J. Clin. Microbiol. doi:10.1128/JCM.02026-14 Copyright ...jcm.asm.org/content/early/2014/11/06/JCM.02026-14.full.pdf · 4 75 The common view is that the emerging global S. Typhi haplotype;

1

For publication in: Journal of Clinical Microbiology 1

2

Genomic Signature of Multi-Drug Resistant Salmonella Typhi related to a Massive 3

Outbreak in Zambia during 2010 - 2012. 4

5

Rene S. Hendriksen 1* , Pimlapas Leekitcharoenphon

1, Oksana Lukjancenko

1, Chileshe 6

Lukwesa-Musyani 2, Bushimbwa Tambatamba

3, John Mwaba

2, Annie Kalonda

4, Ruth 7

Nakazwe 2, Geoffrey Kwenda

4, Jacob Dyring Jensen

1, Christina A. Svendsen

1, Karen K. 8

Dittmann1, Rolf S. Kaas

1, Lina M. Cavaco

1, Frank M. Aarestrup

1, Henrik Hasman

1, James 9

C.L Mwansa 2 10

11

1 WHO Collaborating Centre for Antimicrobial Resistance in Foodborne Pathogens and 12

European Union Reference Laboratory for Antimicrobial Resistance, National Food Institute, 13

Technical University of Denmark, Kgs. Lyngby, Denmark 14

2Department of Pathology and Microbiology, University Teaching Hospital, P/B RW 1X, 15

Lusaka, Zambia 16

3Department of Public Health,

Zambia Ministry of Health, Ndeke House, 205305, Lusaka , 17

Zambia. 18

4Department of Biomedical Sciences, School of Medicine, University of Zambia, P.O Box 19

50110, Lusaka 20

21

*: Corresponding author: Rene S. Hendriksen, 22

National Food Institute, Technical University of Denmark 23

Kemitorvet, Building 204, DK-2800 Kgs. Lyngby, Denmark 24

Phone: +45 35 88 60 00 25

JCM Accepts, published online ahead of print on 12 November 2014J. Clin. Microbiol. doi:10.1128/JCM.02026-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Fax: +45 35 88 60 01 26

E-mail: [email protected] 27

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Running title: Resistant Salmonella Typhi in Zambia 29

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Key words: Salmonella Typhi, epidemiology, whole genome sequencing, haplotype H58, 31

antimicrobial resistance genes, replicon incQ1, replicon incHI1, antimicrobial resistance 32

islands, mobile genetic elements, chromosomal translocation, class 1 integron, mer-operon, 33

plasmid, sub-Saharan Africa, Zambia, MIC determination, outbreak, SNP analysis, 34

phylogenetic tree, deletions. 35

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Abstract 51

Retrospectively, we investigated the epidemiology of a massive Salmonella enterica serovar 52

Typhi outbreak in Zambia during 2010 to 2012. 53

54

Ninety-four isolates were susceptibility tested by MIC determinations. Whole genome 55

sequence typing (WGST) of 33 isolates and bioinformatic analysis identified the MLST, 56

haplotype, plasmid replicon, antimicrobial resistance genes, and the genetic relatedness by 57

Single Nucleotide Polymorphism (SNP) analysis and genomic deletions. 58

59

The outbreak affected 2,040 patients with a fatality rate of 0.5%. Most isolates (83.0%) were 60

multi-drug resistant (MDR). The isolates belonged to MLST ST1 and a new variant of the 61

haplotype; H58B. Most isolates contained a chromosomally translocated region containing 62

seven antimicrobial resistance genes; catA1, blaTEM-1, dfrA7, sul1, sul 2, strA, and strB, 63

fragments of incQ1plasmid replicon, class 1 integron, and the mer operon. The genomic 64

analysis revealed an overall 415 SNPs difference and 35 deletions among 33 of the isolates 65

whole genome sequenced. In comparison with other genomes of H58, the Zambian isolates 66

separated from genomes from Central Africa and India with 34 and 52 SNPs, respectively. 67

68

The phylogenetic analysis indicates that 32 isolates of the 33 sequenced belonged to a tight 69

clonal group, distinct from other H58 genomes included in the study. The small numbers of 70

SNPs identified within this group are consistent with short-term transmission that can be 71

expected over a period of 2 years. The phylogenetic analysis and deletions suggest that a 72

single MDR clone was responsible for the outbreak during which occasional other S. Typhi 73

lineages including sensitive ones continued to co-circulate. 74


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The common view is that the emerging global S. Typhi haplotype; H58B, containing the 75

MDR incHI1 plasmid is responsible for the majority of typhoid infections in Asia and sub-76

Saharan Africa; we found that a new variant of the haplotype harbouring a chromosomally 77

translocated region containing the MDR islands of incHI1 plasmid emerged in Zambia. This 78

could chance the perception of the term “classical MDR typhoid” currently being solely 79

associated with the incHI1 plasmid. It might be more common than anticipated that S. Typhi 80

haplotype; H58B harbour either the incHI1 plasmid and /or a chromosomally translocated 81

MDR region. 82

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Introduction 100

Typhoid fever is an ancient, but still important cause of human illness, mostly in developing 101

countries with poor sanitation and lack of potable water (40). It is estimated that typhoid 102

fever is responsible for up to 26,9 million annual cases of which 223,000 cases have a fatal 103

outcome predominately among children (9). Typhoid fever has been reported to have the 104

highest incidence in Asia, whereas the incidence rate has decreased in Latin America due to 105

improved sanitation and economic development (10,16,34). In sub-Saharan Africa, non-106

typhoidal Salmonella have been reported to predominate in contrast to typhoid fever (10,36). 107

Despite this, large outbreaks of typhoid fever are still frequently reported from the sub-108

Saharan African region (1,30,37,38). 109

Beginning in early November 2010, there was a large typhoid fever outbreak in Zambia, 110

Zimbabwe, and the Democratic Republic of Congo. These outbreaks have to a large extent 111

been overlooked in a region plagued by many needs and few resources. 112

http://www.theatlantic.com/health/archive/2012/04/controlling-the-typhoid-epidemic-113

plaguing-sub-saharan-africa/255243/ 114

For future surveillance and control of S. Typhi infections in the region, it is important to 115

know whether the outbreak in 2010 to 2012 was a result of a single novel introduction or 116

reflects an endemic situation with multiple clones and lineages. Thus the main purpose of the 117

present study was to determine the genetic relatedness of contemporary clinical S. Typhi 118

isolates using whole genome sequence typing. In addition, another purpose was to determine 119

if this was the result of the expanding globally dominant haplotype H58 containing the 120

incHI1 plasmid type associated with multi-drug resistant (MDR) by investigating the 121

occurrence and genetic mechanisms of antimicrobial resistance and plasmid replicons. 122

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Materials and Methods 124


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Epidemiological information 125

Data associated with a typhoid fever outbreak covering the full outbreak period from January 126

2010 to September 2012 were extracted from the laboratory records at the University 127

Teaching Hospital (UTH) in Lusaka, Zambia and used to describe the distribution of cases 128

among the genders, age and time. 129

130

Samples and bacterial isolates 131

In general, stool and blood samples were subjected to standard microbiological procedures 132

for isolation (Supplementary Text). All presumptive positive suspected S. Typhi colonies 133

were identified by biochemical tests and serogrouping (Supplementary Text) and 134

subsequently subjected to a S. Typhi specific PCR assay for confirmation (26). Only a small 135

number of S. Typhi isolates; 94 were available for further analysis of the 2,040 cases 136

identified during the outbreak. The remaining isolates were either not stored or not viable in 137

Zambia. The 94 S. Typhi isolates available and included the study were sent to the Technical 138

University of Denmark, National Food Institute (DTU-Food) for further characterization. 139

140

Antimicrobial susceptibility testing 141

Minimum inhibitory concentration (MIC) determination was performed on the 94 S. Typhi 142

isolates using a commercially prepared dehydrated panel (Sensititre; TREK Diagnostic 143

Systems Ltd., East Grinstead,England), agar dilution technique, and E-tests (7,8). The tested 144

antimicrobials and classes have been listed in Table 2. Clinical and Laboratory Standards 145

Institute (CLSI) (7) clinical breakpoints interpretative criteria for resistance (R) were used 146

except for a few antimicrobials for which epidemiological cut-off values was applied 147

according to EUCAST recommendations (Supplementary Text) (http://www.eucast.org). 148


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Quality control was performed by using reference strain E. coli ATCC 25922 according to 149

CLSI guidelines (7,8). 150

151

Whole genome sequencing, multilocus sequence typing, antimicrobial resistance genes and 152

plasmid replicons. 153

Due to sequence costs at DTU, it was only possible to further investigate a subset of 33 154

isolates which was conveniently selected for whole genome sequencing typing (WGST) 155

based on the criterion to ideally cover all antimicrobial resistance phenotypes, the different 156

specimen types; stool and blood isolates, and different years of isolation; 2010, 2011, and 157

2012, respectively (Table 1). 158

Genomic DNA was extracted from the 33 isolates using an Invitrogen Easy-DNATM

Kit 159

(Invitrogen, Carlsbad, CA, USA) and DNA concentrations were determined using the Qubit 160

dsDNA BR assay kit (Invitrogen). The genomic DNA was prepared for Illumina pair-end 161

sequencing using the Illumina (Illumina, Inc., San Diego, CA) NexteraXT® Guide 162

150319425031942 following the protocol revision C 163

(http://support.illumina.com/downloads/nextera_xt_sample_preparation_guide_15031942.ht164

ml). A sample of the pooled NexteraXT Libraries was loaded onto a Illumina MiSeq reagent 165

cartridge using MiSeq Reagent Kit v2 and 500 cycles with a Standard Flow Cell. The 166

libraries were sequenced using an Illumina platform and MiSeq Control Software 2.3.0.3. 167

Twenty-four isolates were pair-end and six isolates were single-end sequenced. Pair-end 168

sequences ranged in insert size from 11 to 129 with an average of 68. The read depth of the 169

sequences was between 147 to 497 with an average of 259. 170

Five previously published genomic sequences of haplotype H58; AG3, E02-2759, ISP-04-171

06979, E03-9804, ISP-03-07467 were obtained from GenBank and Sanger Institute (accessed 172

5/4/2013). Full genomic information is shown in Supplementary Table 1A. 173


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Raw sequence data have been submitted to the European Nucleotide Archive 174

(http://www.ebi.ac.uk/ena) under study accession no.: PRJEB7179 and PRJEB7182. The raw 175

reads were assembled using the Assemble pipeline (version 1.0) available from the Center for 176

Genomic Epidemiology (CGE) http://cge.cbs.dtu.dk/services/all.php which is based on the 177

Velvet algorithms for de novo short reads assembly. A complete list of genomic sequence 178

data is available in the Supplementary Table 1A. The assembled sequences were analyzed to 179

identify the MLST sequence type (ST) for Salmonella enterica, plasmid replicons, and 180

acquired antimicrobial resistance genes using the pipelines; MLST (version 1.7), 181

PlasmidFinder (version 1.2), and ResFinder (version 2.1) available from CGE (6,23,54). 182

183

Transferability of incQ1 plasmid replicon by conjugation and electroporation 184

Due to the detection of an incQ1 plasmid replicon, plate-mating experiments were attempted 185

with four donor S. Typhi isolates; # 31, #34, #54, and #71 selected based on pylogenetic 186

clustering (same or distant) and plasmid-free, rifampicin and nalidixic acid resistant E. coli 187

MT102RN as recipients (47). In addition, the four S. Typhi isolates were subjected to plasmid 188

purification using Qiagen kits (Venlo, the Netherlands) and attempted electroporated into 189

electrocompetent E. coli DH10B cells (Supplementary Text). 190

191

Identification of the chromosomally translocated MDR region including the incQ1island. 192

The lack of plasmids in the four stains could be a result of chromosomal translocation of the 193

genomic region containing the incQ1 plasmid replicon why genomic DNA was extracted 194

from the isolates # 31, #34, #54, and #71 using an Invitrogen Easy-DNATM

Kit for further 195

analysis. The genomic DNA was prepared for Illumina mate pair sequencing using the Gel-196

Free version of the Illumina Nextera® Mate Pair Sample Preparation Kit strictly following 197

the protocol revision D and sequenced using an Illumina platform. 198


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Prior to assembly, the data quality was assessed using FastQC quality control tool 199

http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ and reads with quality score of 200

below 20 were filtered out. The genomes of the four strains were assembled using 201

SOAPdenovo2 software (31) by combining both paired-end and mate pair raw reads. 202

Subsequently, the two integration points of the chromosomally translocated region were 203

verified by PCR amplification and Sanger sequencing (Supplementary Text). Amplicons 204

produced of the four strains were selected for sequencing and shipped to Macrogen Inc., 205

(Macrogen, Amsterdam, the Netherlands) for sequencing using the same primers as in the 206

PCR analysis. The genomes of the four strains were re-assembled using CLC Bio Workbench 207

by combining the Sanger sequences with the previously assembled scaffold. The raw 208

sequence data have been submitted to the European Nucleotide Archive 209

(http://www.ebi.ac.uk/ena) under accession no.: In progress. 210

Open Reading Frames (ORFs) were predicted on the scaffolds using Prodigal software (19) 211

and were subsequently functionally annotated by constructing functional profiles for all 212

proteins using the PanFunPro tool (29). 213

A functional profile is the combination of all non-repeating functional domains in each ORF. 214

The profiles were created by using InterProScan software to scan the annotated proteins 215

against the collections PfamA, TIGRFAM and Superfamily based on Hidden Markov Models 216

(HMMs) to identify non-overlapping functional domains with an E-value below 0.001 (29). 217

Through this annotation and analysis, the position of the incQ1 replicon fragment was 218

identified in every strain. The respective scaffolds containing this fragment were further 219

compared to the complete genome and plasmid pHCM1 of the reference strain S. Typhi CT18 220

(National Center for Biotechnology Information, accession: AL513382, length of 4,809,037 221

bp) in order to determine the exact insertion site and homology between the strains 222

(Supplementary Table 1E, 1F). 223


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224

Screenings for mutations in DNA Gyrase and DNA topoisomerase IV genes 225

Each of the 33 S. Typhi genomes was examined for the presence of mutation in the DNA 226

gyrase; gyrA and gyrB genes, and the DNA topoisomerase IV; parC and parE genes (49) by 227

determining SNPs in comparison with S. Typhi CT18. Additionally, the gyrA sequences of 228

quinolone resistant strains (#269, #748) were extracted and translated. 229

230

Phylogenetic structure of S. Typhi using Single Nucleotide Polymorphisms, calculation of 231

dN/dS, identification of S. Typhi haplotypes, and genomic deletions 232

SNPs were determined using the pipeline; SnpTree (version 1.1) available on the CGE (24). 233

Fundamentally, each of the assembled genomes or contigs were aligned against the reference 234

genome; S. Typhi CT18 using the application “Nucmer” of MUMmer version 3.23 (12). 235

SNPs were identified from the alignments using “Show-snps” (using option “-Cl1rT”) from 236

MUMmer. Subsequently, SNPs were selected when meeting the following criteria: 1) a 237

minimum distance of 20 bps between each SNP, and 2) all indels were excluded. The 238

selected SNPs from assembled genomes were confirmed by SNPs being called by mapping 239

raw reads to the reference genome using BWA (27) and SAMTools (28). 240

The qualified SNPs from each genome were concatenated to a single alignment 241

corresponding to position of the reference genome using an in-house Perl script. In case SNPs 242

were absent in the reference genome, they were interpreted as not being a variation and the 243

relatively base from the reference genome was expected (24). The concatenated sequences 244

were subjected to multiple alignments using MUSCLE from MEGA5 (51). The final 245

phylogenetic SNP tree was computed by MEGA5 using the maximum likelihood method of 246

1,000 bootstrap replicates (13) using Tamura-Nei model for inference (50). All 415 SNPs 247

related to the outbreak isolates of haplotype H58B are listed in the Supplementary Table 1B. 248


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249

The ratio of the number of non-synonymous substitutions per non-synonymous site (dN) to 250

the number of synonymous substitutions per synonymous site (dS) is a measurement of 251

stabilizing selection (17). A ratio of 1 is expected in the absence of selection, a low ratio 252

(dN/dS<1) indicates stabilizing selection, while a high ratio (dN/dS>1) indicates positive 253

selection (44). The dN/dS ratio, was calculated for each core gene (the genes found in all S. 254

Typhi genomes in this study) using codeML from the package PAML (52). The 255

approximation of the dN/dS ratio was an average of dN/dS from all core genes. 256

257

In contrast to previously methods by Roumagnac et al. (46), a whole genome sequencing 258

approach was used to assigned biallelic polymorphisms positions (BiP) to all the genomes 259

included this study based on BiPs in the reference genome; S. Typhi CT18 using a python 260

script. All BiPs are listed in the Supplementary Table 1C. The haplotype of each genome was 261

determined by the combination of assigned BiPs using the haplotype dendrogram by 262

Roumagnac et al. (46) such as haplotype H58 being defined by BiP36, BiP48, BiP56, and 263

BiP33. Additionally, node B of haplotype H58 lineage I was determined based on SNP 264

position 1,193,220 as defined by Kariuki et al. (21). 265

266

Indels and deletions were excluded from the SNP analysis, why a BLAST atlas based on 267

BLASTP (14) was used to visualize the homology in a comparison of the genomes against 268

the reference genome; S. Typhi CT18 in order to identify potential deletions. The putative 269

deletions were aligned against Zambian genomes using execrate (48). The hit score was 270

calculated by multiplying percent identify with deletion’s alignment length and dividing with 271

deletion’s sequence length. The presence of deletions in the Zambian genomes was 272

confirmed based on the hit score with a threshold of at least 95%. The presence and absence 273


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of the deletions were finally visualized in a heatmap sorted for comparison according to the 274

position of the stains in the pylogenetic tree. Details of the genomic deletions detected in this 275

study are listed in the Supplementary Table 1D. 276

277

Results 278

Epidemiological data 279

2,040 cases were identified of a population of 14.2 million inhabitants during the outbreak 280

from January 2010 to September 2012 with a median of 48 cases per month (Figure 1). The 281

number of cases ranged from one in February 2010 to 246 cases in February 2012. Among 282

the 41 peri-urban health centres in Lusaka (2.1million inhabitants), cases ranged from one to 283

369 cases per centre with a median of 10 cases per centre (3). 284

The overall case fatality rate was estimated to be 0.5% during the outbreak period (3). The 285

majority of cases (n = 1,771; 87%) occurred in children less than 15 years of age ranging 286

from 22 (1.1%) cases in children of less than eight months of age to four cases (0.2%) among 287

patients older than 61 years (Figure 2). Most cases (n = 1,200; 58.8%) were observed within 288

the age group between six to 15 years of age. The cases were evenly distributed by gender 289

with 1,078 (52.8%) male cases (Figure 2) (3). 290

291

Antimicrobial resistance, antimicrobial resistance genes, plasmid replicons, and the 292

chromosomally translocated MDR region. 293

The MIC determination of the 94 S. Typhi isolates revealed a high frequency of antimicrobial 294

resistance. Most (83%) of the S. Typhi isolates exhibited resistance to a core of five 295

antimicrobials: ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and 296

trimethoprim (Table 2). Four (4.3%) of the isolates showed low level (reduced susceptibility) 297

resistant to ciprofloxacin. Of those, three (3.2%) isolates were also resistant to nalidixic acid. 298


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None of the isolates were resistant to the following antimicrobials: apramycin (only approved 299

for veterinary use), azithromycin, cefotaxime, ceftiofur (only approved for veterinary use), 300

colistin, florfenicol (only approved for veterinary use), gentamicin, neomycin, spectinomycin, 301

and tetracycline. One isolate (#551) was pan-susceptible (Table 2). The frequency of 302

antimicrobial resistance were similar for submitted stool (n = 8) and blood (n = 86) samples 303

(Table 2). 304

Among, the 33 WGST S. Typhi isolates, all but the pan-susceptible isolate (#551) (97.0%) 305

exhibited phenotypic antimicrobial resistance (MIC) to the following antimicrobials: 306

ampicillin, streptomycin, sulfamethoxazole, and trimethoprim. In addition, 27 (81.8%), four 307

(12.1%), and two (6.1%) of the isolates conferred also resistance to chloramphenicol, 308

amoxicillin + clavulanic acid, and ciprofloxacin / nalidixic acid, respectively (Table 2). 309

310

All 32 WGST resistant isolates, contained the following genes; strA, strB, ΔaadA1 311

(aminoglycoside: streptomycin), and blaTEM-1 (beta-lactam: ampicillin). Among the 32 312

resistant isolates, some harboured different resistance genes within the same drug class such 313

as for sulfamethoxazole where isolate #6, #14, #35, #739, and #1341 contained only the sul2 314

gene in contrast to isolate #5 which only harboured the sul1 gene. The remaining resistant 315

isolates all contained both sul1 and sul2 genes. For trimethoprim, isolates: #6, #14, #35, 316

#739, and #1341 contained the dfrA14 gene in contrast to the remaining isolates which all 317

harboured the dfrA7 gene. Of the 32 resistant isolates, all except isolates; #6, #14, #35, #739, 318

and #1341 presented resistance to chloramphenicol and harboured the catA1 gene. 319

320

All 32 WGST resistant isolates were investigated for the presence of fluoroquinolones 321

resistance associated with mutations in the Quinolone Resistance Determinant Regions 322

(QRDR) of the gyrase and DNA topoisomerase IV genes; gyrA, gyrB, parC, and parE and 323


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Plasmid-Mediated Quinolone Resistance (PMQR) genes; qnrA, qnrB, qnrC, qnrD, qnrS, 324

qepA and aac(6′)-lb. In two strains, #269 and #748, different single mutations in gyrA QRDR 325

were detected leading to an amino acid substitution in codon Asp87 (Asp-Asn) in strain #269 326

and in codon Ser83 (Ser-Tyr) in strain #748. This correlates well with their phenotype as 327

these were found decreased susceptibility to ciprofloxacin and resistant to nalidixic acid. The 328

two remaining isolates showing decreased susceptibility to ciprofloxacin were not among the 329

isolates selected for WGST and therefore not investigated for the presence of above 330

mentioned genes nor mutations. 331

332

Importantly, none of the 33 WGST S. Typhi isolates appeared to possess the globally 333

dominating plasmid replicon type incHI1 normally found in haplotype H58 when the 334

sequencing data were analyzed using the PlasmidFinder. However, the analysis revealed 27 335

isolates containing an incQ1 plasmid replicon sequence (repC and ΔrepA in Figure 3). In 336

addition, five isolates; #6, #14, #35, #739, and #1341 contained of an incFIB plasmid 337

replicon. One isolate; the pan-susceptible #551 did not reveal any plasmids replicons. 338

339

It was confirmed that ΔrepA and repC; a truncated incQ1 region were present in the plasmid 340

DNA region of all four strains; #31, #34, #54, and #71 in the in silico comparison with the 341

reference stain; S. Typhi CT18. Despite the presence of plasmid replicons in the genomes of 342

the strains, none of them seemed to harbor complete plasmids that could be transferred nor 343

extracted by several commercially available kits in vitro. Bioinformatic analysis of the 344

combined paired-end, mate-pair, and Sanger sequencing of the four isolates revealed a 345

translocation of the plasmid replicon; incQ1 and the antimicrobial resistance islands from an 346

ancestral incHI1 plasmid to identical positions in the chromosome of the present Zambian 347

Typhi isolates (Figure 3). The chromosomally translocated region had a size in range of 348


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23,376 bp (isolate #54) and inserted the chromosome between position 3,470,424 bp and 349

3,472,059 bp and flanked by the genes; cyaY and cyaA according to the reference genome S. 350

Typhi CT18 (Figure 3). The gene content of the four strains was highly similar. The 351

translocated region carried, a complete mercury resistance (mer) operon, a tnpM, and an IS3 352

element flanked the truncated incQ1replicon which was succeeded downstream by genes 353

encoding resistance to sulfonamides; sul2 and streptomycin; strA, strB. These resistance 354

genes were flanked by a transposase gene; tnpB and another resistance gene to β-lactams; 355

blaTEM-1. In between two inserted sequences IS26; tnpR, a class 1 integron consisting of an 356

integrase gene; int, ΔaadA1, and dfrA7 was localized and flanked by genes encoding 357

quaternary ammonium compounds resistance; qucE, sulfonamide resistance; sul2 (upstream), 358

and a transposase; tnpM (downstream). Yet, another IS26, tnpA element flanked the integrin 359

cassette further downstream alongside the genes GNAT and catA1 encoding an 360

acyltransferase and chloramphenicol resistance, respectively (Figure 3). In addition, the PCR 361

amplification of the two integration points of the chromosomally translocated region on the 362

remaining strains revealed that all except for the six isolates; #6, #14, #35, #739, #1341, and 363

#551produced amplicons. This indicates that all except for the six isolates similarly contained 364

the chromosomally translocated region. 365

366

MLST, haplotypes, dN/dS ratio, and population structure of S. Typhi based haplotypes, 367

Single Nucleotide Polymorphisms and genomic deletions. 368

Of the 33 S. Typhi genomes, 32 belonged to MLST ST1 and a new minor variant of 369

haplotype H58 node B of the Kenyan S. Typhi lineage I. We do not propose a number nor a 370

name for this variant; H58B var. as suggested by Dr. Mark Achtman (Personal 371

communication). The genetic evolution of the 32 H58B var. genomes seemed to have a 372

neutral stabilizing selection based on core genes as the dN/dS ratio was 0.94 indicating 373


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limited adaptive evolution and recombination. The pan-susceptible isolate #551 belonged to 374

MLST ST2 and haplotype H14. 375

376

A phylogenetic SNPs tree rooted to the reference genome; S. Typhi CT18 belonging to 377

haplotype H1 with the inclusion of the available non-outbreak genomes from sub-Saharan 378

Africa and Asia of haplotype H58 was reconstructed to investigate the evolutionary 379

relationships (Figure 4A). The tree revealed 1,744 high quality whole genome SNPs. Two 380

synapomorphic (clade specific) SNPs were detected among the Zambian genomes (excluding 381

the genome of strain #551) defining the new minor variant; H58B var. of haplotype H58B 382

(Figure 4A, marked in blue). One of the two synapomorphic SNPs was synonymous at 383

positions 4,638,263 causing substitution C-T. The other synapomorphic SNP identified was 384

located at position 789,347 in the intergenic region leading to the substitution G-A. 385

The topology of the reference genome S. Typhi CT18 rooted tree showed that the closest 386

haplotype H58 out-group neighbor to the 32 Zambian S. Typhi haplotype H58B var. genomes 387

were ISP-04-06979 from Central Africa and E02-2759 from India separated by 34 and 52 388

SNPs, respectively (Figure 4A). 389

390

A similar reference genome rooted phylogenetic SNPs tree including the outbreak genomes 391

from Zambia belonging to haplotype H58B var. (excluding #551; H14) contained an overall 392

415 high quality whole genome SNPs. Among those, 47 were autapomorphic SNPs (genome 393

specific, marked in red) and three synapomorphic SNP (clade specific, marked in blue; two 394

SNPs defining H58B var. and one at positions 2,024,187 causing substitution Gly-Ser 395

representing a clade consisting of four genomes; #1, #15, #46, and #53) (Figure 4B). The 396

topology of the SNP tree revealed three clades of four to six genomes each pairwise separated 397

by less than 15 SNPs. 398


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Overall, SNPs were relative frequent among the 32 S. Typhi isolates of haplotype H58B var. 399

separating individual isolates from the nearest neighbor with two to 62 SNPs pairwise 400

separation. The phylogenetic analysis provided evidence for clonal diversity among the 401

WGST population, with a large monophyletic substructure (subclades) that displays clear 402

differentiations. There was no obvious clustering related to time (year) within the S. Typhi 403

phylogenetic groups. However, one of the monophyletic subclades were observed containing 404

the four isolates; #14, #35 #1341, and #739 - all in concurrence with the variation of 405

antimicrobial resistance genes and plasmid replicon compared to the remaining WGST 406

isolates (Figure 4B). 407

408

Deletions were relative common among the 32 WGST genomes; excluding strain #551; H14 409

in the comparison with the reference genome; S. Typhi CT18. Thirty-five deletions were 410

detected among the genomes ranging in number of deleted genes and size from one gene (29 411

deletions); #D19 (173bp, STY2210) up to six genes (one deletion); #D14 (4038bp, STY2181 412

- STY2186) (Supplementary Table 1D, Figure 5). The majority (n = 26; 74%) of the deletions 413

affected more than 28 of the genomes (Figure 5). A spare clustering was observed among the 414

genomes where in particular, genome; #7 and #12 (11-05-2010) and #15 and #9 all lacked 415

the deletions; #D2, #D6, #D28, #D29, and #D30. This clustering is in agreement with the 416

topology of the pylogenetic SNP analysis whereas the overall clustering based on deletion 417

was not consistent (Figure 4B, Figure 5). One genome; #8 were in comparison with the other 418

genomes more conversed lacking 17 out of the 35 deletions. 419

420

Discussion 421

Among the 33 WGST strains, we did not find any replicon for an incHI1 plasmid, normally 422

associated with the “classical MDR haplotype H58” (21). Apart from the plasmid replicon 423


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incHI1, only a few other replicons have been observed in S. Typhi – all in isolates originating 424

from Pakistan (33). From here, the plasmid replicon incFIA seemed to be predominant but 425

also incFIIA, incP, and incB/O replicons were identified. 426

In this study, we identified 27 S. Typhi isolates containing an incQ1plasmid replicon 427

fragment and five isolates with an intact incFIB plasmid replicon. We confirmed by 428

additional Sanger sequencing that four out of 27 incQ1-positive isolates harboured a 429

chromosomally translocated antimicrobial resistance region. Acquisition of chromosomally 430

translocated regions containing antimicrobial resistance islands, integrons, and mercury 431

resistance genes have previously been observed in S. Typhimurium. A unique 82 Kb genomic 432

island; GI-DT12 was recently reported in S. Typhimurium isolated from a human 433

gastroenteritis case. The region was believed acquired by horizontal gene transfer and 434

harboured a class one integron, a mer operon, and several resistance genes believed to 435

contribute to the ability of survival in adverse environment (20). This raises the question if 436

the acquisition of the chromosomal translocated region of the Zambian S. Typhi isolates is 437

the result of an adverse environment due to poor sanitation. Similar acquisition of fragments 438

of the plasmid replicon; incQ1 and antimicrobial resistance islands as identified in this study 439

has been reported in S. Typhimurium and S. Enteritidis evolving from the R27/incHI1 440

plasmid through co-integration with the pHCM1/incHI1 plasmid of ancestral S. Typhi’s 441

(11,32). However, none of the none-typhoid Salmonella serovars contained the islands the 442

catA1 gene and mer operon as observed in this study nor did the authors describe how and 443

from where the class 1 integron originated. Interestingly, we found the entire region from the 444

H58 S. Typhi plasmid; pHCM1 (Genbank accession number NC_003384; originating from 445

strain CT18) (18) containing seven antimicrobial resistance genes, fragments of 446

incQ1plasmid replicon (repΔA, repC), class 1 integron and the mer operon chromosomally 447

translocated but recombined according to the structure described by Miriagou et al. (32). We 448


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suggest that the class 1 integron containing the dfrA7 gene, and first described in the incHI1 449

plasmid from S. Typhi in 2003 (43), recently has integrated the entire MDR region from 450

another position in an incHI1 plasmid prior to the chromosomal translocation. This is to the 451

best of our knowledge the first time this chromosomal translocation has been observed in S. 452

Typhi. However, it might be more common than anticipated that S. Typhi haplotype; H58B 453

harbour either the incHI1 plasmid and /or chromosomally translocated antimicrobial 454

resistance islands. It has been hypothesized that a milestone of incHI1 plasmid evolution had 455

been reached around 1996 by the acquisition of increased fitness that has outcompeted all 456

others plasmid types in S. Typhi (42). However, it has also been debated that plasmid fitness 457

cost must play a role in maintaining incHI1 plasmids in S. Typhi. By the chromosomal 458

translocation of the incHI1 plasmid region in this new variant of S. Typhi H58, we can only 459

speculate in what effect this will have in virulence, transmission, and acquisition of 460

antimicrobial resistance gene e.g. coding for extended spectrum β-lactamase. 461

462

Since historical data on S. Typhi haplotypes are not available from Zambia, the origin, 463

possible introduction and transmission to Zambia is unknown. The intensive presence of and 464

travel to and from India and an influx of Indian immigrants into Zambia could have 465

introduced S. Typhi H58B var. carrying the chromosomally translocated region containing 466

the MDR islands of incHI1 plasmid and the fragment of the incQ1 plasmid replicon to 467

Zambia. However, a more plausible hypothesis is that an ancestral S. Typhi H58B spread 468

from Kenya to Zambia where it early on evolved to S. Typhi H58B var. and acquired the 469

region containing the MDR islands including the class 1integron and the fragment of the 470

incQ1 plasmid replicon by translocation from the incHI1 plasmid. To prove this hypothesis, 471

historical and contemporary isolates from Kenya, the sub-Indian continent, and Zambia needs 472

to be further investigated. 473


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474

The high resolution pylogenetic SNP analysis of the Zambia outbreak isolates in relation to 475

the out-group of non-outbreak strains demonstrated that the Zambian S. Typhi outbreak 476

genomes belonged to a monophyletic group derived from a single recent common ancestor 477

consistent with a genetic bottleneck with a subsequent radiation into an open niche. The 478

speculation that such a common ancestor could originate from India or a neighboring country 479

was supported by the relatedness to H58 genomes from Central Africa and India separated by 480

only 34 and 52 SNPs, respectively. 481

The SNP analysis revealed 2 - 62 SNPs separating the isolates causing disease, suggesting 482

multiple lineages circulating at the point of introductions. Overall this data appears to suggest 483

that the outbreak was caused by a single MDR clone (83% of isolates) persisting during the 484

outbreak period which occasional other S. Typhi lineages including sensitive ones continued 485

to co-circulate. This hypothesis is supported by several studies indicating mutation rates for 486

none-typhoidal Salmonella or SNP differences among outbreaks. Thus, Hawkey et al. 487

suggests a mutation rate of 3 -5 SNPs per year which is consistent with the differences 488

observed among genomes in several of the clades representing the outbreak period from 2010 489

to 2012 (15). This is also supported by the SNP differences of up to 30 SNPs observed among 490

strains in six outbreak investigated by Leekitcharoenphon et al., (25). In contrast, a lower rate 491

was suggested of 1 – 2 SNPs by Okoro et al. which seems to be a bit low in relation to our 492

data (39). It was suggested that the outbreak in Zambia was a result of environmental 493

changes, poor sanitation or a high influx of infected people from neighboring countries. This 494

hypothesis is also supported by the epidemiological data that typhoid fever is endemic and 495

that the outbreak had been ongoing for several years with minimum intervention and control 496

programs. 497

498


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The high level of resistance to first-line antimicrobials for treatment of typhoid fever is 499

worrisome as 83% of the isolates were resistant to five antimicrobial drug classes; 500

aminoglycoside, beta-lactams, phenicols, sulphonamides, trimethoprim and classified as 501

being MDR (4). A similar resistance pattern has also been described to be emerging around 502

the world (4,21,53). Additionally, high level of resistance has also been reported in S. Typhi 503

towards quinolones and fluoroquinolones including single mutations in the QRDR of gyrA 504

gene (5,18,21,40,53). In this study, we found 4.3% of the isolates being classified as nalidixic 505

acid resistant due to single mutations in gyrA at codon Ser83 or codon Asp87. This is the first 506

time this phenotype has been observed in Zambia. This has resulted in clinicians trying 507

alternative antimicrobial treatment regimens such as using ceftriaxone and azithromycin (10). 508

Treatment with ceftriaxone has in shortened courses shown significant relapse rates but a 509

realistic and alternative drug with clinical cure and good reliability (5). However, it has been 510

debated if resistance to third generation cephalosporins has emerged (2,22,35,41,45). Due to 511

MDR and quinolone resistant isolates, it has been recommended that developing countries 512

should use azithromycin as first priority (4,5,40). The development of resistance calls for 513

restrictive use, to avoid over-the-counter usage and to establish real time global antimicrobial 514

surveillance to monitor the development of antimicrobial resistance and enabling to take 515

action in an early stage as possible. 516

517

In the study, there is a disconnection between the presentation of epidemiological data and 518

the selection of only 94 isolates for MIC and 33 for WGST. Only a limited number of the 519

2,040 outbreak strains were in fact available for further analysis as often encountered 520

working with developing countries as only few isolates normally are stored. Despite the low 521

number of strains susceptibility tested and WGST, the authors believe the results and findings 522


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are valid but should be interpret with care. Thus, the determination of the genetic relatedness 523

was believed to be valid due to the selective criteria of the limited number of isolates. 524

525

The common view is that the emerging global S. Typhi haplotype; H58B, containing the 526

MDR incHI1 plasmid is responsible for the majority of typhoid infections in Asia and sub-527

Saharan Africa; we found that a new variant of the haplotype harbouring a chromosomally 528

translocated region containing the MDR islands of incHI1 plasmid emerged in Zambia. This 529

could chance the perception of the term “classical MDR typhoid” currently being solely 530

associated with the incHI1 plasmid. It might be more common than anticipated that S. Typhi 531

haplotype; H58B harbour either the incHI1 plasmid and /or a chromosomally translocated 532

MDR region. 533

The phylogenetic analysis and deletions suggest that a single MDR clone was responsible for 534

the outbreak during which occasional other S. Typhi lineages including sensitive ones 535

continued to co-circulate. 536

In addition to the isolates being MDR, a moderate number of the isolates were also resistant 537

to fluoroquinolones. In general, there is an urgent need for the global community to take on 538

the responsibility and action to assist the developing countries to control emerging infectious 539

diseases such as typhoid fever by improving sanitation, living condition or possible 540

vaccination trials. Additionally, to develop easy to use real time whole genome sequencing 541

tools to assist tracking those emerging infectious diseases patterns and clonal spread for 542

control measurements and ideally predicting local disease hot-spots. 543

544

Acknowledgement 545


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Thanks to the Ministry of Health, Zambia, for permission to send the isolates. The authors are 546

grateful to Dr. Mark Achtman and Dr. Zhemin Zhou, Warwick Medical School University of 547

Warwick Coventry, Ireland for providing biallelic polymorphisms positions. 548

This work was supported by the Danish Council for Strategic Research (grant number: 09-549

067103) and by the World Health Organization Global Foodborne Infections Network 550

551

Reference List 552

553

1. 2012. Notes from the field: Salmonella Typhi infections associated with contaminated 554

water--Zimbabwe, October 2011-May 2012. MMWR Morb.Mortal.Wkly.Rep. 61:435. 555

2. Al, N. N., B. Zwart, M. C. Rijnsburger, R. Roosendaal, Y. J. bets-Ossenkopp, J. A. 556

Mulder, C. A. Fijen, W. Maten, C. M. Vandenbroucke-Grauls, and P. H. 557 Savelkoul. 2008. Extended-spectrum-beta-lactamase production in a Salmonella 558

enterica serotype Typhi strain from the Philippines. J.Clin.Microbiol. 46:2794-2795. 559

3. Anonymous. 2012. National Epidemic Prevention, Preparedness, Control Committee 560

report, Ministry of Health, Zambia. report . 561

4. Bhan, M. K., R. Bahl, and S. Bhatnagar. 2005. Typhoid and paratyphoid fever. 562

Lancet. 366:749-762. 563

5. Butler, T. 2011. Treatment of typhoid fever in the 21st century: promises and 564

shortcomings. Clin.Microbiol.Infect. 17:959-963. 565

6. Carattoli, A., E. Zankari, A. Garcia-Fernandez, L. M. Volby, O. Lund, L. Villa, F. 566

M. Aarestrup, and H. Hasman. 2014. PlasmidFinder and pMLST: in silico detection 567

and typing of plasmids. Antimicrob.Agents Chemother. 568

7. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial 569

Susceptibility Testing. M100-S24. 24th Informational Supplement. 2014. Wayne, PA, 570

USA. 571

8. Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial 572

Susceptibility Tests for Bacteria That Grow Aerobically. M07-A9. 9th 573

Edition[Approved Standard]. 2012. Wayne, PA, USA. 574

9. Crump, J. A., S. P. Luby, and E. D. Mintz. 2004. The global burden of typhoid fever. 575

Bull.World Health Organ. 82:346-353. 576

10. Crump, J. A. and E. D. Mintz. 2010. Global trends in typhoid and paratyphoid Fever. 577

Clin.Infect.Dis. 50:241-246. 578


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

24

11. Daly, M., L. Villa, C. Pezzella, S. Fanning, and A. Carattoli. 2005. Comparison of 579

multidrug resistance gene regions between two geographically unrelated Salmonella 580

serotypes. J.Antimicrob.Chemother. 55:558-561. 581

12. Delcher, A. L., A. Phillippy, J. Carlton, and S. L. Salzberg. 2002. Fast algorithms for 582

large-scale genome alignment and comparison. Nucleic Acids Res. 30:2478-2483. 583

13. Felsenstein J. 1985. Confidence limits on phylogenies: An approach using the 584

bootstrap. Evolution 39:783-791. 585

14. Hallin, P. F., T. T. Binnewies, and D. W. Ussery. 2008. The genome BLASTatlas-a 586

GeneWiz extension for visualization of whole-genome homology. Mol.Biosyst. 4:363-587

371. 588

15. Hawkey, J., D. J. Edwards, K. Dimovski, L. Hiley, H. Billman-Jacobe, G. Hogg, 589

and K. E. Holt. 2013. Evidence of microevolution of Salmonella Typhimurium during 590

a series of egg-associated outbreaks linked to a single chicken farm. BMC.Genomics. 591

14:800. doi: 10.1186/1471-2164-14-800.:800-814. 592

16. Hendriksen, R. S., A. R. Vieira, S. Karlsmose, D. M. Lo Fo Wong, A. B. Jensen, H. 593

C. Wegener, and F. M. Aarestrup. 2011. Global Monitoring of Salmonella Serovar 594

Distribution from the World Health Organization Global Foodborne Infections Network 595

Country Data Bank: Results of Quality Assured Laboratories from 2001 to 2007. 596

Foodborne.Pathog.Dis. 597

17. Holt, K. E., J. Parkhill, C. J. Mazzoni, P. Roumagnac, F. X. Weill, I. Goodhead, R. 598

Rance, S. Baker, D. J. Maskell, J. Wain, C. Dolecek, M. Achtman, and G. Dougan. 599

2008. High-throughput sequencing provides insights into genome variation and 600

evolution in Salmonella Typhi. Nat.Genet. 40:987-993. 601

18. Holt, K. E., M. D. Phan, S. Baker, P. T. Duy, T. V. Nga, S. Nair, A. K. Turner, C. 602

Walsh, S. Fanning, S. Farrell-Ward, S. Dutta, S. Kariuki, F. X. Weill, J. Parkhill, 603 G. Dougan, and J. Wain. 2011. Emergence of a globally dominant IncHI1 plasmid 604

type associated with multiple drug resistant typhoid. PLoS.Negl.Trop.Dis. 5:e1245. 605

19. Hyatt, D., G. L. Chen, P. F. Locascio, M. L. Land, F. W. Larimer, and L. J. 606

Hauser. 2010. Prodigal: prokaryotic gene recognition and translation initiation site 607

identification. BMC.Bioinformatics. 11:119.:119. 608

20. Izumiya, H., T. Sekizuka, H. Nakaya, M. Taguchi, A. Oguchi, N. Ichikawa, R. 609

Nishiko, S. Yamazaki, N. Fujita, H. Watanabe, M. Ohnishi, and M. Kuroda. 2011. 610

Whole-genome analysis of Salmonella enterica serovar Typhimurium T000240 reveals 611

the acquisition of a genomic island involved in multidrug resistance via IS1 derivatives 612

on the chromosome. Antimicrob.Agents Chemother. 55:623-630. 613

21. Kariuki, S., G. Revathi, J. Kiiru, D. M. Mengo, J. Mwituria, J. Muyodi, A. 614

Munyalo, Y. Y. Teo, K. E. Holt, R. A. Kingsley, and G. Dougan. 2010. Typhoid in 615

Kenya is associated with a dominant multidrug-resistant Salmonella enterica serovar 616

Typhi haplotype that is also widespread in Southeast Asia. J.Clin.Microbiol. 48:2171-617

2176. 618


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

25

22. Kumarasamy, K. and P. Krishnan. 2012. Report of a Salmonella enterica serovar 619

Typhi isolate from India producing CMY-2 AmpC beta-lactamase. 620

J.Antimicrob.Chemother. 67:775-776. 621

23. Larsen, M. V., S. Cosentino, S. Rasmussen, C. Friis, H. Hasman, R. L. Marvig, L. 622

Jelsbak, T. Sicheritz-Ponten, D. W. Ussery, F. M. Aarestrup, and O. Lund. 2012. 623

Multilocus sequence typing of total-genome-sequenced bacteria. J.Clin.Microbiol. 624

50(4):1355-1361. 625

24. Leekitcharoenphon, P., R. S. Kaas, M. C. Thomsen, C. Friis, S. Rasmussen, and F. 626

M. Aarestrup. 2012. snpTree--a web-server to identify and construct SNP trees from 627

whole genome sequence data. BMC.Genomics. 13 Suppl 7:S6. doi: 10.1186/1471-628

2164-13-S7-S6. Epub;%2012 Dec 13.:S6-13. 629

25. Leekitcharoenphon, P., E. M. Nielsen, R. S. Kaas, O. Lund, and F. M. Aarestrup. 630

2014. Evaluation of whole genome sequencing for outbreak detection of Salmonella 631

enterica. PLoS.One. 9:e87991. 632

26. Levy, H., S. Diallo, S. M. Tennant, S. Livio, S. O. Sow, M. Tapia, P. I. Fields, M. 633

Mikoleit, B. Tamboura, K. L. Kotloff, R. Lagos, J. P. Nataro, J. E. Galen, and M. 634 M. Levine. 2008. PCR method to identify Salmonella enterica serovars Typhi, 635

Paratyphi A, and Paratyphi B among Salmonella Isolates from the blood of patients 636

with clinical enteric fever. J.Clin.Microbiol. 46:1861-1866. 637

27. Li, H. and R. Durbin. 2009. Fast and accurate short read alignment with Burrows-638

Wheeler transform. Bioinformatics. 25:1754-1760. 639

28. Li, H., B. Handsaker, A. Wysoker, T. Fennell, J. Ruan, N. Homer, G. Marth, G. 640

Abecasis, and R. Durbin. 2009. The Sequence Alignment/Map format and SAMtools. 641

Bioinformatics. 25:2078-2079. 642

29. Lukjancenko O, Thomsen, M. C., Volby, Larsen M., and Ussery, D. W. PanFunPro: 643

PAN-genome analysis based on FUNctional PROfiles. [2], 1-13. 2014. F1000Research. 644

30. Lunguya, O., M. F. Phoba, S. A. Mundeke, E. Bonebe, P. Mukadi, J. J. Muyembe, 645

J. Verhaegen, and J. Jacobs. 2012. The diagnosis of typhoid fever in the Democratic 646

Republic of the Congo. Trans.R.Soc.Trop.Med.Hyg. 106:348-355. 647

31. Luo, R., B. Liu, Y. Xie, Z. Li, W. Huang, J. Yuan, G. He, Y. Chen, Q. Pan, Y. Liu, 648

J. Tang, G. Wu, H. Zhang, Y. Shi, Y. Liu, C. Yu, B. Wang, Y. Lu, C. Han, D. W. 649

Cheung, S. M. Yiu, S. Peng, Z. Xiaoqian, G. Liu, X. Liao, Y. Li, H. Yang, J. Wang, 650 T. W. Lam, and J. Wang. 2012. SOAPdenovo2: an empirically improved memory-651

efficient short-read de novo assembler. Gigascience. 1:18-1. 652

32. Miriagou, V., A. Carattoli, and S. Fanning. 2006. Antimicrobial resistance islands: 653

resistance gene clusters in Salmonella chromosome and plasmids. Microbes.Infect. 654

8:1923-1930. 655


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

26

33. Mirza, S., S. Kariuki, K. Z. Mamun, N. J. Beeching, and C. A. Hart. 2000. Analysis 656

of plasmid and chromosomal DNA of multidrug-resistant Salmonella enterica serovar 657

typhi from Asia. J.Clin.Microbiol. 38:1449-1452. 658

34. Mogasale, V., B. Maskery, R. L. Ochiai, J. S. Lee, V. V. Mogasale, E. Ramani, Y. 659

E. Kim, J. K. Park, and T. F. Wierzba. 2014. Burden of typhoid fever in low-income 660

and middle-income countries: a systematic, literature-based update with risk-factor 661

adjustment. Lancet Glob.Health. 2:e570-e580. 662

35. Morita, M., N. Takai, J. Terajima, H. Watanabe, M. Kurokawa, H. Sagara, K. 663

Ohnishi, and H. Izumiya. 2010. Plasmid-mediated resistance to cephalosporins in 664

Salmonella enterica serovar Typhi. Antimicrob.Agents Chemother. 54:3991-3992. 665

36. Morpeth, S. C., H. O. Ramadhani, and J. A. Crump. 2009. Invasive non-Typhi 666

Salmonella disease in Africa. Clin.Infect.Dis. 49(4):606-611. 667

37. Mtove, G., B. Amos, S. L. von, I. Hendriksen, A. Mwambuli, J. Kimera, R. 668

Mallahiyo, D. R. Kim, R. L. Ochiai, J. D. Clemens, H. Reyburn, S. Magesa, and J. 669 L. Deen. 2010. Invasive salmonellosis among children admitted to a rural Tanzanian 670

hospital and a comparison with previous studies. PLoS.One. 5:e9244. 671

38. Neil, K. P., S. V. Sodha, L. Lukwago, S. Tipo, M. Mikoleit, S. D. Simington, P. 672

Mukobi, S. Balinandi, S. Majalija, J. Ayers, A. Kagirita, E. Wefula, F. Asiimwe, V. 673

Kweyamba, D. Talkington, W. J. Shieh, P. Adem, B. C. Batten, S. R. Zaki, and E. 674 Mintz. 2012. A large outbreak of typhoid fever associated with a high rate of intestinal 675

perforation in Kasese District, Uganda, 2008-2009. Clin.Infect.Dis. 54:1091-1099. 676

39. Okoro, C. K., R. A. Kingsley, T. R. Connor, S. R. Harris, C. M. Parry, M. N. Al-677

Mashhadani, S. Kariuki, C. L. Msefula, M. A. Gordon, P. E. De, J. Wain, R. S. 678

Heyderman, S. Obaro, P. L. Alonso, I. Mandomando, C. A. Maclennan, M. D. 679 Tapia, M. M. Levine, S. M. Tennant, J. Parkhill, and G. Dougan. 2012. 680

Intracontinental spread of human invasive Salmonella Typhimurium pathovariants in 681

sub-Saharan Africa. Nat.Genet. 44:1215-1221. 682

40. Parry, C. M., T. T. Hien, G. Dougan, N. J. White, and J. J. Farrar. 2002. Typhoid 683

fever. N.Engl.J.Med. 347:1770-1782. 684

41. Pfeifer, Y., J. Matten, and W. Rabsch. 2009. Salmonella enterica serovar Typhi with 685

CTX-M beta-lactamase, Germany. Emerg.Infect.Dis. 15:1533-1535. 686

42. Phan, M. D., C. Kidgell, S. Nair, K. E. Holt, A. K. Turner, J. Hinds, P. Butcher, F. 687

J. Cooke, N. R. Thomson, R. Titball, Z. A. Bhutta, R. Hasan, G. Dougan, and J. 688 Wain. 2009. Variation in Salmonella enterica serovar typhi IncHI1 plasmids during the 689

global spread of resistant typhoid fever. Antimicrob.Agents Chemother. 53:716-727. 690

43. Ploy, M. C., D. Chainier, N. H. Tran Thi, I. Poilane, P. Cruaud, F. Denis, A. 691

Collignon, and T. Lambert. 2003. Integron-associated antibiotic resistance in 692

Salmonella enterica serovar typhi from Asia. Antimicrob.Agents Chemother. 47:1427-693

1429. 694


.asm.org/

Dow

nloaded from

http://jcm.asm.org/

27

44. Rocha, E. P., J. M. Smith, L. D. Hurst, M. T. Holden, J. E. Cooper, N. H. Smith, 695

and E. J. Feil. 2006. Comparisons of dN/dS are time dependent for closely related 696

bacterial genomes. J.Theor.Biol. 239:226-235. 697

45. Rotimi, V. O., W. Jamal, T. Pal, A. Sovenned, and M. J. Albert. 2008. Emergence of 698

CTX-M-15 type extended-spectrum beta-lactamase-producing Salmonella spp. in 699

Kuwait and the United Arab Emirates. J.Med.Microbiol. 57:881-886. 700

46. Roumagnac, P., F. X. Weill, C. Dolecek, S. Baker, S. Brisse, N. T. Chinh, T. A. Le, 701

C. J. Acosta, J. Farrar, G. Dougan, and M. Achtman. 2006. Evolutionary history of 702

Salmonella typhi. Science. 314:1301-1304. 703

47. Sirichote, P., H. Hasman, C. Pulsrikarn, H. C. Schonheyder, J. Samulioniene, S. 704

Pornruangmong, A. Bangtrakulnonth, F. M. Aarestrup, and R. S. Hendriksen. 705

2010. Molecular characterization of extended-spectrum cephalosporinase-producing 706

Salmonella enterica serovar Choleraesuis isolates from patients in Thailand and 707

Denmark. J.Clin.Microbiol. 48(3):883-888. 708

48. Slater, G. S. and E. Birney. 2005. Automated generation of heuristics for biological 709

sequence comparison. BMC.Bioinformatics. 6:31.:31. 710

49. Song, Y., P. Roumagnac, F. X. Weill, J. Wain, C. Dolecek, C. J. Mazzoni, K. E. 711

Holt, and M. Achtman. 2010. A multiplex single nucleotide polymorphism typing 712

assay for detecting mutations that result in decreased fluoroquinolone susceptibility in 713

Salmonella enterica serovars Typhi and Paratyphi A. J.Antimicrob.Chemother. 714

65:1631-1641. 715

50. Tamura, K. and M. Nei. 1993. Estimation of the number of nucleotide substitutions in 716

the control region of mitochondrial DNA in humans and chimpanzees. Mol.Biol.Evol. 717

10:512-526. 718

51. Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei, and S. Kumar. 2011. 719

MEGA5: molecular evolutionary genetics analysis using maximum likelihood, 720

evolutionary distance, and maximum parsimony methods. Mol.Biol.Evol. 28:2731-721

2739. 722

52. Yang, Z. 2007. PAML 4: phylogenetic analysis by maximum likelihood. 723

Mol.Biol.Evol. 24:1586-1591. 724

53. Zaki, S. A. and S. Karande. 2011. Multidrug-resistant typhoid fever: a review. 725

J.Infect.Dev.Ctries. 5:324-337. 726

54. Zankari, E., H. Hasman, S. Cosentino, M. Vestergaard, S. Rasmussen, O. Lund, F. 727

M. Aarestrup, and M. V. Larsen. 2012. Identification of acquired antimicrobial 728

resistance genes. J.Antimicrob.Chemother. 67:2640-2644. 729

730

731

732


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Figure 1. Number of cases with the outbreak strain of Salmonella serovar Typhi by 733

month in Zambia in the period from January 2010 to September 2012 (n = 2,040). 734

735

736

Reference: (3) 737

738

739

740

741

742

743

744

745

17 1

20 16 16 4 3

19 8

83 74

104

62

81

101

81

47 49 36 30

70

34

87

190

246

109

134 128

76

43

23

48

0

50

100

150

200

250

300

Jan

Feb

Ap

r

Maj

Jun

Jul

Au

g

Sep

Okt

No

v

Dec Jan

Feb

Mar

Ap

r

Maj

Jun

Jul

Au

g

Sep

Okt

No

v

Dec Jan

Feb

Mar

Ap

r

Maj

Jun

Jul

Au

g

Sep

2010 2011 2012


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Figure 2. Distribution of age and gender in number of cases among Zambian patients 746

infected with Salmonella serovar Typhi in January 2010 to September 2012 (n = 2,040). 747

748

Reference: (3)749

0

200

400

600

800

1000

1200

1400

< 8months

9-24months

3-5 yrs 5-15 yrs 16-30 yrs 31-44 yrs 45-60 yrs >61 yrs

Females Males


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Figure 3. Overview of the incHI1 plasmid translocated region of Salmonella serovar Typhi to the chromosome of Salmonella serovar 750

Typhi from Zambia. 751

752

The top genetic structure illustrate the multidrug resistent island of pHCM1/incHI1in the S. Typhi haplotype H58. The secound structure 753

illustrate the recombined structure of the multidrug resistent island of the four Zambian S. Typhi strains whereas the bottom structure indicate the 754

chromosomal translocation site of the multidrug resistent island related to Zambian strains. 755

756

Figure 4. Phylogenetic reconstruction of the evolutionary relationships among the Salmonella serovar Typhi genomes from Zambia. 757

758

Numbers marked: in red indicate autapomorphic SNPs (for Figure 4B), in blue indicate synapomorphic SNPs, and green indicate the total SNP 759

difference between isolates. In Figure 4A, the genomes belonging to H58B var. from Zambia are marked in pink. 760

761

Figure 5. Genomic deletions detected in the Salmonella serovar Typhi genomes from Zambia. 762

763

Deletions (marked in black) are based on a 95% hit score. Affected genes are partially or entirely deleted.764


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Table 1. Epidemiological features of the 33 whole genome sequenced typed isolates. 765

Isolate no. Gender Patient age Hospital Ward Clinical details Specimen Date of isolation

1 M 9 A08 Enteric fever Blood 06-05-2011


6 F 10 A03 Enteric fever Blood 11-02-2011

7 F 4 A03 Septicaemia Blood 11-02-2010

8 M 10 A05 Fever Blood 14-01-2010


12 M 5 A08 Typhoid Blood 11-05-2010

13 M 9 A05 Typhoid Blood 16-11-2011

14 M 5 A01 Fever Blood 24-11-2010

15 M 3 A08 Septicaemia Blood 11-08-2010

31 M 9 A08 Fever Blood 26-11-2010



36 M 1 11/12 A05 Enteric fever Blood 29-11-2010


46 M 6 A06 UTI/Malaria Blood 17-11-2010


53 M 3 6/12 A08 Enteric fever Blood 08-04-2010


70 F 4 6/12 A05 Enteric fever Blood 14-02-2011


12 F 20 Clinic 7 Enteric fever Blood 01-10-2012


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225 M Adult Ngombe HC Enteric fever Blood 17-02-2012

269 F 15 E02 Enteric fever Blood 13-11-2010

279 M 4 OPD Enteric fever Stool 23-01-2012



674 F 6 A03 Enteric fever Stool 15-02-2012

739 F 4 11/12 A01 Enteric fever Stool 23-02-2012

748 M <14 A01 Enteric fever Blood 07-11-2011

911 F 1 11/12 Adm Enteric fever Stool 01-03-2012


1341 M 20 AFC Enteric fever Stool 03-04-2012

M: male, F: female, Age in year and months, -: no data available. 766

OPD: Out Patient Department, AFC: Adult Filter Clinic, Adm: Admission Ward. 767

768


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Table 2. Frequency of resistance per variable; specimen, year of isolation, and whether 769

the isolates have been chosen of whole genome sequence typing Salmonella serovar 770

Typhi in Zambian patients. 771

aAbbreviations:AMC, amoxicillin + clavulanic acid; AMP, ampicillin;; CHL, 772

chloramphenicol; CIP, ciprofloxacin; NAL, nalidixic acid; SPT, spectinomycin; STR, 773

streptomycin; SMX, sulfamethoxazole; TMP, trimethoprim 774

WGST: whole genome sequence typing, *: Antimicrobials interpret according to EUCAST 775

based on epidemiological cut-off values, **: Apramycin was interpreted according to 776

research results from DTU-Food, ***: Ciprofloxacin was also interpreted according to 777

EUCAST based on epidemiological cut-off values to detect decreased susceptibility. 778

No resistance observed for apramycin** (R >32 mg/L), Azithromycin* (R >32 mg/L), 779

cefotaxime (R ≥4mg/L), ceftiofur* (R >2 mg/L),.ciprofloxacin (High level R ≥1mg/L), 780

Variable

No. of

isolates

No. (%) of isolates resistant to various antimicrobial agents and indicated

CLSI clinical breakpoints values (mg/L)

AMC

≥32

AMP

≥32

CHL

≥32

CIP***

>0,064

NAL

≥32

STR*

>16

SMX

≥512

TMP

≥16

Blood 86 (91.5) 1 (1.2) 85 (98.8) 73 (84.9) 4 (4.7) 3 (3.5) 85 (98.8) 85 (98.8) 85 (98.8)

Stool 8 (8.5) 0 8 (100.0) 5 (62.5) 0 0 8 (100.0) 8 (100.0) 8 (100.0)

2010 35 (37.2) 0 35 (100.0) 29 (82.9) 2 (5.7) 2 (5.7) 35 (100.0) 35 (100.0) 35 (100.0)

2011 33 (35.1) 1 (3.0) 33 (100.0) 28 (84.8) 2 (6.1) 1 (3.0) 33 (100.0) 33 (100.0) 33 (100.0)

2012 26 (27.7) 0 25 (96.2) 21 (80.8) 0 0 25 (96.2) 25 (96.2) 25 (96.2)

WGST 33 (35.1) 0 32 (97.0) 27 (81.8) 2 (6.1) 2 (6.1) 32 (97.0) 32 (97.0) 32 (97.0)

No WGST 61 (64.9) 1 (1.6) 61 (100.0) 51 (83.6) 2 (3.3) 1 (1.6) 61 (100.0) 61 (100.0) 61 (100.0)

Total 94 (100.0) 1 (1.1) 93 (99.0) 78 (83.0) 4 (4.3) 3 (3.2) 93 (99.0) 93 (99.0) 93 (99.0)


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colistin* (R >2 mg/L), florfenicol* (R>16 mg/L), gentamicin (R ≥16mg/L), neomycin* (R>4 781

mg/L), spectinomycin* (R>64 mg/L), and tetracycline (R ≥16mg/L). 782

783


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J. Clin. Microbiol. doi:10.1128/JCM.02026-14 Copyright ...jcm.asm.org/content/early/2014/11/06/JCM.02026-14.full.pdf · 4 75 The common view is that the emerging global S. Typhi haplotype;