Genome-wide analysis of multi- and extensively drug-resistant Mycobacterium tuberculosis

  • Nature Geneticsvolume 50pages307316 (2018)
  • doi:10.1038/s41588-017-0029-0
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To characterize the genetic determinants of resistance to antituberculosis drugs, we performed a genome-wide association study (GWAS) of 6,465 Mycobacterium tuberculosis clinical isolates from more than 30 countries. A GWAS approach within a mixed-regression framework was followed by a phylogenetics-based test for independent mutations. In addition to mutations in established and recently described resistance-associated genes, novel mutations were discovered for resistance to cycloserine, ethionamide and para-aminosalicylic acid. The capacity to detect mutations associated with resistance to ethionamide, pyrazinamide, capreomycin, cycloserine and para-aminosalicylic acid was enhanced by inclusion of insertions and deletions. Odds ratios for mutations within candidate genes were found to reflect levels of resistance. New epistatic relationships between candidate drug-resistance-associated genes were identified. Findings also suggest the involvement of efflux pumps (drrA and Rv2688c) in the emergence of resistance. This study will inform the design of new diagnostic tests and expedite the investigation of resistance and compensatory epistatic mechanisms.

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The project was supported by the KAUST faculty baseline research fund (BAS/1/1020-01-01) to A.P. The authors wish to thank members of the KAUST Bioscience Core laboratory who sequenced samples. We thank the Wellcome Trust Sanger Institute core and pathogen sequencing and informatics teams who were involved in the Malawi and Uganda studies. The work was funded in part by the Wellcome Trust (grant numbers WT096249/Z/11/B, WT088559MA, WT081814/Z/06/Z and WT098051) and the Wellcome Trust–Burroughs Wellcome Fund Infectious Diseases Initiative grant (number 063410/ABC/00/Z). F.C. was the recipient of a Bloomsbury College PhD Studentship and was supported by the Wellcome Trust (201344/Z/16/Z); J. Perdigão received a Fundação para a Ciência e a Tecnologia (Portugal) postdoctoral fellowship fund (SFRH/BPD/95406/2013). The Calouste Gulbenkian Foundation, the Institute Gulbenkian in Lisbon and the European Society of Clinical Microbiology and Infectious Diseases supported the research of C.P., J. Perdigão, I.P. and M.V. J. Phelan is funded by a BBSRC PhD studentship. T.G.C. is funded by the Medical Research Council UK (grant numbers MR/K000551/1, MR/M01360X/1, MR/N010469/1 and MC_PC_15103). N.F. is funded by the Medical Research Council UK (grant number MR/K020420/1). T.M. is supported by the Ministry of Health, Labor and Welfare of Japan (H21-Shinkou-Ippan-008 and H24-Shinkou-Ippan-010). We thank N. Mistry (Foundation for Medical Research, Mumbai) for contributing Mtb archived strains and drug sensitivity testing data. We wish to thank G. Moniz at the Laboratorio Central de Saúde Pública for supporting the collection of samples in Brazil and the South African National Health Laboratory Service for their contribution providing access to clinical Mtb isolates. The MRC eMedLab computing resource was used for bioinformatics and statistical analysis. The authors declare no conflicts of interest. The work has been performed as part of the TB Global Drug Resistance Collaboration (see URLs).

Author information

Author notes

  1. Francesc Coll and Jody Phelan contributed equally to this work. Ruth McNerney, Arnab Pain and Taane G. Clark jointly directed this work.


  1. Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK

    • Francesc Coll
    • , Jody Phelan
    • , Kim Mallard
    • , Stephanie Portelli
    • , Yaa Oppong
    • , Susana Campino
    • , Nicholas Furnham
    • , Martin L. Hibberd
    • , David J. Moore
    • , Ruth McNerney
    •  & Taane G. Clark
  2. Pathogen Genomics Laboratory, BESE Division, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia

    • Grant A. Hill-Cawthorne
    • , Mridul B. Nair
    • , Shahjahan Ali
    • , Abdallah M. Abdallah
    • , Mona Alsomali
    • , Zineb Rchiad
    •  & Arnab Pain
  3. Sydney Emerging Infections and Biosecurity Institute and School of Public Health, Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia

    • Grant A. Hill-Cawthorne
  4. Laboratory Medicine Department, Faculty of Applied Medical Sciences, Umm Al-Qura University, Makkah, Saudi Arabia

    • Saad Alghamdi
  5. Department of Microbiology, Faculty of Medicine, Umm Al-Qura University, Makkah, Saudi Arabia

    • Abdallah O. Ahmed
  6. Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, Victoria, Australia

    • Stephanie Portelli
  7. National Mycobacterium Reference Laboratory, Porto, Portugal

    • Adriana Alves
    •  & Anabela Miranda
  8. Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Brazil

    • Theolis Barbosa Bessa
    •  & Erivelton Oliveira Sousa
  9. Liverpool School of Tropical Medicine, Liverpool, UK

    • Maxine Caws
  10. Pham Ngoc Thach Hospital for TB and Lung Diseases, Ho Chi Minh City, Vietnam

    • Maxine Caws
    •  & Dang Minh Ha
  11. Foundation for Medical Research, Mumbai, India

    • Anirvan Chatterjee
  12. Faculty of Epidemiology and Population Health, London School of Hygiene and Tropical Medicine, London, UK

    • Amelia C. Crampin
    • , Judith R. Glynn
    •  & Taane G. Clark
  13. Karonga Prevention Study, Chilumba, Karonga, Malawi

    • Amelia C. Crampin
    •  & Judith R. Glynn
  14. Lung Infection and Immunity Unit, UCT Lung Institute, University of Cape Town, Groote Schuur Hospital, Cape Town, South Africa

    • Keertan Dheda
    • , Patricia Sheen
    •  & Ruth McNerney
  15. Laboratorio de Enfermedades Infecciosas, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru

    • Louis Grandjean
    •  & David J. Moore
  16. Department of Pathology and Laboratory Medicine, Aga Khan University, Karachi, Pakistan

    • Rumina Hasan
    •  & Zahra Hasan
  17. Department of Medical Microbiology, Makerere University College of Health Sciences, Kampala, Uganda

    • Moses Joloba
  18. Section of Infectious Diseases, Department of Medicine, Boston Medical Center and Boston University School of Medicine, Boston, MA, USA

    • Edward C. Jones-López
  19. Osaka Anti-Tuberculosis Association, Osaka Hospital, Osaka, Japan

    • Tomoshige Matsumoto
  20. Reference Laboratory of Tuberculosis Control, Buenos Aires, Argentina

    • Nora Mocillo
  21. National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria

    • Stefan Panaiotov
  22. Wellcome Trust Sanger Institute, Hinxton, UK

    • Julian Parkhill
  23. Instituto Gulbenkian de Ciência, Lisbon, Portugal

    • Carlos Penha
  24. iMed.ULisboa–Research Institute for Medicines, Faculdade de Farmácia, Universidade de Lisboa, Lisbon, Portugal

    • João Perdigão
    •  & Isabel Portugal
  25. Corporación para Investigaciones Biológicas, Universidad Pontificia Bolivariana, Medellín, Colombia

    • Jaime Robledo
  26. Regional Laboratory Directorate of Health Affairs, Makkah, Saudi Arabia

    • Nashwa Talaat Shesha
  27. Division of Molecular Biology and Human Genetics, SAMRC Centre for Tuberculosis Research, DST/NRF Centre of Excellence for Biomedical Tuberculosis Research, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa

    • Frik A. Sirgel
    • , Elizabeth M. Streicher
    • , Paul Van Helden
    •  & Robert M. Warren
  28. Institute for Integrative Cell Biology, CEA, CNRS, Université Paris–Saclay, Orsay, France

    • Christophe Sola
  29. Laboratorio Central de Saúde Pública Professor Gonçalo Moniz, Salvador, Brazil

    • Erivelton Oliveira Sousa
  30. Unidade de Microbiologia Médica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (UNL), Lisbon, Portugal

    • Miguel Viveiros
  31. Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University, Sapporo, Japan

    • Arnab Pain


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R.M., A.P. and T.G.C. conceived and directed the project. G.A.H.-C., K.M. and R.M. coordinated sample collection and undertook DNA extraction. S. Alghamdi, A.M.A., A.O.A., A.A., T.B.B., M.C., A.C., A.C.C., K.D., L.G., J.R.G., D.T.M.H., R.H., Z.H., P.V.H., M.J., E.C.J.-L., T.M., A.M., N.M., D.J.M., S. Panaiotov, I.P., C.P., J. Perdigão, J.R., P.S., N.T.S., F.A.S., C.S., E.d.O.S., E.M.S., P.V.H., M.V. and R.M.W. undertook sample collection, DNA extraction, genotyping and phenotypic drug resistance testing. G.A.H.-C., M.B.N., M.A., Z.R. and S. Ali prepared libraries for Illumina sequencing. J. Parkhill led the generation of Malawian and Ugandan sequencing data. F.C. and J. Phelan performed bioinformatic and statistical analyses under the supervision of T.G.C. S. Portelli and Y.O. performed additional confirmatory analysis under the supervision of M.L.H., N.F. and T.G.C. F.C., J. Phelan, S. Portelli, S.C., N.F., M.L.H., R.M., A.P. and T.G.C. interpreted results. F.C., J. Phelan, R.M. and T.G.C. wrote the first draft of the manuscript. All authors commented to and edited various versions of the draft manuscript. F.C., J. Phelan, R.M. and T.G.C. compiled the final manuscript. All authors approved the final manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Ruth McNerney or Arnab Pain or Taane G. Clark.

Integrated supplementary information

  1. Allele frequency spectra for SNPs (left) and small indels (right)

  2. Principal-component analysis confirms lineage- and sublineage-based population structure (total variation explained across five components is 82.7%)

  3. Supplementary Figure 3 Protein structure for Alr.

    Alanine racemase mutational map showing the position and effect of mutations based on measure of protein stability by DUET. Unfavourable mutations are depicted in blue and favourable mutations are depicted in red, where color intensity reflects the extent of effect. The PLP cofactor is shown as a stick representation in green. a, The protomer structure of alanine racemase depicted as a cartoon with the PLP cofactor shown as sticks. b, The active site with residues that have been identified in the GWAS depicted as sticks and their hydrogen bonding propensity shown as dashed black lines.

  4. Supplementary Figure 4 Polymorphisms in regions surrounding ethA (top left), thyA (top right), pncA (bottom left) and katG (bottom right) using the complete dataset (n = 6,465).

    The top panel shows the density of SNPs per kilobase (green, nonsynonymous; black, all). The red crosses show the location of the small indels. The middle panel shows the location of the large deletions found in samples used in this study. The lower panel shows the location of the candidate regions and flanking genes.

  5. The analytical workflow, including procedures adopted for raw sequence data processing and the GWAS approach

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–5 and Supplementary Tables 1–9

  2. Life Sciences Reporting Summary

  3. Supplementary Data 1

    List of mutations found in candidate genes. A list of mutations found in drug resistance candidate genes. Mutations are separated by low/high frequency and bolded if they were significantly associated through GWAS or PhyC.

  4. Supplementary Data 2

    ENA accessions and DST phenotypes. A list of all in-house strains sequenced for this study with ENA accessions and DST phenotypes.