Abstract

To examine the transmission dynamics of Mycobacterium tuberculosis (Mtb) isolated from tuberculosis patients in Ho Chi Minh City, Vietnam, we sequenced the whole genomes of 1,635 isolates and compared these with 3,144 isolates from elsewhere. The data identify an underlying burden of disease caused by the endemic Mtb lineage 1 associated with the activation of long-term latent infection, and a threefold higher burden associated with the more recently introduced Beijing lineage and lineage 4 Mtb strains. We find that Beijing lineage Mtb is frequently transferred between Vietnam and other countries, and detect higher levels of transmission of Beijing lineage strains within this host population than the endemic lineage 1 Mtb. Screening for parallel evolution of Beijing lineage-associated SNPs in other Mtb lineages as a signal of positive selection, we identify an alteration in the ESX-5 type VII-secreted protein EsxW, which could potentially contribute to the enhanced transmission of Beijing lineage Mtb in Vietnamese and other host populations.

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Acknowledgements

We would like to thank the clinical staff who recruited patients into our study from the following District TB Units (DTUs) in HCMC, Vietnam: Districts 1, 4, 5, 6 and 8, Tan Binh, Binh Thanh and Phu Nhuan; and also our colleagues from Pham Ngoc Thach Hospital for Tuberculosis and Lung Disease, HCMC Vietnam. This work was supported by the National Health and Medical Research Council, Australia (project grant no. 1056689 to S.J.D., Fellowship no. 1061409 to K.E.H., Fellowship no. 1061435 to M.I., Fellowship no. 1072476 to D.B.A.), A*STAR Biomedical Research Council, Singapore (12/1/21/24/6689 to Y.Y.T.) and the Wellcome Trust UK (research training fellowship no. 081814/Z/06/Z to M.C.) and as part of their Major Overseas Program in Vietnam (089276/Z/09/Z to J.F. and 106680/B/14/Z to G.T.).

Author information

Author notes

  1. These authors contributed equally: Paul McAdam, Phan Vuong Khac Thai, Maxine Caws, Sarah J. Dunstan.

Affiliations

  1. Department of Biochemistry and Molecular Biology, Bio 21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia

    • Kathryn E. Holt
    • , Paul McAdam
    • , David J. Edwards
    •  & David B. Ascher
  2. Pham Ngoc Thach Hospital for Tuberculosis and Lung Disease, Ho Chi Minh City, Vietnam

    • Phan Vuong Khac Thai
    • , Dang Thi Minh Ha
    • , Nguyen Ngoc Lan
    •  & Nguyen Huu Lan
  3. Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam

    • Nguyen Thuy Thuong Thuong
    • , Nguyen Thi Quynh Nhu
    • , Hoang Thanh Hai
    • , Vu Thi Ngoc Ha
    • , Guy Thwaites
    •  & Jeremy Farrar
  4. Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, Oxford University, Oxford, UK

    • Guy Thwaites
    •  & Jeremy Farrar
  5. Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia

    • Artika P. Nath
  6. Systems Genomics Lab, Baker Heart and Diabetes Institute, Melbourne, Victoria, Australia

    • Artika P. Nath
    •  & Michael Inouye
  7. Department of Clinical Pathology, University of Melbourne, Parkville, Victoria, Australia

    • Kym Pham
    •  & Michael Inouye
  8. Genome Institute of Singapore, Singapore, Singapore

    • Chiea Chuen Khor
  9. Singapore Eye Research Institute, Singapore, Singapore

    • Chiea Chuen Khor
  10. Department of Statistics and Applied Probability, National University of Singapore, Singapore, Singapore

    • Yik Ying Teo
  11. Saw Swee Hock School of Public Health, National University of Singapore, Singapore, Singapore

    • Yik Ying Teo
  12. Department of Public Health and Primary Care, University of Cambridge, Strangeways Research Laboratories, Cambridge, UK

    • Michael Inouye
  13. Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, UK

    • Maxine Caws
  14. Birat-Nepal Medical Trust, Kathmandu, Nepal

    • Maxine Caws
  15. Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria, Australia

    • Sarah J. Dunstan

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Contributions

S.J.D., K.E.H., M.C., M.I., Y.Y.T. and C.C.K. are the study principal investigators who conceived and obtained funding for the project. S.J.D. provided overall project coordination; M.I. organized and supervised the DNA sequencing and K.E.H. devised the overall analysis plan and wrote the first draft of the manuscript along with P.M. M.C. and S.J.D. established the TB cohort for this genetics study by working with P.V.K.T., D.T.M.H., N.N.L., N.H.L., N.T.Q.N., N.T.T.T., G.T. and J.F. to coordinate the collection of clinical samples and phenotypes. K.P. performed DNA quality checks and genome sequencing on all Vietnamese samples, while V.T.N.H. performed Sanger sequencing on selected samples. D.B.A. performed protein structure analyses, and H.T.H. and N.T.T.T. performed the macrophage growth and infection experiments of EsxW variants. K.E.H., P.M., M.I., D.J.E. and A.P.N. analyzed the data. All authors critically reviewed manuscript revisions and contributed intellectual input to the final submission.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Kathryn E. Holt or Sarah J. Dunstan.

Integrated supplementary information

  1. Supplementary Figure 1 Association between Mtb genetic distance and spatial proximity of patients.

    For all pairs of Mtb isolates, the probability of both originating from patients living in the same district was calculated (y-axis) and stratified according to genetic distance between the Mtb genomes (expressed in number of SNPs, x-axis). P-values indicate result of testing for difference in proportions of isolate pairs originating from same district, for isolate pairs with SNP distance <10 vs >20, calculated separately for Beijing and non-Beijing isolates. (Note it was not possible to further stratify non-Beijing pairs into L1 pairs vs L4 pairs for statistical tests, due to low sample size.).

  2. Supplementary Figure 2 Proportion of clusters not explained by transmission of antimicrobial resistance mutations.

    Each cluster (defined at various thresholds of maximum patristic distance between tips) was checked to determine whether all members of the cluster shared any of the antimicrobial resistance mutations identified by Mykrobe Predictor. Clusters in which no known antimicrobial resistance mutation was conserved in all members of the cluster were considered not driven by antimicrobial resistance. For each lineage and at each maximum patristic distance threshold value for clustering, the proportion of clusters not explained by antimicrobial resistance are shown below. Colour legend for lineages is the same as throughout the paper: pink, lineage 1; dark blue, lineage 2.1; orange, lineage 2.2.1; light blue, lineage 2.2.2; red, lineage 4.

  3. Supplementary Figure 3 Homoplasic mutations detected in the HCMC Mtb population and located in known drug resistance-associated genes.

    Mutations arising on more than one branch in the HCMC Mtb tree in the following genes are included: embB (ethambutol resistance), gidB (streptomycin resistance), gyrA (fluoroquinolone resistance); inhA (isoniazid resistance), rpoB (rifampicin resistance).

  4. Supplementary Figure 4 Homoplasic nsSNPs that are fixed within Beijing lineage and also arise independently in other branches in the HCMC phylogeny.

    Tree shown is the ML phylogeny for 1,635 HCMC isolates with lineages highlighted, reproduced from Figure 1. Outer rings indicate the presence of derived alleles for the three nsSNPs that were found to be fixed within the Beijing lineage (2.2; including 2.2.1 and 2.2.2).

  5. Supplementary Figure 5 Map of HCMC showing location of district TB units where patients were recruited.

    Yellow regions show districts included in the study; red dots indicate location of the district TB units where participants were recruited; red star indicates location of Pham Ngoc Thach Hospital for Tuberculosis and Lung Disease. Blue regions indicate other urban HCMC districts, green are greater HCMC districts (not included in recruitment).

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–5 and Supplementary Tables 3 and 4

  2. Reporting Summary

  3. Supplementary Table 1

    Details of the M. tuberculosis isolates sequenced for this study

  4. Supplementary Table 2

    Details of the publicly available M. tuberculosis genomes included in this study

  5. Supplementary Table 5

    Repetitive regions of the M. tuberculosis genome that were excluded from SNP analysis

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DOI

https://doi.org/10.1038/s41588-018-0117-9

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