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Genomic analysis identifies targets of convergent positive selection in drug-resistant Mycobacterium tuberculosis

Abstract

M. tuberculosis is evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including genetic changes favored by selection in resistant isolates, are incompletely understood. Using 116 newly sequenced and 7 previously sequenced M. tuberculosis whole genomes, we identified genome-wide signatures of positive selection specific to the 47 drug-resistant strains. By searching for convergent evolution—the independent fixation of mutations in the same nucleotide position or gene—we recovered 100% of a set of known resistance markers. We also found evidence of positive selection in an additional 39 genomic regions in resistant isolates. These regions encode components in cell wall biosynthesis, transcriptional regulation and DNA repair pathways. Mutations in these regions could directly confer resistance or compensate for fitness costs associated with resistance. Functional genetic analysis of mutations in one gene, ponA1, demonstrated an in vitro growth advantage in the presence of the drug rifampicin.

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Figure 1: Characteristics of sequenced tuberculosis isolates.
Figure 2: Candidate genes under selection in resistant M. tuberculosis.
Figure 3: Evolutionary convergence at the gene level in rpoC.
Figure 4: M. tuberculosis ponA1 mutant survival in the presence of the drug rifampicin.

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NCBI Reference Sequence

Sequence Read Archive

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Acknowledgements

We thank the technical staff of the British Columbia Centre for Disease Control Public Health Microbiology and Reference Mycobacteriology Laboratory in Vancouver, M. Bosman from the National Health Laboratory Service in Cape Town and L. Fattorini from the Istituto Superiore di Sanita in Rome. This work was funded by a Senior Ellison Foundation Award (M.M.) and in part by a contact from the National Institute of Allergy and Infectious Diseases (HHSN266200400001C to B.B.), the Department of Pulmonary and Critical Care at Massachusetts General Hospital (M.R.F.), a postdoctoral fellowship from the Harvard MIDAS Center for Communicable Disease Dynamics (B.J.S.) and a Packard Foundation Fellowship (P.C.S.). S.G. was supported by the Swiss National Science Foundation (PP0033_119205).

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Authors

Contributions

This study was designed and conducted by M.R.F. and M.M. M.R.F. wrote the first drafts of the manuscript. B.J.S., P.C.S. and E.S.L. provided conceptual input on the evolutionary testing, analysis support and key manuscript edits. K.J.K. and E.J.R. constructed the ponA1 mutants and measured their MICs. R.S. provided bioinformatics support and K.R.J. helped with the curation of the isolate phenotypes. R.M.W., E.M.S., T.C.V. and A.C. conducted molecular epidemiological studies and performed molecular characterization, drug susceptibility testing and selection of isolates from South Africa. A.S. and D.K. performed molecular characterization and drug sensitivity testing and selected isolates from Peru and Russia. B.P. and J.E.P. performed molecular characterization, drug sensitivity testing and selection of isolates from the Centers for Disease Control and Prevention. M.R.O. identified the individual with progressively resistant tuberculosis and performed molecular characterization and selection of serial isolates from this individual in Italy. J.L.G., J.C.J., M.R. and P.K.C.T. conducted the tuberculosis outbreak investigation in British Columbia and performed molecular characterization, drug susceptibility testing and sequencing of these isolates. M.K.-M. conducted the epidemiological study of tuberculosis transmission in San Francisco, and M.L.B. and B.M. performed molecular characterization and sequencing of these isolates. B.N.K. and N.K. characterized the W-148, Haarlem and C isolates. S.G. collected the 24 drug-sensitive M. tuberculosis diversity strain set. J.G. and B.B. provided oversight for sequencing and bioinformatics support.

Corresponding authors

Correspondence to Maha R Farhat or Megan Murray.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Tables 1–10 and 12–20, and Supplementary Note (PDF 7052 kb)

Supplementary Table 11

Description of the M. tuberculosis isolates studied (XLSX 47 kb)

Supplementary Table 21

Multiple alignment of nucleotide sequence at all variable sites within the targets of independent mutation (XLSX 243 kb)

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Farhat, M., Shapiro, B., Kieser, K. et al. Genomic analysis identifies targets of convergent positive selection in drug-resistant Mycobacterium tuberculosis. Nat Genet 45, 1183–1189 (2013). https://doi.org/10.1038/ng.2747

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