST5711:EC143-Feed:ThailandST3485:China



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

48

MLST FOR EPIDEMIOLOGICAL STUDY - MUT

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