Abstract
The emergence of multidrug-resistant (MDR) typhoid is a major global health threat affecting many countries where the disease is endemic. Here whole-genome sequence analysis of 1,832 Salmonella enterica serovar Typhi (S. Typhi) identifies a single dominant MDR lineage, H58, that has emerged and spread throughout Asia and Africa over the last 30 years. Our analysis identifies numerous transmissions of H58, including multiple transfers from Asia to Africa and an ongoing, unrecognized MDR epidemic within Africa itself. Notably, our analysis indicates that H58 lineages are displacing antibiotic-sensitive isolates, transforming the global population structure of this pathogen. H58 isolates can harbor a complex MDR element residing either on transmissible IncHI1 plasmids or within multiple chromosomal integration sites. We also identify new mutations that define the H58 lineage. This phylogeographical analysis provides a framework to facilitate global management of MDR typhoid and is applicable to similar MDR lineages emerging in other bacterial species.
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Acknowledgements
This work was supported by the Wellcome Trust. We would like to thank the members of the Pathogen Informatics Team and the core sequencing teams at the Wellcome Trust Sanger Institute (Cambridge, UK). We are grateful to D. Harris for his superb work in managing the sequence data. We also thank L. Fabre for her excellent technical assistance.
This work was supported by a number of organizations. The authors affiliated with the Wellcome Trust Sanger Institute were funded by Wellcome Trust award 098051; N.A.F. was supported by Wellcome Trust research fellowship WT092152MA. N.A.F., R.S.H. and this work were supported by a strategic award from the Wellcome Trust for the Malawi-Liverpool Wellcome Trust Clinical Research Programme (101113/Z/13/Z). C.M.P. was funded by the Wellcome Trust Mahidol University Oxford Tropical Medicine Research Programme, supported by the Wellcome Trust (Major Overseas Programmes–Thailand Unit Core Grant), the European Society for Paediatric Infectious Diseases and the University of Oxford–Li Ka Shing Global Health Foundation. D.D., P.N. and V.D. were supported by the Wellcome Trust (core grant 089275/H/09/Z). K.E.H. was supported by the National Health and Medical Research Council of Australia (fellowship 1061409) and the Victorian Life Sciences Computation Initiative (VLSCI; grant VR0082). C.A.M. was supported by a Clinical Research Fellowship from GlaxoSmithKline, and P.J.H. was supported by a UK Medical Research Council PhD studentship. This work forms part of a European Union Framework Programme 7 Marie Curie Actions Industry Academia Partnerships and Pathways (IAPP) Consortium Programme, entitled GENDRIVAX (Genome-Driven Vaccine Development for Bacterial Infections), involving the Wellcome Trust Sanger Institute, KEMRI Nairobi and the Novartis Vaccines Institute for Global Health. The authors affiliated with the Institut Pasteur were funded by the Institut Pasteur, the Institut de Veille Sanitaire and the French government 'Investissement d'Avenir' program (Integrative Biology of Emerging Infectious Diseases Laboratory of Excellence, grant ANR-10-LABX-62-IBEID). C.H.W. was supported by the UK Medical Research Council (MR/J003999/1). C.O. was supported by Society in Science and the Branco Weiss Fellowship, administered by ETH Zurich. A.K.C. was supported by the UK Medical Research Council (G1100100/1). J.J. was supported by the antibiotic resistance surveillance project in the Democratic Republic of the Congo, funded by project 2.01 of the Third Framework Agreement between the Belgian Directorate General of Development Cooperation and the Institute of Tropical Medicine (Antwerp, Belgium). F.M. was supported by a research grant from the Bill and Melinda Gates Foundation. The findings and conclusions contained within this publication are those of the authors and do not necessarily reflect positions or policies of the Bill and Melinda Gates Foundation. J.A. Crump was supported by the joint US National Institutes of Health–National Science Foundation Ecology and Evolution of Infectious Disease program (R01 TW009237), the UK Biotechnology and Biological Sciences Research Council (BBSRC; BB/J010367/1) and UK BBSRC Zoonoses in Emerging Livestock Systems awards BB/L017679, BB/L018926 and BB/L018845. S.K. was supported by US National Institutes of Health grant R01 AI099525-02. S.B. is a Sir Henry Dale Fellow, jointly funded by the Wellcome Trust and the Royal Society (100087/Z/12/Z). S.O. was supported by the National Institute of Allergy and Infectious Diseases of the US National Institutes of Health (R01 AI097493). The content is solely the responsibility of the authors and does not necessarily represent the official views of the US National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. C.D. was supported by the University of Oxford–Li Ka Shing Global Health Foundation.
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Study design: V.K.W., S.B., J. Parkhill , N.R.T., K.E.H. and G.D. Sequencing data generation: A.J.P., J.A.K. and E.J.K. Data analysis: V.K.W., K.E.H., J. Parkhill, N.R.T., A.J.P., J.A.K., D.J.E., J. Hawkey, S.R.H., A.E.M., A.K.C., J. Hadfield, C.O., R.A.K., E.J.K., D.A.G. and D.J.P. Isolate acquisition and processing and clinical data collection: D.J.P., S.B., N.A.F., N.R.T., F.-X.W., P.J.H., N.T.V.T., R.F.B., C.H.W., S.K., M.A.G., R.S.H., J.J., O.L., W.J.E., C.M., J.A. Chabalgoity, M.K., K.J., S. Dutta, F.M., J.C., C.T., S.O., C.A.M., C.D., K.H.K., A.M.S., C.M.P., A.K., E.K.M., J.I.C., S. Dongol, B.B., M.D., D.B., T.T.N., S.P.S., M.H., P.N., R.S.O., L.I., D.D., V.D., G.T., L.W., J.A. Crump, E.D.P., S.N., E.J.N., D.P.T., P.T., S.S., M.V., J. Powling, K.D., G.H., J.F. and K.E.H. Manuscript writing: V.K.W., S.B., K.E.H. and G.D. All authors contributed to manuscript editing. Project oversight: S.B., K.E.H. and G.D.
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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.
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.
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.
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.
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.
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.
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Supplementary Figures 1–6 and Supplementary Tables 2–7. (PDF 1126 kb)
Supplementary Table 1
Isolates used in the study. (XLSX 118 kb)
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Wong, V., Baker, S., Pickard, D. et al. Phylogeographical analysis of the dominant multidrug-resistant H58 clade of Salmonella Typhi identifies inter- and intracontinental transmission events. Nat Genet 47, 632–639 (2015). https://doi.org/10.1038/ng.3281
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DOI: https://doi.org/10.1038/ng.3281
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