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Supplementary Figure 1
Terminal branch lengths of H58 versus non-H58 isolates.
The number of SNPs between each isolate and its last common ancestor was determined from the phylogenetic tree in Figure 1. The
frequency of each terminal branch distance was calculated and adjusted for the number of isolates in each of the lineages. All branch
lengths are shown in the main panel, and those with lengths of less than 25 SNPs are shown in the inset.
Nature Genetics: doi:10.1038/ng.3281
Supplementary Figure 2
Temporal analysis.
(a) Time-dependent accumulation of SNPs in the whole genomes of S. Typhi isolates. Root-to-tip branch lengths extracted from the
maximum-likelihood tree of S. Typhi are plotted against the year of isolation. Points representing H58 isolates are colored red. Lines
indicate linear regression of branch lengths on isolation dates, for H58 (red), all S. Typhi (black) and all S. Typhi isolated since 1992
(dashed). (b) Changes in the effective population size of the H58 lineage over time. The central black line indicates the median
estimates, and shaded areas represent confidence limits expressed as 95% highest posterior probability densities (HPDs). The dashed
red vertical line corresponds to the year in which the H58 lineage appeared to disseminate to multiple geographical locations in the
corresponding H58 BEAST analysis.
Nature Genetics: doi:10.1038/ng.3281
Supplementary Figure 3
Association of S. Typhi H58 and multidrug resistance.
The frequency of H58 among MDR and non-MDR isolates and their associated country of origin are displayed (odds ratio and P values
were calculated). All countries shown have 2 MDR isolates. OR, odds ratio; Inf, infinite; CAR, Central African Republic; DRC,
Democratic Republic of the Congo; S. Africa, South Africa. *P < 0.01.
Nature Genetics: doi:10.1038/ng.3281
Supplementary Figure 4
Phylogenetic distribution of acquired resistance genes and DNA gyrase and topoisomerase IV mutations found in the 1,832 S. Typhi isolates.
The phylogeny of 1,832 S. Typhi isolates constructed using 22,145 SNPs is depicted in the center and surrounded by colored band
circles representing (1) The geographical region the isolate is from and the number of (2) resistance genes, (3) gyrA mutations, (4) gyrB
mutations, (5) parC mutations and (6) parE mutations present in the isolate. A red arc represents the H58 lineage, and the phylogenetic
position of the CT18 (R) reference (AL513382) is indicated. Branch lengths are indicative of the estimated substitution rate per variable
site. A, alanine; R, arginine; N, asparagine; D, aspartic acid; Q, glutamine; E, glutamic acid; G, glycine; I, isoleucine; L, leucine; K,
lysine; F, phenylalanine; S, serine; Y, tyrosine. *Rare SNP.
Nature Genetics: doi:10.1038/ng.3281
Supplementary Figure 5
Antimicrobial resistance trends of H58 S. Typhi isolates.
Numbers of H58 S. Typhi that were MDR on genotyping and/or harbored at least one gyrA mutation conferring nalidixic acid resistance
and reduced fluoroquinolone susceptibility, among isolates from (a) Southeast Asia, (b) South Asia and (c) Africa.
Nature Genetics: doi:10.1038/ng.3281
Supplementary Figure 6
Phylogenetic distribution of novel phage regions identified in the S. Typhi H58 lineage.
The maximum-likelihood phylogeny of 853 S. Typhi H58 isolates constructed using 1,534 SNPs is depicted in the center, rooted using
an S. Typhi isolate from the nearest neighboring cluster of non-H58 isolates as an outgroup (black circle; isolate 10060_5_62_
Fij107364_2012) and surrounded by colored band circles representing (1) country of isolation and (2) phage regions. Each of the phage
regions is detailed in Supplementary Table 6. Branch lengths are indicative of the estimated substitution rate per variable site.
Nature Genetics: doi:10.1038/ng.3281
Supplementary Table 2. Public reference strains used in this study. Key: *= S. Typhi isolates used in study by
Holt, K. E. et al. (2008) 1 R = Reference strain used in chromosomal phylogenetic analyses.
Public reference strains of S. Typhi
Isolate name Tree name Accession number Year of isolation
Continent Region within continent
Country
BL196 BL196_2005 SalTypBL196v1 2005 Asia Southeast Asia Malaysia
CR0044 CR0044_2007 SalTypSTCR0044v1 2007 Asia Southeast Asia Malaysia
CR0063 CR0063_2007 SalTypCR0063 v1 2007 Asia Southeast Asia Malaysia
P-stx-12 P-stx-12_2012 ASM24553v1 2012 Asia South Asia India
ST0208 ST0208_2008 YKP860805.1 2008 Asia Southeast Asia Malaysia
UJ308A UJ308A_2012 SalTypUJ308Av1 2012 Australia & Oceania Oceania Papua New Guinea
UJ816A UJ816A_2012 SalTypUJ816Av1 2012 Australia & Oceania Oceania Papua New Guinea
E98_3139* E98_3139_1998 ASM18037v1 1998 North America North America Mexico
J185SM* J185SM_1985 ASM18031v1 1985 Asia Southeast Asia Indonesia
CT18*R CT18_S.Typhi_CT18_1993 AL513382.1 1993 Asia Southeast Asia Vietnam
Ty2* 10349_1_84_RusTy2_1916 ERR343332 1916 Europe Eastern Europe Russia
404ty* 10349_1_90_Indo404ty_1983 ERR343338 1983 Asia Southeast Asia Indonesia
E00-7866* 10349_1_88_MorE00-
7866_2000
ERR343336 2000 Africa North Africa Morocco
E02-1180* 10349_1_89_IndE02-
1180_2002
ERR343337 2002 Asia South Asia India
E98-0664* 10349_1_86_KenE98-
0664_1998
ERR343334 1998 Africa East Africa Kenya
E98-2068* 10349_1_87_BanE98-
2068_1998
ERR343335 1998 Asia South Asia Bangladesh
M223 * 10425_1_10_UnkM223_1939 ERR349340 1939 Unknown Unknown Unknown
150(98)S* 10561_2_47_Vie150_98_S_1
998
ERR357622 1998 Asia Southeast Asia Vietnam
8(04)N* 10349_1_95_Vie8_04_N_200
4
ERR343343 2004 Asia Southeast Asia Vietnam
E02-2759* 10349_1_91_IndE02-
2759_2002
ERR343339 2002 Asia South Asia India
E03-4983* 10540_1_4_IndoE03-
4983_2003
ERR352601 2003 Asia Southeast Asia Indonesia
Public reference plasmids and phages
IncHI1
plasmids Tree name Accession number
R27 AF250878_R27 AF250878
pHCM1 AL513383_pHCM1 AL513383
pAKU1 pAKU1 AM412236
Other plasmids Tree name Accession number
pHCM2 pHCM2 NC_003385.1
Nature Genetics: doi:10.1038/ng.3281
Supplementary Table 3. Plasmids identified in S. Typhi H58 isolates. A summary of the known and novel
plasmids identified in 853 H58 isolates. The number of isolates that contain the plasmid and their geographical
origins are described. The phylogenetic distribution of each of the plasmids is shown in Figure 6.
Tn2670-like composite transposon (catA1, sul2, dfrA7, blaTEM-1, strAB, sul2) Location Number of isolates Countries of isolation Plasmid - IncHI1 PST6 185
140 43 15 9 6 4 1 1
Cambodia Vietnam Kenya Laos
Pakistan India
Tanzania Sri Lanka Unknown
Chromosome – yidA 13 10 3 1 1
Bangladesh Iraq
Pakistan Palestine
India Chromosome – cya 72
19 4 6 3 3 1 1 1 1
Malawi India
South Africa Tanzania
Bangladesh Cambodia
Afghanistan Africa
Australia Nepal
Chromosome - STY4438 11 Fiji Chromosome – fbp 1 India Other acquired resistance genes Location (genes) Number of isolates Countries of isolation IncN plasmid (sul1, aadA1, dfrA15) 1 India IncN plasmid (Tn6029: blaTEM-1, strAB, sul2; sul1, aadA1)
1 India
IncFIB(K) plasmid (blaTEM-1, sul2, qnrS1) 5 1 1
Bangladesh South Africa
Unknown IncFIB(K) + IncN plasmids (Tn6029: blaTEM-1, strAB, sul2; dfrA5, blaCARB-6, catB, aac3)
13 Tanzania
Novel plasmid (strAB, sul1, blaOXA-23) 1 Bangladesh
Nature Genetics: doi:10.1038/ng.3281
Supplementary Table 4. Isolates sequenced using PacBIO RS II Platform. S. Typhi H58 isolates selected for sequencing on the
PacBIO RS II platform (Pacific Biosciences, CA, USA).
Name of isolate (tree name) Laboratory number
Accession number
Year of isolation Continent Region within
continent Country
10349_1_74_Ind12148_2012 ERL12148 ERR343322 2012 Asia South Asia India
10060_6_83_Tan129-0238-M_2008 129-0238-M ERR331380 2008 Africa East Africa Tanzania
10349_1_79_Ind12960_2012 ERL12960 ERR343327 2012 Asia South Asia India
9475_6_19_Mal1016889_2011 1016889 ERR279116 2011 Africa Southern Africa Malawi
10607_2_36_Mal1036491_2012 1036491 ERR360828 2012 Africa Southern Africa Malawi
Nature Genetics: doi:10.1038/ng.3281
Supplementary Table 5. Amino acid substitutions in the quinolone resistance-determining regions of DNA
gyrase and topoisomerase IV genes. (a) Combinations of coding changes detected in the QRDR and their
frequency amongst H58 and non-H58 lineages. (b) Nucleotide substitutions resulting in QRDR coding changes.
Key to derived base: A = adenine; C = cytosine; G = guanine; T = thymidine and amino acid abbreviations: Ala =
alanine; Arg = Arginine; Asn = asparagine; Asp= aspartic acid; Gln = glutamine; Glu= glutamic acid; Gly=
glycine; Ile = isoleucine; Leu = leucine; Lys = lysine; Phe = phenylalanine; Ser = serine; Tyr = tyrosine. *Rare
SNP (see B).
(a) Combinations of coding changes detected in the QRDR and their frequency amongst H58 and non-H58
lineages.
gyrA gyrB parC parE H58 Other Ser83Phe - - - 199 (23%) 57 (6%) Ser83Phe - Glu84Gly - 6 (1%) 1 (0.1%) Ser83Phe - Ser80Ile - 1 (0.1%) 0 Ser83Phe - - Leu416Phe 1 (0.1%) 6 (0.6%) Ser83Phe - - Asp420Asn 153 (18%) 1 (0.1%) Ser83Phe Gln465Arg - Asp420Asn 6 (0.7%) 0 Ser83Tyr - - - 74 (9%) 8 (0.8%) Ser83Tyr - - Asp420Asn 1 (0.1%) 0 Asp87Asn - - - 0 5 (0.5%) Asp87Tyr - - - 8 (0.9%) 12 (1%)
Asp87Tyr* - - - 2 (0.2%) 1 (0.1%) Ser83Phe, Asp87Tyr - Glu84Lys - 1 (0.1%) 0 Ser83Phe, Asp87Tyr - Ser80Ile - 19 (2%) 0 Ser83Phe, Asp87Tyr - - Asp420Asn 1 (0.1%) 0 Ser83Tyr, Asp87Tyr - Ser80Ile - 1 (0.1%) 0
- Gln465Leu - - 0 1 (0.1%) - Ser464Phe - - 27 (3%) 5 (0.5%) - Ser464Tyr - - 0 2 (0.2%) - Ser464Phe, Gln465Leu - - 0 1 (0.1%) - - - - 353 (41%) 879 (88%)
(b) Nucleotide substitutions resulting in QRDR coding changes. Gene Nucleotide change Amino acid change gyrA C248T Ser83Phe
C248A Ser83Tyr G259A Asp87Asn A260G Asp87Tyr G259T Asp87Tyr*
gyrB C1391T Ser464Phe C1391A Ser464Tyr A1394T Gln465Leu A1394G Gln465Arg
parC G239T Ser80Ile G250A Glu84Lys A251G Glu84Gly
parE C1246T Leu416Phe G1258A Asp420Asn
Nature Genetics: doi:10.1038/ng.3281
Supplementary Table 6. Novel phage sequences identified in S. Typhi H58 isolates. A summary of the intact
prophage regions, and the corresponding closest sequenced phage reference genomes, that were identified in 853
H58 isolates using PHAST 2. The number of isolates that contain the phage sequence and their geographical
origins are described. The phylogenetic distribution of each of the phages is shown in Supplementary Figure 6.
Novel phage regions
Number of genes
Regions size (kb)
Closely related phage family Number of isolates
Countries of isolation
1 24 27.8 P4-like phage 3,4 1 1
Laos Malawi
2 80 68.4 SP4 (P2-like phage) 5 1 Malawi
3 58 52.8 SE-OLF-10058 (P2-like phage) 6 24 India
4 44 35.9 EC026_P13 (P2-like phage) 7 1 India
5 79 50.1 P1-like phage (cryptic plasmid) 8,9 54 8 1
Vietnam Cambodia Pakistan
Nature Genetics: doi:10.1038/ng.3281
Supplementary Table 7. Non-synonymous SNPs that define the H58 lineage. The non-synonymous SNPs that are present in 99%
of H58 isolates and not in the non-H58 isolates. Gene annotation was performed using the S. Typhi CT18 database at NCBI
file://localhost/(http://www.ncbi.nlm.nih.gov/; Accession number AL513382). Functional categories are as annotated in the S. Typhi
CT18 genome (Accession number: AL513382). Key for derived base: A = adenine; C = cytosine; G = guanine; T = thymidine. Key
for ancestral AA (amino acid) and derived AA: A = alanine; R = arginine; N = asparagine; D = aspartic acid; C = cysteine; E =
glutamic acid; Q = glutamine; G = glycine; H = histidine; O =hydroxyproline; I = isoleucine; L = leucine; K = lysine; M =
methionine; F = phenylalanine; P = proline; S = serine; T = threonine; W = tryptophan; Y = tyrosine; V = valine; * = stop codon.
Coordinate in CT18
Base in H58
Base in non-H58
Amino acid substitution Gene name Functional Category Function
2750755 A G A2421T STY2875 Pathogenicity/adaptation/chaperones Large repetitive protein
1629304 A G A25V ssaP Pathogenicity/adaptation/chaperones SPI2 type III secretion system protein SsaP
2875160 A G Q185* sptP Pathogenicity/adaptation/chaperones Pathogenicity effector tyrosine phosphatase protein SptP
4192687 T C A32V gph Degradation of small molecules Phosphoglycolate phosphatase 3843665 T C G204D dsdA Degradation of small molecules D-serine dehydratase 89102 G A K201R etfB Degradation of small molecules Protein fixA 3152879 A G M286I uxuA Degradation of small molecules D- mannonate dehydrolase 3004181 C T E337G recB Degradation of macromolecules Exonuclease V subunit 1360939 T C Q30* dbpA Pseudogenes Putative ATP-dependent RNA helicase
3360344 C T H97R nanE2 Pseudogenes Putative N-acetylmannosamine-6-phosphate 2-epimerase 2
4196909 A G A56V bigA Pseudogenes Putative surface-exposed virulence protein 3824631 G T D60E torC Pseudogenes Cytochrome c-type protein 1286044 C T D372G trpE Central/intermediary metabolism Anthranilate synthase component 1 3144053 C A V203G puuB Central/intermediary metabolism Putative oxidoreductase 693560 T C M100I rlpB Central/intermediary metabolism Rare lipoprotein B precursor
4273783 A C R1019S metH Central/intermediary metabolism Putative B12-dependent methionine synthase
2401233 A G R116C yfbT Central/intermediary metabolism Putative phosphoglycolate phosphatase 40159 A G G11E betC Central/intermediary metabolism Putative secreted sulfatase 880083 G A T201A iaaA Central/intermediary metabolism Putative L-asparaginase 387595 T C T204I rtn Central/intermediary metabolism Putative rtn protein 2972433 T C M51I csrB stable RNA CsrB regulator 4665891 A G A245V arcA Regulators Arginine deiminase 2002943 A G L63F sirA Regulators Invasion response-regulator 3398551 A G A620V yhdA Membrane/surface structures Putative lipoprotein
3659647 T C G251E lsrC Membrane/surface structures Putative ABC transporter permease protein
529155 G A K590E kefA Membrane/surface structures Integral membrane protein AefA
1270888 A G R252Q kcsA Membrane/surface structures Putative membrane transport protein (voltage-gated potassium channel)
2202853 T C R334C yegT Membrane/surface structures Putative nucleoside permease 461438 A G R4C yajI Membrane/surface structures Putative lipoprotein 4020211 T C T44M lip1 Membrane/surface structures Putative membrane protein 2388057 A G T530I nuoG Information transfer Putative NADH dehydrogenase I chain G 1810914 A G A99T SBOV18161 Information transfer Hydrogenase-1 operon protein HyaE 3484294 T C V213I wecF Conserved hypothetical Putative 4-alpha-L-fucosyl transferase 4775254 A C P237Q yjjV Conserved hypothetical Putative deoxyribonuclease 4253640 A G A315T dprA Conserved hypothetical Hypothetical protein 387082 A G
Intergenic
1055966 T C
Intergenic 2288504 T C
Intergenic
2348633 A G
Intergenic 2662406 G A
Intergenic
3182059 G A
Intergenic 3693688 T C
Intergenic
3863384 G T
Intergenic 4214165 T C
Intergenic
Nature Genetics: doi:10.1038/ng.3281
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Nature Genetics: doi:10.1038/ng.3281