Parallel bacterial evolution within multiple patients identifies candidate pathogenicity genes


Bacterial pathogens evolve during the infection of their human host1,2,3,4,5,6,7,8, but separating adaptive and neutral mutations remains challenging9,10,11. Here we identify bacterial genes under adaptive evolution by tracking recurrent patterns of mutations in the same pathogenic strain during the infection of multiple individuals. We conducted a retrospective study of a Burkholderia dolosa outbreak among subjects with cystic fibrosis, sequencing the genomes of 112 isolates collected from 14 individuals over 16 years. We find that 17 bacterial genes acquired nonsynonymous mutations in multiple individuals, which indicates parallel adaptive evolution. Mutations in these genes affect important pathogenic phenotypes, including antibiotic resistance and bacterial membrane composition and implicate oxygen-dependent regulation as paramount in lung infections. Several genes have not previously been implicated in pathogenesis and may represent new therapeutic targets. The identification of parallel molecular evolution as a pathogen spreads among multiple individuals points to the key selection forces it experiences within human hosts.

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Figure 1: Whole-genome sequencing of 112 epidemic B. dolosa isolates recovered from 14 subjects shows the steady accumulation of mutations over years.
Figure 2: Bacterial phylogeny reveals a likely network of transmission between individuals and between organs.
Figure 3: Pathogenic phenotypes are associated with point mutations in key genes.
Figure 4: Parallel evolution identifies a set of genes under strong selection during pathogenesis.


  1. 1

    Suerbaum, S. & Josenhans, C. Helicobacter pylori evolution and phenotypic diversification in a changing host. Nat. Rev. Microbiol. 5, 441–452 (2007).

    CAS  Article  Google Scholar 

  2. 2

    Smith, E.E. et al. Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc. Natl. Acad. Sci. USA 103, 8487–8492 (2006).

    CAS  Article  Google Scholar 

  3. 3

    Musher, D.M. et al. Emergence of macrolide resistance during treatment of pneumococcal pneumonia. N. Engl. J. Med. 346, 630–631 (2002).

    Article  Google Scholar 

  4. 4

    Wong, A. & Kassen, R. Parallel evolution and local differentiation in quinolone resistance in Pseudomonas aeruginosa. Microbiology 157, 937–944 (2011).

    CAS  Article  Google Scholar 

  5. 5

    Zdziarski, J. et al. Host imprints on bacterial genomes—rapid divergent evolution in individual patients. PLoS Pathog. 6, e1001078 (2010).

    Article  Google Scholar 

  6. 6

    Yang, L. et al. Evolutionary dynamics of a bacteria in a human host environment. Proc. Natl. Acad. Sci. USA 108, 7481–7486 (2011).

    CAS  Article  Google Scholar 

  7. 7

    Kennemann, L. et al. Helicobacter pylori genome evolution during human infection. Proc. Natl. Acad. Sci. USA 108, 5033–5038 (2011).

    CAS  Article  Google Scholar 

  8. 8

    Mwangi, M.M. et al. Tracking the in vivo evolution of multidrug resistance in Staphylococcus aureus by whole-genome sequencing. Proc. Natl. Acad. Sci. USA 104, 9451–9456 (2007).

    CAS  Article  Google Scholar 

  9. 9

    Harris, S.R. et al. Evolution of MRSA during hospital transmission and intercontinental spread. Science 327, 469–474 (2010).

    CAS  Article  Google Scholar 

  10. 10

    Goodarzi, H., Hottes, A.K. & Tavazoie, S. Global discovery of adaptive mutations. Nat. Methods 6, 581–583 (2009).

    CAS  Article  Google Scholar 

  11. 11

    Pleasance, E.D. et al. A comprehensive catalogue of somatic mutations from a human cancer genome. Nature 463, 191–196 (2010).

    CAS  Article  Google Scholar 

  12. 12

    Moxon, E.R., Rainey, P.B., Nowak, M.A. & Lenski, R.E. Adaptive evolution of highly mutable loci in pathogenic bacteria. Curr. Biol. 4, 24–33 (1994).

    CAS  Article  Google Scholar 

  13. 13

    van der Woude, M.W. & Bäumler, A.J. Phase and antigenic variation in bacteria. Clin. Microbiol. Rev. 17, 581–611 (2004).

    CAS  Article  Google Scholar 

  14. 14

    Croucher, N.J. et al. Rapid pneumococcal evolution in response to clinical interventions. Science 331, 430–434 (2011).

    CAS  Article  Google Scholar 

  15. 15

    Holt, K.E. et al. High-throughput sequencing provides insights into genome variation and evolution in Salmonella Typhi. Nat. Genet. 40, 987–993 (2008).

    CAS  Article  Google Scholar 

  16. 16

    Pallen, M.J. & Wren, B.W. Bacterial pathogenomics. Nature 449, 835–842 (2007).

    CAS  Article  Google Scholar 

  17. 17

    Elena, S.F. & Lenski, R.E. Evolution experiments with microorganisms: the dynamics and genetic bases of adaptation. Nat. Rev. Genet. 4, 457–469 (2003).

    CAS  Article  Google Scholar 

  18. 18

    Woods, R. et al. Tests of parallel molecular evolution in long-term experiment with Escherichia coli. Proc. Natl. Acad. Sci. USA 103, 9107–9112 (2006).

    CAS  Article  Google Scholar 

  19. 19

    Barrick, J.E. et al. Genome evolution and adaptation in a long-term experiment with Escherichia coli. Nature 461, 1243–1247 (2009).

    CAS  Article  Google Scholar 

  20. 20

    Lipuma, J.J. The changing microbial epidemiology in cystic fibrosis. Clin. Microbiol. Rev. 23, 299–323 (2010).

    Article  Google Scholar 

  21. 21

    Vermis, K. et al. Proposal to accommodate Burkholderia cepacia genomovar VI as Burkholderia dolosa sp. nov. Int. J. Syst. Evol. Microbiol. 54, 689–691 (2004).

    CAS  Article  Google Scholar 

  22. 22

    Lipuma, J.J. Update on the Burkholderia cepacia complex. Curr. Opin. Pulm. Med. 11, 528–533 (2005).

    Article  Google Scholar 

  23. 23

    LiPuma, J.J., Dasen, S.E., Nielson, D.W., Stern, R.C. & Stull, T.L. Person-to-person transmission of Pseudomonas cepacia between patients with cystic fibrosis. Lancet 336, 1094–1096 (1990).

    CAS  Article  Google Scholar 

  24. 24

    Biddick, R., Spilker, T., Martin, A. & LiPuma, J.J. Evidence of transmission of Burkholderia cepacia, Burkholderia multivorans and Burkholderia dolosa among persons with cystic fibrosis. FEMS Microbiol. Lett. 228, 57–62 (2003).

    CAS  Article  Google Scholar 

  25. 25

    Kalish, L.A. et al. Impact of Burkholderia dolosa on lung function and survival in cystic fibrosis. Am. J. Respir. Crit. Care Med. 173, 421–425 (2006).

    Article  Google Scholar 

  26. 26

    Morelli, G. et al. Microevolution of Helicobacter pylori during prolonged infection of single hosts and within families. PLoS Genet. 6, e1001036 (2010).

    Article  Google Scholar 

  27. 27

    Sibley, C.D. et al. A polymicrobial perspective of pulmonary infections exposes an enigmatic pathogen in cystic fibrosis patients. Proc. Natl. Acad. Sci. USA 105, 15070–15075 (2008).

    CAS  Article  Google Scholar 

  28. 28

    Guss, A.M. et al. Phylogenetic and metabolic diversity of bacteria associated with cystic fibrosis. ISME J. 5, 20–29 (2011).

    Article  Google Scholar 

  29. 29

    Mowat, E. et al. Psuedomonas aeruginosa population diversity and turnover in cystic fibrosis infections. Am. J. Respir. Crit. Care Med. 183, 1674–1679 (2011).

    Article  Google Scholar 

  30. 30

    Wilder, C.N., Allada, G. & Schuster, M. Instantaneous within-patient diversity of Psuedomonas aeruginosa quorum-sensing populations from cystic fibrosis lung infections. Infect. Immun. 77, 5631–5639 (2009).

    CAS  Article  Google Scholar 

  31. 31

    Weigel, L.M., Steward, C.D. & Tenover, F.C. gyrA mutations associated with fluoroquinolone resistance in eight species of Enterobacteriaceae. Antimicrob. Agents Chemother. 42, 2661–2667 (1998).

    CAS  Article  Google Scholar 

  32. 32

    Reyna, F., Huesca, M., Gonzalez, V. & Fuchs, L.Y. Salmonella typhimurium gyrA mutations associated with fluoroquinolone resistance. Antimicrob. Agents Chemother. 39, 1621–1623 (1995).

    CAS  Article  Google Scholar 

  33. 33

    Silhavy, T.J., Kahne, D. & Walker, S. The bacterial cell envelope. Cold Spring Harb. Perspect. Biol. 2, a000414 (2010).

    Article  Google Scholar 

  34. 34

    Vinion-Dubiel, A.D. & Goldberg, J.B. Lipopolysaccharide of Burkholderia cepacia complex. J. Endotoxin Res. 9, 201–213 (2003).

    CAS  PubMed  Google Scholar 

  35. 35

    Ortega, X. et al. Reconstitution of O-specific lipopolysaccharide expression in Burkholderia cenocepacia strain J2315, which is associated with transmissible infections in patients with cystic fibrosis. J. Bacteriol. 187, 1324–1333 (2005).

    CAS  Article  Google Scholar 

  36. 36

    Crosson, S., McGrath, P.T., Stephens, C., McAdams, H.H. & Shapiro, L. Conserved modular design of an oxygen sensory/signaling network with species-specific output. Proc. Natl. Acad. Sci. USA 102, 8018–8023 (2005).

    CAS  Article  Google Scholar 

  37. 37

    Worlitzsch, D. et al. Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients. J. Clin. Invest. 109, 317–325 (2002).

    CAS  Article  Google Scholar 

  38. 38

    Marteyn, B. et al. Modulation of Shigella virulence in response to available oxygen in vivo. Nature 465, 355–358 (2010).

    CAS  Article  Google Scholar 

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We are grateful to M. Caimano, M. Cendron, P. Kokorowski, S. Lory, C. Marx, N. Delany, S. Walker, M. Waldor and R. Ward for insightful discussions and comments, to O. Iartchouk, A. Brown, M. Light and their team at Partners HealthCare Center for Personalized Genetic Medicine (PCPGM) for Illumina sequencing, to J. Deane and L. Williams for technical assistance, to S. Vargas for assistance with IRB protocols and to M. Baym, M. Ernebjerg, A. Palmer, E. Toprak, K. Vetsigian, Z. Yao and all of the Kishony lab members for helpful discussions and general support. J.B.M. was supported by the Foundational Questions in Evolutionary Biology Prize Fellowship and the Systems Biology PhD Program (Harvard Medical School). G.P.P. was supported in part by The Mannion Fund for Research of the Center for the Critically Ill Child of Children's Hospital Boston. J.J.L. was supported by the Cystic Fibrosis Foundation. This work was supported in part by US National Institutes of Health grants (GM080177 to the Systems Biology Department, Harvard Medical School and GM081617 to R.K.), by a grant from the New England Regional Center of Excellence for Biodefense and Emerging Infectious Diseases (NERCE; AI057159 to R.K.) and by a Harvard Catalyst grant (to R.K., A.J.M. and M. Cendron).

Author information




J.-B.M., A.J.M. and R.K. conceived of the study. J.J.L., A.J.M. and G.P.P. collected the clinical samples. T.D.L. and N.L. performed resistance phenotyping. J.B.G., D.R., M.R.D., D.S. and G.P.P. performed LPS phenotyping and complementation. M.A., G.P.-B., A.J.M. and G.P.P. conducted chart review and provided medical information. T.D.L., J.-B.M. and R.K. performed whole-genome sequencing and data analysis. T.D.L., J.-B.M., J.J.L., A.J.M., G.P.P. and R.K. interpreted the results and wrote the manuscript.

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Correspondence to Alexander J McAdam or Gregory P Priebe or Roy Kishony.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Tables 1, 4 and 5 and Supplementary Note. (PDF 1715 kb)

Supplementary Table 2

Polymorphic loci among 113 B. dolosa isolates (XLSX 217 kb)

Supplementary Table 3

Genes mutated during an epidemic of B. dolosa (XLSX 26 kb)

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Lieberman, T., Michel, JB., Aingaran, M. et al. Parallel bacterial evolution within multiple patients identifies candidate pathogenicity genes. Nat Genet 43, 1275–1280 (2011).

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