• A Corrigendum to this article was published on 30 March 2017
  • An Erratum to this article was published on 26 May 2017

This article has been updated

Abstract

Microbial pathogenesis studies are typically performed with reference strains, thereby overlooking within-species heterogeneity in microbial virulence. Here we integrated human epidemiological and clinical data with bacterial population genomics to harness the biodiversity of the model foodborne pathogen Listeria monocytogenes and decipher the basis of its neural and placental tropisms. Taking advantage of the clonal structure of this bacterial species, we identify clones epidemiologically associated either with food or with human central nervous system (CNS) or maternal-neonatal (MN) listeriosis. The latter clones are also most prevalent in patients without immunosuppressive comorbidities. Strikingly, CNS- and MN-associated clones are hypervirulent in a humanized mouse model of listeriosis. By integrating epidemiological data and comparative genomics, we have uncovered multiple new putative virulence factors and demonstrate experimentally the contribution of the first gene cluster mediating L. monocytogenes neural and placental tropisms. This study illustrates the exceptional power in harnessing microbial biodiversity to identify clinically relevant microbial virulence attributes.

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Change history

  • 06 March 2017

    In the version of this article initially published, in Figure 2b, in the panel called "CNS infections" the bar of CC3 should have been represented in red and the one of CC121 should have been represented in blue. The errors have been corrected in the HTML and PDF versions of the article.

  • 06 March 2017

    In the version of this article initially published, the titles of the x axes in Figure 5b and 5c should have been “Brain/blood CFU ratio” instead of "Blood/brain CFU ratio," and the title of the z axis in Figure 2c should have been "% of isolates" instead of "Number of isolates." The errors have been corrected in the HTML and PDF versions of the article.

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References

  1. 1.

    , & The interaction of bacteria with mammalian cells. Annu. Rev. Cell Biol. 8, 333–363 (1992).

  2. 2.

    , , & Cellular microbiology emerging. Science 271, 315–316 (1996).

  3. 3.

    et al. Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli. Proc. Natl. Acad. Sci. USA 99, 17020–17024 (2002).

  4. 4.

    et al. Complete genomes of two clinical Staphylococcus aureus strains: evidence for the rapid evolution of virulence and drug resistance. Proc. Natl. Acad. Sci. USA 101, 9786–9791 (2004).

  5. 5.

    et al. Simultaneous identification of bacterial virulence genes by negative selection. Science 269, 400–403 (1995).

  6. 6.

    et al. Comparative analysis of the genome sequences of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Nat. Genet. 35, 32–40 (2003).

  7. 7.

    & Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes. J. Cell Biol. 109, 1597–1608 (1989).

  8. 8.

    Human listeriosis and animal models. Microbes Infect. 9, 1216–1225 (2007).

  9. 9.

    Illuminating the landscape of host-pathogen interactions with the bacterium Listeria monocytogenes. Proc. Natl. Acad. Sci. USA 108, 19484–19491 (2011).

  10. 10.

    et al. Genetic characterization of clones of the bacterium Listeria monocytogenes causing epidemic disease. Proc. Natl. Acad. Sci. USA 86, 3818–3822 (1989).

  11. 11.

    et al. Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential. Infect. Immun. 65, 2707–2716 (1997).

  12. 12.

    , & Listeria monocytogenes lineages: genomics, evolution, ecology, and phenotypic characteristics. Int. J. Med. Microbiol. 301, 79–96 (2011).

  13. 13.

    & in Bergey's Manual of Systematic Bacteriology Vol. 2 1235–1245 (Williams & Wilkins, 1986).

  14. 14.

    , , , & Differentiation of the major Listeria monocytogenes serovars by multiplex PCR. J. Clin. Microbiol. 42, 3819–3822 (2004).

  15. 15.

    et al. A new perspective on Listeria monocytogenes evolution. PLoS Pathog. 4, e1000146 (2008).

  16. 16.

    et al. Worldwide distribution of major clones of Listeria monocytogenes. Emerg. Infect. Dis. 17, 1110–1112 (2011).

  17. 17.

    , , , & The ubiquitous nature of Listeria monocytogenes clones: a large-scale Multilocus Sequence Typing study. Environ. Microbiol. 16, 405–416 (2014).

  18. 18.

    Distribution of serovars of Listeria monocytogenes isolated from different categories of patients with listeriosis. Eur. J. Clin. Microbiol. Infect. Dis. 9, 210–213 (1990).

  19. 19.

    et al. Listeria monocytogenes isolates from foods and humans form distinct but overlapping populations. Appl. Environ. Microbiol. 70, 5833–5841 (2004).

  20. 20.

    , , , & Multilocus genotyping assays for single nucleotide polymorphism–based subtyping of Listeria monocytogenes isolates. Appl. Environ. Microbiol. 74, 7629–7642 (2008).

  21. 21.

    et al. A molecular marker for evaluating the pathogenic potential of foodborne Listeria monocytogenes. J. Infect. Dis. 189, 2094–2100 (2004).

  22. 22.

    , , , & Select Listeria monocytogenes subtypes commonly found in foods carry distinct nonsense mutations in inlA, leading to expression of truncated and secreted internalin A, and are associated with a reduced invasion phenotype for human intestinal epithelial cells. Appl. Environ. Microbiol. 71, 8764–8772 (2005).

  23. 23.

    et al. Conjugated action of two species-specific invasion proteins for fetoplacental listeriosis. Nature 455, 1114–1118 (2008).

  24. 24.

    et al. Optimized multilocus variable-number tandem-repeat analysis assay and its complementarity with pulsed-field gel electrophoresis and multilocus sequence typing for Listeria monocytogenes clone identification and surveillance. J. Clin. Microbiol. 51, 1868–1880 (2013).

  25. 25.

    et al. Comparative genomics of Listeria species. Science 294, 849–852 (2001).

  26. 26.

    et al. Pathogenomics of Listeria spp. Int. J. Med. Microbiol. 297, 541–557 (2007).

  27. 27.

    et al. Comparative genomics of the bacterial genus Listeria: genome evolution is characterized by limited gene acquisition and limited gene loss. BMC Genomics 11, 688 (2010).

  28. 28.

    et al. Reassessment of the Listeria monocytogenes pan-genome reveals dynamic integration hotspots and mobile genetic elements as major components of the accessory genome. BMC Genomics 14, 47 (2013).

  29. 29.

    & Analysis of comparative data using generalized estimating equations. J. Theor. Biol. 218, 175–185 (2002).

  30. 30.

    et al. A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier. Science 292, 1722–1725 (2001).

  31. 31.

    et al. Listeriolysin S, a novel peptide haemolysin associated with a subset of lineage I Listeria monocytogenes. PLoS Pathog. 4, e1000144 (2008).

  32. 32.

    et al. The role of L. monocytogenes serotype 4b gtcA in gastrointestinal listeriosis in A/J mice. Foodborne Pathog. Dis. 6, 39–48 (2009).

  33. 33.

    , , & Carbon metabolism of intracellular bacterial pathogens and possible links to virulence. Nat. Rev. Microbiol. 8, 401–412 (2010).

  34. 34.

    et al. A chromosomally integrated bacteriophage in invasive meningococci. J. Exp. Med. 201, 1905–1913 (2005).

  35. 35.

    et al. “Epidemic clones” of Listeria monocytogenes are widespread and ancient clonal groups. J. Clin. Microbiol. 51, 3770–3779 (2013).

  36. 36.

    A simple sequentially rejective multiple test procedure. Scand. J. Stat. 6, 65–70 (1979).

  37. 37.

    Teoria statistica delle classi e calcolo delle probabilità. Pubblicazioni del R Istituto Superiore di Scienze Economiche e Commerciali di Firenze 8, 3–62 (1936).

  38. 38.

    et al. Modeling human listeriosis in natural and genetically engineered animals. Nat. Protoc. 4, 799–810 (2009).

  39. 39.

    & AlienTrimmer: a tool to quickly and accurately trim off multiple short contaminant sequences from high-throughput sequencing reads. Genomics 102, 500–506 (2013).

  40. 40.

    et al. Reordering contigs of draft genomes using the Mauve aligner. Bioinformatics 25, 2071–2073 (2009).

  41. 41.

    et al. MicroScope: a platform for microbial genome annotation and comparative genomics. Database (Oxford) 2009, bap021 (2009).

  42. 42.

    et al. Organised genome dynamics in the Escherichia coli species results in highly diverse adaptive paths. PLoS Genet. 5, e1000344 (2009).

  43. 43.

    et al. The genomic diversification of the whole Acinetobacter genus: origins, mechanisms, and consequences. Genome Biol. Evol. 6, 2866–2882 (2014).

  44. 44.

    phytools: an R package for phylogenetic comparative biology (and other things). Methods Ecol. Evol. 3, 217–223 (2012).

  45. 45.

    , & New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria. Appl. Environ. Microbiol. 70, 6887–6891 (2004).

  46. 46.

    , & Tools for functional postgenomic analysis of listeria monocytogenes. Appl. Environ. Microbiol. 74, 3921–3934 (2008).

  47. 47.

    , , , & Development of multiple strain competitive index assays for Listeria monocytogenes using pIMC; a new site-specific integrative vector. BMC Microbiol. 8, 96 (2008).

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Acknowledgements

We thank C. Soto Alvarez, G. Pontdeme, T. Cantinelli and L. Diancourt for their contributions to MLST data production and analysis, and S. Roche for providing low-virulence strains for genome sequencing. We also thank D. Mornico (Center of Bioinformatics, Biostatistics and Integrative Biology of the Institut Pasteur) for his help with the submission of genome reads and assemblies. This study was funded by the Institut Pasteur, INSERM, from the French government's Investissement d'Avenir program, Laboratoire d'Excellence 'Integrative Biology of Emerging Infectious Diseases' (grant ANR-10-LABX-62-IBEID), the European Research Council (ERC), ERANET Proantilis, the Programme Hospitalier de Recherche Clinique MONALISA and the Programme de Recherche Translationnelle (PTR) ANSES–Institut Pasteur. Listeriosis surveillance in France is funded by the Institut de Veille Sanitaire (InVS) and the Institut Pasteur.

Author information

Author notes

    • Mylène M Maury
    •  & Yu-Huan Tsai

    These authors contributed equally to this work.

    • Sylvain Brisse
    •  & Marc Lecuit

    These authors jointly supervised this work.

Affiliations

  1. Institut Pasteur, Microbial Evolutionary Genomics Unit, Paris, France.

    • Mylène M Maury
    • , Marie Touchon
    • , Eduardo P C Rocha
    •  & Sylvain Brisse
  2. CNRS, UMR 3525, Paris, France.

    • Mylène M Maury
    • , Marie Touchon
    • , Eduardo P C Rocha
    •  & Sylvain Brisse
  3. Paris Diderot University, Sorbonne Paris Cité, Cellule Pasteur, Paris, France.

    • Mylène M Maury
  4. Institut Pasteur, Biology of Infection Unit, Paris, France.

    • Yu-Huan Tsai
    • , Caroline Charlier
    • , Viviane Chenal-Francisque
    • , Alexandre Leclercq
    • , Charlotte Gaultier
    • , Olivier Disson
    •  & Marc Lecuit
  5. INSERM Unit 1117, Paris, France.

    • Yu-Huan Tsai
    • , Caroline Charlier
    • , Charlotte Gaultier
    • , Olivier Disson
    •  & Marc Lecuit
  6. National Reference Centre for Listeria, Paris, France.

    • Caroline Charlier
    • , Viviane Chenal-Francisque
    • , Alexandre Leclercq
    •  & Marc Lecuit
  7. World Health Organization Collaborating Center for Listeria, Paris, France.

    • Caroline Charlier
    • , Viviane Chenal-Francisque
    • , Alexandre Leclercq
    •  & Marc Lecuit
  8. Paris Descartes University, Sorbonne Paris Cité, Institut Imagine, Necker–Enfants Malades University Hospital, Division of Infectious Diseases and Tropical Medicine, Assistance Publique–Hôpitaux de Paris (AP-HP), Paris, France.

    • Caroline Charlier
    •  & Marc Lecuit
  9. Institut Pasteur, Center of Bioinformatics, Biostatistics and Integrative Biology, Paris, France.

    • Alexis Criscuolo
  10. Paris-Est University, ANSES (Agence Nationale de Sécurité Sanitaire de l'Alimentation, de l'Environnement et du Travail), Food Safety Laboratory, Maisons-Alfort, France.

    • Sophie Roussel
    •  & Anne Brisabois

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Contributions

M.L. and S.B. conceived, supervised and directed the project. L. monocytogenes isolates were collected and characterized by A.L. in the context of French National Reference Center for Listeria activities, with the help of V.C.-F., as well as A.B. and S.R. Methods for clone identification were developed by M.M.M. and S.B. Epidemiological analyses were performed by M.M.M. and S.B. Clinical data collection and analysis was conducted by C.C. and M.L. Statistical analyses were performed by M.M.M. and E.P.C.R. Comparative genomics analyses were performed by M.M.M., M.T. and E.P.C.R. Phylogenetic analyses were performed by M.M.M., A.C. and M.T. Y.-H.T. generated mutant CC4 strains. In vivo experiments were performed by Y.-H.T., O.D. and C.G. M.M.M., S.B. and M.L. wrote the manuscript, with contributions from Y.-H.T., C.C., A.L., M.T. and E.P.C.R.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Sylvain Brisse or Marc Lecuit.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7 and Supplementary Note.

Excel files

  1. 1.

    Supplementary Table 1

    Distribution of genotypic categories in food and clinical sources.

  2. 2.

    Supplementary Table 2

    Distribution of genotypic categories in bacteremia, CNS and MN infections.

  3. 3.

    Supplementary Table 3

    Bacterial strains used for in vivo tests.

  4. 4.

    Supplementary Table 4

    Stepwise multiple regression of the parameters recorded during the in vivo experiments on the clinical frequencies of clones.

  5. 5.

    Supplementary Table 5

    Genomes used in this study.

  6. 6.

    Supplementary Table 6

    Gene families of the core genome.

  7. 7.

    Supplementary Table 7

    Virulence gene products shown in Supplementary Figure 5.

  8. 8.

    Supplementary Table 8

    Pan-genome of the 104 genomes.

  9. 9.

    Supplementary Table 9

    Correlation of the pattern of presence/absence of gene families of the pan-genome with the clinical frequency of clones.

  10. 10.

    Supplementary Table 10

    Primers used for CC4-specific PTS mutagenesis.

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DOI

https://doi.org/10.1038/ng.3501

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