www.sciencemag.org/content/358/6364/789/suppl/DC1

Supplementary Materials for

Integrated view of Vibrio cholerae in the Americas

Daryl Domman,* Marie-Laure Quilici, Matthew J. Dorman, Elisabeth Njamkepo, Ankur Mutreja, Alison E. Mather, Gabriella Delgado, Rosario Morales-Espinosa, Patrick A. D.

Grimont, Marcial Leonardo Lizárraga-Partida, Christiane Bouchier, David M. Aanensen, Pablo Kuri-Morales, Cheryl L. Tarr, Gordon Dougan, Julian Parkhill, Josefina Campos, Alejandro

Cravioto, François-Xavier Weill,† Nicholas R. Thomson*†

*Corresponding author. Email: dd6@sanger.ac.uk (D.D); nrt@sanger.ac.uk (N.R.T.) †These authors contributed equally to this work.

Published 10 November 2017, Science 358, 789 (2017)

DOI: 10.1126/science.aao2136

This PDF file includes:

Materials and Methods Supplementary Text Figs. S1 to S11 Tables S3 and S4 Captions for Tables S1 and S2 References

Other Supplementary Materials for this manuscript include the following: (available at www.sciencemag.org/content/358/6364/789/suppl/DC1)

Tables S1 and S2 (Excel)

2

Materials and Methods

Bacterial isolates

The 164 seventh pandemic Vibrio cholerae El Tor (7PET) isolates and the 88 non-7PET isolates

were obtained from the collections of Institut Pasteur, Paris, France (n = 122); ANLIS, Buenos

Aires, Argentina (n = 20); and the Universidad Nacional Autónoma de México, Mexico City,

Mexico (n = 110). Metadata, such as biotype and serotype, were obtained from the various

providers of strains.

Antimicrobial susceptibility testing

Antimicrobial susceptibilities for selected isolates was assessed at the Universidad Nacional

Autónoma de México by the disk diffusion method and by determination of minimal inhibitory

concentrations (MIC) both according to CLSI (Clinical Laboratory Standards Institute)

guidelines (M23-A3 2008; M02-A11 2012) and using Müller-Hinton (MH) agar plates (BD

Difco, USA). The isolates were categorized as being sensitive, intermediately resistant, or

resistant to each antimicrobial. All the antimicrobial disks were sourced from BD BBL Sensi-

Disc, USA. The following antimicrobial drugs were tested: azithromycin (15 μg),

chloramphenicol (30 μg), ciprofloxacin (5 μg), doxycycline (30 μg), gentamicin (120 μg),

imipenem (10 μg), nalidixic acid (30 μg), tetracycline (30 μg), ticarcillin (75 μg) and

trimethoprim/sulfamethoxazole (1.25 μg / 23.75 μg / 75 μg). Pseudomonas aeruginosa ATCC

27853, Escherichia coli ATCC 25922, E. coli ATCC 35218, Staphylococcus aureus ATCC

29213 and Enterococcus faecalis ATCC 29212 were used as controls in the susceptibility tests.

3

Whole genome sequencing

Whole genome sequencing was performed at the Wellcome Trust Sanger Institute and at Institut

Pasteur using the Illumina HiSeq and MiSeq platforms to generate 75-250 bp paired-end reads.

Short read data are available in the European Nucleotide Archive (ENA) database (accession

numbers are provided in Table S1-S2). Short reads were assembled using Spades v3.8.2 (40) and

annotated using Prokka v1.5 (41) Genome completeness estimates and checks for contamination

were performed using CheckM (42).

Additional genomes

We supplemented our analyses of both 7PET and non-7PET datasets with previously published

genomes, listed in Supplementary Tables S1 and S2. For genomes that were only available as

assemblies, we simulated 100 bp paired-end Illumina reads using wgsim v.0.3.2 (-e 0 -r 0 -X 0 -1

100 -2 100) for uniformity in our data analysis pipelines.

Read alignment and detection of SNPs within 7PET

A reference based alignment for the 7PET analysis was obtained by mapping paired-end Illumina

reads for each of the 518 isolates to the Vibrio cholerae O1 El Tor reference N16961 (accession

numbers LT907989/LT907990) using SMALT v.0.7.4

(http://www.sanger.ac.uk/science/tools/smalt-0). Variant detection was performed using

samtools mpileup v0.1.19 (43) with parameters “-d 1000 -DSugBf”, and bcftools v0.1.19 (43), to

produce a BCF file of all variant sites. High quality SNPs were determined as previously

described (44). Putative recombinant regions were detected and filtered from the alignment using

4

Gubbins (45) with default parameters, resulting in a final alignment of 5,645 variable (SNP)

sites.

Core gene alignment for non-7PET

A pan-genome was created using the assembled genomes of 148 V. cholerae and three outgroup

sequences (Vibrio metoecus 07_2435; Vibrio metoecus RC341; Vibrio sp. RC586) using Roary

(46) with options “-e --mafft -s -cd 97”. A core gene alignment of 1,006,857 sites was produced

from 2,105 core genes. Poorly aligned and gap-rich sites were filtered from the alignment using

trimAl v1.4.rev15 (47) with the “-automated1” option, resulting in an alignment of 983,205 sites.

SNP-sites v2.3.2 (48) was used to identify the variable sites only, yielding a final alignment of

169,738 SNP sites.

Phylogenetic analysis

Maximum likelihood (ML) phylogenetic trees were constructed using RAxML v7.8.6 (49) under

the GTR model with the gamma distribution to model site heterogeneity (GTRGAMMA), using

500 bootstrap replicates. The ML analysis for the 7PET (518 isolates) dataset was constructed

using an alignment of 5,645 variable sites with putative recombinant sites removed. ML analysis

for the non-7PET dataset (152 isolates) was performed using the core-gene alignment of 169,738

variable sites. Phylogenetic trees were visualized with ggtree (50) and iTOL (51).

Temporal analysis

The temporal signal within the 7P dataset ML tree was investigated using TempEst v.1.5 (52) by

calculating a linear regression between the root-to-tip distance and the year of isolation for each

5

sample. Randomization of the dates of isolation removed any association between time of

sampling and root-to-tip distance. A time-scaled phylogeny for the 7PET was produced by

applying the LSD algorithm (53), which can scale to a large number of taxa. The genome-wide

mutation rate (6.1 x 10-7 substitutions site-1 year-1) obtained by BEAST (54) from our companion

paper (16) was employed here. LSD version 0.3beta was run with constrained mode (“-c”), using

variances from the estimated branch lengths (“-v 2”), and confidence intervals computed from

1,000 simulated trees (“-f 1000”). The time to most recent common ancestor for all 7P El Tor

was estimated to be 1914.784 (95% CI, 1911.531 - 1917.973). This corresponds well with the

BEAST estimate from Weill et al. (16), which was 1917 (95% CI, 1912 - 1922).

Phylogenetic clustering

The 148 non-7PET V. cholerae isolates were clustered into lineages guided by a hierarchical

Bayesian analysis of population structure (BAPS) (55) and the maximum likelihood phylogeny.

BAPS was run on an alignment of 115,789 phylogenetically informative sites (i.e., private SNPs

were removed) derived from the final core gene alignment with the following parameters: L = 3;

K = 500.

Comparative genomic analyses

The output from Roary (46), BLAST (56) and mapping analyses were used to determine and

characterize the analyzed genomes. Gene families were computed for both 7PET and non-7PET

datasets using Roary. For in silico typing of the 7PET Ogawa/Inaba serotypes, ARIBA (57) was

used to call variants against the wild type Ogawa V. cholerae V06-92 sequence (ENA accession:

AEN80191). Similarly, ARIBA was used to call variants of the ctxB allele against the V.

6

cholerae N16961 El Tor sequence. The presence of genomic features and islands was determined

using a combination of data from BLAST analyses, mapping to V. cholerae reference genomes,

and data from ARIBA.

Antibiotic resistance genes (ARGs) or ARG-containing structures (Fig. S11 and Table S1) were

identified within the 7PET dataset using ResFinder version 2.1

(https://cge.cbs.dtu.dk/services/ResFinder/), by BLAST analysis against GI-15 (58), Tn7

(GenBank accession number CP000038; coordinates, 4098721-4113720) and SXT/R391 ICEs

(59), and by using PlasmidFinder (60) v1.3 (https://cge.cbs.dtu.dk/services/PlasmidFinder/).

ARIBA was used to call variants of the quinolone resistance-determining region of the DNA

gyrase and topoisomerase IV genes, and to determine ARGs for the non-7PET dataset (against

the ResFinder database).

Supplementary Text

Supplementary Note 1: Sampling framework

A total number of 2,049,525 cholera cases were reported by 23 countries within Latin America

and the Caribbean to the WHO between 1991 to 2014. Our dataset for 7th Pandemic El Tor

comprises 15 of these 23 countries, and captures 1,851,451 (90.3%) of those cases reported.

Despite covering over 90% of cases, our sampling is limited within Central America and the

Caribbean (with the exception of Haiti). However, as we can link our genomic data with the

previous ribotyping, PFGE, and electrophoretic typing data, we can extend our coverage which

7

allows for the description of 99.8% of the all cholera cases reported to the WHO between 1991-

2014.

We attempted to represent previously described cholera isolates within Latin America to place

these into a holistic framework in which to understand the relationships between these isolates.

In total, we analyzed 665 Vibrio cholerae isolates, which included 519 7th pandemic El Tor

(7PET) isolates as well as an additional 146 non-7PET strains. We have sequenced 10 of the 19

reference strains originally typed by Popovic et al. (12), which includes all strains from Latin

America from that reference collection. In addition, we sequenced the collection of isolates from

Mexico (11), which were also typed according to the scheme of Popovic et al. (12). Thus, we can

now link the historical typing schemes to whole-genome data (Table S4).

Initial ribotyping and electrophoretic typing studies demonstrated that the Latin American

epidemic was a clonal expansion of ribotype 5 and electrophoretic type 4 (12). Popovic et al.

typed 108 toxigenic isolates from 14 Latin American countries from 1991-1992, of which 104

isolates were ribotype 5 (and ET 4) and four isolates were ribotype 6 (and ET 3). Further

characterization of 352 toxigenic isolates from 13 Latin American countries by Evins et al. (13)

demonstrated that 323 were ribotype 5 and ET 4. Twenty-three isolates from Guatemala and

Mexico were ET 3, ribotype 6a and resistant to furazolidone, sulfisoxazole, and streptomycin

(13). A more detailed study within Guatemala demonstrated that initial isolates from 1991 had

identical PFGE patterns to the Latin American epidemic strain, but by 1993 isolates were

multidrug resistant (furazolidone, sulfisoxazole, streptomycin) and had a PFGE pattern similar to

isolates from Mexico from 1993 (61).

8

We also included previously described Vibrio cholerae O1 “variants” from Latin America. This

included the “Amazonia” variants isolated in the western part of the Brazilian Amazon between

1991 to 1992 (19) and the “Tucumán” variants from Argentina isolated between 1992 to 1998

(18). We also included isolates from previously described cholera in Mexico, which included

Classical biotype isolates (20, 21), El Tor isolates (20, 27), and the Mexican O1 variant ribotypes

Mx1-3 (11). Only those of our samples which had defined provenance were sequenced for

inclusion in our analysis.

A single isolate (F99/W) from Formosa, Argentina sampled from the environment in 1992 was

placed phylogenetically into the 7th Pandemic El Tor (7PET) lineage, clustering among early

wave 1 isolates from Iraq and Iran from the mid 1960s. This isolate is non-toxigenic (lacks

CTXφ) and does not harbor any signatures of the LAT-1 (WASA and VSP-II variant) or LAT-2

(GI-15) sublineages. These data match the description in the thesis that first characterized this

isolate (62). Although this isolate has a curious position within the phylogeny, the phylogenetic

analyses and epidemiological data do not support any relationship between the 1990s epidemic

and this isolate.

Supplementary Note 2: Patterns of disease seen across Latin America

Our sampling of 7PET and non-7PET lineages enables us to describe three overarching patterns

of cholera disease in Latin America. The first is that of very localized disease, whereby local

lineages enter into the human population in sporadic short-term outbreaks, and in which

secondary infections are rare. This pattern is often associated with foodborne infections, and are

9

often localized to coastal regions within known local foci of V. cholerae populations (11). This

disease pattern is exemplified by the MX-3 lineage, which only appeared in 2000 along the

Mexican Gulf Coast (Fig. 1; S4), and by an outbreak by a non-toxigenic serogroup O12 isolate

(strain 1587) seen only in Lima, Peru in 1994 (63) which is unrelated to other Latin American

isolates (Fig. S2).

A second pattern is that of localized lineages causing disease over much longer time-periods and

large geographic areas. This is exemplified by the Gulf Coast, ELA-1, and MX-2 lineages (Fig.

1; S2; S4). While cases associated with the Gulf Coast lineage are seen in the coastal regions of

Mexico and the United States, isolates from this lineage have also been found in Peru and

Argentina (Fig. 1). ELA-1 isolates span a period of 36 years (1978-2014) and are associated with

distinct and disparate geographic areas (Fig. 1; S2; S4). These lineages represent long-term,

stable local foci, and the large geographic spread of several lineages may indicate transport via

human populations. As sampling continues to increase, it seems likely that lineages or isolates

associated with the first pattern of disease may transition into the second.

The third pattern is that of pandemic cholera, which is defined by massive, explosive epidemics

in short periods of time (Fig. S10). Epidemic cholera has been shown to spread in a manner

similar to that of aggressive forest fires (64). There are vast differences in the burden of disease

caused by infection with pandemic lineages versus local lineages.

Epidemic cholera in Latin America appears as explosive events for which the number of cases

falls sharply after introduction events (Fig. S10). The dramatic decrease in cases and the eventual

10

disappearance of both LAT-1 and LAT-2 pandemic sublineages in nearly all Latin American

countries by 2010 (Fig. 2C) suggests that these sublineages did not establish a long-term

environmental reservoir in Latin America. Thus, in contrast to true local lineages, some of which

have been isolated since the 1970s (Gulf Coast), pandemic cholera appears to have a limited

capacity to be maintained in Latin America. The patterns seen across lineages may be due to the

different modes by which local versus pandemic clones move into and through human

populations, i.e., via foodborne mechanisms versus a human-to-human transmission route.

11

Fig. S1

Spatiotemporal distribution of V. cholerae from Latin America. Circle size is scaled by the

number of isolates from a given location per year.

12

Fig. S2

Maximum likelihood phylogeny of 151 genomes (148 V. cholerae) estimated from a core-

genome alignment of 2,105 genes. The tree is rooted on outgroup sequences of Vibrio metoecus

and Vibrio sp. RC586 (see Fig. S3). Select lineages present in Latin America are highlighted.

The scale bar denotes substitutions per variable site.

0.05

TMA_21 Brazil 1982NHCC008D Bangladesh 2010

HE−25 Haiti 2010AM_19226 Bangladesh 2001

1587 Peru 1994623−39 Bangladesh 2002

S−18 Australia 2009

KNE109A Kenya 2009KNE109B Kenya 2009

2806 Mexico 2010KNE114 Kenya 2009EM1676A Bangladesh 2011

MZO−2 Bangladesh 2001

MZO−3 Bangladesh 20011582 Mexico 2003

644 Mexico 20021835 Mexico 2003

12129_1 Australia 1985LMA3984_4 Brazil 2007

C 7965 Bolivia 1991354 Bolivia 1991

HE_48 Haiti 2010

HC_1A2 Haiti 2010HC_56A1 Haiti 2010HE_39 Haiti 2010HE_46 Haiti 2010

T1437 Argentina 1997T717 Argentina 1998

HE−16 Haiti 2010HE_09 Haiti 2010

Mex1 Mexico 199187211 Mexico 1991

1148 Mexico 2004CP1035 Mexico 2004

98/660 Mexico 199898/659 Mexico 1998

98/1453c Mexico 1998

98/2222 Mexico 19981474 Mexico 2007

00/51.7 Mexico 2000

99/19C Mexico 199999/1468 Mexico 1999

2370 Mexico 2001

CM 91−3 Mexico 19832709 Mexico 2001

HE_45 Haiti 2010

HC_43B1 Haiti 2010HC_41B1 Haiti 2010

T610 Argentina 1994

T13074 Argentina 1994T522 Argentina 1998

VC591_14 Argentina 2014T5957 Argentina 1993

T12550 Argentina 1995T2220 Argentina 1994

1074−78 Brazil 1978

116063 Brazil 1978TM11079_80 Brazil 1980

C 8481 Brazil 1991

Amazonia Brazil 1991CNRVC140120 Brazil 19913222 Brazil 1991CNRVC140119 Brazil 1992

VL426 UK NA

T777 Argentina 1994CT5369_93 Brazil 1993

J31_W Argentina 2000 C 8108 Trinidad 1992

V51 USA 1987CP110 USA 2010

NI16 Nicaragua NA

BJGO1 USA 2010RC385 USA 1998

00/3259 Mexico 2000

00/3240 Mexico 200000/3260 Mexico 2000

848 Mexico 2000S−29 Australia 2010

1354 Mexico 20013145 Mexico 2007

2908 Mexico 2008909 Mexico 1999

C 7654 USA 1991

C 7641 Guatemala 199199/2114 Mexico 1999

T2734 Argentina 2004

00/3242 Mexico 20003271 Mexico 2009

2012Env_9 Haiti 2012

CP1037 Mexico 20031146 Mexico 2004

97/670 Mexico 1997

2710 Mexico 20012174 Mexico 2001

Me3 Argentina 19952012EL_1759 Haiti 2012

930125_S5 Sudan 1969V52 Sudan 1968

150136_S4 Sudan 1969150137_S6 Sudan 1969

RC27 Indonesia 1991

93154 Mexico 199697639_1 Mexico 1995

O395 India 1965

GP8 India 1970GP16 India 1971

A389 Bangladesh 1987A70 Bangladesh 1969

Mex16 Mexico 199795412 Mexico 1997Mex15 Mexico 199795409 Mexico 1997

C6706_CDC Peru 1991N16961 Bangladesh 1975

2010EL_1786 Haiti 201092/33213 Mexico 1992

NCTC_8457 Egypt 1930BX330286 Australia 1986

MAK757 Celebes_Islands 1937M66 Indonesia 1937

3247_CN China 1977 2583−87 Peru 1987

SF366 Argentina 1999 C 7724 El Salvador 1991

A213 USA 1984A219 USA 1986

E506 USA 19743569_08 USA 2008

A209 USA 19802740−80 USA 1980

2284 Mexico 20102283 Mexico 2010

391 Mexico 2010

586 Mexico 2010667 Mexico 2010

819 Mexico 2010

1596 Mexico 20041876 Mexico 1999

99/2118 Mexico 199900/3275 Mexico 2000

99/2110 Mexico 199900/3079 Mexico 2000

99/1127 Mexico 19991127 Mexico 1999

VC_2283_05 Argentina 2005VC_521_09 Paraguay 2009

VC849_07 Argentina 2007VC182_14 Argentina 2014

MX-1

ELA-1

MX-3

ELA-2

ELA-3

Classical

7PET

Gulf Coast

MX-2

ELA-4

ELA-5

85

79

100

100

100

100

100

100

99

97

97

100

100

100

100

100

100

100

100

100

100

97

13

Fig. S3

The full maximum likelihood phylogeny from Figures 1 and S2 with outgroups is shown. Nodes

are colored by the clinical or environmental origin of the isolate. Detailed descriptions of the

isolates can be found in Table S2. The scale bar denotes substitutions per variable site.

0.2

Vibrio parilis RC586 USA 1999

Vibrio metoecus RC341 USA 1998Vibrio metoecus 07_2435 USA 2004

TMA_21 Brazil 1982NHCC008D Bangladesh 2010

HE−25 Haiti 2010AM_19226 Bangladesh 2001

1587 Peru 1994623−39 Bangladesh 2002

S−18 Australia 2009

KNE109A Kenya 2009KNE109B Kenya 2009

2806 Mexico 2010KNE114 Kenya 2009EM1676A Bangladesh 2011MZO−2 Bangladesh 2001

MZO−3 Bangladesh 20011582 Mexico 2003

644 Mexico 20021835 Mexico 2003

12129_1 Australia 1985LMA3984_4 Brazil 2007

C 7965 Bolivia 1991354 Bolivia 1991

HE_48 Haiti 2010

HC_1A2 Haiti 2010HC_56A1 Haiti 2010HE_39 Haiti 2010HE_46 Haiti 2010

T1437 Argentina 1997T717 Argentina 1998

HE−16 Haiti 2010HE_09 Haiti 2010

Mex1 Mexico 199187211 Mexico 1991

1148 Mexico 2004CP1035 Mexico 2004

98/660 Mexico 199898/659 Mexico 1998

98/1453c Mexico 1998

98/2222 Mexico 19981474 Mexico 2007

00/51.7 Mexico 2000

99/19C Mexico 199999/1468 Mexico 1999

2370 Mexico 2001

CM 91−3 Mexico 19832709 Mexico 2001

HE_45 Haiti 2010

HC_43B1 Haiti 2010HC_41B1 Haiti 2010

T610 Argentina 1994

T13074 Argentina 1994T522 Argentina 1998

VC591_14 Argentina 2014T5957 Argentina 1993

T12550 Argentina 1995T2220 Argentina 1994

1074−78 Brazil 1978

116063 Brazil 1978TM11079_80 Brazil 1980

C 8481 Brazil 1991

Amazonia Brazil 1991CNRVC140120 Brazil 19913222 Brazil 1991CNRVC140119 Brazil 1992

VL426 UK Unknown

T777 Argentina 1994CT5369_93 Brazil 1993

J31_W Argentina 2000 C 8108 Trinidad 1992

V51 USA 1987CP110 USA 2010

NI16 Nicaragua Unknown

BJGO1 USA 2010RC385 USA 1998

00/3259 Mexico 2000

00/3240 Mexico 200000/3260 Mexico 2000

848 Mexico 2000S−29 Australia 2010

1354 Mexico 20013145 Mexico 2007

2908 Mexico 2008909 Mexico 1999

C 7654 USA 1991

C 7641 Guatemala 199199/2114 Mexico 1999

T2734 Argentina 2004

00/3242 Mexico 20003271 Mexico 2009

2012Env_9 Haiti 2012

CP1037 Mexico 20031146 Mexico 2004

97/670 Mexico 1997

2710 Mexico 20012174 Mexico 2001

Me3 Argentina 19952012EL_1759 Haiti 2012

930125_S5 Sudan 1969V52 Sudan 1968

150136_S4 Sudan 1969150137_S6 Sudan 1969

RC27 Indonesia 1991

93154 Mexico 199697639_1 Mexico 1995

O395 India 1965

GP8 India 1970GP16 India 1971

A389 Bangladesh 1987A70 Bangladesh 1969

Mex16 Mexico 199795412 Mexico 1997Mex15 Mexico 199795409 Mexico 1997

C6706_CDC Peru 1991N16961 Bangladesh 1975

2010EL_1786 Haiti 201092/33213 Mexico 1992

NCTC_8457 Egypt 1930BX330286 Australia 1986MAK757 Celebes Islands 1937M66 Indonesia 1937

3247_CN China 1977 2583−87 Peru 1987

SF366 Argentina 1999 C 7724 El Salvador 1991

A213 (917−84) USA 1984A219 (2538−86) USA 1986

E506 USA 19743569_08 USA 2008

A209 (2741−80) USA 19802740−80 USA 1980

2284 Mexico 20102283 Mexico 2010

391 Mexico 2010

586 Mexico 2010667 Mexico 2010

819 Mexico 2010

1596 Mexico 20041876 Mexico 1999

99/2118 Mexico 199900/3275 Mexico 2000

99/2110 Mexico 199900/3079 Mexico 2000

99/1127 Mexico 19991127 Mexico 1999

VC_2283_05 Argentina 2005VC_521_09 Paraguay 2009

VC849_07 Argentina 2007VC182_14 Argentina 2014

ClinicalEnvironmental

Origin of isolate

14

Fig. S4

Geographic distribution of samples for V. cholerae lineages. Each panel shows the location of

isolates; circle size is scaled by the number of isolates from a given location. Only those isolates

were included for which explicit data on their place of origin were available. The name of each

lineage is given in the bottom left corner of each panel.

15

Fig. S5

Serogroup of V. cholerae. The phylogeny is the same as that shown in Figures 1 and S2. The

hash mark denotes a branch that was artificially shortened for visualization purposes. The

serogroup based on the O-antigen is reported. The designation “non-O1” is given to isolates that

were not of the O1 serogroup when tested phenotypically and not defined further.

16

Fig. S6

Presence of genomic islands associated with pathogenicity across the V. cholerae phylogeny.

The phylogeny is the same as that shown in Figures 1 and S2. The hash mark denotes a branch

that was artificially shortened for visualization purposes. The seventh pandemic lineage is

highlighted.

17

Fig. S7

Presence of the toxin co-regulated pilus (TCP) and ctxB alleles across the V. cholerae phylogeny.

The phylogeny is the same as that shown in Figures 1 and S2. The hash mark denotes a branch

that was artificially shortened for visualization purposes. ctxB variants are denoted.

18

Fig. S8

Temporal signal within the 518 seventh pandemic V. cholerae El Tor isolates. (A) Regression,

as performed by TempEst (52), of the year of isolation versus root-to-tip divergence derived

from the maximum likelihood tree of the 7PET lineage in Fig S10. (B) Three tip-randomization

experiments over the whole dataset demonstrate the temporal signal is not artefactual.

19

Fig. S9

Maximum likelihood phylogeny of 518 seventh pandemic V. cholerae El Tor isolates. Scale bar

denotes number of mutations per variable site. Branches in the tree are colored according to the

waves defined by Mutreja et al. (26), where blue is wave 1, red is wave 2, and purple is wave 3.

The geographic origin of the sample is denoted by the first ring. The other rings (2-5) display the

distribution of allele or genomic region variants across the 7PET lineage.

0.01

1. Geographic area (GEO)AfricaAsiaEuropeLatin AmericaOceaniaUnknown

2. ctxB allele

12345

ctxB1ctxB3ctxB5ctxB7frame-shift/partialctxB new

3. VSP-IIwild typevar WASAvar. 1 (VC_0495::ISVch4)var. 2 (∆ VC_0495-VC_0498)var. 3 (∆ VC_0495-VC_0500)var. 4 (∆ VC_0495-VC_0512)var. 5 (∆ VC_0513-VC_0516)

4. WASA-1Present

5. GI-15Present

100

96100

100

100 100

100

99

98

100

100

20

Fig. S10

Number of cholera cases reported to the World Health Organization (WHO). (A) The number of

reported cases per year for various geographic regions. (B) Map of countries reporting cases to

the WHO from 1990 to 1991.

21

Fig. S11

Genomic characterization of antimicrobial resistance in seventh pandemic V. cholerae El Tor.

Resistance to eight antibiotics was determined (TET: tetracycline; CHL: chloramphenicol; SXT:

sulfamethoxazole-trimethoprim; SUL: sulfonamides; STR: streptomycin; NAL: nalidixic acid;

CIP: ciprofloxacin).

22

Table S3. Characteristics of expanded Latin American lineages

( ) number of environmental isolates; for details see Tables S1-S2

+ = present

/ = absent

Lineage Number,of,isolates Countries Dates Serogroup Biotype CTX

7PET 518((28) Worldwide 195752013 O1 El(TorctxB1,'ctxB3,'ctxB7

Classical 12((0) Asia,(Mexico 196551997 O1 Classical ctxB1ELA51 15((2) Argentina,(Brazil 197852014 O1 El(Tor /ELA52 8((4) Mexico,(USA 199152008 O1 El(Tor /ELA53 8((4) Mexico,(Haiti 199752012 O1 El(Tor /ELA54 3((3) Mexico 198352001 O1 El(Tor /ELA55 3((3) Brazil,(Bolivia 199152007 O1 El(Tor /

Gulf(Coast 10((3)

Mexico,(USA,(Peru,(Argentina,(El(Salvador,(

China

197452008 O1 El(TorctxB1,'ctxB12

MX51 10((0) Mexico 199852007 O1 El(Tor /

MX52 18((7)Mexico,(Argentina,(Paraguay

199952014 O1 El(TorctxB1,'ctxB2

MX53 3((0) Mexico 2000 O1 El(Tor /

23

Table S4. Historical typing schemes matched to phylogenic lineages.

The strains listed are those sequenced that link directly to previously-described typing schemes.

We have included the various naming conventions given by different groups where possible.

Phylogenetic,Lineage Previous,typing Strain Location Year SourceLAT$1 Popovic,et,al.,ET,4,,ribotype,5 C,6706 Peru 1991 Human

Koblavi,et,al.,ribotype,B5Dalsgaard,et,al.,ribotype,R1

Lizarraga$Partida,and,Quilici,ribotype,M5

LAT$2 Lizarraga$Partida,and,Quilici,ribotype,M6 92/33213 Mexico 1992 HumanPopovic,et,al.,ET,3,,ribotype,6aTamayo,et,al.,ribotype,B21a

Gulf,Coast Popovic,et,al.,ET,2,,ribotype,11 ,2583$87 Peru 1987 HumanPopovic,et,al.,ET,2,,ribotype,14 ,C,7724 El,Salvador 1991 Water

ELA$1 Popovic,et,al,,ET,6,,ribotype,13;,Evins,et,al.,PFGE,62 ,1074$78 Brazil 1978 SewagePopovic,et,al.,ribotype,19;,Evins,et,al.,PFGE,62 ,C,8481 Brazil 1991 HumanCoelho,et,al.,zymovar,99Koblavi,et,al.,ribotype,B18b 3222 Brazil 1991 Human

ELA$2 Popovic,et,al.,ribotype,15 C,7641 Guatemala 1991 WaterPopovic,et,al.,ribotype,16 C,7654 Alabama,,USA 1991 Water

ELA$4 Popovic,et,al.,ET,5,,ribotype,12 ,CM,91$3 Mexico 1983 Sewage

ELA$5 Popovic,et,al.,ribotype,17 ,C,7965 Bolivia 1991 River,waterKoblavi,et,al.,ribotype,B18c 354 Bolivia 1991 Environmental

MX$1 Lizarraga$Partida,and,Quilici,ribotype,Mx1 98/660 Mexico 1998 HumanMX$2 Lizarraga$Partida,and,Quilici,ribotype,Mx2 99/2118 Mexico 1999 HumanMX$3 Lizarraga$Partida,and,Quilici,ribotype,Mx3 00/3259 Mexico 2000 Human

24

Additional Data table S1 (separate file)

Details of the 7th Pandemic El Tor (7PET) Vibrio cholerae isolates and genomes used in this

study.

Additional Data table S2 (separate file)

Details of the non-7PET Vibrio cholerae isolates and genomes used in this study.

25

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