Use of whole genome sequencing to estimate the mutation rate of Mycobacterium tuberculosis during latent infection


Tuberculosis poses a global health emergency, which has been compounded by the emergence of drug-resistant Mycobacterium tuberculosis (Mtb) strains. We used whole-genome sequencing to compare the accumulation of mutations in Mtb isolated from cynomolgus macaques with active, latent or reactivated disease. We sequenced 33 Mtb isolates from nine macaques with an average genome coverage of 93% and an average read depth of 117×. Based on the distribution of SNPs observed, we calculated the mutation rates for these disease states. We found a similar mutation rate during latency as during active disease or in a logarithmically growing culture over the same period of time. The pattern of polymorphisms suggests that the mutational burden in vivo is because of oxidative DNA damage. We show that Mtb continues to acquire mutations during disease latency, which may explain why isoniazid monotherapy for latent tuberculosis is a risk factor for the emergence of isoniazid resistance1,2.

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Figure 1: Experimental protocol for assessing mutational capacity in different disease states.
Figure 2: WGS identifies SNPs in strains isolated from animals with active, latent, and reactivated latent infection.
Figure 3: The mutational capacity of strains from latency and reactivated disease is similar to that of strains from active disease or in vitro growth.
Figure 4: Mutations in Mtb isolated from macaques with latent infection and related human isolates are putative products of oxidative damage.


  1. 1

    Balcells, M.E., Thomas, S.L., Godfrey-Faussett, P. & Grant, A.D. Isoniazid preventive therapy and risk for resistant tuberculosis. Emerg. Infect. Dis. 12, 744–751 (2006).

    CAS  Article  Google Scholar 

  2. 2

    Cattamanchi, A. et al. Clinical characteristics and treatment outcomes of patients with isoniazid-monoresistant tuberculosis. Clin. Infect. Dis. 48, 179–185 (2009).

    Article  Google Scholar 

  3. 3

    Denver, D.R., Morris, K., Lynch, M. & Thomas, W.K. High mutation rate and predominance of insertions in the Caenorhabditis elegans nuclear genome. Nature 430, 679–682 (2004).

    CAS  Article  Google Scholar 

  4. 4

    Haag-Liautard, C. et al. Direct estimation of per nucleotide and genomic deleterious mutation rates in Drosophila. Nature 445, 82–85 (2007).

    CAS  Article  Google Scholar 

  5. 5

    Lynch, M. et al. A genome-wide view of the spectrum of spontaneous mutations in yeast. Proc. Natl. Acad. Sci. USA 105, 9272–9277 (2008).

    CAS  Article  Google Scholar 

  6. 6

    Capuano, S.V. III et al. Experimental Mycobacterium tuberculosis infection of cynomolgus macaques closely resembles the various manifestations of human M. tuberculosis infection. Infect. Immun. 71, 5831–5844 (2003).

    CAS  Article  Google Scholar 

  7. 7

    Lin, P.L. et al. Quantitative comparison of active and latent tuberculosis in the cynomolgus macaque model. Infect. Immun. 77, 4631–4642 (2009).

    CAS  Article  Google Scholar 

  8. 8

    Li, H., Ruan, J. & Durbin, R. Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res. 18, 1851–1858 (2008).

    CAS  Article  Google Scholar 

  9. 9

    Ioerger, T.R. et al. Genome analysis of multi- and extensively-drug-resistant tuberculosis from KwaZulu-Natal, South Africa. PLoS ONE 4, e7778 (2009).

    Article  Google Scholar 

  10. 10

    Hernandez, D., Francois, P., Farinelli, L., Osteras, M. & Schrenzel, J. De novo bacterial genome sequencing: millions of very short reads assembled on a desktop computer. Genome Res. 18, 802–809 (2008).

    CAS  Article  Google Scholar 

  11. 11

    Gutierrez-Vazquez, J.M. Studies on the rate of growth of mycobacteria. I. Generation time of Mycobacterium tuberculosis on several solid and liquid media and effects exerted by glycerol and malachite green. Am. Rev. Tuberc. 74, 50–58 (1956).

    CAS  PubMed  Google Scholar 

  12. 12

    Gill, W.P. et al. A replication clock for Mycobacterium tuberculosis. Nat. Med. 15, 211–214 (2009).

    CAS  Article  Google Scholar 

  13. 13

    Muñoz-Elías, E.J. et al. Replication dynamics of Mycobacterium tuberculosis in chronically infected mice. Infect. Immun. 73, 546–551 (2005).

    Article  Google Scholar 

  14. 14

    Barry, C.E. et al. The spectrum of latent tuberculosis: rethinking the biology and intervention strategies. Nat. Rev. Microbiol. 7, 845–855 (2009).

    CAS  Article  Google Scholar 

  15. 15

    Lin, P.L. & Flynn, J.L. Understanding latent tuberculosis: a moving target. J. Immunol. 185, 15–22 (2010).

    CAS  Article  Google Scholar 

  16. 16

    Sarkar, S., Ma, W.T. & Sandri, G.H. On fluctuation analysis: a new, simple and efficient method for computing the expected number of mutants. Genetica 85, 173–179 (1992).

    CAS  Article  Google Scholar 

  17. 17

    Lang, G.I. & Murray, A.W. Estimating the per-base-pair mutation rate in the yeast Saccharomyces cerevisiae. Genetics 178, 67–82 (2008).

    CAS  Article  Google Scholar 

  18. 18

    Boshoff, H.I.M., Reed, M.B., Barry, C.E. & Mizrahi, V. DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis. Cell 113, 183–193 (2003).

    CAS  Article  Google Scholar 

  19. 19

    Werngren, J. & Hoffner, S.E. Drug-susceptible Mycobacterium tuberculosis Beijing genotype does not develop mutation-conferred resistance to rifampin at an elevated rate. J. Clin. Microbiol. 41, 1520–1524 (2003).

    CAS  Article  Google Scholar 

  20. 20

    Telenti, A. et al. Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet 341, 647–650 (1993).

    CAS  Article  Google Scholar 

  21. 21

    Nathan, C. & Shiloh, M.U. Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc. Natl. Acad. Sci. USA 97, 8841–8848 (2000).

    CAS  Article  Google Scholar 

  22. 22

    Ng, V.H., Cox, J.S., Sousa, A.O., MacMicking, J.D. & McKinney, J.D. Role of KatG catalase-peroxidase in mycobacterial pathogenesis: countering the phagocyte oxidative burst. Mol. Microbiol. 52, 1291–1302 (2004).

    CAS  Article  Google Scholar 

  23. 23

    Sassetti, C.M. & Rubin, E.J. Genetic requirements for mycobacterial survival during infection. Proc. Natl. Acad. Sci. USA 100, 12989–12994 (2003).

    CAS  Article  Google Scholar 

  24. 24

    Boshoff, H.I., Durbach, S.I. & Mizrahi, V. DNA metabolism in mycobacterium tuberculosis: implications for drug resistance and strain variability. Scand. J. Infect. Dis. 33, 101–105 (2001).

    CAS  Article  Google Scholar 

  25. 25

    Fenhalls, G. et al. In situ detection of Mycobacterium tuberculosis transcripts in human lung granulomas reveals differential gene expression in necrotic lesions. Infect. Immun. 70, 6330–6338 (2002).

    CAS  Article  Google Scholar 

  26. 26

    Saint-Ruf, C., Pesut, J., Sopta, M. & Matic, I. Causes and consequences of DNA repair activity modulation during stationary phase in Escherichia coli. Crit. Rev. Biochem. Mol. Biol. 42, 259–270 (2007).

    CAS  Article  Google Scholar 

  27. 27

    Bjedov, I. et al. Stress-induced mutagenesis in bacteria. Science 300, 1404–1409 (2003).

    CAS  Article  Google Scholar 

  28. 28

    Cohen, T., Lipsitch, M., Walensky, R.P. & Murray, M. Beneficial and perverse effects of isoniazid preventive therapy for latent tuberculosis infection in HIV–tuberculosis coinfected populations. Proc. Natl. Acad. Sci. USA 103, 7042–7047 (2006).

    CAS  Article  Google Scholar 

  29. 29

    Perriëns, J.H. et al. Increased mortality and tuberculosis treatment failure rate among human immunodeficiency virus (HIV) seropositive compared with HIV seronegative patients with pulmonary tuberculosis treated with “standard” chemotherapy in Kinshasa, Zaire. Am. Rev. Respir. Dis. 144, 750–755 (1991).

    Article  Google Scholar 

  30. 30

    Lee, J., Remold, H.G., Ieong, M.H. & Kornfeld, H. Macrophage apoptosis in response to high intracellular burden of Mycobacterium tuberculosis is mediated by a novel caspase-independent pathway. J. Immunol. 176, 4267–4274 (2006).

    CAS  Article  Google Scholar 

  31. 31

    Kurtz, S. et al. Versatile and open software for comparing large genomes. Genome Biol. 5, R12 (2004).

    Article  Google Scholar 

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This work was supported by a New Innovator's Award, DP2 0D001378 from the Director's Office of the National Institute of Health to S.M.F., by a subcontract from National Institute for Allergy and Infectious Diseases (NIAID) U19 AI076217 to S.M.F., by the US National Institutes of Health (NIH) RO1 HL075845 to J.L.F. and by the Bill and Melinda Gates Foundation (J.L.F.). The genome sequencing has been funded in part with federal funds from the National Institute of Allergy and Infectious Disease, US NIH, US Department of Health and Human Services, under contract no. HHSN266200400001C. The project described was supported in part by Award Number U54GM088558 to M.L. from the National Institute of General Medical Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health. We thank D. Gurgil and J. Xu of the Enterprise Research IS group at Partners Healthcare for their support and for provision of the HPC facilities and E. Klein for necropsy and pathology of the infected monkeys, as well as the veterinary technical staff for care of the animals. We also thank E. Rubin, C. Sassetti, B. Bloom, T. Rosebrock and B. Aldridge for helpful feedback.

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C.B.F. performed molecular studies, conducted the data analyses, prepared the figures and drafted the manuscript; P.L.L. and J.L.F. conducted the infection of the cynomolgus macaques, determined clinical state and acquired bacterial strains on necropsy; M.R.C. analyzed sequence data and directed validation of SNPs; R.R.S. performed molecular and fluctuation analyses; O.I. oversaw sequencing of isolates sent to Partners Healthcare Center for Personalized Genetic Medicine (PHCPGM); J.G. oversaw sequencing of isolates sent to the Broad Institute; N.M., T.R.I. and J.C.S. oversaw sequencing and analysis of isolates sent to Texas A&M University; M.L. supervised and advised statistical analyses; S.M.F. initiated the project, performed molecular studies, supervised preparation and analysis of the data and drafted the manuscript.

Corresponding author

Correspondence to Sarah M Fortune.

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The authors declare no competing financial interests.

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Ford, C., Lin, P., Chase, M. et al. Use of whole genome sequencing to estimate the mutation rate of Mycobacterium tuberculosis during latent infection. Nat Genet 43, 482–486 (2011).

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