the use of genomics for understanding introduction and

The use of genomics for understanding introduction and propagation routes of

cholera in Africa during the seventh pandemic

François-Xavier WEILL, MD, PhD Enteric Bacterial Pathogens Unit

Infection & Epidemiology Department

June 9th 2017

Cholera

•  Clinical-epidemiologic syndrome

•  Vibrio cholerae, usually O1 with CTX toxin

•  watery diarrhea (rice water) that rapidly lead to dehydration

•  Explosive outbreaks/true pandemics

•  Often in the wake of wars ,civil conflicts, climatic events, famine, refugees

•  1.03 billion people at risk (Asia and Africa)

•  Estimated 2.86 million cases and 95,000 deaths/year (Ali et al. Plos NTD 2015)

•  Treatment: rehydration +++ and antibiotics

History

Classical •  1817-1823 : Asia, Middle East, East Africa •  1829-1851 : Global •  1852-1859 : Global •  1863-1879 : Global •  1881-1896: Global •  1899-1923 Asia, Middle East, Eastern Europe

1st-6th pandemic

El Tor 1961, Indonesia

7th pandemic

R. Koch F. Pacini

Vibrio cholera Vibrio comma 1883, Egypt 1854, Italy

Classical laboratory methods

•  O antigen serogrouping (O1, O139, O37, …) •  Biotyping (El Tor vs Classical) •  O1 serotyping (Ogawa, Inaba, Hiwojima) •  Phage typing •  Multilocus enzyme electrophoresis (MLEE) •  Ribotyping •  Pulsed-field gel electrophoresis •  ctxB (B subunit of cholera toxin) RFLP or sequencing •  tcpA (toxin coregulated pilus A) sequencing •  Sequencing of other virulence genes ….. •  Multiple loci VNTR analysis (MLVA)

Confusion !!!

Vc O1 El Tor (ET) genomes •  N16961 (Heidelberg et al., Nature 2005)

4.03 Mb, 3885 CDSs 2 circular chromosomes (2.96 Mb, 1.07 Mb) CTXΦ phage on CHR1 Large integron island (125 kb) on CHR2

•  22 other genomes, including 12 O1 ET (Chun et al. PNAS 2009)

•  7P isolates contain VSP-1 and VSP-2 •  CTXΦ can be carried on CHR2

Bangladesh 1975

73 GI identified

136 global isolates sequenced including 123 O1 ET

A pioneer genomic study Lineage L2 El Tor (7th pandemic)

•  3 waves •  Role of the Bay of Bengal •  Identification of intercontinental transmission events

Lineage L1 Classical (1st to 6th pandemic)

•  2005 : incidence in sub-Saharan Africa was 95 and 16,600 times higher than that in Asia and Latin America, respectively

•  2005 : CFR in sub-Saharan Africa (1.8%) was 3 times higher than that in Asia (0.6%); no cholera deaths were reported in Latin America.

•  2007-2012, >95,000 cases/y notified by >20 countries in Africa

Why a genomic study on cholera in Africa ?

28 African isolates studied but all but one from East Africa : Angola 1989 (n=1); Djibouti 2007 (n=3); Kenya 2005-2007 (n=17); Mozambique 1991, 2005 (n=6); Tanzania 2009 (n=1).

?

Objectives of our study

Analysis of 1,070 genomes, including 631 from Africa

(45/54 countries)

•  Identify the introduction and transmission routes of V. cholerae O1 ET in Africa, 1970-2014

•  Linkage between the different outbreaks •  Emergence of antimicrobial resistance

742 sequenced isolates (558 from IP) 328 published genomes

Material

0"

50"

100"

150"

200"

250"

1931(1940" 1941(1950" 1951(1960" 1961(1970" 1971(1980" 1981(1990" 1991(2000" 2001(2010" 2011(2014"

Asia"Africa"Europe"America"Oceania"Unknown"

Methodology

1. Illumina sequencing (75-250 bp PE short reads) 2. Mapping against reference genome N16961 (CHR1 and CHR2) 3. SNPs calling and filtering (SMALT), assembling (SPAdes) 4. Phylogenetic and phylogeographic analyses and estimatation of the divergence times of the different lineages/strains (RAxML after Gubbins, Path-O-Gen, BEAST) 5. Extraction of antibiotic resistance genes and their genetic support (Resfinder on read assemblies, PacBio sequencing)

Maximum Likelihood phylogeny

40.0

1070 Vibrio cholerae, 1937-2014

9,300 SNPs Hypermutators

M66 Indonesia 1937

Wave 1

Wave 2

Wave 3

Reference N16961 Bangladesh 1975

Analysis on 3,970,515 nt (98.4%)

RAxML

11 introductions to Africa T1, T3-T12

BEAST

ML

228 isolates (2,961 SNPs); strict clock rate; Bayesian skyline population size

1917

M66

CNRVC960188 ACTGAT L002

CNRVC961190 CATGGC L002

5174 7 5

ERR576982

ERR576981

ERR576972

ERR579055

ERR579057

ERR579060

ERR576980

ERR579058

CNRVC930061 TGACCA L002

ERR579066

5174 7 7

ERR579053

CNRVC960976 CACGAT L002

16244 6 40

CNRVC930060 CGATGT L002

CNRVC940044 ACTTGA L002

ERR579059

ERR576971

ERR579056

ERR577143

ERR579074

ERR579050

CNRVC950218 GTGGCC L002

CNRVC930067 GCCAAT L002

CNRVC961129 CAGGCG L002

CNRVC950165 GTCCGC L002 CNRVC950161 GTAGAG L002

CNRVC950172 GTGAAA L002

ERR579063

ERR579054

CNRVC961077exCNRVC961090 TCGAAG L001

ERR579052

ERR576983

CNRVC930036 TCGGCA L001 CNRVC930066 ACAGTG L002

CNRVC950100 CCGTCC L002

CNRVC930029 TCGAAG L001

CNRVC950641 GTCCGC L001

960871 S20 CNRVC960799 GTGAAA L001

CNRVC930076 CAGATC L002

CNRVC960842 AGTCAA L001

960872 S21

ERR576979

CNRVC961005 CACTCA L002

CNRVC960800 GGCTAC L001

CNRVC960768 TAGCTT L001

960778 S19

ERR579051

THSTI GP160bis

ERR577142

ERR577140

150131 S8

961017 S1 961018 S2 930030 S3

930033 S15 930044 S17 6353 7 21

6353 7 17

16244 7 20

RKI-ZBS2-CH8 CTCAGA L002

CNRVC150244 CTCAGA L001

16244 6 94

16244 7 1

16244 7 2

CNRVC960922 CACCGG L002

5174 7 3

16244 7 55

16244 6 41 16244 7 15

16244 7 54

16244 7 3

16244 7 4

16244 7 1

9 16244 7

16 16244 6

88

16244 6

50

16244 6

90

16244 6

93

16244 6

89

16244 6

91 16244 7

14

16244 7

24 16244 6

46

16244 6

92

16244 6

54

16244 7

56

16244

6 87

16244

6 49

CNRVC150228

CACGAT L001

16244

6 84

CNRVC950250

GTTTCG L0

02

16244

7 18 16

244 7 1

7

16244

7 6

RKI-ZBS2-C

H19 GCGCTA L0

02

16244

7 5

16244

7 57

16244

7 59

NICD 69147

NICD 69173

NICD 69175

16244

6 36

16244

6 34

16244

6 39

16244

6 35 16

244 7 7

2

CNRVC900

125 GCCAA

T L001

16244

6 37

4056

6 5

4056

6 3

CDCC8178 re

p

4056

6 4

THST

I GP1

06bis

9 7 44261

16244

7 8 16244

7 13

16244

6 73

16244

6 72

CNRV

C9300

79 TC

GAAG L0

02

16244

6 61

16244

6 78

16244

6 79

200L GCATGC 043059CVRNC

16244

6 77

16244

6 76

16244

6 62

16244

6 85

16244

6 86

16244

6 43

36 6 44261

16244

6 67

16244

6 65

32 7 44261

96 6 44261

07 6 44261

16244

6 71

16244

7 22

16244

7 21

16244

6 45

86 6 44261

1624

4 7 7

1624

4 6 74

1624

4 6 75

1624

4 6 66

95 6 44261

46 6 44261

1624

4 6 44

85 6 44261

38 6 44261

28 6 44261 18 6 44261

08 6 44261

CNRV

C150

217 G

TGGC

C L00

1

100L ACTCAC 922051CVRNC

65 6 44261

23 7 44261

per 531FCDC

1624

4 6 51

84 6 44261

92 7 44261

03 7 44261

ERR5

7697

8

82 7 44261

43 7 44261 16

244 7

35

25 7 44261

94 7 44261

4 7 4715

per 100L ACCAGT 12 emoT oaS 100L GCACTA 671029CVRNC

15 7 44261

84 7 44261

74 7 44261

05 7 44261

17 7 44261

18 7 44261

28 7 44261

01 8 65361

9 8 65361

100L ATCGCG 542051CVRNC

06 7 44261

100L GGCCAC 722051CVRNC

CNRVC150231 C

ATGG

C L001

16 7 44261

100L AACTGA 012051CVRNC

83 7 44261

2 6 6504

21 6 6504

01 6 6504 9 6 6504 01 7 4715

1 8 4715

2 8 4715

3 8 4715

ERR576950

379675RRE

200L ACTGTA 410059CVRNC

14 7 44261

22 2 8317

per 8978CCDC

0714FCDC

CDCC8799 rep

ERR580006

54 7 44261

73 7 44261

64 7 44261

34 7 44261

44 7 44261

8 8 6504

24 7 44261

100L AGCCCT 520039CVRNC

200L CCTTGA 381049CVRNC

8 7 6504 11 7 6504 1 7 6504

7 7 650 4

01 7 6 504

2 1 7 6 504

8 1S 81 906 9

150132 S9

21 7 44261

24 6 44261

61S 198069

02 7 3536 200L ATCAAC 998069CVRNC

100L CCTTGA 119069CVRNC

8 7 4715

2v 16961N rts rotle ravoib 1O earelohc oirbiV

ERR579083

780975RRE

919975RRE

599675RRE

ERR579070

789675RRE

699675RRE

270975RRE

ERR577139

141775RRE

260975RRE

560975RRE

889675RRE

ERR579088

11 3 5704

2 3 5704

940975RRE

9 7 4715

3 8 6504

100L AAAGTG 612051CVRNC

100L GAGATG 412051CVRNC

200L GCTAAT 99HC-2SBZ-IKR

100L GCTTTG 812051CVRNC

100L TTTTAC 432051CVRNC

13 7 44261 72 7 44261

6 3 5704

100L ACAACC 632051CVRNC

9CR

63 7 44261

35 6 44261

33 7 44261

74 6 44261

37 7 44261

57 7 44261

07 7 44261

47 7 44261

7915FCDC

8915FCDC

per 1369BCDC

per 2369BCDC

75 6 44261

200L TTTTAC 650079CVRNC

66 8 65361

76 8 65361

4 8 65361

39 7 44261

95 8 65361 06 8 65361

16 8 65361

85 8 65361

87 8 65361

86 8 65361 96 8 65361

CDCC8465 rep

CDCC8466 rep

97 7 44261

21 8 65361

98 7 44261

62 8 65361

41 8 65361

31 8 65361

77 7 44261

08 7 44261

87 7 44261

CDCF3397

CDCF3403

81 3 6308

02 3 6308

71 7 7346

CDCF3835

97 8 65361 4 8 44261

2 8 44261CDCF5097 6905FCDC

32 3 6308

91 3 6308

8036 3 17

7 3 5704

ERR579067

100L CTAGAC 040019CVRNC

ERR576970

100L CGACAT 171029CVRNC

1 8 6504

9 7 6504ERR579064

6 7 4715 4 8 6504

3 3 5704

200L ATGTTC 471039CVRNC

THSTI V12

THSTI V108

THSTI V149 1 2 6504

11 2 6504

MO10

ERR579061

CNRVC150222 ACTGAT L001

100L TCCTTA 422051CVRNC

11S 431051

01S 331051

100L ATGTTC 461019CVRNC

100L GCTAAT 614019CVRNC

3931FCDC

100L GACTAG 103039CVRNC

16244 8 52

35 8 44261

200L CGATGG 241069CVRNC

200L TCCTTA 672069CVRNC

CNRVC150252

CNRVC150251

CNRVC150211 AGTTCC L001

16244 8 51


16244 8 3

16244 8 19

16244 8 5

CNRVC150213 CCGTCC L001

CNRVC150254

16356 8 77

16244 8 37

CNRVC030392 CNRVC030391

CNRVC010046 CTTGTA L001 CNRVC010048

4056 6 11

ERR579082

ERR579080

ERR579081

16244 6 52 16244 6 55

16244 6 38

16244 7 25

76 7 44261 16244 7 66

16244 7 90 16244 7 76

16244 7 69

16356 8 6

16356 8 5

62 7 44261 CNRVC900108 ACAGTG L001

16244 7 62 16244 7 83

16244 7 85

16244 7 95

16244 7 94

16244 7 68

16244 8 36 16244 8 35

16356 8 17

16356 8 16

16244 7 63

16244 7 64

16244 7 65

16356 8 11

16356 8 1

16356 8 7 16244 8 34

16244 7 84

16356 8 76 16356 8 72

16356 8 73

16244 8 22

16244 8 24

16244 8 15

16244 8 16 CNRVC990329 CGATGT L001

16356 8 8 16356 8 86

CNRVC990256 GTGGCC L001

16356 8 75

16356 8 91

16356 8 74 16244 8 14

16244 8 1

16244 8 13

16356 8 43

16356 8 39

16356 8 42

16356 8 41

16356 8 80

16356 8 92

16356 8 89

16356 8 90

16356 8 93

16356 8 83

16356 8 82

16356 8 81

CNRVC150220 GAGTGG L001

CNRVC940047 GATCAG L002

CNRVC150221 GGTAGC L001

CNRVC150239 CGGAAT L001

ERR579313

ERR576986

ERR576977

ERR579311

ERR579986

ERR579073

CNRVC930155 TCGGCA L002

CNRVC010100

5174 5 9

3002 8 2

ERR576951

ERR579309

ERR579085

5174 5 7

3002 8 1

ERR576985

ERR579068

ERR576975

ERR579091

ERR579092

ERR579086

3002 8 6

ERR579078

ERR579114

ERR579077 CNRVC990209 CTAGCT L002


ERR576976

ERR579093

ERR579079

ERR579069

ERR579076

ERR576984

3002 8 5

6437 7 2

6437 7 9

6437 7 8

7138 2 1

8014 8 9

8014 8 10

8014 8 8

6353 8 3

6437 7 6

7138 2 3

7138 2 7

8014 8 12

8014 8 11

6437 7 4

7138 2 2

6437 7 14

THSTI V157

THSTI V156

4056 2 4

4056 2 3

CNRVC930003 TCATTC L001

4056 2 10

MJ1236

4056 8 6

CNRVC150223 ATGAGC L001

CNRVC150215 GTCCGC L001

CNRVC150230 CAGGCG L001

CNRVC940149 AGTCAA L002

RKI-ZBS2-CH218 TATAAT L002

6353 7 1

6353 7 5

6353 7 10

CNRVC150219 CGTACG L001



CNRVC950798 CTAGCT L001

CNRVC950792 GAGTGG L002

CNRVC950800 CTATAC L001


NICD 14794

NICD 15070

NICD 16230

NICD 14191

NICD 1405

NICD 1264

NICD 1241

NICD 1242

NICD 273

NICD 16879

NICD 4076

NICD 15104

CDCF8478

CDCF8479 CDCF5149

16244 8 39

16244 8 40

16244 8 41

NICD 15959

NICD 16756

NICD 16211

NICD 15907

NICD 16744

6437 7 23

6437 7 22 B33

6437 7 20

5174 7 1

5174 5 1

5174 6 10

4056 6 7

4056 6 6

THSTI V8 4056 2 6

4056 2 12 5174 8 9

4056 2 5

THSTI VC63


THSTI CO327

THSTI CO368

16356 8 2

16356 8 15

RKI-ZBS2-CH234 TCATTC L002

16244 7 86

16356 8 20

16244 7 53

16356 8 30

16244 7 88

16244 7 87

16244 7 91

16356 8 22

16356 8 21

CDCF5144 16356 8 64

16356 8 63

16356 8 65

16356 8 62

960263 S14

960260 S13

16356 8 35

16356 8 36

CNRVC150226 CAACTA L001

16356 8 40

16356 8 55

16356 8 38

16244 7 92

16356 8 3

16356 8 24

16356 8 23

16356 8 29

16356 8 44

CNRVC010042

CNRVC010038 CAGATC L001

CNRVC030567

CNRVC020404

CNRVC030485

16244 8 28

CNRVC030477

CNRVC030475

CNRVC020393

CNRVC020062

CNRVC080496

10868 2 85

10868 2 47

10868 2 39

CNRVC120195

CNRVC120194

CDCK6975

CDCK6979

CNRVC040206

CNRVC040203

CNRVC040257

CNRVC040314

CNRVC060010

CNRVC070136

CNRVC070144

CNRVC040303

CNRVC060009

CNRVC070142

CNRVC070124

16356 8 49

16356 8 27

16356 8 32

16356 8 33

16356 8 18

16356 8 19

82 8 65361

16356 8 31

16356 8 95 16356 8 94

16356 8 34

16356 8 7

1

48 8 65361

16356 8

85

16244 8

9

16356 8

87

16356 8

88

16244 8

8

CDCF4169

16356 8

47

16356 8

51

16244 8

31

CNRVC980374

TCCCGA L002

16244 8

33

16244 8

30

16244

8 32

16356

8 56 163

56 8 7

0

16356

8 48

16356

8 57

16356

8 54

16356

8 46

16356

8 45

16356

8 37

16356

8 50

16356

8 52

16356

8 53

16244

8 27

16244

8 26

100L TGTAGC 500000CVRNCCNRVC010

023

CNRVC000

323

CNRVC990

234 CTA

TAC L0

02

CNRVC010

108

CNRVC010

205

CNRVC010

206

CNRVC040

210

CNRVC050

334

CNRVC050

323

CNRVC010

123 AC

TTGA L

001

CNRVC010

221

CNRV

C0101

20

CNRV

C0102

03

CNRV

C0500

73

CNRV

C0500

60

CNRV

C0700

12

CNRV

C0700

11

CNRV

C0400

61

CNRV

C0807

64

CNRV

C0807

62

CNRV

C0601

11

CNRV

C0500

11

CNRV

C0400

82

CNRV

C0401

10

CNRV

C0400

74

CNRV

C0500

08

CNRV

C0300

84

CNRV

C0203

67

CNRV

C0203

61

CNRV

C0300

82

CNRV

C0401

27

CNRV

C0402

09 CD

CK010

7

CDCK

0108

CNRV

C0401

32

CNRV

C0602

73

CNRV

C0701

64

CNRV

C0501

40

CNRV

C050

139

CNRV

C080

497

CNRV

C070

334

CNRV

C070

316

CNRV

C060

272

CNRV

C060

279

CNRV

C060

280

CNRV

C080

124

CNRV

C080

122

CNRV

C100

176

1685

3 4 56

CNRV

C100

183

96 4 17921

1297

1 7 78

1297

1 4 61

95 4 17921

1297

1 4 58

THST

I D31

153

THST

I AM3

05

CDCF

5332

100L CATATC 342051CVRNC

100L TCGATC 042051CVRNC 100L ACTGTA 212051CVRNC

CNRV

C030

593

CNRV

C030

594

CNRV

C020

003

4370

2 4

8 2 0734 3 2 0734

11 2 0734

7 2 0734

21 2 0734 2 2 0734

01 2 0734

4370 2 9

6084 8 20

7298 7 27

22 7 8927

7 7 8927

7298 7 5

71 7 8927 62 7 8927

92 7 8927

13 7 8927

82 7 8927

81 8 4806 21 7 8927 51 7 8927

91 8 4806 68 6 8927 58 6 8927

3335FCDC

32 8 44261

160010CVRNC

260010CVRNC

CNRVC010254

351060CVRNC

482050CVRNC

12971 6 75

383080CVRNC

64 4 17921 47 7 17921

4 6 4715

52 4 17921

17 2 86801

35 2 86801

84 7 17921

100L GACTAG 342010CVRNC

CNRVC020284

392030CVRNC

33 3 6308

52 3 6308

62 3 6308SRR227307

63 3 6308

82 3 6 308

903722RRS

81 7 7346

915 0 30C VRNC

300060CVRNC

CNRVC070045

46 2 86801

CNRVC080370

331080CVRNC 260001CVRNCCN

RVC100057

CNRVC070530

47 6 17921

95 7 17921

310041CVRNC

05 4 17921CNRVC140012

24 2 86801

44 2 86801 73 2 86801

1 4 17921

97 2 86801

01 2 86801

54 7 17921

01 7 17921

57 7 17921

272011CVRNC

662011CVRNC

53 4 17921

39 2 86801

96 2 86801

91 7 17921

29 2 86801

82 2 86801

67 2 86801

71 8 44261

81 8 44261

21 8 44261

11 8 44261

01 8 44261

05 8 44261

94 8 44261

100L GCACTA 150010CVRNC 100L CGGATT 550010CVRNC

100L TAACCG 800010CVRNC

580000CVRNC

CNRVC010045

34 8 44261 44 8 44261

24 8 44261

64 8 44261

54 8 44261

84 8 44261 74 8 44261

92 8 44261 52 8 44261

83 8 44261

CNRVC000043

100L ACCAGT 440000CVRNC

7 8 44261

6 8 44261

02 8 44261

12 8 44261

CNRVC000209

CNRVC000212 ACAGTG L001

964030CVRNC

CNRVC030032

770050CVRNC

CNRVC040299

CNRVC060120

020070CVRNC

CNRVC060119

21 8 6504

33 7 8927

01 8 6504 2 8 6504

14 7 8927

51 5 8927

3 7 8927

42 7 8927

31 7 8927

93 7 8927

34 7 8927

6 7 8927 21 5 8927

01 5 8927

15 8 6437

760070CVRNC

CNRVC070066

3 5 8927

02 5 8927 02 7 8927

41 5 8927 74 7 8927 11 7 8927

05 8 6437 44 7 8927 64 7 8927

04 7 8927

83 7 8927

1 3 0734

6 3 0734

8 3 0734

11 3 0734

7 3 0734

CNRVC960215 ATGAGC L002

232MA ITSHT

100L CCTGCC 992099CVRNC

100L ACTGTA 892099CVRNC

THSTI C18932

9 3 5704

200L ACAACC 770079CVRNC

76021E ITSHT

THSTI CRC155

10811H ITSHT

THSTI CRC198

21CR ITSHT

THSTI I17260

6 6 4715

2 6 6592

6 5 4715

THSTI J4770

THSTI J16173

9 2 6504

3 8 2003

3 6 4715 2 5 4715

2 6 4715

THSTI IDH0002

1 6 6592

4 5 4715

423722RRS

ERR576974

7 8 2003

ERR579075

SRR491153

5 6 4176

3 6 4176

32 5 4176

12 5 4176

91 6 4176

2 6 4176

1 6 4176

1 6 4715

5 6 4715

63100HDI ITSHT

CNRVC080397

76320HDI ITSHT

ERR351208

ERR351210

ERR351188

9 6 4715

3 5 4715

THSTI IDH00563

527803RRS

SRR308724

327803RRS

SRR308706

SRR308708

SRR308713

SRR308720

SRR308692

SRR308691

SRR308709

SRR308707

SRR308693

SRR308717

SRR308690

SRR308704

SRR308665

SRR308705

THSTI CO908

THSTI CO1089

THSTI NLC8

THSTI G27875

51661I ITSHT

THSTI H12381

THSTI AM120

THSTI CO783

THSTI CO553

THSTI D16133

3 6 6592

6 6 6592

4056 2 7

5174 6 7

4056 8 9

2956 6 5

CIRS101

4 6 6592

THSTI VC4

ERR579090

ERR579089

THSTI K12759

CNRVC050280

CNRVC090123

CNRVC090143

CNRVC090144

6714 5 8

6714 6 10

6714 5 4

22 6 4176

6714 5 1

2011EL 1137

6437 7 21

SRR227311

0074 L002 R1

0034 L008 R1

1R 800L 5300

121 L008 R1

1019828 5 L007 R1

1020229 6 L007 R1

152 L008 R1

0014 L008 R1

6714 6 23

02 6 4176

6714 5 14

6714 5 2

6714 5 11

6714 5 7

6714 6 18

6714 6 16

21 6 4176

6714 6 4

6714 5 17

81 5 4176

31 6 4176

6714 5 12

6714 5 5

6714 5 20

6714 6 17

6714 6 21

6714 6 6

6714 5 19

8 6 4176

6714 6 9

6714 6 14

6714 6 15

51 5 4176

6714 5 16

THSTI L9496

CNRVC060089

THSTI K9398

7346 8 53

4056 2 2

5174 6 8

THSTI L19089

5174 5 5

CNRVC060333 THSTI IDH01405

THSTI IDH01272

SRR308722

SRR308703

4075 3 12

SRR227303

SRR227335

SRR308721

SRR308716

THSTI IDH02959

THSTI IDH02834

2010EL 1786

2010EL 1792

2010EL 1798

CNRVC100278

SRR135545

SRR135540

SRR191363

SRR191381

SRR308727

SRR308715

SRR308726

7298 7 34

ERR351205

ERR351191

ERR351206

ERR351179

ERR351203

ERR351174

ERR351207

ERR351192

ERR351199

ERR351175

ERR351185

ERR351193

CNRVC090175

CNRVC090182

CNRVC090177

CNRVC140160

CNRVC100224

CNRVC100159

CNRVC110128

CNRVC140167

2010EL 1749

CNRVC100186

CNRVC100240

CNRVC100246

CNRVC110241

CNRVC110230

12971 4 77

12971 4 67

12971 7 77

12971 4 72

12971 4 79

12971 4 76

12971 7 80 CNRVC110096

CNRVC110113

CNRVC110120

CNRVC110058

CNRVC110060

CNRVC110262

CNRVC140027

CNRVC140150

16853 4 62

16853 4 59

16853 4 64

T1 T2

T3

T4

T5

T12

T11

T10

T8

T9

T7

T6

1 2

3

N16

961

0.02

Haiti

2014 1980 1990 1970 2000 1960 1950 1940 1930

GEO

T12

T11

T10

T9

T8

T7

T6

T5 T4 T3

T1

T2

1

2

3

N16961

Haiti

1987 (1986-1988)

1980 (1979-1982)

1958 (1957-1959)

M66

O139

Geographic area (GEO) Africa Asia Middle East Americas Oceania

Keys

Europe Unknown

World Health Organization (WHO)

1970

Ogawa

Ogawa

Inaba

Inaba

Guinea 1970, origin ?

-Guinean students returning from the Black Sea -Pilgrims or soldiers from the Middle East Angola 1971 origin ? -Portugal West Africa

Africa, 1970-2014

11 introductions to Africa Guinea 70 Middle East Angola 71 West Africa

Five introductions to West Africa and six to East Africa Middle East acting as a springboard during six introductions.

Followed by up to 28 years of regional circulation Two separated and persistent foci (West Africa and the Great Lakes-Horn of Africa region). Rare exceptions.

Introduction of the cholera in America in 1991

A5, Angola 1989

Latin America

Different hypotheses:

-a ship from China -El Niño

Martinez-Urtana et al. Nat Microbiol 2016

West African origin ?

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600 610 620 630 640 650 660 670 680 690 700 710 720 730 740 750 760 770 780 790 800 810 820 830 840 850 860 870 880 890 900 910 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200 1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 1350 1360 1370 1380 1390 1400 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500 1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 1610 1620 1630 1640 1650 1660 1670 1680 1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800 1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010 2020 2030 2040 2050 2060 2070 2080 2090 2100 2110 2120 2130 2140 2150 2160 2170 2180 2190 2200 2210 2220 2230 2240 2250 2260 2270 2280 2290 2300 2310 2320 2330 2340 2350 2360 2370 2380 2390 2400 2410 2420 2430 2440 2450 2460 2470 2480 2490 2500 2510 2520 2530 2540 2550 2560 2570 2580 2590 2600 2610 2620 2630 2640 2650 2660 2670 2680 2690 2700 2710 2720 2730 2740 2750 2760 2770 2780 2790 2800 2810 2820 2830 2840 2850 2860 2870 2880 2890 2900 2910 2920 2930 2940 2950 2960 2970 2980 2990 3000 3010 3020 3030 3040 3050 3060 3070 3080 3090 3100 3110 3120 3130 3140 3150 3160 3170 3180 3190 3200 3210 3220 3230 3240 3250 3260 3270 3280 3290 3300 3310 3320 3330 3340 3350 3360 3370 3380 3390 3400 3410 3420 3430 3440 3450 3460 3470 3480 3490 3500 3510 3520 3530 3540 3550 3560 3570 3580 3590 3600 3610 3620 3630 3640 3650 3660 3670 3680 3690 3700 3710 3720 3730 3740 3750 3760 3770 3780 3790 3800 3810 3820 3830 3840 3850 3860 3870 3880 3890 3900 3910 3920 3930 3940 3950 3960 3970 3980 3990 4000 4010 4020 4030 4040 4050 4060 4070 4080 4090 4100 4110 4120 4130 4140 4150 4160 4170 4180 4190 4200 4210 4220 4230 4240 4250 4260 4270 4280 4290 4300 4310 4320 4330 4340 4350 4360 4370 4380 4390 4400 4410 4420 4430 4440 4450 4460 4470 4480 4490 4500 4510 4520 4530 4540 4550 4560 4570 4580 4590 4600 4610 4620 4630 4640 4650 4660 4670 4680 4690 4700 4710 4720 4730 4740 4750 4760 4770 4780 4790 4800 4810 4820 4830 4840 4850 4860 4870 4880 4890 4900 4910 4920 4930 4940 4950 4960 4970 4980 4990 5000 5010 5020 5030 5040 5050 5060 5070 5080 5090 5100 5110 5120 5130 5140 5150 5160 5170 5180 5190 5200 5210 5220 5230 5240 5250 5260 5270 5280 5290 5300 5310 5320 5330 5340 5350 5360 5370 5380 5390 5400 5410 5420 5430 5440 5450 5460 5470 5480 5490 5500 5510 5520 5530 5540 5550 5560 5570 5580 5590 5600 5610 5620 5630 5640 5650 5660 5670 5680 5690 5700 5710 5720 5730 5740 5750 5760 5770 5780 5790 5800 5810 5820 5830 5840 5850 5860 5870 5880 5890 5900 5910 5920 5930 5940 5950 5960 5970 5980 5990 6000 6010 6020 6030 6040 6050 6060 6070 6080 6090 6100 6110 6120 6130 6140 6150 6160 6170 6180 6190 6200 6210 6220 6230 6240 6250 6260 6270 6280 6290 6300 6310 6320 6330 6340 6350 6360 6370 6380 6390 6400 6410 6420 6430 6440 6450 6460 6470 6480 6490 6500 6510 6520 6530 6540 6550 6560 6570 6580 6590 6600 6610 6620 6630 6640 6650 6660 6670 6680 6690 6700 6710 6720 6730 6740 6750 6760 6770 6780 6790 6800 6810 6820 6830 6840 6850 6860 6870 6880 6890 6900 6910 6920 6930 6940 6950 6960 6970 6980 6990 7000 7010 7020 7030 7040 7050 7060 7070 7080 7090 7100 7110 7120 7130 7140 7150 7160 7170 7180 7190 7200 7210 7220 7230 7240 7250 7260 7270 7280 7290 7300 7310 7320 7330 7340 7350 7360 7370 7380 7390 7400 7410 7420 7430 7440 7450 7460 7470 7480 7490 7500 7510 7520 7530 7540 7550 7560 7570 7580 7590 7600 7610 7620 7630 7640 7650 7660 7670 7680 7690 7700 7710 7720 7730 7740 7750 7760 7770 7780 7790 7800 7810 7820 7830 7840 7850 7860 7870 7880 7890 7900 7910 7920 7930 7940 7950 7960 7970 7980 7990 8000 8010 8020 8030 8040 8050 8060 8070 8080 8090 8100 8110 8120 8130 8140 8150 8160 8170 8180 8190 8200 8210 8220 8230 8240 8250 8260 8270 8280 8290 8300 8310 8320 8330 8340 8350 8360 8370 8380 8390 8400 8410 8420 8430 8440 8450 8460 8470 8480 8490 8500 8510 8520 8530 8540 8550 8560 8570 8580 8590 8600 8610 8620 8630 8640 8650 8660 8670 8680 8690 8700 8710 8720 8730 8740 8750 8760 8770 8780 8790 8800 8810 8820 8830 8840 8850 8860 8870 8880 8890 8900 8910 8920 8930 8940 8950 8960 8970 8980 8990 9000 9010 9020 9030 9040 9050 9060 9070 9080 9090 9100 9110 9120 9130 9140 9150 9160 9170 9180 9190 9200 9210 9220 9230 9240 9250 9260 9270 9280 9290 9300 9310 9320 9330 9340 9350 9360 9370 9380 9390 9400 9410 9420 9430 9440 9450 9460 9470 9480 9490 9500 9510 9520 9530 9540 9550 9560 9570 9580 9590 9600 9610 9620 9630 9640 9650 9660 9670 9680 9690 9700 9710 9720 9730 9740 9750 9760 9770 9780 9790 9800 9810 9820 9830 9840 9850 9860 9870 9880 9890 9900 9910 9920 9930 9940 9950 9960 9970 9980 9990 10000 10010 10020 10030 10040 10050 10060 10070 10080 10090 10100 10110 10120 10130 10140 10150 10160 10170 10180 10190 10200 10210 10220 10230 10240 10250 10260 10270 10280 10290 10300 10310 10320 10330 10340 10350 10360 10370 10380 10390 10400 10410 10420 10430 10440 10450 10460 10470 10480 10490 10500 10510 10520 10530 10540 10550 10560 10570 10580 10590 10600 10610 10620 10630 10640 10650 10660 10670 10680 10690 10700 10710 10720 10730 10740 10750 10760 10770 10780 10790 10800 10810 10820 10830 10840 10850 10860 10870 10880 10890 10900 10910 10920 10930 10940 10950 10960 10970 10980 10990 11000 11010 11020 11030 11040 11050 11060 11070 11080 11090 11100 11110 11120 11130 11140 11150 11160 11170 11180 11190 11200 11210 11220 11230 11240 11250 11260 11270 11280 11290 11300 11310 11320 11330 11340 11350 11360 11370 11380 11390 11400 11410 11420 11430 11440 11450 11460 11470 11480 11490 11500 11510 11520 11530 11540 11550 11560 11570 11580 11590 11600 11610 11620 11630 11640 11650 11660 11670 11680 11690 11700 11710 11720 11730 11740 11750 11760 11770 11780 11790 11800 11810 11820 11830 11840 11850 11860 11870 11880 11890 11900 11910 11920 11930 11940 11950 11960 11970 11980 11990 12000 12010 12020 12030 12040 12050 12060 12070 12080 12090 12100 12110 12120 12130 12140 12150 12160 12170 12180 12190 12200 12210 12220 12230 12240 12250 12260 12270 12280 12290 12300 12310 12320 12330 12340 12350 12360 12370 12380 12390 12400 12410 12420 12430 12440 12450 12460 12470 12480 12490 12500 12510 12520 12530 12540 12550 12560 12570 12580 12590 12600 12610 12620 12630 12640 12650 12660 12670 12680 12690 12700 12710 12720 12730 12740 12750 12760 12770 12780 12790 12800 12810 12820 12830 12840 12850 12860 12870 12880 12890 12900 12910 12920 12930 12940 12950 12960 12970 12980 12990 13000 13010 13020 13030 13040 13050 13060 13070 13080 13090 13100 13110 13120 13130 13140 13150 13160 13170 13180 13190 13200 13210 13220 13230 13240 13250 13260 13270 13280 13290 13300 13310 13320 13330 13340 13350 13360 13370 13380 13390 13400 13410 13420 13430 13440 13450 13460 13470 13480 13490 13500 13510 13520 13530 13540 13550 13560 13570 13580 13590 13600 13610 13620 13630 13640 13650 13660 13670 13680 13690 13700 13710 13720 13730 13740 13750 13760 13770 13780 13790 13800 13810 13820 13830 13840 13850 13860 13870 13880 13890 13900 13910 13920 13930 13940 13950 13960 13970 13980 13990 14000 14010 14020 14030 14040 14050 14060 14070 14080 14090 14100 14110 14120 14130 14140 14150 14160 14170 14180 14190 14200 14210 14220 14230 14240 14250 14260 14270 14280 14290 14300 14310 14320 14330 14340 14350 14360 14370 14380 14390 14400 14410 14420 14430 14440 14450 14460 14470 14480 14490 14500 14510 14520 14530 14540 14550 14560 14570 14580 14590 14600 14610 14620 14630 14640 14650 14660 14670 14680 14690 14700 14710 14720 14730 14740 14750 14760 14770 14780 14790 14800 14810 14820 14830 14840 14850 14860 14870 14880 14890 14900 14910 14920 14930 14940 14950 14960 14970 14980 14990 15000 15010 15020 15030 15040 15050 15060 15070 15080 15090 15100 15110 15120 15130 15140 15150 15160 15170 15180 15190 15200 15210 15220 15230 15240 15250 15260 15270 15280 15290 15300 15310 15320 15330 15340 15350 15360 15370 15380 15390 15400 15410 15420 15430 15440 15450 15460 15470 15480 15490 15500 15510 15520 15530 15540 15550 15560 15570 15580 15590 15600 15610 15620 15630 15640 15650 15660 15670 15680 15690 15700 15710 15720 15730 15740 15750 15760 15770 15780 15790 15800 15810 15820 15830 15840 15850 15860 15870 15880 15890 15900 15910 15920 15930 15940 15950 15960 15970 15980 15990 16000 16010 16020 16030 16040 16050 16060 16070 16080 16090 16100 16110 16120 16130 16140 16150 16160 16170 16180 16190 16200 16210 16220 16230 16240 16250 16260 16270 16280 16290 16300 16310 16320 16330 16340 16350 16360 16370 16380 16390 16400 16410 16420 16430 16440 16450 16460 16470 16480 16490 16500 16510 16520 16530 16540 16550 16560 16570 16580 16590 16600 16610 16620 16630 16640 16650 16660 16670 16680 16690 16700 16710 16720 16730 16740 16750 16760 16770 16780 16790 16800 16810 16820 16830 16840 16850 16860 16870 16880 16890 16900 16910 16920 16930 16940 16950 16960 16970 16980 16990 17000 17010 17020 17030 17040 17050 17060 17070 17080 17090 17100 17110 17120 17130 17140 17150 17160 17170 17180 17190 17200 17210 17220 17230 17240 17250 17260 17270 17280 17290 17300 17310 17320 17330 17340 17350 17360 17370 17380 17390 17400 17410 17420 17430 17440 17450 17460 17470 17480 17490 17500 17510 17520 17530 17540 17550 17560 17570 17580 17590 17600 17610 17620 17630 17640 17650 17660 17670 17680 17690 17700 17710 17720 17730 17740 17750 17760 17770 17780 17790 17800 17810 17820 17830 17840 17850 17860 17870 17880 17890 17900 17910 17920 17930 17940 17950 17960 17970 17980 17990 18000 18010 18020 18030 18040 18050 18060 18070 18080 18090 18100 18110 18120 18130 18140 18150 18160 18170 18180 18190 18200 18210 18220 18230 18240 18250 18260 18270 18280 18290 18300 18310 18320 18330 18340 18350 18360 18370 18380 18390 18400 18410 18420 18430 18440 18450 18460 18470 18480 18490 18500 18510 18520 18530 18540 18550 18560 18570 18580 18590 18600 18610 18620 18630 18640 18650 18660 18670 18680 18690 18700 18710 18720 18730 18740 18750 18760 18770 18780 18790 18800 18810 18820 18830 18840 18850 18860 18870 18880 18890 18900 18910 18920 18930 18940 18950 18960 18970 18980 18990 19000 19010 19020 19030 19040 19050 19060 19070 19080 19090 19100 19110 19120 19130 19140 19150 19160 19170 19180 19190 19200 19210 19220 19230 19240 19250 19260 19270 19280 19290 19300 19310 19320 19330 19340 19350 19360 19370 19380 19390 19400 19410 19420 19430 19440 19450 19460 19470 19480 19490 19500 19510 19520 19530 19540 19550 19560 19570 19580 19590 19600 19610 19620 19630 19640 19650 19660 19670 19680 19690 19700 19710 19720 19730 19740 19750 19760 19770 19780 19790 19800 19810 19820 19830 19840 19850 19860 19870 19880 19890 19900 19910 19920 19930 19940 19950 19960 19970 19980 19990 20000 20010 20020 20030 20040 20050 20060 20070 20080 20090 20100 20110 20120 20130 20140 20150 20160 20170 20180 20190

CNRVC950014_Ecuador_1995

CNRVC930170_Democratic_Republic_of_the_Congo_1984

CNRVC150218_Ethiopia_1985

CNRVC930428_Tanzania_1993

CNRVC930044_Lebanon_1970

CNRVC960951_Libya_1970

CNRVC930046_Chad_1971

GP143_Bahrein_1978

CNRVC150244_Italy_1973

CNRVC950189_Cote_d'Ivoire_1973

CNRVC920176_Sao_Tome_1989

GP140_Malaysia_1978

CNRVC950396_Burkina_Faso_1974

CNRVC930061_China_1961

CNRVC960627_Ghana_1970

N16961_Bangladesh_1975

CNRVC150131_Ukraine_1970

688_Mexico_2006

F4170_Nigeria_1997

CNRVC950235_Nigeria_1970

CNRVC940012_Angola_1994

CNRVC930030_Slovakia_1970

A232_Mexico_1991

CNRVC910040_Cambodia_1991

CNRVC930167_Algeria_1982

CNRVC950007_Angola_1995CNRVC150245_Cote_d'Ivoire_1988

PRL5_India_1980

CNRVC950345_Senegal_1971

RKI-ZBS2-CH19_Angola_1972

CNRVC150216_Turkey_1976

CNRVC980046_Benin_1985

CNRVC150229_Tunisia_1981

3538-CDC_China_1981

CNRVC150217_Ghana_Nigeria_1982

CNRVC920049_Malawi_1992

CNRVC930042_Cameroon_1970

CNRVC940051_Rwanda_1988

M66_Indonesia_1937

CNRVC961077_Thailand_1963


CNRVC930067_Cambodia_1963

A10_Bangladesh_1979

A397_Bangladesh_1987


CNRVC960768_Iraq_1966


CNRVC950558_Tunisia_1971

A177_Colombia_1992

3735-CDC_China_1988



1_1970_Israel_1970

CNRVC960800_Iran_1965

863-CDC_Mauritania_1986

C8798_Uganda_1992

1551-CDC_China_1979

CNRVC960928_Liberia_1970

CNRVC930037_Sierra_Leone_1970

CNRVC980040_Burkina_Faso_1984

CNRVC960623_Ghana_1970

CNRVC940183_French_Guiana_1994

CNRVC960911_Israel_1970

CNRVC961190_Indonesia_1937

CNRVC930060_Philippines_1961CNRVC930036_India_1962

CNRVC960312_Rwanda_1996

CNRVC960971_Niger_1970

CNRVC140139_Portugal_1974

CNRVC961005_Pakistan_1964

A231_Mexico_1991

CNRVC150236_Somalia_1985


A6_Indonesia_1957

A201_Argentina_1992

CNRVC930127_Guinea_1970

CNRVC140118_Brazil_1991

CNRVC950340_Senegal_1974


CNRVC960596_Ethiopia_1970CNRVC960918_Jordan_1970

A27_Peru_1991

3024-CDC_China_1973

CNRVC961129_Viet_Nam_1964

CNRVC150132_Ukraine_1974

CNRVC930075_Mali_1970

CNRVC930425_Burundi_1993

A5_Angola_1989

CNRVC950100_Malaysia_1973

CNRVC980041_Mali_1984

GP60_India_1973



CNRVC920170_Malawi_1989A152_Mozambique_1991

CNRVC960575_Benin_1970

CNRVC140124_Bolivia_1992

177_03_Zambia_1997

CNRVC950535_Comoros_1975

CNRVC950476_Morocco_1972

CNRVC930174_Sri_Lanka_1981


CNRVC960976_New_Guinea_1962


CNRVC150234_Somalia_1985

CNRVC950172_Viet_Nam_1969

Wave 1

VSP-II var.

WASA1 America 1991-2006

1-20 SNPs

Asia Middle East Africa Europe America

The cholera was introduced to Peru from West Africa in 1991

http://www.cdc.gov/

1970-1984 96.3% isolates wild-type (77/80)

0.2 ARG/isolate (0-11)

1985-1999

2000-2014

37.2% isolates wild-type (100/269) 4 ARG/isolate (0-12)

0% isolates wild-type (0/281) 5.9 ARG/isolate (1-17)

Africa

ARG, antibiotic resistance gene

Antibiotic resistance

AMX, amoxicillin STR, streptomycin CHL, chloramphenicol TET, tetracyclin SUL, sulfonamides SXT, cotrimoxazole NAL, nalidixic acid CIP, ciprofloxacin AZI, azithromycin CTX, cefotaxime

Num

ber o

f AR

G p

er is

olat

e

Year

African isolates n=631

Only 16.6% (105/631) of the African isolates are inferred to be resistant to tetracyclins

Figure 3

Phase 1 (1970s-1980s)

Figure 3

Large incA/C plasmids bla strAB aad aph(3')-I cat1 tetB tetC

sul1 sul2 dfrA15 dfrA15 Acquired in Africa

Phase 2 (after 1980s) Chromosomal determinants Genomic islands: GI-15 aad_new sul1 29kb SXT/R391 (5 variants) strAB sul2 (floR) (dfrA1) (tetA) (tet_new) (qnrVC1)

(dfrA31) 100kb gyrA and parC mutations Acquired in South Asia

Conclusions

•  Iterative introductions of cholera to Africa from Asia (arguing against an environmental reservoir of Vibrio cholerae O1)

•  Separated Western and Eastern African foci

•  Role of the Middle East as a springboard (in particular in 1970) but recently direct transmission from South Asia

•  Confirmation of the West African origin of the South American outbreak of

1991 •  Accumulation of chromosomally-encoded multidrug resistance elements,

as observed previously with human pathogens Shigella dysenteriae type 1 and Salmonella Typhi H58

C Tarr

Collaborators

Paris: ML Quilici E Njamkepo N Fawal J Rauzier JM Fournier PAD Grimont C Bouchier H Salje Bangui: S Breurec R Bercion Yaoundé: A Ngandjio Dakar: B Garin Niamey: F Sidikou Abidjan: M Dosso

S Moore R Piarroux

D Domann G Dougan J Parkhill N Thomson

A Mukhopadhyay T Ramamurthy

F Luquero AL Page

K Keddy

Thank you for your attention !

L Bonte M Henkens

C Pazzani

R Grunow

JL Koeck

M Damian

M Hamze

B Lafourcade M Mengel

E Monakhova

the use of genomics for understanding introduction and

Documents